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Full text of "Report of program activities"



J, 1. WUm^^ ^0, Co Jo ^m^^c aael 

Othsr laipestisatorsi mss M. M. CJullea^ Mp. A, A. .^nisrscm^ mil 
Mts, D. P. Maarsoa 

Coose3?atisg ttedtsj 3Ip„ 6, M„ Brig^, Besgaycii Si^laisg B3f^aeh„ 

Br. M. Potter, laJKxmtory of Blolt^';^ mi 

XSr. lo H, Seissbacfe, febomtor^r of Clinical 
Biodsemistryp SHI 



l^a Ys^urs ((^Isa^r yssx 1959) J 
tCtotai; 8 

Erofessiosai; 4 
<>5i&S2'S ' U«l/3 

Project Xteseripticsis 

p!iSr3iolos;i(%X.z>Ql® of essentia autri@nt£ for es^sriisia^tal asdjsslao 
To S@fii2@ the ss&tstboXic ftmctics of @er(^:ln euitriei%'t@ sssd to @tu^ 
lzit®rr!Sls,tioziisbdps s^ascog th@s@ smtk£^@sa.ts. 



aEOuats of e&eh. asitrlest kswwjs to fe© s^^uired ibjy the psrfcleaiar 
species. T^ effects of spseifie dsfiGieaeies sad isajslgmces skt©-^ 
by EB^tstur^s^it of ^:iQrsiolQ3ical} cli«!si«tl; ecd eisis^'^liri^QS^ 
ia tl£e ^2i!ssl,its tissi}@Sj,3^ @scret-a» In eoopss^tiqai ^t& 
Of otl^r i^feosrarlwel^s, tl^ relatioiMilp of smtriti^a to 



to 



«. 2 » 

Serial m>o ,„HIAMI} - I 

A. ISatsi&oMeia s^sS fusaetim of f&t-soltiba^ vltsssLaso 

lo ¥itsaaia A. ¥ltssda A elQdbol Is isstsrSfled Mrectly with 

!S&is ^®tm. Is 3^asti"^2^ SKas^eific ^tli regret to ^t^ ®cl<^ .sua 
sssy ifepacesffisct as fsSSi"fel^^il. ^elsraiisa for tls® ©sl^riS^^s^tic® ojf ■yit'SSia 

tMJScsus S3flei®i3cy o? vit^^ A. For tMs r^oa^ s^s©ci^ iietss^ 
precautioas xs®re tslaea to sseur© iMe<s©li^^d Ssfleisaeies csf ^ ^msSj. 
tfitsasla. flas tes^iass' of i^tasdu A assfieieat eMcks s&ewed seat'^rea 

eeH 2m^/^» of t^ c^rsltellKm, In YitaHisi E ^flciesj^, . l©?®© ^tse-otlG 

^i® %srMs. ta tl^ eo^Jj^. vitsasii& A and £ ^^£±el®xtm» msi^ 

Bslimlxm. &^ a low l&^X of xmsatus^t^ fal!%- aei^;> Imt so "^tia^isi 
dsv@lop @SBet3,y lil^ tl^ @t^ISBi@a;l:@d ecatx-oXs, FolXoi^is^ artdflei^ 

til© ©ggseamss^ of ®siaa•tJl'?^^ dlafe^sis, Al-^iot^ -^s© albraMaajglc^sulia 
sstlo dj-qps, -fclssre is Mttls <^^S!Sg8 ia totai aes-um proteiaic S^aetcsr. 
other ^sm cteages ia sssnaa ^^3t@iia aost Is® ia"eol^'^5. is. -^^ parecipS-totis 
•>e€nxrr<ss.ee of tSa® e^sag^, A fe-- eMftke s®eov@r spost^ssmisS^j ham%^^. 

f4artSjsr stus^ on tits effeet of eer^Ja 0is?g»lG soXveEts ea r®s-Bi?««- 
t02:y eaagfiBBs ij|gieffl.t©s t&at tbs soivests cosfeisje t?lt& iOta satgiffiB to 

vitssia S smd o<tla®r lipi&s a3?s eoaslfered to l^ das to reiosml of tfes 
sclveat fiposa th« sai^stBg amtlserthaa to a eofacte? fsaaetloa. B?©sa:^- 
ticms of c^tochs'G^ z«auc(r«s@3 ttot az<@ i^ite i^^i'v® 1^ agOssg ^n !^> 
yisactlnated Isy the aMiti^ei of toeo:^^70l or &s^ of smm^l l3®^yosylat«sd 
ecsspsKiisfi®. It is t!3£}u^t timt isihlMtors fosis ^srisig ©gisg '^ich €^S2 
fe« ^stsw^sfi l!^ tte Is^to^Lats* lipids, fess© fisaOax^s e^^ggsarfc -Jte 
▼tew ^mt tccojterol ao@s siot ]^?tleipat® aiyect3^ iia @l®cts?®a taraaspj^^^ 



° 3 - 



;&is c&s@?mti€^ a^K-s^^'^th eosB@3a2Si<;g3s eibo^ tis&t tSae e^lusi-9® 



Bo ^ist^boldeaa mS. fuz^rtisss of -^ll^ai^ B!|g enS folia @isiS. 

Sifisi^is^ in oM^ s^M.e;?@S 1^ t^ tm® of a Si©t ^sorSiS'jlis^ la 
ss^i^ii^ Slid M^ 1& i^to ¥it^£i£ %g Ms :^ ©ff€et-u^3ss tSi@ 
od^wsiticm of ito eMeks' «^fmssss ^■^ resg^st ■^ ss'^cfteia^ fat^ 
fflsli, ^(^ msistwffi^. ■ la eosrfesi^t to ■^ s^aalts ^tls <ai@t® boa?te:'« 

visasi t^ f&t eosfegst of t^ ^@t is s^ssd. i^^sff^tOy^ ^» is 
& 8^€lf is irit^Sjs Bjg-^gtMc^iss E^l^solie i^elatij^s^p is <^iieks 



C2f formissiaaglu^^isLc ©eld {JltS^) la ^^is us>ii^ '^^ i® elMlsr to 

tS^ fiBiisig as satSo S^pS^^sSati^^ ^tU ^tmla Big esa®ss tbs 
@:aes?3tiGsi @f tMs &i®ti^£@ s@tg^ilt!@ -to f@tQs?B to i^ss^^ le^ 1@-?@Z@ 
eflffix' @@^H3S'®1. ^i^^o A Si.^'ts^ si:^pI^^iS£% of isst^o^£ii@ f€^ g@@ ^^ 
closes sm issas^to't^ S^^ Sa FX^U @2^^i3tissi| tismi foUm^ ^ i^^sm^^ 

es£6sr@ti«m of ^C^:^ is ■m^'^ -m&ti&l in est^lisMa^ '^^ ^?^;@^Me ii;t@r«> 

of ■^■fe^ffllB Bjgj iSssesfi^^ Ssa l^lslssteeJs^ ^^Js^'p aisd ofsJie^s hss^'S "beta 

aM Ba3^m>). '^^m m^Lomm^ i^adch se€ tlsou^st: to Is® e@@%s^i^ foi^ 
of tins vitm^j^g ®m l3@!]isii3i^sol@«sobas:^Ss C^) &^m^m sad Sis@ti^l» 
b@2&sd@i«^sol@»co'bs&dSs C^BC) emas^^', t&3 latter @M£mM ^ tl^ 

©iBKSSt ^ well as -eltaffiiii 3ig t^Meh is Isette? ^ssa tSie I^ activity- 

of ths ^a^sat coffiatoui&i» «3®s4iJi^tSyl vlt^a^a Big. ^is ^&^tia«pxsgd4:las 
aetivity sM iS^ pot&s^ Ije sas-^srisg SKSOJ s®er®ti<aii to ^ssts^ ©f 
tbs BSC eceatgrge is »jj2f.-?2l22t to that of vltesla ^5^= 

?. Folle aeid, Sccfi pxi'«-tSi^ ess Ijs ^-estcs^ t® foSie asid assficisat 
aice'ter Ite fiiefcag^ a^aati<^ of pcP9«ai^ pesaieillia G. Bo'Si tias 
«rcK®dm esid t&e imiciXUa sfeietiss imiS. scti-sritgrs tli® effect of t&® 
iatter ms p«>Mb3iy as^aiatea. Is^ the iatestiassi flora. SStjeslae, wMch 
is sts\ietixs®i:^ siallj^ to pag^a-aaiaffllseBaoic seiS, is la^oba^Jy coaoverted 
to this eo^ptsaafi ia tlae feo^. Xa srepSseiag Sistasy foUe seid, procsdiae 
aM i^^«^sisis^easoi8 asid i^'m etml ®cti^^% alxsut mss-teath that 
of ptssrc^iailw^a^® acid. Mlc© ttet ^ra ffi®^ geveralgr teficieat 1^ 
dietas^ oEstsaicsa of folic ssiS pl^ss 0„5^ sulf®susiaiS!S is Ite diet. 



- 4 - 

Seslal 56, SIAHP - 1 

were injected with leuk^ia cellB (ia eo^p^ratioa vitSi Br. Itottar), 
Vlth elt^sr a sormsl straia or a ^f olie aeid saxtagoaist-resiatsat" 
stmin 0!f ceUss d^iOi Is fielajfed ccosi&rebly csTner «»at of eoatrd 
!3iee s^eeiidiig foUe seido ^Sbe^ x^ssul^ l@s& subpart to ^ss-lie? 
data to issdieatd tl^t i!@taS»oliGal2^ a folie aeid ^fioi^ss^ ^id the 
effects of afeaiaistsilfflg a felie aeid ^tagc^st ei?e ©sits dlffareat. 

Co Sist^i^ ^x»-^la iis^ks end saist^i^mee of &s^es fussticao 



1. ^roteia defiei@2e;f . T^ effect of ^r^oteia S^^-^tios tgooe 
t33s e@^p2ste soeeiMe osdLSaee B^t&s. &A its izidiTiSs:^ esi^jssests £a 
2St lii^er is bei&g iir^sstigated. ^smliMs^^ results^ vkma. based ost 
Uver W9t veii^t, indicate iS^t af^ar thirty €a^ a psrofcsia dgficieae;^ 
Iffo&iees osOy a sli^t deereese izi tls initial esi^^m soseisia de^e^x)'' 
ga®s©i, ao fSms^ ia tfee e3rt;od[a'c^i@<>b«^to^ms@«C3. eo^es^ a ?0^ 
of c^rt«:iffi«^ o^Sase^ a sli^t ler^s is eueei^,te»<!srt<^i»rai!@«>e 
sad a ^ Issss of acti^t^ of t^ ^jole sueeiaie asi^ssm 
HHth ^se esse^tioa of sueeiMe oosid^se; ^teebraseoC; ^id 
<^rtcK^rcse oxidase; ssv isstSsods ijsere devised for 



0. li^iXsr purified diets &^ issiSs^tified factors o 

1. Purified diel^. A Mf^il^ purified diet ia ^Meh pstiteisi Ssast 
eol^0Iate3y repilaeed bgr assizio aei^ tias dsvelciped f «r t^ guisiea 
pig6 Skis diet supports son^a growth and de^Ippisesst^ at a ra-^ 
e^ual to ttet eibtaix»d ^th a diet coataisiiig wsH Isalasced ps^yteia. 
It is expected tbat iad.6 diet ^siU Is® pirtiealar3jr ^isefuX ia dster- 
l» esMs&tialitsr of trace eXes^ts that ase^ ^ 
ia purified protsias. 



2o ^sideatified flastorsc ^Sis aSiiticsi of t^rious ^attJOi^ materials 
to a SQfatliiStie diot Goeitssiaias ade^^sate sssoissts cf slX wstTi.'Ssfts ks^^& 
to 'be required \s^ tlie ^sisea pi® c8j:@@s am i&;sQs<ov^8S3t ia growth ratSo 
ij^c goiasa pi^ oaSy tOsree to fii^ ^s old are fed ths diet eoatain<° 
isg a soiBree of tSie f^stosr for ts^ %9s@ks tbsir t^eigtst sv@r@ses eb^zt 
a? to ^ grssas Mgber t^oi t!mt of ^^ ecat^olSo ^Ms effeet ^sa 1^ 

Issr a vide -^ariet^ of z^tursa sateai^isasoo^^s^^lje, cereal^ $sM 
products. TI& tsBst c«»steat aaS riebest eams^ scmree is aXfeS^a 
?t^aetio3^tio6i ais%d at isoXatiosi of t^ aeti^^ priaeiptLe is 



Si@aifieage@ to MMQ B@8@Brcla s A i^re cosset® tssidarst^mdiss 
of tiae smtritioa, Mod^isistsarp @ad ise^^Kdisia of essential eMao acidS; 
proteiaSy 'vi.'i&ss&ts&g ndsss^^j^g f&ltisif saids^ 8Si& 'uaid^xtif ied factors ia 
different liviag orgsais^s gs& be esgected to eoataribute still ftirther 
to our &a0»2j8dse of the ro2«s of these esseatial autri^sts ia lassssiB. 
beiags. It is t^ll estahlls!3@d iSss,t tlse autritioa of ^m plisgrs a role 



- 5 - 

Serial Ho. NIAi® - 1 



ia t3^ etiolo^ of naa^ Ss^i^^j^tix^ aatd i^t^ollc cUseasss^ ^aptsia 
Basle studies in tmtsdtias said bicdl^sMsts^ cf sutrie&ts ^^ prc^Ss 



2® iiaes of 






Bart B iaduSefis Yes 



Se3?iffll lo, MIAM) - 1 



XsdiTriduffll Project Paport 
(XLeaflay Imx- 1959 



Bart B : BMorS;, A'wsasf^s^ assd Ptfeilcaticms 

Pttolicaticas other i^n E^'^?aets fe«E tMs psx>5<®cts 



Agsmioffy Bo W., ©sM S'osj, M. B« ^i'VQjc Mtagsmlsm of cfeollB© and 
Uaositol. jfetur® iS^s 1259-1260^ 1959. 

in -fcissuas &sfiei@Bt la rttamiJi S. Ifetwe ^s ll48~ia.^, 1959 « 
Bieri, Jo S.# Bri^ffiS^ a. M.j, ^ll®3?a, C. J., aaa fCK, M, Bo Spiw^o 

da^pslopsffia-b of feinsle ebie^sas for estea&d periods '^tSiesut 

vlteMa E o? etteff ©sstlo^aaat©. J. ®atrltS.083, (in pross). 



Bien, J. 0., «aad Poilaj^d^ C, J. SsFum laroteto eimags© ia vitassia E 
^ficl«mt cMc&s. J. latritioa ^s 301-^5* 1959. 

adL^e, e. M. I^iteatifled STibstosees, la "Food, ISse Yesrljook of 
Ji^rieultisr®" 162-.167, 1959 . 

i&lggSi, S, M. Sutarttlc®?. m& aiseaeses Folic aeia studies ia ti^ 
!28S5us©„ Jtoc J„ dlB. Sutaritica Js 390-396^ 1959. 

a^^sfeaI3.j, R, E., aad Bissrf., J. G. Batfeology of tlje hr&dsx tst aissple 
m^ saLsssd deficiaieies of vitalm A amia S ia tte daick. J. 
Sutritioa (ia jaresB). 



Fox, Mr, R, %iwy;, sM Iri^gs, S. M, Effect of dietery lactose tiposa 
eMcte fed a puyifted ai®t, Poultiy Sci. ^s 96i^»96!8, 1959. 

FoK, M,. B. SpiiP^jp mid MiefeelTOa, 0« Salt Bdxfeasss for purifi@d- 
t^rpe diets, X. Effect of salts in ®e©el®snatiBg oKidative 
rsaaciditgr. J. IStrtxitioaa f£8 123-13^;, 1959- 

RsaSj M, B. Spivej, and Mlc3a©le.@a, 0» Salt s3El;stU2?®s tat purifl@d« 

t§tM &lmt&. IX o Effect of salts m. th® S&illaM hnmdm 
reactim. J. latsltioa ffls £69-.g96, 1959. 

Fox, M. R. Spi?®y, CBPtiz, L. 0.^ ead Briggss, G. M. Sa® «ff«ct of 
disktst;^ f&^ on vi'^M^ Bi2'*^^'^^^^3^i^ intdxrolSitiosiBliiipSo ^, 

SSatritiosi^s 3Tl-3Slj 1959- 

Bsiaas-dj, C. J., fimd Bierijf J. G. flis festructieaa of vitaato A Isj 
Mood. Brit. J. mitH-tioa ^s 359-3^^ 1958, 



Serial Ho. NLftM) - I 

'PoUaxiLg C> Jo, aad Bi®ri, J, ©. C5a tlxe oeei:mTenee of Titassia A 

alfieilss^ ija fish sad feog c?m. BiocMm. BiouSs^. Acta Us 558" 
559. 1959. 

PollaM, Co J,, «sad Bi«ri^ J„ 6. Furt&sr obs«rTO.ti<aas ;^ tte effect 
of isooctaa® oa respijatosy essyE^s. Jo Biol.. CSj®m, £3^s 2907- 
19n, 1959» 

Bollard^ C. *F., aad Bieslj J. S, StuSi^s os& tto Mologiml ftmetioa 

of vitsEELa E„ I, fbeojtorol aa& reSucsd ai:^3a39]^$^iiai!a® JsuclsotiSe-- 

PoJJLwd, C. J., asd Mea.*!, J, S. J^tesflUcatioa of vi:\msdn &. 1^ ®s®t<^ie 
poifiSss* firosst p33£C£^sss« .Arabia Blocbeia» Biopl^srSo (ixi pv^ss). 



Pollardj Co J,, aad Bierij ^. S. ©feudies of t&s biological fasictioa 

csf vit^dn S. n. ®ts jmture of t&s speoific ®eti"s?atissg ®ff®«t 
of too<^3erol in a@e& pretm^ticas of Gytooiaxsm reSuctss^Sc 



r 



J. Blol« Cte®, (in 

Keid^ H, E„ CSwtasa pig smtritioao Eroc, i^aiml Cas« r&rol 8? 23-33,. 
1958. 

B&ia.. Mo E., and l^ai;!:^; M. Q, Slul^itioQsa studies vith Urn goiism pig. 
V» Effects of deficiaacy of fat oar vossatm^teS fatty ®.cifls» J. 
Bfutritioa ^s 6ll-^, 1959 « 

Sxsaef^a emd Avm^ r@latisig to tMe px>o^eet3 

Dto John Q» Bieri i&ee@pteS a FuHbrigJit Award ^4iieh eovsrs ts^velisis 
e3£3peas©8 t© Cop^ategetij, XtezsMrk^ to voTk ^rlth ^?of , Ssarik Ifesa, ^c^>®l 
^arlge wjtaier cmd world autisorL-^ in the "bioehfflalstsy @S£d satritic^ of 

vitssssiiis in ths DcpBX'tsBSHt of BiocJissBlsts^ s^d ^jts^tloi&j Pol^tsoh&ic 

XtlStltutGv 



2. Hutritioa 

3. Betlissds 



Calendar Tear 1959 



fSssrfc At 



Fro jest Titles Ma&etes— Eff©<gt of bsnaossss cm ^rte1»clS.saa of 
fat mid capboi^^'ate. 

arlafiipal toTOSti®atojrss E?s. B. 0. Seo*? ©isd S. S. Ossnaiek 






Totiali 5-1/3 

ProfesBiomlj S 
Ofcfeers 3-1/3 

I^^ct Ztescr£pti<m; 

ObjactiTcs s ¥0 ^^terMss tM iaflueace of hssswmal &&& other 
ftotosrd osi tlie i^tabollsm of fat s^d am^»o3:^t!rat@ isi no3nE@l asd 



l^limds are treated ^th vai^ous boansiesjits. ^3i& effs^cts 
of esstjixpatioa azad honiecisal sdffiiaietriatioi& are studied is, .yjw aod 
In vitro tuslstg cossvCTitiaml <md Isotopic tdchni^tiss. 



fejpy FXadtogs g 2te stu^y of tte !so3?ss«ai»8 involired to tJis 
de^lcpannt of die^tlc ketosie Ims basxi co-atimied. Last j^m- it 
vsfi reported timt ia p£mci''®@.t@ctmil!^& rats ^prlved of ■UiQ pituitszy 
or thet adz-^sa^ the o&ji^ torac^us is.eeSeS for tlss ^^^lopamut of I^tosla 
^^leai i!3suliQ yma vithhsld va3 a glucocorticoid. (Ss^o^th hsxmsmt Issd 
oo effect mi& MSSE vans ketossnlc &xl^ if tiie adr^ia.1 gl&odls vere 
intact. In tsubs^taasit esp®rliisente; de^aststhascme hs^ h®en vsb®&. &b th^ 
glucocorticoid ijecBus® it pro^eaa the sajss effects lae eorfeisoa© ead 
oaO^ 01^ thousmidth as midh. xb nse^d. T^ isiniiml @ffectii^ doe^i of 
ia & 150 i®. 2?a.t ia 1 Mis. p«r degr. 



' ^^ ketog^ic actio:^ of Ssi3Cgm»t&^0!3!& is seesi imssM&ti^ 
&ft€»r injdetiosii ia iasuli^ dsficlimt rata bi^pqpl^risectoMised for oas 
to two hours . Bcnusmsr ti^ i^to^Biet effect is dslajsredy &t Isast 



.=. 2 » 

S«rial Bb. miamd - 2 

2k hsisrsg ia a»limXs !^x»p£^fs@GtOB2i2@& for cn^r e v^sTs. astd gaint6isi@d 
-with ifflsuXig& and tub@»f@$diag up to IT hm^^ b®for® gi'viBg SsisxBaatlmsGsii}. 
If growth haewsm is giwa ^tU aaxBBEfetisasoui® to t!j® iBtt©? aMmls 
kstosis Sevsljops at <ssce; s?o»tl& bermcgis aIoa» £®s ao effect. In 
diabetics l^n^i^^saeto^zad for cs^ to ttjo hours^ @?(»itSi hosnasaste givisa. 
alosi® or witli ^saLl doses of desssa^-atescsss (0.5 fj^.) Sas *» appfeci^le 
sffsct cm ketcaje bckl^ foy^itiosa» AdMaistratlea of iasuMa with 2.5 
ng. of tesaiBBthas<ase d@lays the oas^t of ketoeis for at Ijeast sevea 
hours. If growth bori^sne is also gi-vesi kstosis daireiops. Se^i^ez^ 
6be®rmtiOKis soesi to i&aicate tli&t g^'otrth borsxme acts as a ket<^^&ic 
agent ibj ioMblting Insulin ^otivil^ but tMs @eti<m p«eur@ osq34^ ^iriben 
the insulin c<mtes3it Is lo^» lisaaurdB^sits of tlte imt® of ^to^sss&sts 
in n^tsr slices aod of th@ li^r fls,t ecatmt in t^ sbove iygisiiOs 
9ugS88t8 tl^t the actiCBi of glucoeorticoiSa emfi tlis anti«inmilin 
scti'^i^ of gpfo^ii'tls^ hos^s^^sS; In tl£@ producti^^ of kstosiS; ocsurs 
prlraria^ la adipose tissue o E^pssrlswits ©x® being as6igs®e to test 

Attn J! ,oa lMiHak.A.>M«.<aBln.(*^A«^ <ni 

tnjl.3 l3QP|iQ^tra@SlS o 

^ne: x%t@ of Tssto^smssia in liii^Eira of diabetic and nastml sutCf 
stuMed ^th slic@8 i^d yfi.m tlss |)«pfused qs^S; laas bean found to 
■be (sloml^ i^lat©S to tS^ assoisit of aaaphospfeolipia. flat in ths li*TOr» 
For a si-v«a fat content tfeare is no dlffereac® in tte rat® of ket©« 
®S3SjS3is betuissn ths nonml and dis^tic ;mt. Tl3s x^te of I^togenesis 
was t!^ same igfestimr- it vm tSsteraiiaea m liv®r slices or in the 
perfused or^». Ifetlgr liimrs of pancreateet«MBi2s®a. 2®ts, perfused 
for at li^tst tlaree bosrs^ asOse 1.6 Big. of ketoae bc^ee (ra^isured as 
a<^t«aa®) ^&s gama of livisr eaela iiour.. Additicsa of regulir iasuliai, 
36 Tjnits in 60 al. of perfusate, had so effsct on ketcaoe bo^ for^« 
ti«m or on tiss glwcoee content of ti^ perfusate. Ute perfueat© at 
the aad of tte runs hsd at least 50^ ©:? tijs insvOln addud, 

T5s® E^taboliSEi of fat by liver is being studied in tiss per5^2S@^. 
organ. ^3@ findings to ^te, using radioactive tripalMtln^ indi(^,te 
tMt tcciglyoeri^s ar^ reaSil^ -^&!@n isp 2»:id incorporated into ^Bos^to- 
lipiSs; ketdse bodies, and carbon dio^ds. fteere is evidanee tiist." 
x^dii^etivs £^t otter tS^n tripsJiQitin is l^ing seereted into tlae 
blood. 

®i© effect of iBSuiia and its Inek on utilisatioaoi of tetoa© bodies 
has been studied in dietetic z^ts Imving a ver^ l^^r xiate of enSo^enous 
Isetogaaesis. ^^le rats vere depleted of bodr ^^t "^ pralos^d fasting 
prior to p&ncreeitect<%^. Utilisation ^^as studied 1^ s^asuring tlie Sisap- 
pesraac© of laetoae bodies from the bieod f olloiJiag a single ixitraveaou® 
inJeeticM of eitiaer D(")~"b«tai®rtoox^b^!.t:prate or aeetoacetate. Ufcilias* 
ti«m of betafegrdpa^butgFJ^ta is reduced 6o^ ia inmxlia def icleat rats 
end c&n be resuSily restored to ncs^s^l "b^ insulin siicbiKinistratiQn. 
Acetoaeetate ntiUaation is also isiiaired to the diabetic rat but to 



- 3 - 

Serf.®! Ito» MIAMD 



la (xmtrast to t&s ^s&sr^&ZJj accepted vle^ timt iasmlia M» so e£f act 

QQ I^tOdS bO^ Utm^&tlOSl. ^SS studies iXl ^^& j^aC3?<S8tGCt(^l2€Sd SlSlt 

sls^v tl@^t &gr]^f!^t<:m»&sla, or ketosie^ la iBStOia ^tici&s^ is tl^ 
r@3u3.t of bot^ istc^'@as®d pHsSuctiosK Sisd Ssss^^tised utlli.S3^tloE of 

^toias bodies. 



S® fBBtisi^ Isstosis of pne^E^sscy ija r®.ts also aj^sars to T:® 'Ws.® 
result of mx isssiaHa Jtoak ia tl:® tissu®s, ^0 IsetosiSy vMeii to^slop® 
<aal§r SuriE@ tfe© last t3»@® ^^ of pr®®^s«^, is rssifiiisr assrected l5j 
saall sMmats of isxmOia «3? Oxrijiffi®®, ISsese rats are -mgy s®asitiYe 
to Jasulia ead Ortease, Bos©s of Orismse iiMeh Imd -rexy Mt'U.® ©ff®et 
OK tins blood sta^r of ]ios3;^'@g;i^£t asHsssils ■!^^c^a&&. amf&^e l^po^^^ssds. 
imd disath in th3 prspimst rats. It vould s^^ tlmt tisa M^ priority 
of t^ fetu^s for gkaoom causes tl^ blood glucose co»ceatratl<^ lis 
t&s f^tSag aottess" to nEs^ia at a l©v©l too low to stjtoilate seci^stioss 
of iiigulin. Stoll aaMJUKts of gluccse ©iicsM^ cor^rect tfes laetosiSo 

Zs^t ^ssc it V8S r^pcs^tsS t^t ^ucosortlcolSs @ii3 gron^t^ ^SE^si^iii^ 
gl^@2k Bissgl^fV&ve aot k@to@saic l£i ^^pl!^B®ct€3Sd2@d f^tlag pr@^@£it 
r&ts. It £ies aov''b@®a obser^S tl^t ^®ffi tbs borsKMaass aa?« gi^if^aa to^t^^sr 
s@v®2% Isi@tosi8 issasdiately Sr^lopSc ^Shsss fisSisigs ez% Ha ag^resma^t 

with ti»9S@ la tM l;^pQ^:j^ei€!etOEaiS!s@d°psiies?^i.t@ctos^^S r@.t giv®si ijmy 
of IsisuHjIo 



JDi^poBiticm i^d s^et^rbioa of 3;^MocaIeitaa and f Itioaride ia th© 
lioie of @roi^^ v®.tB Mma stuped, with tli@ coUabos^tiaei of D?@. B« 
C. MMas aiad I, Mjpklsa of ti^ ECBB. ^ issasytiag a stsal pia isto 
tlie tiM£ mS. taMsg serial romtgeao^rsss; it -ms possible at th@ 
ead of ties ©agsrisisntal psrlod to divid© «1^ tibia iato sssveafal 
8«p@sits for ©.mljiseso Oo® of t^ss sstst Important aiid intss^stiBg 
fiaais^ of tMs stu^ isa© that imch of tlie Ca^5 @M F r®lssa®d dus-- 
lag rsffisffls^lijag of t!j« ejsds of tb® grovriag bos^ did aot enter tfe© 
gjassral cireuSstioa but, ^^s ^Ispc^itea ia tSe laasadiste vlc^ni-^c 

of fiat 'm&oUM's&m S1i^i^'°^llltus sroy be reapoosibl® f os* 
ths esrljr esaset of imseM.ljar <feg©rz^trat:l<m ®s©oeiat®d witJi tMs d3,is©as@„ 
Bjs gmture of tlsa cSisttK.'teac®© in lipid His"te3boiiS!iB is poorJ^ tssiderstood.-, 
S3sE( sM®f acti'slily of tl33.s ^arotiect lias bsea dirseted to -Kte stu% of 
«68B'2j effects of iJiumllja defici«mc^ «a fat laetabolissi, aassal^, aKjMiiisa- 
tl<m of fat frosa adiposci tissts© to tlie liver^ its cosvsraiosa to lsgt<s^ 
bodi@s @ad tteir ■utiliafitloa "^ paris&issuL tieaissa . "Sim pricey 
l»»r@3@i3s iimral-uM 3mv@ l>3e»L i^ars cle^rl^ defisied. Studies issing 
isol&t@d e^^ans ®ad tiB&asia mr& in pro^c^ss to elucidate pa^ si-U 
fijBd asode of ®ctioa ©f ti-ass® Ijostksjss, 

gmpos®d Cy ayg of ggo^eetg Ste stud^ of feepitic ssst^3>olisai 
of fat ii& i^ psrfiisos cs^aa ei^lo^lsig ^radioisotopic tccbssi^vnErs ^?111 
bs ©srt^m^d to d©tsyjaiaKS tbe iats-^yjelluasr sit® of s^ta&olism of 



„ 4 -■ 

Se^i&l m>. KIAMK - 2 

t^^)4rc@ri^s sssaS. msasteyifiefi fatl:^ acids, tJte s$q,\3msc« of events 
leaSizig to tl^ir iscorporaticm into ketou® bodies eM iilxd ssatur® of 
the fat b®iag ssereted into i^ 'blood (perftisat©). At aii early 
date^ -i^ effect of izasulin as&d *aaip6kis@tic ' honaooes oa ttsese 
phesoBSSisa win be iawsti@ited. A lagthod for pea?fuisissg isolated 
adipose tlBSQ^ ±& beissg i^v@lopsS. 'Bd.s teclmlqius vill be t2sed to 
ataiS^ t3£s effect of h<xsmss^B q& tbs uptake ^sd tl^ T@l^^se of f^t^, 
israsel;^ ua©6t9rifi@& :^tt^ sei^ sM tsfiglycssldes. ^ vivo ex^sl° 
sisats wLU. b® ccatissuied to detsnsia© the rol^ of growtsTE^aE^ is 
the distusfbed asst^boliem of fs&t in tite diabetic o 



itert B iacludfidg Yes 



Serial lOo NAXTO, ■:,„2,_..^.,.,_.. 
PHS-SIH 
CWLen&sr yeap 1959 
Part Bs loaors, AwsrdS;, e^isd Piiblic&tio&s 
Publicsatleajs other ttoa abstrset® tress, this pro^cts 

1. CStemickj S. S., aad Scow, H. 0. Sferly ef:fect« of "total'' 
pasep^tect?^^ cm fat B»taJ>olisa la the rmt. te. J. Hacfsiol. 
Ig6! 2J25-131, 2559. 

2. Sccw, B. 0», CterMck, S. S.^ aad Ouarco, B. A.j Sfe-fco^mic 
aetiosi of pitwltayy aad edr^cssl Isosracmes iaa p®jer^,tectoKls®d 
rate, J51^bet®8 8? iSSt-lkSf 1959« 

3. Scow, B. 0. Effect of gro^Jth hgxassasss and tls^rosdae ost groiftli 
mkd Qhsmi&&l eoa^sitioa of muscl®;, haas, and otiasr tissues is 
tl^5?3roiaect<S!da®d-&ypopJ3gn5ect<23ilg®<a rats. to. J. Jl^iolo i^g 
859-865. 1959. 

4» Id.kis^;p R. C, Scov^ R. 0,, ZipkisSs I., ®sxd Ste®y«, A. C. 
ISftposititaj a&d retcmtioQ of fltiospia® aM raaiocalelxjss te tfea 
safowlag yat. Abj, J. Hsyloi, 3^: tJ-So, 1959^ 

5. Sco!/; B. 0. F&t s^tstbolies in eseperisssffi^kl diaSsstae, in 
''Ei?ogress in Clinical Saaoeriaolc^''* edited by E» Bo Ast??«^df 
(Irvaae emd Str^tt<»&, liew York (iM piress) o 

6, Scc/tr, R, 0., ood (SasrEiels, S. S, Bos^saosaal coatrol of protsia 
&nd f&t aetaixilieffl in tbs pgtacr^itectasBised 7e,t« I^ceat 
Srogrees jLa Exeaaas Bsss&rcii 16; , 19^> ('Exis ^per 
was B3?ese3sted st tli© 1959 Isuroatiaa Bostssma Coaferesaeeo) 



Icaiors ^id At^sTds relatistg to tMs pxi^sc'&i 



S«riai lo. miamd - 3 
3. Bethes^ 



JndXvldmX Project Bepart 



E&rt A« 



Rrojeet TitJ^s Stradies oa ^ra-fyee sad obseo aMisaa^. 

PrlacipO. Xffivasti^torsg SJrs, F. S. Baft, B. S. ©sstafsscm, 

0. mckels«a, Z. M. T&l®ay, B, So 

of Batliolo^ 81^ HistoGtosists^y IXAMD 

l^a f@ars (csJ^endaa' yesr 1959) s 
Soia&ls 3^ 

Otiiers 8 

Cfojeeti-y^s ? To ^teareda© tb® sutriti<s^ly feiochiSEslealf mid 
^bgrslolQ^cai (^mrsctezlstics of ^t£m-fr@e sM^Xs; to stu^ tbs 
pfa^iologi<^l and bioetssMcal cima^^ associated vd.ish cSaesil^ in 
esq^szlassatsa aaixsalsj -to stuc^ espeyiiseatail^ a ccsa8d.ti.<m (Mlsa^ita 
disease) asJsociatsd witia th« iagesfcioj of sea fooS fycsa a special 
erea ia J^a.^^; to 3tu%- tJ^s zzutriticoel T&qvdTsasmtts of raS»'&its. 

Metbods B igplo|yed2 Gana-free sats sM guimsa pigs are ^a1^tlE®d 

OQ diets of kwtwa eca^iiticsi ia st®ril» -^sls©, Tfeeir gposrtilij Mood 
^ctu3?@^ syaS otlSier pla^iolcgical fuactlcass m^ studied at vssrious tiMss 
®f1«sr tbsy Imw beea started oa tte spgcial dl®tSo @a:m-fip©® esoia^ls 
are ijaoeula.t©d with siagle stsssijas of bactssl«m to 6&ti&mism tte 
or^^aas x^spossibie far various phencGBssa s©sa in ccareieatiiCKsal 
asainsuLs. 



^e urins fr^sn v&t& iib&t bad 1b®«3i imde obese ^ f«edi)Q^ a M^ 
fat diet vas eaaarined for a -variety of proteins. Ste iaflxieacs of di@tai^ 
altd£%ticms on tbe e!«i3;°©tioa of tbese prol^eiae veB studied <> 



« 2 - 

S©riBi IJOo tilAMD " 3 

Gats asd chicks Jmm "bsesa fe& diets tc ¥Mcb 'Hex« asd^deS & 
of ses^gileB of ^apazDSse s@a fo«^c ^lis i»s^ food teas 8ee»x«d trsm. tte 

ffisd ori^mfi of the asilfflials ¥©r® ©sBaisaisa MstoXogimiS^ ashS scssss c^gaas 



5au?e® v©®l£ old rabbits hm® Ts«sea f«d parified dlsts feileisat la 
thiasaim with eszS vittoist a thissaiis© saatagcmist. Orlas m& fseal 
easiples bavo bsen tested for tM@ffilsi@ tb?ou^bcfut tha liwa of iOae snimle 
8&a vbi^ tha aaiBsOs m«re sacrifice, tMawisiis smJijma ^®r& ^orleS 
out OQ bs^iaj liver, cooal eoatesitej, esi& a sius^b)®r of ot2£@r tissues « 

lo G®rsB»f^r-ee ^aimOs. Pnelisdmafy results indicate timt sasitljsr 
8ffitiMotic8 sor TlteMB C hs& ats^ ■^It^oiB-spsrixig effect la ^@£m»fm® 
rats suggestiog tli&t -Uj^ action obsertmd la th@ caE£<?@ntiox^l aaiatsXs 
is nadiatsd ttoou^ the flom ia tte ^stroiatestlsal tract. ^Jtea 
geria^free jrats ■wore tetea out of tls© l^als: essd eontasainated, tiagy Sid 
aot iihov a paatot&eiiic acid'-spas'iag effect &v&n. thou^ ths ration ^^®s 
suKPleaeated vith eitfesr x^aiclllia or vitsnin C. Efforts as?® bsissg 
m^ to dstrnteism f^^tfmr tM lack o:^ r^spesmm ia tl»e sso^trm^^rtse 
asisals tJias ©ttsHfiitable to iacOT^a®te coataiatBatlott of th® rat wltij, 
all of the fegact®rla aora»Xly fam& in tJie eoawmtioaial rats' ^ustro- 
iat®Bti»®l tspffict or vte&^mr th©r» m-e isijgraiologricsl ttffersaees 
tSss co£):v@atioa!M3i @ad eX'»S!@rst»fr®3 ratSo 



ConveatiossaX ^ixLosa. pigs ^jiiich Imve ssisnmr beea auz^ad; @:^»r poorly 
for ths first fmr wssfes of lif© wtssa fed a "ccassajete* ratios ^^t 
B£Po*.je<6S essceUeat growa la guiijaa piige ttot Mw Ijeea left with 
their oothers for two or tto-e© 3a|^, Work ie la ps^jgrsss to dsteiMa© 
tia& fiactos- or fsctcs?8 iss<§,utg^^ tj tbs uasH^iaiea gutesa pig. T«ro litters 
of ®s2ris^fr@@ guisM^ pig@ Imvie Isstm bora ia tte starile tmi&s. ^te first 
llttar of two pi@5 Sled three weIss csf t@r hlrth followlag -ab&t ajpspmrea 
to ba mmmX ssecfur^ m& dmi®lopj^it, Autoip^ showed ys&lom ara^^stiv® 
of a Td-lMJia K d©flci«iscy. ©is diet for- th® secca&a litter of ^jres 
pigs is 'beiag ©uspteMatsd \ilth @ ec®spl®t® vit^sfja M5Etere» 

A darvlce hBS heea perfected vMch peevasats copropte^ ia coBveatioml 
rats, 9Ms consists of a li^t"Wsl^^ plewstic tals® ishidb fits oror th» 
tall of tb<s rat sad ia vhlda the fec^s are eollectado Wim. mts «wre 
fitted with this d^vice^ th^ d»9elop a folic acid dsf icismey m diets 
that pexmlta SKsrsial gr(%^th wd blood pictures ia smts th&t caa coasu2»s 
their fise®0. ®re( rats ^itfe t&ese '*tail cups" pass f@c?es "^Mch a|?paar 
to Isawa a diffes-eat h®et«lols®l<ml flc«a ttaa rata aofc so fitted. 
gte Xaeto^illi comt ia th© f©e®s of the tsdl-cxipped rats -ms oa3^ 
0.1^^ tlb© 'msS&sr present "before tba teil»cu]^* was' eppliedo 5Hiis fiad- 
ia® subsets that the amrelopsEsat of a vltmia def icieacsf ia the tail» 



- 3 - 



traet. ftee ori{{iaBl. oltsereatiests euggssted t&at t2&^ aefleiesK^ ms 
autt to the ioaibiUI^ of tlse svt to i&gsst its feees m& that the 
vitffiBias 8gnitb@8is@a ia tSe gftstroiistestSiutl trace visre utilised cel^ 
after tbs f @e93 ti@re 



(3srs»-£i?ee rats davelsp <i Vitassm S Sefieieae^ ^th 

asA 100^ isortalitgr vitMa 30 ^i^a aftex* lieies sti^rted as 
a diet defieitnt ia tl^t -AtgsB&SL. Oom^satioml mta fed tSss @a@ di@t 
shoir ito si^as of a def ieleocisrc 1^ sesne-fsr®® TitfcMa E aefiislejit mts 
eouM 1» eured la 2^ bmsr® %^ '«@rlo^zs vitSBsia K cos^omsds or Isgr i£ioa2li^<x^ 
tioa of this gistsroiatestij^&l ts®si^ vith s siJBgls sp8«i«s of teete^iim 
v!k£@Si ^s&& 1}#^& isfolbiitod tstsat cossvoistlooiiX x%t@« ^tiis eSs£@ih?stloa 
su£gp9Sts l^t ^taisia S is mSis &im&liSs>lm to tlM; eQss««siti«sig& ?%t 
]^fl]@3i^X^ ^T osii® strs^ia of 'bsistfipi'tsBio . 



Si» suTvi-val tisis of vltisiaia % dgfioiast ^sg:^Sx^ stits i^ms 
aigoifieaatly 8bort@a»d \^ iass<^»si»s tSse f&t eoanst of tOxe dist. 
It feas te«a Ssotm for b^b©' sr®^^ that a vitsaaSa S a©f ieiaasy €«m "o@ 
pro&2®@d la eoavsatiOBSi^ rats Iby s^SSiag & vmije^ of eoXf cm^iiaiss to 
tha diet. It lias eesusssd tls&t tbe lAttea- coei^OQads wr@ sroSueiag 
tbe d@flei€9s@y \s^ eh^igi]^ tiie iate®tiaal fXes^o ^jbrls ia tMs Isbomtos^ 
abovs that sulf!»smaosBllis® <(dM9& @3Sed to a vit^sia S«fr@s (Si®t s^^oa^g^s 
a ^sesmsTliatsle diathesis ia ger!S»f^r@@ rats ia tiio Ti@eks «M2@ tli@ sulf aE^» 
iaide«f2!«@ di@t proSno@0 t&e 6@fi£gi eH^tet ia fcfur ysoelsB^ Tim pres^it 
ml&ms^ sug^ats timt Siulf ^:i3ias33^(li2^ ng^ set as @ 'tltsaada S 



tSrix^txy oileull dsvialop ia 50^ of t3se gsesm-fefKi asJbe rats -s^eh 
is ^soeiated vith 1^ ^aeerstioa of Xsr@s assfosts of oasOle susido 
Otissr ^jQ3^ has ^b&m that a ssrsldsodas Ssfloieat^ iasssesa&e, tli» «^er@« 
tioa of oafsslie @eid„ Xs^ the jjex^ii^fira® easd ia the tail<»o«^^ esem<m^ 
tiQ8^ smts^ a 3^^i&9si&!8 Sefieirasear do©e aot isssr^^e the iaoi&sass 
of weisiss^ esIeuXi. 

S, Chasit^o SerlsJ. eareass m^3i^me of t^ rats 4^' high f^% 
diets ehoif tiat these rats freas tlH» ^gisssidBg cf i3m dietazy r^sa^ 
hsv@ a higher f&t ecatee^ s»soci&t@d vith their ssore rs^d mt@ of 
growth. OstwsrdSy, '^te@s@ asaii@sls ^g]^^ to bs siisll^ to older r®.%& 
of -i^ s^ae ho^ «el|^ ji^iatsaiaed oa the Imr fiat' di®t. Tlxst Imsmm 
ia fl^t ism foimd &8 esKTly riis fiife ■msks m. tiss di@t. 



of iHm oijee® 3%ts iisre X^r^^sr tSs^ai the^se ia 1^ 
ia spite of thiSj tite eoaseatrstioa of i^ter^ jp^'oteia^ 
tlse sssse la the ti^ gs^es^pso f^se ooBceats^tioBi of tots^ 
•ms^ hii^r la the liwrs of the oihese nste. "Bob ki&ss^ 
sod hearts la the <^»ese rate «@re Xssiger t^ia tlxMse ia the Imu 
ccBBiiseols ts^ the ]^^xl£^te eo^poe>iti<^ ^ss esseatisl. '^le sssias ia ^o^u 




Ssrial 186. wiamp - 3 

f&t tS^a tl^t of t^ leaa asiimls, Sha vei^ts of the a&nsml 
0m^ ia. th@ obe@@ mU. vor® aSsout I.5 tlises msa& in ths lesa mtSo 

ssutraX) ssd ifltaimin Co ^l^s eatsss&ia^bicsk of sStos^oli^S ^sd el^slssterol 



Ss© oba: s y@ts had © slomss' rgjaovssl XBte for ia-toav^aously ia^ectsfi 
gIticose»«>TOS©ji9ai28ig ai^:^ties» !Ss© coiJC®at3?aticHi of -sit^ffilsa Bjjg isi 
pl^sm m& Hv&m of sjbess sats ws^' gres'ser tMa that ia lesaa coa-ferolB 
ev^ -y^erugh Siet^y e(ms@a.tf@.t£os;® of tMs -vltgiaisi ^?@r@ tlis s@bb»„ 

til® ro^uianssirait far vitaala ^i^, ©t l^ast ia -^ss eM^, is iac3e®@s«a 
"bsr a M^ fat diet, ^x@ eS^oirpticm of sa emX Sesss of -^itaiaiii B^js 
ms sreete? i& ^^^ db@s@ tl^i in ^se l@®a z^tSo !e&e €iff@r^iee is 
s(bsai^io& ij@s sob Sue i^sti7@2^ to l^s SjLff^'^Eices is f@,t aout@&t of 
thj» Mets silsos @iult mt& t!mt lm& b®en f @a 1t^ M^ £^t diiet (vMoh 
proSuesd ti^ dsssi^) fop se-ws^l weeks alaeswed t!^ sgsse ^sorptism 
of vit^siB 3]^ &s ^:^ le^ti xats. 



irsts -^^ in t!^ Issm. £^^ fas^Xe z^ts e^ix-@t€ -^exy little s^otai^i 
tSse ©fess© f^2Si© rats do, !Si® iidtei^fy protsiss -msQ a®io2y alSsuasia 
ssd ^«glo'bulisi i^icli diffex^ fsra^ otlss^ fo£?ss cf pirat@i{!u«^& ^^es^ 
^LX iilis s«ruia prateiBs ^rs p^essoto ^!Sij@ d^^'se of p?ot@i£i\2S^@ ia ho^ 

tl^ ol3®se aM tl^ l&m rats ^sss ^os-soaed Issr the pi^seac© ia the diet 
of 15^ of ^ i^rt fill©? (Sol^^floe). 



Altliou^i tiss Sga?©©j®.»lfes?aj^ ®KdllIS sa-fess "bosoi^ dSiss© vixm. f©fi 
a M^ f@t Sietj, a strain (S^/H) ssY9Sue@d t^ eswssis^ tOse t(?o does 
aot ^e<»^ ob©se<. TSsg S^B/^ s®ts eaa aeeisBtely regulate thei^ es3jOTie 
istalse s^^cdless of tl%g f^t soatsat of tb@ di«tp 

3 a Hs&'bitSs Xt tes %>@(ssi z^po£'t@d i^b^t v@l)ibits! do ^ot d@v@lop 
a thismifift ^ttei&asy ^^mx^bl^ sissce tlie;^ emsmB their '^M^f o? 
soft f@e®s tfflsiela coiat^isa l@r@s ssajisisfes ©f vitsaaias. ^laea tinges to 
four v&dk old j^bMts we?® plae@d <m a tM.affiiBB«.free «U,©t, ti*^ 
giaioed ^'®i^t &t sS^out tl3@ 0@agi ?^t@ as tl^l? st;g^@lssss&-bed c^itrols 
lyut ©Kffipetsd coMiferalJly sasHey s^mats of tbe -wit^gda ia t^eir 
^ariaej t^je csoaiceatimticaa of the vitsato ia tfe© fe^ss aud li-ros^ of tlie 
"deficisat" gsftsup ^ras leas th^i tmt la the c«srteols ^a^^re^e tb« c©e^ 
e<mt^2,t8 h&d t^ sssEs ecsK^stx^tiea of tMss^iie, ^ f^r, fcws° out 
of ei^t 3?^Mts isalatMi^d 00 t3» dsf ici®at di^ for cas® to tivo Ismdyed 
dagrs Ssrcel£);^»d atesia ts^ss'^s msm oa tli@ 8usspl@i^Eit@d diet did so. 
M«sa & thlCTis!© 3tit®s!i3£il@t %!^ glvm, tbs f^Mts m t^ dafieieat diet 
shos^d ^f's^t®? '^i^t losses &S.& s^^uix'sd ®l Xs^i^s? ]^?icd oS tiss® f c^ 
r@cov@^ tb^i thos@ CS& tbs si^plssi^sitsd di@t« 



» 5 « 

Serial lo. hiamd 



ko mjssas&i^ dis^s®, ^^len Br. Leocard BarO^od (ISXH!S) ^ms la 
ijiatpsn a mmfiSer of ^®^:'s sgo^ h@ lisvesti^ted a lo(»Qlaed epi&sialc 
of & peculiar sieurolo^cal disease o See food fram tJ^ ad^oiaing 
l^r ^^s iEiplicated as tise caus® since all vlctlsss t^st® fisheriaexi or 
ate s^^ food caugjit Ts^ a ^as^r of the f^giaily. i^ts ^ad birds in t32@ 
area i^ere repctrted to bIssjw tbs s^s@ ctmditicsi. ffc® s^ food brougjit 
back "by Dr. EJjylead was fed to eats, Ti^ developed sever® sieiiroiogieal 
disturtsaaces essosiat^ •te'ltli passal^is of tlie Msjd Hssass imd ccstmasi-ve 
seiEureSo \fe ba;v® secured evicbaee tJmt ths toxicity of tfe© si» fcxjd 
is coasider^lgr Isss ca a bars® !!igs,t regiss^ t^^m cm a -vege'^ble stoc-k-» 
ndlk diet. J^j old cdsieks ar© mxdh nsare s^asltive to tJas toKici-^ 
tbasx «tfeSo Future vrark wiH tse dcs^ witii "^se cMckSo 

Tiae JiEc^^sse reported tlsst & m/xvary ^,ta3^t in tl% ^fflueot fr<^ 
a vijagrl ^Lastics pl^aat oa the edg® of tbs "l^igr vss respoaBible for tl^ 
i^yn^ptdsio AH^JOugk ve ®st seisroloslcal s^i^^tosis in the «!«-iT!i^i,fl fed 
crgaMc asreury con^ouads, tSje coTarse of the ^s^e and the aiaoisit 
of i^rcury required to produce ti® s^i^ito^s are slightly dif f er®at 
f^csii timt seen vith ttse s^ food. 

Si^aiflcaace to MAHD IJesearch g ©s© ■work -with the ^na-fye© 
imiwaTlB vial give us a better uaders^taa^aag of t2s© xEe-fehod ^feareby 
enlBStls utilize those vlteisiss vhlda. are m^ a-vsilsl^ls to Idseai 1^ 
the flora in tSse isitestls^ ts^ct^ ^^ose ^li^Os are developizis 
sffios iaterestiag leads as to the factors timt a^ be iavol^^ in bloc^ 
cosigul&tiosk ixi the iBi^ct aniis&l. 

CStesl-^ is a im^cr health probl^zi in t3as IMited StateSo T^ 
^Msity ^veloped ia the rats saore clu^ely ree^^les timt i& huas^ 
beixigs siace it is product eatiraty by diel^:^ i^&bs. 



Ths studies ca. mmass-ta. disease rs^ be iiai^rtaat for public 
health workers siace at least a doz^i plants in this coimtzy are £ijdw 
usiag the em& p8P<Mess bs the Js:^ffisse for EssEuSl&ctariEg viigrl plastics o 

Rcoposed Gfflgse of Broject s Work oast the obese rats will be 
contiBue^ m m. ettart to dstsi^xm tl» chss^ss ia t^ coispositios^i of 
organs emd tissues as tb« assdxmls beccsss obese, Fuirther studies vill 
be im&e ozt the factors -U^t appaar to iafluence protelxsuria in t^ 
x«to ^Ose ^Ksrk x^th gemtofree aaaiatals -uiU pra>!^tb2y be e^i^^^dsd during 
the ccasdiig y^or to oilier ast^ tl»a l^se nutritiossal studies « 

Stv^es ^11 be assritsd out to deterMiie "s^sethsr t2>@ coa^tion 
ps^odueed in aninsls fed the Jbxsaese sea food is due ^itirel^ to 
s!srcu?y» 



Bart B isieludeds Yea 



Serial ilo» miam) 



lEdividual Bm^eet Iteport 
OaLaaaar Year 19^9 



Bart B » Haaors, Aw^^^ @M Ribli<^tloas 

^bll^itioQS other t!mi absts^ets fxcss. ^xis pfojects 

Esswstiosi of etl^leBS oslSe vlth Bieot;i2^!^& and asicotiaic aeid„ 
J. Biol. Ctesj, l^s 889-894, 1959. 

Mle^lsea, 0.,, &s& Asi^r&oa, A. Ao A ^tSs®a for pre^triisg iatsot 
asilffisas for carcass ®ma^es« J. Xsbo CUa, Ifea. 53s 282-290, 
1959 » 

Miekslsea, 0. mt&r. In "Food, ISse Yesarbook of .Agriculture'* l68-i^^ 
1959o 

gfilckslsesi, 0. !E5se effect of bi^ ealeium iat^kes«>=iatroaucti0a. 
Fe^xatiosi i^'oe. (in ps^ess). 

Honos>s sxi& Av^rds relating to tMs pro^ctg 

m% Olsf Micteslssn ^?as elset©a to ni& Sdltori®! ^sarS for 15s© 
Joun^l of Kutritios for 3 «K«e«^®ar terra. 



2, Kutritica 

3. B^tbssaa 



(XLeaasr tear 1959 



^^LA" 



Bxjject ^tlss Sff@cfe of Sletssy ptospimtss c© ^isatal <^rles ia 

S^rinci:i^ Ia-v®sti^toE'sg Etrs, X, X. ^ip end Oo MLckelssa 

Other In-vesti^tos^s Drs. f « d'. JfcClure, Ho C, MMas, I. Ziplsla, 
A. L. Kussell, aafl ISfo Co L, Whit®, HE 

Br. H. Sebx^er, ©i:® P^sas^lvEBia Stat® TMiverslI? 

Dare, 3, 3osl<^, G. So ^t@r^, imd Miss H. G. 
01s<^, TiivlBtQn of laMaa ^^th 

Misses Mo f^cott ©aS Bo Weinberg, Sofutii BaJcota 
State College of -%rietilt«se ^a l&cS^siic 

Ooep^^tij^ teltss MirlsiQa of Inaiaa Bsaltii 

^s^ Eeonmales Ses@rtisss.t^ South B^sol^ S-^te 

Course of A^elcvUsMSQ b^ M@cl^si<s .Arts 
B@:g@xia@3S,t of iFti^ics, !!!!h!e B^as^^lv^ils Stat® 

Bi]£'@sai of IndiSQ ktHad^ts 

^sa Tesrs (cal^ifiss' jfi^p 1959) s 

totals 1/3 

Fsrof®ssi<sjsls 1/3 

Inject I)esc?i|ri;i<mi 

gbjactivgss Sb dstejraiiffis t^iethiesr tlis addlticm of phosj^jstes 
to ti^ Me?s^°°ehil&?ea will reSuc® tlse i2^iesB.ce of &essMl ciriesj 
■Hlisthsr tte adSittoa of plios:^mtes to the die*fcs of eMlSs^s win ®ff<>ct 
Idle growth assd tovslopjsatj to e^sstluata tljs sj^el^ of laifi! piiositetafa 
Ba& o&lciVDxi adi&sd to tbs 61<@ts; saod to e-salu&t® the efficacy of adSad 
(^ileltim (the otl^sr- eosi^^osnt of t^ i^od]^t® coz!];^$uQd) as a la^gzs of 
redociiig the sS^aor^iosi sasid retasatioa of stroatiiM^, 



« 2 » 

Sarial gbo fXUm - h 

to bs-ead ijs four ms£s?diag schools s^ tlie aSditlc^ of a plae®l>o (fXour) 
to th© "br^ad in four ecsitarol ©ehools. ^ithisr the subjects aor the 
investi^tors toow iOi® idsati-^ of tls© sd!«sois. ftotal susabss" of ckHfirsa 
in the stu% is 1800, SoutlBs ®:^!!imtioias ImluSs ^ ^aaml dm-tail 
esisMimtioa i&elu&l^ biJ&t^:^ Mt@-^«sl^ s^^ogrs^plis ss& all eMldra^;. 
quiorteray n^aeur^^its of l^i^t m^ wei^t <ai all chilSs'SE; ©ma^l 
"borse ^ssssil^ aatermlsiatioas -osisis l®ad x^aic^gm;^ <m a sss^l© of ^K) 
dsiidrea i^rasa both the ct^t^rol @£iid su^^^lsm^g&ted sdzs^lS; samt©! eeOlva, 
coUaetioQ ^sd mja3^ais osa a staple of 200 cMl^r®a fr«Ba both tfe© 
c<mtyoI aad si^pl^^ated sdKsols, regiaSsp food iat^se sur^^^s jKcltid&ag 
collsctioa said aa^Ogrsis ©f s«©iKr®s®a-^tiirs diets ia sXL schools, ©oat2x>ll®a. 
bsOm&cs stu^ oa % sssOl gnsup of eMldri^ in & eoz^ss^l ^id & @up^I@3^nt@cL 
ediool for cslsivm^r x^^^spi^te^ i^^^sium^ t^sd gtrm^ti^is^^^ ^miml pi2^1<^X 
ssss:^ f oi? SEutritiooal status ts& a s^^ls of cblld?^ in both cootsrol 
aad su£!^I@asat®d schools, e^id ssim^l blood iirSss colli@eti<m ob & ssss^a® 
of cMldz'Sss ia lK>tl& grou;^ of schools for bsss^l^in (blood), (^cl^m. 



®6S stud^ats in ei^t boardii:^ sclasols Ib BGrtJi ^id &utla I^Sgota 
©3S& w^lesr tJse jurisdlctiistt of tiss Bureai of Icdi^i Afffeis^o fS^ye is 
s total giroup of 3600 cMld^^i; tlst^e iaaluasd tsk tls© stu% ®2« all 
tlie s«v!sa to foarteea ^ms olds (l800)o 

l^or Fiiadia^ g Bpeliadjsasy deatal eurwgr shows a ajoesrst® 
insideao® of carias emd peridoatsil disuse @^^g tlj^se cMldx-^i. 
A7x^a@Eaa0zite are ccsai|plet®d for ths sdditic^ of tise sup;pl^@mt s^ the 
placebo to tlM bsead ia all schooia. ^ftds s-^ytsd Tilth t^ begiimlag 
of sdsool ia S^?t®i3issr, 1959 • Sto eMiaj-ea ia -a:^ si^pS^sj^ted 
schools kDJ. r@cei-v@ a^^smisKtel^ os<e ^i^s^ of E^dsd c^oium p9r ds^. 
Bii3, tdasy idll rscei-v© tteoi^soat tlj® sdsool 



®SB laMsn eMldren <® eateriisg sefejol (fi-v^ to s^vsa j'®ars of 
s^) ©prpaar to "^ ^K-^dl^ iaa(2sr^\'©leB®d in so ffer as Ssslght sssd 
■ereight ®re c«»se««ttsd. ®«! oldei? clsildre© ctss^^^ fmtm^lj vtth t±e 
sl^md^s^ fosc vhile cMld?^i„ 

SigjoifiesBe® to ,,BI4M> ^Sesaagefeg Altfeou^ fluoria© eddsd to 
driaMog°"^geF's«^dei~^®13ael'K^~©f deatal (^x'ies bgr 50 to 65^, 
tbar© is stiH e las?^ lEcsfe^sat of carles to be tatssaa ^s« of o !ISjss« 
is a piiblic fc^ilth seed to ds^^lop & msmis of reducing t^ iacitoaes 
of dssQt@l ^^@s isioasig the pec^le 'ubo do not liav@ acosss to a 
flisoriefeted t«at®.T Buppl^o !&3 ©dditi«m of s^lci^m offesB sm opportsmity 
to emlsaite tSi© safely- of lossg-term calcium supplants bsbA its effects 

CIEk ^"O^b QBd d@V®lopSSS3lt> Xt Off@?S S^ S^S^S of SSCtH'iSg B3& &&S^S^T 

to & fisidass^t@l pdblic poroblem, ^^ssl^i id.ll sd^d caleirsa reduce 

tfee absorptiosi ®!3d s^tOBcticm of ststmti^s^o 



- 3 - 

Serial Eo» HlfeMD - 4 . 

g^;^s®a Course o f t h® ^gro^ectg 15o srrea^ge a two to tla«e week 
^leace s-feuOy ^Ing tsbe first part of tbs smmr va^tioa i^am, i960) 
in S59l©cted ecatrol ®aS supplei^te^ sclsool©,. BspetitlGa of MLfisee 
stud;L@s vill fe® csx^ea ©ut ia Jus©, 196I, ®3 coatlaue tlie isavssti^" 
tioa sa gibers owtlissfi for &s^ mid possibly two ©Mitica^l jmrs. 



Itot B iselufeSg lo 






3c 



ftfft Ao 



Tm^etg^ fltlQj Bioelaaais^y^ end f^&odoglcsa ISotLs of l^icto? 3 



Priaeipal laweBfcigsfeors S3.«-tiS SclsiJ^a ana Csslvlis Mo F@a.ts 



MEffi i:«ac« Ccfi^^®sadar y®m 1959) s 
»o*als 3-V.*' 
Kro$)ss8lG««ls l«l/3 
Osiers a 

inject De8C2:lg*io«i! 

ObjectA^jgss 1» etuSy tlie bioXosleal efSeete of Steelos? 3 @M grlhsr 

clwmileaLL s^/sS^bttWiiJi csl^ Fqc'^op 3«se^'^ offltigywiP^^ &oA iss isscwssr^dL^K^ 
tl)«ir cliniecl 



§«tmmi3iis& Toy essma. eaa^B^ using Tsaeismffbixm 

agelQst ^L^irleffsr U-rar mexosia ta tiw x^ as t«sfe a^vtaoo A sdesK^ 

«aaljrtieal laerlbod^ raciio acti-rae^eas oaal^Kia^ aoA « sesositiwa 

CHaloBTjUns^^Fle ssseor sra useA for seiLsB&vffi ds^sradnsfeionso ^^^iSfteiifi 

mflfaoias are asj^M for ^b» ssrogpsarK^on ef 'parlous 

•qpacleUy &S smOjmiXBa^^sE^aSssSjasi sulfur m&sao seid S/srlvs^-^^So 

7TOBi%A.<sBte^iitsi& and isolsftdUsn ^cisbiiiQiufis \Gtst&sBi^g>ft^s^^f 

ounrasslr djls^rlbufelon^ ®ICo) sore usad fteoc 

of Faie'^cx' 3 $se^^^>ii&3jO!i!s ftron, fisc^ural sounsntj^ asd also fs? 



aeltmium ecimounds inas corriad oudb in eoUabcnraMon 



"tailosr maafi" oresnoselenlum ccss^Kmide f^ "^lis studST'' 



Past B included "^^ ^ O" 



MIAM3>.5 

Testa of boiisalogpus a@ri<ss of £aix^atic zoosto^ suod diaeXesto 
carbocsylic ecidu have led to the sstebllsluffient; of certain 
x^tles det;emlJQizig Eoctor 3 activity. For iBBtasics, ttus >jsq)Z!o=° 
8ele»0»dlcarlO2j:yllc acids tested havs been far 1«S8 pTt^ntt tiian 
thetr diseleulum Quatjogues. An aptim«a of blofKiteucy trais fouitd 
f^kefi tJse eelenlura yma la '^h^ 7 positi<% fs%m a carbo^l group <> 
All of ^M iscsaeric disclsao-'dibutyric scMs Mve bsea stufli^sd. 
aM xK;!3e were found to be as ©stivs as 7a7*«sdis®lea>'di.«ai-butyrJLc 
acid. Substitution of ths seLeaius^sarrsrlisg carbon atosa vlth a 
meth;?-! gpxsp further increased biqpotency. !I3ie r^sultiag di<» 
8el«i»=7,7'«'di«a=.^^erle ecid siss^sd ae SZE^q ^^ ^'^ 7 ^^ ^®^ 
ae'ieziiumj as cocipar^ to .7 7 l^r c&it si9l€a:iium for ;smtural 
WactoT 3. IMs valeric ecid ^ri'W&%i.v& ie a rsc&sie JsLxixeee, 
It'^t/mp^B at r«SQli£tio]a ecm unSer t«a^ in <^^esr to detamixie tl^e 
bi^.opoteacies of the cpti^^sOly active fc^aiSo AIsq, a large quantity 
of the cossigpouM has b®e& sysstheeised as^d piefe at o;^;' disposal., so 
tbat Z30«r toxici'^ studies s^ therapeutic txlals csn be carried oat° 

Tftsts of othar orgasog^^eaium coeqpouzids again d^aoztstarated 
1^ fact thatj, %i£tile isany tj'pes of orgiE»!oselenium cost^uzids 
exhibit activity^ miiior cban^^es in structure quit^ often produce 
profound changes in Ffeetor S-^i^tarifeSBcy. tSse SDcq's of a series of 
ten Sjl^S^benzoselenadiazoless, obtained frtss Dr. F. So ^sy of 
the Caseer Research Laboratory of the IlDi varsity of florid&j, 
raniPGd freest ^ i&g to ^ pg of s&leniuBi. 

Ttiss iisproved isolatiosx sches.® for ]?actor 3, eio^ployiag i&lld 
conditions^ has been refined furtl'er sod ssost of the prepurified 
starting material has been processed by this m^^toi* 'Bae 
conceotrate froai this ■^rooe&w^ is bsing used for f«^r^er &%tim^^ 
to characterize the biologically active form of l^tor 3 by laeans 
of such techniques as electropjaoresiaji proper dturastatogre^tiys 
column chroanatography and counter»curre^ distribution. A loethod 
to deteniiiae selenium con^unds on psiier chrccaatognsaBS by means of 
neutron activation of the ps^er as a ^(Siolf. is being developed in 
collaboration vltb the Oak Sld£^ national lebwatosE^. It is 
expected that gamoa ray i^pectrootstry of th@ Acti-ffal^d chrc»aato» 
eraaas will permit the location of Factor S^selenium. Bsaults '^us 
far have been ixisconelusive because of the ^cQ&^tuMt of interfering 
radioactive isotopes^, fOir exasiple goM and iroa, in the irradiated 
chroioatogreiQS . 

It was discovered -ymt sulf^jr endno acids hav^' a profound sparing 
effect oa the requirement for vitamin E. A tJKsrougb study of the 
effects of sulflir aiaino ecidxsu^iplQmeatetion gb dietcacy 3Liver necrosis 
showed that these ccsaigounds dio not prevenfe liv«r necrosis by i^ietft^ 
selves; but they delay the onset of the disease, Suift-r aaino acids 
free from biologically active traces of selenium, as esVablished by 
xnadioactivation, vere used. Su^lcsKsafeation of o5 per ccaotfe selenium- 
fSree J^cystine^ or equivalent amounts of hosaocystine or DLp-tEfithioninef, 
reduced the level of vitamin E required for the preveafeioa of liver 
necrosis to 1/lOth. While nanmlly .73 ing per cent of dl>=o?«3copheryl 



i^eo3 



ace&ate in the &i.9^ sf fotrds ^0 per e&it pinstectioa^ ?0 j^g per cezit 
suffice to produce the emus effect in the presence of .^ per ceix& 
Z^cystlias! or «6S per cent DL^^iBetMcmlJse^ d^sts to iav«sti@eKto 
%^e-{^er resorption sterility siaotm t^ s^ae pheapmepoia «r® uaSar 
vayo 

Wiiai selenite as liver profeectijag Jigeafe,, the effect of sulfur 
amino acid addition is wtxeh leae pro£K»^iK;ed< !:Oje reduetioa of the 
SDcQ for selenite aM>utx&& tc about §0 to 70 P®? ceszfe. !!lisae effects 
art only produced by those siiilfur amiisc: acids -t^iich are ia tli® 
pathway of sorsaal s^taboli^m.. Sulfite^ solfate^ tautrififij, etc., 
msre ixiactiinso GwMxt&tlcM of si;dbapti:]al levels of selexdte aad 
dl<»C8»tocopheryl ec«tate^ on the othar luand, showed that there tma 
a ali^t iButual potentiation. For tkm establishment of this efftect, 
a a i n i i a mi of ca o3 jsg -^^ox^ ceat of vit^^ossin E in the diet is neceesaryo 

SifflxLfic ance to HIAM j Res eerch; !!3ie studies have yielded results of 

basic aigaificance for the undsr«> 
standing of bicxdterdcal ctM nutritloii&L phenossaena. I^actor 3, a 
potent biochemical e^eatj, is effecti-ve in se-veral ap^scies in preventing 
fatal necrotic l:<aion8 of liver, hear!;, kidxsfey, issoscle, and of«h^ 
tissues c ^e discovery that Factor 3 i@ a selenitsa coo^uiad has 
opened up nms^rous questiosts of scientific interest and also baa 
practical tis^ALicatioss. Xt aiay lead tfi> a sigr^ficsr^ CQn^:lbutian 
to the unds^rstanding and the treatstes^ of i^icrotising diseases in 



mjM^ 5 






SeO^mlum lipases &a L«(^G;t;iise aad Brntsctiasi 



2» Si^mszs, S., Bogios&JL; So Ea, and Iblts, Co Mo; 



to 7italeto£f«»Z£vil&esli€ga8lEr«a^iseit«!a Vfg 1 



r@le;^»iag to ^teis ssro^ects 



Serial m. msm- J 
?s:oJttet 1!itl@; ^9 Bole <»f Vitaaain S aad I^Beter 3 ia 



Cc&l^i^ter y®®r 1959) s 
FM£»0sio&als 1^1/3 



CI)4ectiT?eflg ^ elueidsfeo the notos of afttlcsa of vitamin E 
Itetoff 3o Tb elari£^ the eibei& of evesto in t 
de-velopHssI of Notary li^nsr zaeerosie sad azi^LiQ^sui 

(bsest i3»;y5Cl« is@cn@sii6^ amseizlasr d^s^ra;)^^ etCo|a 

dtiffesran^al eszit 
of SoiwiffAitesf sod Sbs9itoGifiQ C&Eida6ifiQii 




9trevious3^o It vsb 

to 
& studies GO ^18 rels^coi of 
jBetflOMiUe f a9to» to sneiaiog of ds£iei«a% aad 
it VB0 fouofl "Qiefts tlM fssniifiSf did 
In tbe preeenee of m^g 9W@Uisg ^ame miffi>2Bed2j r®&2e^o 



Fflrt B iaoliaflitd %« ^^' Jfe /°7^ 



2 



It 18 possible Itet \ltemln B m^ ham m efftect on zoeUiboUem 
«!}ich in turn effect® -^is &^tt(itur&L JJO^grity of tise nitocbonari^a 
StJQc® tlas £sr&al)olic Itsjsls for sdtoebossSrlal svslling has b&sn an 
ob4ect of cositrQiverey« studies to eXucidesto scsse of tfas s^cr 
iftBuos «es?e un&esrtebea. Xt ^^as coacxfissaed tliat oxidative :^h<»^^!0« 
rylfttion p»7tsects ^tocbo^firla agetizust s^aiiog in a hgipotassdlc 
Biediusif dB8|^t€i a Zoofoiwa pETOrfescf^vts c£f€ot of gg^mdinitgorii ha nol 
(filiP). 13i« lalter iias siiunai to prevootfe eztd eMscwlerate snellias 
dif!£te3C!Sist* oQsi^litioQSo D^P psraftiCNsts s^stlBst syv^JLisg in 
viusrein oscidati'v^ ]^itosptoEyXstiesi is aot poBSibXcse Xsa 
psradLttlug pijos5i!2i0pyl®l5i®ii BHP sceelsrafees y ^i i -f «£ ^Sii®a 
iHB? is the ecs^tor. la^ea All> is t^ eece^i^Qsr, boeever^ prataetioo 
occurSo 2IbQ0 of ^iQ <yiiiiei? &g6£its tBtc o upliPg oscidBtiwi ^^d^bo^ 
3^l0.tio& (dictsissz'^^ ezkitig so^ tligygOiiKiBft) hSB ho&B, fouQd to 
poseees all th« effects of SilSP im sisellizisa tSie i^rotetstive 
ectioa of SBP has been ^is^m to be iadi^entoit of its oneoiq^liaig 
effect. Xt bas been Jiypotbssised -^sat its site of action ms^ be 
adeasrlate lEioase at the aiitoehoQilrial stxeSaem^ 

La ett€»^»ts to ea^lain the pi'^^v^^zxtion of atitochooSriia 
swelling b^ certain redueing tst^mk^g celling was 8l»iaied iiffl&^ 
conditions in t^ch different z^st^j^atoi^ ensspsiaa tmre in en 
oocidised or reduced state. With ms^leae bliiie it ms shmm 
ths& reduction of the ®axTi@r8 ^as £9t neeessasy fcir pgralsectic^ 
by en^iitQrcin A and c^^anide* SLoeldn^, of the oocidation of ^be 
succinate chain has little effect in i^he preveafeioc of s!ito» 
choadrial sv@lling» as eanpared to thc> effect dbtainsd bsr 
iiMbition of the electron trao^pcn^ c^^ssln of ]]^i»liDted Bvib^ 
strsteso A ^^nerglstic action between AISP and tbe respiratocQr 
inhibitors is thou^ to be a Isey to tl^ uoderstanding of 
mLtoehosdrial stabilityo 

Slonatamotts lack of vitasnin S and l^^ctor 3 in the diet 
results in res^^rator^^ decline^ a defect vS^iida characterizes 
tbe latent phase of dietary necrotic li-ver degeneration. Uver 
slices of such rats are unable to steinteain <3oemal o«ldstion. 
i Btitocbo&drla are prepca?ed trata. these livesrs^ no 

decline is obasarved «ith the various imdbm& of tSte tri'^' 
carbosylie acid cycle as sufbstrate^ vLth the exeeiptiQQ of 
succinate, mm the latter, decline oecurxe^l. vfsaa addition of 
ISB to the mediumj this ^^ammmmm has been ai?lirlbtsted to m 
accumulation of oscalacetateo It 
of livers during the latent i^iase of li\ 
decline of respiration very similaa' to that obseBnTsd in 
slice if c»»lBetoglutarate o 
metary vitamin S iirevented the decline of 
oocidation fuUy^ end thact of succinate oaddatian aoatly-o Cietary 
TSetor 3 Cas selenite) vas without effect on the»ft systems. ^ 
vitgo svq^eaeateatioD of a physiological coaacentsratitHi of 
cs»toccipberol {3 7/30 mg tissue)^, c(»sg^etely prcven^^ied decline^ 




F«ee 3 



6a did tbe asrtloKidazrfe CfFD and a toeq^biarol Bis^bQlit« (tbo 

Since th0 mitochondria, '^seans^lvee do not eaiblbit decline^ 
and tite hQ92»geDatee do^ sosae othier particle or factor in tim 
lusBOgeiiate isuat tte combined vi*^ tba siitoclKnidria to elicit 
dftelJTMS of resj^irationo ^^se aiierosoEBe ^^eetica^ but sot t^ 
eoluible eupenoataot fractios^ «as elioim to have this jigm^geirtyo 
Moraovex' tha McrosoiBias caused a ise?Io&d lowering of tiie P/0 
ratio of stltoclKsaSria vith c»»lBetogXutffl;«te aa aubatrateo Xn 
vitaniln S»cbsficlent bosnogeaates and atltocbondria ?/0 ratios 
irere found to be Io«en^ t^th obe^glutarate as substrate. 
Sosne eocq^onent fs^sa '^[ae 2aieroeamal pre^peratlQQ is inhibitoir^ 
to res9ir8ti(m and tc cKi&^ve sibo^pliorylatioao Shis agta^ 
is sensitive to boiling for one minute. It ie assparentljr 
released in iritaiain Eedeficient bonogiffimteso Bowevor^ siiero°> 
sooal preparations from S^siQ^golemented anjmals also caaise tiais 
effect, particularly vith ageingo Ttm jmture of tibis ixMbitors- 
a^eot is being investigated fUrtl£«^° 

Sigedflcaaee to SlfiMD Reseai'c fa; M elucitoU.on of sorse of the 

"" :^ECtors involved in ndtocbondrial 
stabili^ is of significance in the stud;^ of aseay diseese states 
involTing cell structureo nram the evidence prescmted it seeaaa 
certain that vitamin S cogotrols soaoe zce^bolic mecbfiBfiispa «tiichj 
in turn, are rdsded to the jaaintenasce of structural in^gpity^ 
darificotioo of the xucture of this lastabolic reCLe of vitaBdn S 
sbmxld aid in a bt; .>ter uanriberstandlng of its role in preventing 
dietary Hwe i^scroslOo 

Proipoeed Course o f Rro je ctg !Bie nstuze of iA» agaat in is^crosooea 

vbich asfpears to be respoosibXe for 
the re^iratory decline in homogenates» as veil as the looezlng 
of the P/0 ratio of QN>ketoglutorate^«ill be studied furl^wro Of 
maacimian interest vill be the nstabolie eirctmstaase tihieh accounts 
for the instability of tSam laicrosoBies in the vitaoin SBdefieienfe 
ststeo Based oaa electron nicrosca!Edc evidence indicating t^ 
breakdown of ssicrosomes and aitochcodria in the latent ^^lase cf 
liver necrosis, the sietabolic interrelationship of these cellular 
par^eles vill be investigeted closere It is also planned to 
study the effects of dietazy Vaertor 3 on these phenomena. 






of Suscimte CM-teUose in Bat M-rar 
Jo Moao Ciimo ^ 191 (19593 o 

ItttocUoaarie, Jo Biolo CSs^o^ ^^ 1338 (1959) » 

I4^8e^^ Mo ^o , and Ooorain^ Lo Mo^ Stoaies oq S%a(bilit.y 
of Rttfe I£v®x> llitoi^bottixleis £« ^1« of @9dl4e!feiini 
HioQiioxylatiet' ia @WBlliiis» Jo Biolo 0!i28m« 2^^ ^M 
CI959)<. 

of Ret U^rar mtee^esaaritu: S. Bela^em of ^« EXee^^osi 
C1959).' 



eM Mse?^ r<§Ia.ti2is to tM» ^groi^^z 



Isdlviauaa Project Be^otr^ 
Caleaoto ?e^ 1959 



3. 



Part As 



Project fltlG! mclogical Sigoificesa® of tJhe Qlueoe« 

factc^ C^SmsadumCnX))^ eod its Belaf^ioa to 

Coop^'a^&issg traits: NBiaa 



Q ¥«ers Ccal^iasy 5@ar 1959) s 
Itofeals 5=.2/3 
ProfessioD&I*. l»x/3 



^^j^gUwgi ®o iBveetigBte ISjs ©flfecta of dmjaatasaClIl}" 

aiscltaalgm 1^ i^ch @Cf«e^ive cl^rassdiMCni) cosspoimSs isg^crov® 
glueosft 'toilts^'sscd* "feo asisasr 'y:i<B @£f'efffes ef such cobbp^^sSs qsi 
glucos® u^ts^ in i£ ylfepp jB^rsttsss^ to develop a^imJL v&^ of 
c^llc&tioa of chrosdmCITx) ecsE^^^esj, to study tiselx' «ff<3Ct oa 
diebates in expe£>tB»atal fiixiiiffia.s &aSL in tSie huGssn^ to atui6^ tiie 
roXs of the liver on glucose utiliss^ios; ssA to elucidate the 
eotrxelatioa be^wssn elirosQiufflC^X) ia 1>loo^ a^ "imullB UW' 
padBeipleso 



Mett»d0 Bttgloyed : l^tt rats mr«i lasdUs^eined oa vm>ious x^/kij^&l eM 
seD!i«>s2rD:%ltetic dlefts^ mfift gLueos« ressovBl rat«»s 
are rasasto^sd a£%er iisferawmooius iiijection of 125 ^ glucose per 
100 g of y^^xto ^^ pcsftSQcy is dstemti^ied Isy ct^steriaoea of 
glucose tolereuGce before azid af^«p e^srpliccEt'icn of a single dose of 
QfSF Isy stxaBacb. tubingc OfF preparations are ad&sd to various Q^'^ 
defieieat Oiets^ sM glucose rasmoval ractes er« detezoiittd in r&ts« 
iinuLzitais^l (m such diets for "^ariovis periods of t&mbc 

%idydinsal fafe tissu© is r^aoijed froffi swfes raised sm a CgE?<= 
deficisnrt diet; as veU &s on 0^<»sc^l®!%!ited re^iomso CHucose 
uptake by tMs tissue is lasasured ^tbi and irlthout (!l!?o 



Page 2 



to Etafeixeal so«xrces of GW^ Stabili'tey of QS^ activity ia various 
purified tssustias^ is zeaasusf^do 

^'A iS^etssLmillDo Ta&s el^eszst is iSm acti^ 
of ti^ dietasy f »;t@r ^iSiieh is neessssry for tbe 
of xiosa^ gluicose tolflffezfee® ia •&& rsto 1&& fiMisg 
puts GluraiBiva& os tlis li^ of ts^&sti elaaes^s requiar^ for ^Ol 
being a&d noes^ ^jscticat of tto dioimal ors^iiss^ syod pGS@ibl3r 



Ix& tito eouorse of '^j® pusrifica^on ^rooedtsrs s^lied to 

tS» jmpaiTBwmt of g;i.uc®se tol^ei%$ la rsfes iltit a siaglis dose 
of §0 to lOO pg p^ 2B3 g of 'mi^o Tai69& purified fractioos 
lost tinetr activitgr "f^aisa stored at -^"^ laaetimstioa nam 
eeeoBi^ifiBied t^ 'tto psraeipitstiosi of a fii^^ Iststsai^ ws^iassislLa 
Wsft aaliiQg of csn^dbs @o^xe«€}s of @^^ ®3 ■w&Xl as of ptsrified 
preporatione^ ca tlse o^ie:? h^^» did a&t ^butsra^ <»» activity. 
She findiae iadiceted ti» iawolvosBiffiBl of a tz«8« elsssaat e» 
actiiw ccqpoQffisto Of aU tiso eXemsistfi^ 8<sae ^g«re eliisdoated 
of '^isir aa^ls oceuzre^iMe ia @ai'«da£iciffiat diets^ asd 
because of -^iftir pi^opertieso Salts of 4? el^sessts tnsre 
G0 to -^teir Olf poteneyo Oai^ trivalssxt cisosiiumClIl) %^ 
effective. BssEas^alent Slmsaium salts ^1^9 foiu^ to be inectiv^. 



Screezdjag of a @reat wsi&s&s of cli£«isi«B3C^£££) cw^oiaaiB» a2X 
of Kbich are co<»rdiastiooa eonipleacea^ sboved tltat v^ry stable 
cooopdinstiOQ coa^poKBids ^rasre izKBctivie« Basfflgles are tbs 

r g^byleawdifliniipe e^^^^le^eso Tbsy ore inert aoo 
be utilized. S» oxalato^ salicylslo and bis^bigu^jdds 
the Q'^ser hfind^ sliQi^ed a bii^ de@?e9 of po^®t^^o 
3a are agre labile and can relinquish the chroBdum 
for utilization lii the tissues. Only SD pg of dta^aiuaCni) ere 
req|uir@d per 100 g rat %ei^t to cure the glucose toleoraoiee 
factosr deficiessyc 



A osetbod for the ssroduetiou of etseaaiLwBiin.) eoD^eaoeSj using 
chroBBic acid and sulfur dlcKide^ has beca devieed for the 
of eocardiiffiktion eos^Iexesc A lar@3 varied of cbrcadumCHXl 
cooardinatioot deri'vati'ves of aodjso asids^ pgncimidinss^ purines^ 
nucleosides and nucleotides^ as wsU m of biologieaUy occurring 
bases have been pre[B®red and assayed in tbs glucose tolerance test 
i^th ^le aim to fiM tise ssEssrt potoaxt and therq^eutically most 
suitable chrcstiiumC^^) c@i^?lax. Ste ideotifiea^oa <^ settaally 
occurring eosiplexes of citsea±mi'nj)s f&^ instance in seruKj, also 
has beoi iaitiated. 



7 

3 

Ms@sur«i&SB^ Qt gXv^^e v^t^s^ g>hc^@a iSis^ n^ isoXs^sd f ®t 



to iacrtase gXusose v^STof f!e^ ti@eus in tMs sysrimo A £tin 
9£fect \ies olyleiasd -aith ol pg per fl^ko ^ in -y» a33®9« «sqperi^ 
saea^^ smll sssmm&s ®r iBsulisi «^S!f« i^ecfisis^edo Tbi^ increased gLucos@ 
ugstaks could b® eccoua^ed t<^ lisr ee& iscr@ase of fe^ s^^zs^^ele in 
^M tissue. & €^Ei@x-i2QS23^ uMng z-sdiOQctive gLvnetm^s dbsrca^umCHl) 
8iQlpl!60tBotstio3i djdb^&msQd tSie iiWfOX^potfBfeloB gS Jjiib^XitsSL glusosG 
caorboa Isxto fs& sg^iperaKiassit^Si^ three foX&o 



CSmmLumCm) 'yas fosiad to bs ^iH toOaratedo Zn a<m'^£-»p. 
distisetion to etxvaletxt ^SmsAvm^ n^i^ l^m m (SS? astivitgr^ it 
is z«lati'wly s^stossie« l&eUa^ ec^gOjessea^otion of -vssrious 
cQiqKMiaas of ehrgoiiuc^ni) s-s^nsaled x» signs of to^ci^y ^Imh 1 a 

1 e of the «l«aaesit p«s> kg of ^Ettigtst ^m toX^ic^tsed ^leil ty rsts 



^Sae efl^ect of 6!E?c»ecti-v<» cSsroeduniClIX) <&&i^^lMS»a is bsisig 
•tu&Lfid in «aiaoca& dUbetie amiiSBlSe Bsts mm staibUiBed iaaitlln 
z^8>3lui3?co2fB0fft^ food inta&0 n&d l>Xood susbt l^'VsiLs^ t^io atsssSis ^t@^ 
all(»an treataBasst r^^po&dcd to th» figppHeeticsi of clurasduiaCnx) "i^ 
lomriag of fasting KLood susar Idvels^ i^ch wsre toou^ doien 
fipom 2^ to 1^ lag per c«nt and \fy ceasing U7ii»sy 
QsccsratioQo 



Sig^^eaiMs e to ^lEEAM) Bes^ffi;^; ^s T&sostti^ findings indices ^gt 

^irOB^dlBCHl)^ «V%1 ^30U^ ]pS«ViG»8lj 

considered of no isspoactancs fee "^ui ctzdJssJL o^ipinisiaji is essentiaX snd 
i»ai8pen8a2>Xe fosr t£ee atBin^ansofte of iaorsaal esar'Mia^aratft fisetabolismo 
Siis finOiag may to M^i]^ si^aifiean^ fbtr futtsre r®seafdb in diab^fcos 
and otber disssseso Ism inteavienous gXueo^e tolcrane® is -y^e eerlieet 
and saost 8«nsiti-v« sya^gt/m of incj^^ient diabetes in tlie immano In 
<nst (IQf«>dj6fici$SQrfe diats tljae Xsnf sLusosa tolsrazKsa is ^ses onl^ 
r«co0aized tiius far^ bu^ lasspe semffe i^mifestatioaos i^ b@ 
a di«ts Tsssee rigidly f^-^ of @3^«ssetiV8 chztsaiumClXMo It 
is coaeai-vi^ls tbact clmaaiiimC^^XX) sa^ bo a bittorto "aissins ^iak" 
in diikbateso 13w discovery of chrGBdmaCnZ) as "^ts active pafinciple 
of G!Q? SKigr iutve opensd v^ nev oKp^^ifiiszs^aX and tiisifa^^utic 
to this 



Fas® ^ 



ifi fflpos cfl ^^Oc wagse <^_^^^i& B ^^ffigfe; ^te© ©I'fe® @!s£ 3@fij@ ^ ®e^©ss ®if 
mbsSDoUm, sod ^» e££^ct <i^ ^simssduaiCllX) -^ar«tm tiiUL be 8t<dli®d$ 

viU. be «sdiC3a£ted$ aM -^»3 £«sultB ss^HjsA to l»mm (iifi£)@t®iS sod 

iaves^i«!sted ISims^ '«i2i©h t^ 

aetlvo fiilmsBliaiCZXE} eM aeal ^tH i-% isi -^to 37®g»jds«i@ii of 



7 



on Stoek Diefeso Mo Jo &f Tb^siolo ^ 6lk Cl9?9)o 



of ^bec^b^ol Ustaibolites oa Besfpizsa^kssy SeeHm^ia 
Dietary Sscsro^c Id^ssf Begssoorsblotaa B&oshliSo ^^Si^o 
Actajg, ls81^ C1959). 



Cl959)o 



Kroso S080 3&^o Blo3.p &n& Sisdo C^ ^nsssjo 



is Ko(, aod Msr^s^ Wo^ OUr^aiumClH} find -^te OlMeose 
Cl959)° 



sUseisaf^ ftir ^^rsioSLogiseiM (amaieo Berlia ia§9° 



ffiod J^taras relefUag ts> ^lie ^n>j@et; 



L.-. . Ifeffiar 1959 



Serial SOc BXAMD -j 
lo ISixtritian & BndoezlBOtLoi^" 
2g EadoezlaiaLogy 
3o 



Port A o 



Sro^eet Title: StiaSy of Antezlar Fltultafy 

I84)tlfi.tiea «sf 



F^lseisal Zo»«stieB>toars: 2^o Bobert Wo 

BTo Bster Go Coadlif^s 



Otber Xn^esti^tGETs: Mr. Toloae B. mffmr^g BfriBo Siaxy Mc Sosxistm^ 

Sr. Ao Albert^ Br. Sidney tfenaer. 

Sir. XfiOBard flaszes aiKl Bet. Bietaord A, HLUex'. 



diaiC; ffijid GoIuBibiA QaiTsrsil^o 



Ccaiessaa? y«sr 1^9): 
SoftaO.: 3 2/3 

Sre!£tessi<»Ba: 1 2/3 
Othsr: 2 

B%j8@t Bseex>iptiaa&: 

Ob^get&WBi 'So iaeJjB.'te protsla bsassoaaeSj, espseially antsrioK' 
pitui'^u^ jbaniaees &»« gluids; Ann fVoaeticaaalj* txaa^piLaataMe sKnise <n 
3%% pitoitexy tuiMirS; sud ftcm blood; to stuciy tihe prlsszy and seooBdaa^ 
of parateixi bccncssssy 6^esifl(ljly tfas safterior pitui'tairy 
of tbsas bi09nBea»8 as tl»y eireulats ia 
t, to eeani^late 



Stcbh pituilstzy glaads^ 
zat or aouse pitaltaa^ tunsrsj, aad Itrcm blood. Crude eztmets vliieh nay 
eoBteUbft aevexel taagaoBeg axe fraetlooated by teelaaiqiaeB eflBBonLy laged in 
SS'oteta ebKaistxy^ sueh as mfclt preei:^ta>tl@fi; sfld-vest f)»etiooati<ga; is»° 
electrio pa^eipi'to.tiflfiy prepaapatlfe eleo'tet^gSKsesiS; ioa-esehaage ctoeaio- 
tcgsttpiiy aad Qotsstex^@ucxeiit distributiffift. ¥87iois8 qusustitative biological 
assays axe 3feQ]udxed to de-texsaine th» eoafies^atieai off tb 
ia tte sorotsin fl^awtieeso Iftysieal igir^perties of tbe purified 
by tx&s elee^raslMseBiSj, 8edinezstotioa» diffwiicstt ax 
for tSie study of proteiaso Xdest&fiesticB aad qiaatitatlve 






IXAliD - 8 




5a@B ^ 


@stJasa.ti«ai of iSse m^joi 
mi^ Toy :ge.-psT aad t&i « 


lasebeiase etetaato^^sl^. Elaetle nethods tvee tbe 




|iA'feisi.''toi^«> 



fSS-psradue'^ pitu&iaffy^Bfise's of al@® xs&s tbB Imps ^m,% 'SSBL uaeeffitaaalaat 




i, pmlaetia^ ACSl^ S^ aag IS, 
Shia :f«sr« 3%f<3 AXbeirl: 1»fi1»d mems ttaaer f^i^aetioBS fcr 

A m@i.tiv® mspeass «&s fotasd tfltb a tmm extxaet, althcmelk tbe enoust 
of ^Sl iajeetsd «a@ 1,500 isSma gKCftte? ^ea tliat to & bevias pitoitKry 
& pralt£9a z^spQsasse ffeis eli»ssly 

&£ fJMstlmsmjmo ¥lth tis& (sec^^mtiaa ef lgr» BieMrS Fzups, «S» 
£H MwaeiajS; it Sa&s hmix tmod ^mt at ^ 9 - 9<>? «»»& a 3.«»r salt t 
tmti<3Si» %SH is a;&i«c%e& eta a dietlnrlaMs^etb^l eelltiloee (iSJiS^C) 
^^xUe jyg is a€t ed£»Qr'bed asd ^^ses iSmsv^ tl» eol«Hm« Sfais is 

effieieacsr of iea 




^ a e6aibSmtieB& «3!f ion eaceliflfflse ^Imimteign^ptaQr uelag 2ffiAB-C 
stasis^ ®Bl eleet?Q!sis6seai0« It Ms Toem tmm& that S5H aetlvitr is 
t?i<^ ss9@7el @i££&semt psf^itsias^ Siis fijaOisg gm&tly @4sb- 
|!llsat@s 1^ pirt^slss of iscOatiag 



a£ 'ms. Srm Ismm Itloed pLaam vss® Eseae for Bs*. 
Si&asy 1^iS3ras7 iJise fonnd tin -^jgrstroiMe ef£)eets of tSiess f^metioeSj, 
as mil m l^ums tsm lamm pit«3ite7&SB« t9 te asutsolized by 
to Tamlm plttaitasy -^syrsr&rofpMs. Tids isSiisates a laek ef species 

arg®«if ieitgr to tS® case c^ 



Bl®@a maaea lls1smeti(Mo Sol^ili^ ntuaies Isa^ shtsen ttet 
tto sBe<»^"^nK"^*^F^o"iiF^jie«s«iLaticia) p?cee&a» f«!P «» «acferae» 
tiflB asf ®«yt»to pa-Qri^to Istasnaessiss fSm bleed iSaiffla Oepssds upaa tl» fiaet 
that »08t bLooa Bsporteto® aa» ftgaatsseA to 75-^ «t3aBsi«3L-sal±B»^ so that 
■Oaey assm ao Xoiaser g^iMjs to &«»« afjosoms-etlsaaOIie asdto at sfi 7« 
CMy pa£^ @f "^ffi pla^sa pmlsias aro Ssmt)sm1ffi& at 
of ®thBBal« 






XagMliB flre amcea? By miisa^ I^^ laSmled ia»«lis^ it 
*^i -tite «idl€iasr«&^ to ^t" QXtxaGMo 
1« eia^lif&sd. It is aot ae^ssaarir to l^vf^dJLix» tte meioa 
«« BbGL is aiiasd to tiBi plasaffi; T3ef®K« h 

be flaal @ess^&1a»tlQsi is ^ Bad aed 
_ at «om ta^emtuse fear 30 aiasftos, ^ 
prorteia is «Bse«is«d asei iSas vmsl Im wight yieM e£ 2-^ of 
is «3cteft@t»d trith «&0»t 80^ sf t3» issoLifio 




gpoaaetiffii A traa^piLBStelsils pituitasy ttsaas* ef vKt»g 
se-eallea oassnertfoipie ttaaerj, oibtsdjaed tstm J». Jaeeib Forth^ is toiag 
gTOtni tt»d studied for its iseGcacae eessteactc I!re3isiBBsy stodies ^ha« 

iLC9B> tittt is QBly 1^ of ttAt &r iSam pitsdtsxy. ^sA, 
is ssvezttX lnia£be«d ti»s ^bm size of -»» ^tui-^oy^ t! 
adx^zeOs XO tims osKHsa size^ @xa.i^ 
irith sdHk^ aod iaftotile aisplesj, sbesrias a. lAek of 
lAticsoa of the 




Sialic Acid ; la «QllefbQK»tiea wLtb £9r. 
it Is&B \)«i&a tismi -^i^ vi*^ izwjra&sias daaage of SSI, t(te depil^tiosa 



of sSslio mid fras tiss tl^a?®id gLeads of taSiy eMeks oe@tss ia pamllsl 
with «tt dspiUtiCT of stal»3A iodine caad 1^3X, sialie sMiid is a 
toBut of ttayroeleSnilia, tb» stossge f oim @f «1» -Qofzisid iemam ia 
tl^nroid glswiflo 

g<g»><-fiqaM^ to Ha&ii? 
of pitoitMry taoiMBes is nai€, as 3f«iV 
astailAbilitar «f 

ai^piliei-ly «xr « 
tMs am tats teea seklsvsd by @i 

of bloodlmls of 




a&d pi^sieal 

.ty of SSa sad prolsetia viH lis 
viU be dBtexaiaftdj, tO0>tlser vith 
kiBstie 

of i^t i ffpi lg i m l ^teri'wti'ves viU be oaorled ^»to 
oa the effieet of !Bra on tfayreid pfeQrsioloigr ia tike €kl@k vUX 
ViU be asde oa tlas blood levels of 
ia xttdLortlDroideetQnised itiee insdasted vitit 



^^ 



t® dB-teasilae «tea tbe Ueoa liefrals «£" SSI ^iaso 

ef prdU^titt «B& iBsuUtt saw pilsKiaiaBd^ 

A nofv Bdezo astbcd for da-teetl^ai asd Mofti^ffisr «sf p?<Q>lAetia lis teing 



Bsurt B laeliaaBd» 



]8BMS}' 



BMffc Bi 



B&li^tiCBg tf&eae ^S»& abstmeta tffm iM.m gggi^egts 



ff Sa Wit; QfiffTSAfflU; Ms K»ff S3BA ISSf^SHSu-g ^a Bo 

3s VngpVwKr^pLB X3KHI p9L%!ll>^KE^ giMSHOSf SflQSe pS>MHk^B3r3r 



& mM^^So Acta, ^, i>30Jt38, X959o 




5 1^9 « 



pitialts£^S; ^fmufEajasita^aA pituitszy tsrassrs of sdoe aad ULoed 
a. ir. CSa press) » 



<sf aBSlti:p3« fmm of beviae -3^r3r«y|ire!plae Bioehto. & Bio|%s. A« (in 



aad AMBgde getotaag to liMs project ? 
sa?. B»tes> @« Cosnaiiffte msmpis& & 



aa& 79l»ffaj, sad mlaxy for « 




Oal«i^ar IB^a? 1959 



Serial Hoio HIAM D- 9 
1. Mtritlcm and EBdoeriaelQey 
2o SndoexlBology- 

3« 



pas* Ac 



l^^«€t l^tlss studies @» t!» se®2«tiea aai letaboLisa of 

steroids in aaa and sjii^tlec 



Eriaeipa Xa^estig&ten I^o 3U.a®6i£»d WUson 



Otier iBsresti^ters: £3>« MT11<ftffi C. Sutler {( 

Br. ISsrtlaer C. Llpsett (Knc) 
£»>« SatO. Beaea (aXAMD) 
!^« Dsvid Wo Qjras 



Itaits: €eoreetam Iteiversll^ Seltool of Basdiei» 
Itotlosal Cten^r Xnatitttte 
diBieel Eadeeriaolegy Breneh. BXAMD « IS2C 



1^9)s 
2.5 
: 1.5 

leO 

eorUoal bioesnstte'^^ jrccesees ia 
(3) CoQi^em^iae vltb eliaieal 
islized probl^s of steroid a»l;3rBi6. 




CD Ca) 



mms&s «ad Ifajor gladJ aa^t fl) Ca] 
cteitrelofped lir ^ ^[taatitfttire de^SniaAtien ^ 3 A^ uteroiflB ia 
?, via, A 5»iT.^t(».sB«ga8Bel«o» CS-lT^Bl}, 



wriae, via, A :?»lT^f(»-sB«gasBei«SB» ^-IT'-BS), itsdsief aet^bclite, 

/^S^pregsasaelapiQl C5-rf) «ad dei(ydr«»3>4«aaro8teyoB» CDHfl)c 



55 ^ of 5-b; ssr dey, vMle 2 otiaexw Isad neste. Fi-ra s&tiessts vitli 
adxeBoeeaftieal ear«siaaa esoxeted iaere^i^d SisauBts »© -feo 15 ag., 
vS&He in 3 mflgeets vith Caelsiag<>8 ej^adresBS Cl witb adrenal eareincBa), 
5-l<S %iaa aSMseat. So 5-17 FO ««» ' «aad ia eagr of ttae «« 

vEilBOs^ but aU Sad olemted asKHaits ^ im (6^90 ag.) 



9 



«ff tjieltor im earisiisates tvm 5-17»BS la tin 
eouLd aet be CBs»fei<«d by & sto^ ef the znties &£ 
^=>VS ajafi im la uriffle« <Ste saly fsors^slfttiea nas t^t both iffiz« 
«leTftted ia a&?easl earelB@»o Osr s«8ults do^ hovevvr^ 
tliat $»17 R£ a^r not be a zosiOaffl;^ sgrsttesizttd ps«etHra«r 







ea ®iir partitifin 
» C^ setabQUt»8 
i^tism \sy tl» s®@«^3!3r «f 70^ «f t&a sspliis& sadieeetiTi'^ ia tStes 

'*«ffirtisel sie^bslite" fie«eti«a« aad 3o| is «tSier finetl^s i;mm 



te 



Cb) MsillAxy iB?«seda£«8 f«f tte det8»aiB&tie& sf aaaitl^esH 
stsroia as^aaoll-^s laasfe beea developed aad assdiedo 



I Sios beea fam& t(» be » aaij«e' nsts!b«a.its ia 
, (27 Mgo sea? day Sa aae 85ii}Je@t)o 



I foGESBSxly belMfed te be eseexvted «aly ia 

lp!eatly elevated Cv^ t@ 



20 sgo paar Ssy)e 

be@a stodiedo Zb aU^ tie possibility @f ea edseml toastr nas nded 
oiix^ by @8r as^lyseso 7earlmie erthir adi^aoeQsrbieaa ebnsoanlitlee «e^ 

deline&tedo 




. 9 



Ite|i5 fP0S(^ C^ y^ ^ .,. j^1^» ^? T^il, 







Bsrt B lagilttfiBd. 



9 



Bart Bs 

miems S«5 Lipsstt^ Mo B., 9a& BeUer^ Lo Co 
Off AOm, A sted? la wiaas^ etes^d 










^9taaMllt»a» f^«»eediags of tte AppiUsd Sesdjaur aa Upids 



aSM- 5BS 

1959 



3o 



& 



IC 



A. 



Ssnsjeet SltLes 



KriaeiFa 



Coo!E@3%tlBe ttoits:. 







Sar. 









Sre Ho 9i:Uea, 
ta(HR&IR) 







of 8&»dieiB»^ 



1^9)s 



ft!^J©^% 



90 



fflflp 



lQ^P«?«ha2fiSiieo-pi^tltKsy aeUmtlas 






Hii'8b'g**-1llw <sf 



of Qorisie&SLiSs la dog 



ssodlfieatleB of the vm EtOsr 



ta^^be ai£bm£n ia 
li^r olAetroOytle leslABa la 
wlefluw Is 

tevelCFped la 
bya 



»^3og fiafflLa sgi Ik 8«s8e seellMaeury sta 



m e«.t8«ltiL 
as HOSfek as a 10 fold 



mm- 10 



«M2a ia 






folloifiag 




ifi lit^Le m wo ^mam i» ^b& mnwai 



foUflRdag 



to tite 



stoSarof 



B£A]®: era 



»s 41^ *%& te s^aeed la' tbst"'ibR»ia @f sfjainnil l aoi^te^ aaA 
IMiaazy eertieei^ vUl Im stoaiAd f siLUwiag oleetriaa 



;^3r« B a06 lael^^dc 



1959 



3o 






Bri»eis«I Xs»9st£s&1;^: XIr. 



SstBls 1 1/3 

©Smtj 1/3 












tbe i^I^lBe aois-^. %S» ijpiisftl ees*! tsas iSmn txaasoctsd at the 67 lisv^lo 

tite iBbelsd esiB&tii» 



«QiU0<8t»d 8teue>tS2ig a,t tSs 

tarn— 4<rfcA ^k'EtA julv iHMj. tiiaLJI xifinjiM.* 9 IMM. «m«l ji-»Qii]ifr t»Lit«m »«m^ 4mj^»<mJi 

by 



wla sm^d fosr abcmfe 10 aay» befosne sm^ipsariBe. fl» #5 libsliag of 
t%s» as««si»& ereatitnsrist vas oaly a litr&le lossr tlaaa tisat of tis@ 



vssfimxf esmMMm is Insf^a to bs ^terivod iaasdiatel^ 

bL5 i»1m 



£n)n tbe es«ati2se of mtsiOxg iMa aggiraaclmte laaat&l^ la »l^ Xa1»lJisg 



& 






T 



feOJLo^ing ^pSMl eeeA 



%is% list to mas- 9«%Iva.ti<m» 




-boS^g ]tesso«ilsSs» of inu?i0ys stems of its 
atrlbtxtes to tiaSsrs^Kafiis^ of dlseaussd 8'tetBS« ISts nSiofV! 

tixMas gl'ves ft pceliEd:»asy aas^rar to eas 

to sssHaIb ^is siiseljs atro;^ aM xs^alysSs f OkLLotflag iso2&tioa of 

]^xl]^a3»I tissias fir€B t!» essxtssa i^^^^oas myntmsi msMr» abore tSm 

lewel. of i^^£»I eard 






Sgo3®gt; H«BS 8X9 teiag aade to sepsftt 



of SQT ^i^e@« St^Si £ttaaifts ad^^ Ute esteaied to 



SfefftB 



1959 



Serial lo. MMB ° 12 
3» 



Basrt A: 






©ifesr Ssi?©sti^-fe«Mj fee J^s ^sris (Ml), 2J?, Millisa Te l^psMS^ 



CCM^a^o? Tsesp 1959): 
z 1 

J 1 
; 







^id ^ gg Ffcafliag^; Si^^sl S@gs 



f ^d ^^ass a li@&t;s% is 
tea 

Sasr tlto loft aax^a^ w^ is msml&^sS, esd tlae x&te of alde- 

e^«se®tica is ss^stss^S.. ^@ f^Mii^s sOae^ tl:at ^ffi miss es? alSs- 









Birt B mst isel-isSsd. 







SesrtA, 



Sff@^®t mM^'.A^ 






: 1 

1/3 






ss^^set so tax fea 
of 

is SstaaKl^d by & ^peetsro» 

OCT M^ tts l^r ig@ 50 '^ ismssss @i sigsdflcm&t ^i^ is -^ 




@!f KSI is %lc®d em ^ %;^tea mt<. 






@i^' ^ G^"ia W^lia 



isto^» Ijleed 



"Sssst 3 &ot i^lisisdo 



1959 



Serial Bo. m^m- U 
1. BrtxltlaB& 

3o 



Bert A. 



BP9j«et Vitle: XamOis 






I^o Ac So Besfild ana Sk-o Jkmis B. Field 
iSSAUi maswsA msmrsli^ sad IX&lffi Cgb).^ 145C 



ss CCaiaaflsar !«» 1959) 
hotels 1 1/3 



£rai;3«e« 






A MTtibod for tie bi^taas^ cdT wleso 



Isvei of iasiHia 1» tte s<aa»tatie wis lalood of 
tffisg aad igmedi&tely f «iUwlas 
of QSM ef 't3» foOLlcwdisg: groir^ hsmamif ■ssninefS.ng 
fMs has tecsn em^sm& vitb tlstt iamlia liffvel f ^Uoving 
salisKt and of 




yiaflij aga; Bunslia emtsat ®r sariptenl Uood ef «te dsg 
_ fiurlilag 18 A5pregcSii&«fa^ 37 vKKAbI. pSAemj sraeveetie ULood: 60 tAFMi 
foUewlag iafosim of c^vteeee 275 vSI^3.. jtl 

is ao rim ia tits le««l of iasiaija. »i® @«asjslvieia& is tint ca 

la Ifes <s(til9r kztmra alOiseHsia^ td «a^ laeinaaed sats of laouOLlei 



A c o il Jl B l Kswfetero s^ttdjr tets teon CMonrifBd cpstt wI^Ik 



14 






iwt dlagihraga sad His fst TxtO^e TvspvaasBUSBg asli 
iathls 



o MMIBP BsaeaKP^ It is -ro^ 

stuily of ^Sbetaa to fifSEro zttliaials InfOEnAtisa « lm«Is ef iamlSa la 
Uood la dialiet&e 



eZcas* i;is> 






BBTt B aofe Inelutea. 



MB •» ME 

1959 



1. 

2. 
3. 



Smrlal »». liXABSD - IS 



Buft A^ 



KfOjeet fi-y^ss 


eS fficrteiirlly ia : 


Bptneipal Satvesid 


^tOFS STo Ri^n: 


Otiurr XDrajT^^te 


rs: Or. Botbert W. 


Total: 

otter: 


kr Ycsr 1959): 
2/3 
1/3 
1/3 


Smo^et ^sexlF&S 


.&m (Bin X) 


0l»J«etl-9«83 


T& &»tsaasS3m -^m wk 



@3C^S% J^JQ^^piQWHll 



A. mUer 



distnliB. 



otf t 



for % to 7 ^eys into 
, <3r (t) iSam sars distelis 

IV^eted vtiUkism&hk aad lit ainilBrly 
VX9 detexnlxttdo MstoSLoslaal study «Rf 
<^ tbfs Sena tinrei@ft fflsit adbrsi^ls aam bel^g 



J^^ 



£a tto Isiltial 

wi#it ^il»»a @^ly 



<^»» fsrs distcais Wffi 



te tte gr«wiag field 



toSZAMD 



%iiU 



aad to 



Bfopeced ^Bnafm of gajojeet: X»£t£al 



foUosias 



viU 1»e 

@f tlH 



HiailD^ 15 



iw u refc ypo sgiyBis •1000 aad fbUowiag leaiosw i& tte brain itself. Xte 
ISQ^potbaLLBwis aad utxraaorsd sarto odT Ite l^isflfplvsis vlll be stuaied 
taistoLogieally asA ths f iadiags vlll d8-bnsdn» tte eowrae of e9Rperl°° 



Projeet Bewsriptien (itet 2)t 

Ob^^ctlyi lb dev«l6p «i sensitive aetltod fear 
IiffolAetiB la blood aad urlaeo 

Mtthods eaigiLeyied ; BKslaetia ms iajected iBtzKnatouaOy 
iato &OXBU or fiOFpofiifaQ^secitaBized pi^scsss 1a a divlfisd doaey ixo bemv 
apart. AnSaals wre killed fota: benars after tte laltial lajestiea 
aad tte naber of aitoses (axreeted la aetapteee bgr ealebielaa} 
soanted la staadard axeaa ef iriuile aanwBits of tte epittelixn &£ tte 
esrap saeso 

JhjQg fladjb agg; Ste initial resulta eboir ttet aitoais vas 
iaereased by a total 1& of 0.02 uaita Cl»0 iigo ) of pra(Laetla« b»t aert 
proportiotaately iaereased b^ doses 10 to 100 fold greater. 

Slgnlflcunee to BEEftMD research ; Xt Is obriausly dssireblfe to 
be able to detect iaad assay jorolaetia la blood wad urlae of eUaieal «ad 
esperiaeatel aaterlalo B» loeal orop aethed la oareat use is tsusoitable 
for blood aad urlae extxacte, sinse toscie substaaees aad 
to eaaae a re^poese slaulatlag 'tint of prelaetla. 



be csplored to flsd a preeedore vbnw tte rnwpaae is ia jrapartioa to 
tte dose of prolaetia. Srats vUl be esteadied to ladiide extmets of 
blood aad wrlaeo 



I^xt B as^t ladudede 



1959 



1. 
2. 

3. 



> 16 



XsoiUtlm 



T!Ktt A. 



VP0^e^Tit3Mi S 


tafii>8 OB 


F«lis Aeifl 


Briaeifal l^nrsstieaten 


b: M. SSJ 


LTexasBB oad J» Co 


Otter £»f«stS€R>ters: 


B. Gs3 


r€d»r» K. 0. Sen 


Ce9|?sx%ttsg l&aits: 


Bi9»5 




Itaft Iters CCBlesietas' %< 
Sertel: 


.: 1 

1 




0@»7S 





Bo SlsUuk 



SfOJ96% 



ettfoUe ae£a: 




of if flSBBlteiSlSHSSlAMB 



Q(f foOLis &«id Cpa»f«3Lie)o 
SS«f(9iU« acid to ksMsm fooaso 
(in tSiB nt) f«ar 



eello 



aeifi; praTslie A, 
*o«iBg i»v«8ti0kted» 

1]® esidUssS la 



to 



«f folie 
£sm 2»em livw^ Xts ^salstry is 
larvtaB vSsiek eenrerts it to t8tsa» 
(satR2^«io maaasstti etf SAD tmd « 
, it appears ttaat fOECf alle A 
to tetralsQrarardLie acid. 



MAM)- i«^ 




far ^.^ ^, 

IMm apjaxieatly Is » zvaxat of iBMbitl«i 

vlth liviKr aisorterse ibis nrts^bolits oif w<«^^fffn 



lAUk I&Vo MsxTiii sad BaekBaigsl^ IQE9K)o 



» «te aetal>«iUffla «af McrUdtoe Dgr «aid)lijag ttas ntsMLiaB of fiaimlaia®» 
seM to oeeur ia folle aeifl aad vitmia B^^ dsfieieat za,ta» 
is oa iaexviassd iasflvperetiaa isS^leailM Ssnm I»»Ms«ifiiw->2->(A» 
issto sois&roa CQs £nm L-iai8tiaim<»S°C^. SsaoeiystBiitt sdiinistmtifin 

zaSissetl'TO CQge TS& <Si££tapaiae»& ia tSie netabetLisn of Mi fft l^l T«* eooLd 
lae daaegsotEatsa ia tto tao gre«ipe «r mts Cio «»# feiLie aeid vso -vit 



It tes teea f oiad that Tilania Bi2 «^s »«« setivats ttHije aeid ia 

xmt Cio «of ntm&a Bxa ^^flelABi^ tms tritboofe iaeUaaee on tte @sa« 

«f fells seid td tftt3nilcspd3r«Kreila.te das^-vatiras ia tte iateet 



fear 
t) Mm aeir teea SKsrifSM 20 fold 
aspesrs to bs a 'ritsoBi 



B ae^ xosdi^ -li» Bj^ 



talil^SBd 



aBta3»«ai«98'~^?Qlie aeid vllX z«s«lt la a 
of 
l)Sr 'sMeh f ^siLie aeid 

Iseiag TeiMly stufttefi. Ude prai^set is ais«ete& at dis<» 
of f @11« aesid «si8t la tl» body and bsiir 
to 




Cl) Sto is^lats tl» teo f aaes of ps^«Ale aeid ead 



liirs" 



edT ethl«a£att for aamxal weks^ «aEes«te 
SMS appiziently 1« & zvaUIt of iBhibitLoB 

vith liver disordfianso Slds att^bciLite oif MBtiaiiwr 
taM l)e«m fotaad la «b» vcriae of ttro (2) eterhstles (la MUAbasntl^ 




(To. e@lla!bae&tiea isi1& X2r. D. X). Bsraoa^ B£MS«) 
» tie aelnliallw of MsUaias Dqt «an»Iisg tte 

aeM to «»eiir ta ti&te aeUL aad vitioda B}9 dsfieieat xata» 
is oa Iaex«a88d lasfltc^^oratlcB is£ loltlt firm lRliifltifiiw-2<^3U^ 
iato «tcpirea COg fjram L-)ai0tiflSae>2°caA. SoBuse^rstslaB cArilaistmtiQn 

zttfii^Mnefeiw CQs» ll» aiffteosees ia tits i»tal»eaifa of kLstidiBe eoiO^ 

»LS a»fietoiit)» 



It tea teea f oraad -»»t Titusia Bj^ eUws acrt aetlrots r«(Il« aeid la 
the mt Cio «e» Titsoaia 1^ ikatiebB&By ^«« vithoot 

t tsSLie ««id t9 tstXBliyarcif eilate dsri^tL^s la 



) tea a«»r l3««a& pxrified 20 foM frmi eartsnets tf S. eeiLi 



aspesra to te a vitaaia %o scoteia. fStLs Bi« 




t@ aSftlP gageardhs 11« iMlAtlea «ad iteati- 
fioatiflB ai lib mriflua arbalboiliisi d^ f'olie aeid vm z««Hlt in a 
of tfa 
1)Sr irlii«h foUs aeid 
am teSag wi&Isr studlad.. »iie jie&S96% ia dix«ete& at dia» 
Bier t3» van^nas tmm &S f elie aeid esiat la tbe iKsdy aad bisisr 



CD a 

e8ta>hli8l& ttelr 






(2) 9e fflndy tte xvaeticms eeaaeenMd in Hkm txtatsScemtim. 
tKt jcvfoiLic seiA to eUaranraonai Autor «Bd to jnacity aim CBzgpso 

(3) Si» sttaq^ to establlah^ at Ite bioehodaa lanml, tte 
iatnr-xvlKtlsBateip of foLie aeld oasd Titanla Bj^o 

^) Setgyalmtfwn ef the ans^bialaft 1^ «9d«h MetTHIowlwt in 
«» mt aetlTfttes ti» «failisati(aB <tf «te C-1 unit of ffflnriaiwogTttfcMde 
aoido 



Itert B Inelwflftdo 



- 16 



Barfc Bs 







1959 




& EaAeeriBOiLQi^ 










Otis^ Xanwat£®ti-^gr0: 


So ] 




Warn 




BT 1959): 
3o5 
1»5 
S 



£rQljSe% B39@3Pi>£>%i4l3&S 




la liffi s^odi^s m f dLie aeido Fear ^i^^.t-*"; 

folleaeid 

., ««. ««««.»^^««. «f 5- ««a 10- 

is 




Mftjcr fjaflias^ IfiBlSteiSa «Ev%ilsai3;s f «sk- «te detezaimtlQsi cf f «lie 



aeid in fisdnai tisaiiilSipsBd «m (1) 

lat^r by 




17 






ie«w9 «f -vttlvtt in aftnyiag tlasuss vbidki hsvc a loir eoatftat ef 
f«lie aeid dsiMmtifes. 



gj '^gyrt ^ fniwff to yiT-^^Mpi xttassan^ Stew BlespdblsiLogieBiL 
prooedisiiMi (s^b & tvzy iaopooftBiic^ tooiL for Ite iuBBiiiiinwiinBv*tl> of tiie < 
tmtiau of TBTiotts esU eoestitxwErto^ 1. Co^ aodao aelds aad r±' 



Ei'OPMOtt COUPMi off jgtfQJOIC^i So OOttfeiSnB filDRHMlt idLOP^iOtLoglLSI^ 



«ad e@U«3}oe«to iriissx ealXed vqpoa vitSi other sraagpo ob prejeeto 
xv^pdrias adearobiologleaa asaaqri of viiaBSjas asid 



jl^set B laeluaod. 



17 



Bttgt B: 

Publi^&'^ass @tb&£' l&sa aba-^srlis trm. tMs protest: 






CSBlendar Ktsax* 1959 



Serial Boo BXAiO- ia 
le Bstrltim 6 SadoeriafliLosir^ 
2o RvotiOB&tlott & ISQlatieaa 
3o 



RUPt A o 

Proij«et Title: Iaz«»<-Male sreeessins of biolae&eal 

na'terlalo 
Sristelpal Inrestis&tQK': J«Sm Ce Buessteey 

Otter IeErasti0i1sGor8t Baas 

Ceepevatiag Itoits: BBS - B»tae«l Bradnets LabwatOKy 

Mbb Iters COalsBter Year 1959): 
Sortel: lo5 

fraeeaaSJOBtO.', 0,50 
Olter: 1»00 

lirQjeet SeseripUees 

Ob^etawta s ShsQr 3?e^^iLeH» (3^ liqpertaaiee i£ tl» bioelseKlstEsr 
of dloeaae reqiaire Ite isolAtlsa a»a laeatlfleation <sf aubvtenoes 
«kLeh are ipccsent iA oolsr iaca» aaeoBts is tte atttussl predueto 
Xt teeows aeeeseaxy^ la erter to oibtaia wMtfiisimttt ^goHatltLes 
of -Ihe desired em^em&g to praeess Isxse 01^10*0 a£ bialeeleal 
KiterielS; sueli as liv^ery lizftiiWy exemtfisy prodttets^ pLsat 
sftterielS; etc. 

Mittoda eaajiLayeds 'Bm l^beontesy is eqtiisped vith 
lar0i<-8Qal* ajBsarettva^ sti^ as stills^ filters, x«a«tieu saad 
eztxttetioa tettles^ etc. Xb »eat isolstian pnAOsu tte ori«lBftl 
aaall-seale joroeess hes to te nodified sad dsvelopsd; so ttet it 
««a te eerried out offieieaiay oa tte lS3e$^ scale. Bis adapla- 
ti«e or soroeess develQiEneat is aa isportsat fw»ti4» of -ia» lib- 
(smteary. tte degree of sartieipatien of -tte l^bcomtosy ^rarSos 

to -ttae needs of tte speeifie 



lagoir flaaiBiy ; She Ittege-soele lebon&tecy flMllitiies 
^ be vsed^off^aa 



of BXR iswstl^tiOBSc 






llr&r v&m sroesswa toe sK«f dLie A 
. A oodSfictttlcn of t3» pi^ag of tli 



ftteilitifsa 






w9^^^ ^htt mi will iHiiiiiftiyri' ftnA •frm^Tnwi/l *tiMV>afflMi^tt1 Mf ^Int Ittvnn su^l^ 

as tin ifgeiL&tlcft «f Ipbs pc«7«lie ae£ds 



"to &CStiT 






ia iMrgfi-weaiM enpesfttlona as »eqfalzttd liy sueh xrojaete as 
foUe aeid aaft ^» alkaloia svciblm CiB)o 



Burt B aob ineluasa. 



PHS-NIH 

individual Project Report 

Calendar Year 1959 



Serial NOo NIAMP^i o 

1, Siochesaistry & Metabolisia 

2o Enieyines & Cellular Biocfee^iscry 

3„ Bethesda 

Part A. 

Prejeet Title: Carbohydrate Mstaboiises 

Principal Investigator; fo-, Gilbert Ashwell 

Other Investigators: Dr. Jean Hietoan 

Mr, William Fricer 

Cooperating Units; Dr. J, Do Sssiley^ Beseareh Associate 
DTo Ho Ao GyiOcin, Cancer Fellow 
Offo Jo Jo Bums, National Heart Institute, 
Laboratory of Cbasieal Pharmacology 

Han Years (calendar year 1939) s 
Total: S years 

Professional: 4 1/3 years 
Other: 2/3 year 

Project Descriptions 

O bjectives : The present studies are an outgrovth of earlier work 

on the aetabolism of uronic acids in aumaaalian and 
bacterial tissues as veil as an investigation of the biosynthesis 
and degradation of ascorbic acid. This vork has been extended to 
cover the taechaniee of formation of soaoe of the oore cce^lex sugars 
found in naoBaaliaQ mucopolysaccharides and in the endotoxin fonii«> 
ing lipopolysaccharides of bacterial origino 

Mathoda ia^loyed : In ^neral, the i^thods eaiployed involve the 

use of radioactive ineorporatioas of precursor 
sugars into the ooe^^ounds being studied. This is usually followed 
by participation of the ensyme systesa involved, Xnterotediates are 
detenained by specific enzyaatie or colorimetric procedures and 
isolated by eolussn or paper chrcMaatography, Final identification 
is made by tl^ preparation of appropriate crystalline derivatives. 



Part B included 2 Yes 



NIAMD*io 

Page 2 """ 

Major Findings ; The outlines of a nev patfatwy for uronlc acid 

saetabollsB in bacceria taeve recorded in the last 
annual report., This pathway has been intensively studied during 
the ensuing year and purification of all the enzymatic reactions 
achieved. This w»rk has been cotopleted and has been suboitted 
for publication o 

Studies on the biosynthesis of L»iduronie acid in aniaal 
tissues have been undertaken in conjunction with Dro Jean Hickman,, 
Preltatnary results have shewn that raouae skin hoeogenates contain 
an enzymatic system capable of Incorporating variously labelled 
sugar precurabrs into chondroitin sulfate B, an iduronic acid con« 
taining ottcopolysaeeharide. Comparison of the relative efficiency 
of incorporation revealed glucuronie»6<>C to be a far better pre«> 
cursor then inffGAo6<»C and UDFG<>6»C te be completely inerto 
These unejcpected findings cannot be readily explained on the basis 
of our present knowledge and suggest the presence of a heretofore 
unrecognized pathway of glucurmiic acid metabolisaio This presisaptioa 
has been somewhat strengthened by the very recent observatiec that 
the same enzyme preparation catalyzos a rapid exchange of P »P 
in the presence of glucuronic acid»l»phosphate and a mixture of 
nucleotide diphosphatse o 

An alternate approach to the problem of L»iduronic acid 
metabolism has been undertaken together with DTo James Smiley and, 
mere receBtly» with Mr, William Fricer, These studies have shewn 
that chemically synthesized L»idu7onic acid is rapidly metabolized 
in the presence of TPN5 by both liver and kidney preparations with 
the resultant formation of L»ido8»ic acid. This enzyme, which has 
been purlfiod eb®*xt 200«fold from beef kidney is not specific for 
L>iduronic acid, HcNiever, the determinations indicate the affinity 
of the enzyme for this substrate to be significantly greater Chan 
that for any other compound so far es^ainsd. The subsequent 
metabolism of the L»idoiiic acid formed is under investigation. 

In a continuing collaboration with Or, Burns and Mr, Sanfer 
of the Heart Institute, further progress on the enzymatic degrad««> 
tion of ascorbic acid in metsmaXtsn tissues has been made, A 
partially purified rat kidney enK;^Be has been obtained which 
eatalyaes the decarbo:qrlatioa of diketogulonie acid, a naturally 
occurring oxidation product of ascorbic acid. The products of 
this reaction hAve been identified as L«lyxonie and L-xylonic 
acido L»:qrloaieol«C and L<»lyxaniC'»l<»G have been synthesized 
and exasBined for further otetabolifra by Ja vivo experiments with 
rats, lASs than 5% of injected C app^re^'in the e:^£red C0» 



WIAMP» IQ 
Page 3 



Ma log Ftndiny a, 5f*'ft^ » 



in the first 24 hours, the bulk of the radioactivity being recovered 
in the urine o At the preaent tiota, neither the oechanisai nor the 
biological significance of this reaction is knowuo 

An alternate enzymatic degradation of diketogulonic acid in 
tnannalian kidney has been observed by Dr. Seileyo Ih this case, a 
DPII*linked dehydrogenase catalyzes the formation of 2»keto*L->gulonic 
acido This enzysne has been purified free frent contamination with 
the above described deearbosylaseo The significance of this findo 
ing, as veil as the subsequent fate of the 2»keto hexonie acid, is 
being pursued, 

!n collaboration with Oto Morris Cynkin, an investigation 
of the biosynthesis of a new group of rare sugars, the 3,6 dideo^ 
hexoees, has been initiatedo These sugars have been shown to be 
present in the endotoalns (o«>antigen8) of Salmonella and Escherichia 
and appear to be located on the ends of polysaccharide chains. They 
have been demmstrated to be involved in the innHsnological specificity 
of the o<=>antigens<, 

A otodifieation of the malonaldehyde^thiobarbituric acid re« 
action has been developed which permits the detection end quanti^ 
tative determination of those sugars in quaoitities of lees than 
0.01 \au>leB^. Preliainairy results of growth esperisents involving 
glucose«l<>C indicate that glucose is converted to 3,6-dideoa7->L° 
Jiyloheamae (oolitose) in S, coli Olll with^t rearrangement or in- 
version of the carbon skeTetono 

Sifpaifie yace to H I AMI)_ ge ee arch ; Kaowledge of the metabolic processes 

of the sugars and sugar acids 
described in this report is essential for the understanding of the 
mechanism of formation of the ceupleac polysaccharides and their role 
in normal and pathological states » ttis present studies are directed 
toward an understanding of the basic problems of mucopolysaccharide 
biosynthesis „ Onlj in the light of this knowledge can a rational 
approach to the more specific problems of pathology be made, 

^Pr o pos ed Course ^^^ of ^^ Pr o j e e t : It is planned to continue along the 

outlines described above in the ex." 
pectation that relevant information concerning the biosynthesis 
and metabolism of the biologically is^ortant muco» and 
l&popolysaeeharides will be obtained. 



PHS-NIH 

IndtviduAl Project Beport 

Calendar Year I9S9 



NXAMD»19 



Page 4 

Part D o 

Pttbllcattons durlntj ^ ^ P ' 

Bums, J. Jo and Aslwell, 6c: L»Asc«rbie acid, the enzyiaes, edited 
by Lardy, et al. Aeademle Press, to preaso 

Asfauell, Go, Sanfer, Jo, and Burns, Jo JoS Studies on the neehanlsa 
of L-syluloae formation by kidney eosysaeso J, Biol» Chesio 234? 472, 
1959 o 

Kanfer, Jo, Bums, J, J., and Aahwell, Got IfAseorbic acid synthesis 
in a soluble ensynae aystea froa rat liver utierosoaeso BioehiiBo 
BioplriFSc Acta 31: S56, 1959 « 

Hiekwm, Jo x:^ Asbimll, Qoi A sensitive end stereoapeeific enxyseatic 
assay for xylulose^ Jo Biolo Chemo 234: 758, l959o 



PHS->Nm 

iQdividwil Project Mpmt 

CaleadAT Year 1959 



Serial NOo MIAMD» 10 

lo Bioeheuis try '& Ha tab-oi isra 

2. Bnzymes & Cellular BioclieBtistry 

3c Bethesda 



Part Ao 



Project Titles OMwicAl and Bnsyaic Studies ftelated t® tbe Structure 
and ^tabQlten of Mbmucieic Acid and its Ognstitu^ 
entS;, 



Principal Investigators Dto Maxine P. Singer 

Other Investigators s Dr, Audrey Stevens 

Cooperating Units; Dto Ciuli® L„ Gan£isni» Laboratory of Cellular 

Phanaacalogy^ National Institute of HsnCal 
Health (Serial NOo M>CP22) 

Man Years (calendar year 1959 }s 
Totsls 11/3 years 
Professionals I year 
Otber; 1/3 year 

Ob^ectiyeffig tbet ^ject of the main part of the work in the year 

1958 vas to study enxyaies that catalyze the breakdonm 
of rib»nucleie acid and then to use soaie of these enzymes , in con« 
junction with chemical methods, to elucidjste the structure of the 
sio»called "sGlubLe" ribonucleic add that is presumably involved 
in protein biosynthesis^ Becently studies hove been begun on the 
biosynthesis o£ S^ribosylursciloS'^aaoophosphate* a newly discovered 
constituent of SKA, 

>^^!»^«i ^^^g^° Colwan and paper chresiatographic techni<^les hove 

been utilised to study the products of iNA degrad«o 

ti«mo Assays of onsymic activities have involved chendeal and isotope 
tracer teehnicpeso Tractsr methods are also being used to study 
S<»ribosyluracil biosynthesis o 

Mffi^or gjndinfljs ; A new nuclease hasi been diseowered in extracts of 

mouse, Ehrlich Aaeites t^nor ceils » This nuclease 
has been partly purified and shewn to hydrolyze polysdenylic end 
polyuridylic acids to S'«AMP and S^^UHP, respectively , 



Part B included ^ Yes __ N© 



PHS»MIH 

Individual Project Rapmt 

Calendar Year 1959 



Serial »©» MIAMP» ?p 

lo Biochemistry '"& 'MeCaboiism 

2. Bnzynee & Cellular Biochemistry 

3c Bethesda 



Part Ac 



Project Title; Cheeieal and Enaynic Studies Related m the Stmeture 
and MetAboliem of Mbonucieic Acid and its Ggnetitu^ 
entSo 

Principal Xmnestigators Dtd Maxine Po Singer 

Other Investigators s Us, Audx-ey Stevens 

Cooperating Units; Dr^ Giulia Lo Canteni, Laboratory of Cellular 

Pharmacology, National Institute of Hantal 
Health (Serial Noo M-CP22} 

Man Years (calendar year 1959 }s 

Totals 1 1/3 years 
Pr<D£e8sioti4ils 1 year 
Others 1/3 year 

Objective« s tbet object of the main part of the work in the year 

1958 vas to study enzymes that catalyze the breakd«wn 
of ribonucleic acid and then to use some of these enzymes, in c«in» 
junction with chenical methods, to elucidate the structure of the 
eo»c^lled "soluble" ribonucleic acid that is presuaably involved 
in protein biosynthesis „ Becently studies hafve been begun on the 
biosynthesis of 5«ribosyluracil<»5'-»iB0ooph&8phate, a newly discovered 
constituent of SHA^ 

M tethods ^Sap loy ed g Colusn and paper ehrosaatographic technitiues have 

been utilized to study the pro((hscts of SNA degradao 
ti<smo Assays of ensymic activities hev@ involved cheadcal and isotope 
tracer technic^eso Tracer avsthods are also being used to study 
S'^ribosyluracil biosynthesis . 

Maj or Pi ndings; A new nuclease has been diseofvered in extracts o£ 

sfficmse, Shrlich Ascites tums eeilSo This nuclease 
has been partly purified and shown to hydrolyze polyadenylie and 
polyuridylie acids to S'«AMP and S^'^UHF, respectivelyo 



Part B included ^ Yes __ N© 



Page 2 



The alkaline de!gradati<9n of "soluble" RNA froa rabbit liver 
has yielded adenosine and 3*,5*oguano8ine diphosphate (adxed with 
the 2*,5*o>lsosBer) ( 6 ) as uell as the 2* and 3* iseners of the 
four usual tnostoaucleocideso This indicates that the chains in this 
preparation are terminated, at the "nucleoside" end by adenosine, 
and by S**guanylic acid at the other end^ The approxiaate ratios 
of the total ttssunts of adenosine, uridine, guanos ine, cytidine and 
S-ribosyluraeil in this soluble WA vere found to be 1:1: 1,8s l»7:Oo2o 

Extensive %7&tk on the mechanists of polynucleotide phospbory'^ 
laae action on polyribonucleotides in previous years allowed the use 
of this enzyaee to elucidate aspects of "soluble" Wh structure » 
Soluble BNA is phoephorolyzed very slowly by polynucleotide phose 
phitfylase. Moreover , the reaction stops vfaen frooi 20 to 30 percent 
of the "soluble" WA has been converted to nucleoside diphosphates » 
Our previous vork bad deaonstrated that polynucleotide pbosphorylase 
acts on a polynucleotide chain by stepwise reooval of eaononudeotide 
units starting at the "nucleoside" end of the chain (that end bear<> 
ing unesterified C«2* and Co3^ faydroe^l8)„ Since it is also known 
that amisso acids are bound to "soluble" RM through eithar this 
€«2* or C^S" hydros^l group, it vas expected that the ability of 
"soluble" BSIA to act as an acceptor for assiino acids would be altered 
by polynucleotide pbosphorylase, Xt was found, honyever, that poly» 
nucleotide pbosphorylase does not destr^ the amino acid acceptor 
ability of "soluble" mk after 20«30 percent phesphorolysis. The 
aaino acid acceptor activity per nucleotide residue of "soluble" 
BNA is unchftnged for several aodno acids o Preliminary experiments 
suggest that the nature of the secondary structure of the chains 
may detenoine their resistance to degradatione 

Xt has been found that growing yeast cells utilize orotic 
acid as a precursor for S-ribosyluraeil o 

S ignificance to MIAMfi Research : These studies contribute to our 

knowledge of the structure and 
synthesis of the "soluble" WA. and othar BNASo Thereby, they are 
directly concerned with the mechanism of protein synthesis, as 
well as SN^ structure and consequently are significant for many 
problems concerning normal or abnormal cellular functiooo 



r«ga 3 

Propoaed Oawroe of Project ; The pur££ieAti«o and study of the ascites 

nucleoae will be continued as a neana 
torard a new tool for the study of BliA structure. The studies on 
polynucleotide phoephorylase action on "soluble" RMA will be eono 
tinued in order to detenalne precisely the structural features re» 
sponsible for its resistance to ens^yiaie attaeko In addition, it is 
planned to use other enayises to elucidate further the structural 
features of "soluble" mk^ 

The studies «t S«ribosyluraeil biosynthesis are being cone 
tinued with ia l£l£&& experinents. 



PBSoNXH 

2a<i£vidual Project Bi&p&tt 

Celondar Year 1959 



NXAMP»20 



Page 4 

Part B , 

Btoblteattans during 19S9s 

Slager, He Fo , Heppel, Lo Ao » Hilaoe, R. J., Odaaa, So, and Mil, S,i 
Ensynatic synthesis of polyribonucleotides. In Begg, R. Wo (edo): 

atog ^gdinsft , og t^. "^^T^ <r*«*t'*^^, <e?^£f Cyiference. »et; York, 
Acadeffiic Press, lec,, 1959, po 41o 

Heppel, L, Ao, Stager, Mo F^, and Hilactt, R. Jot The aechanisa of 
action of polynucleotide phosphorylasoo AaUo Bo Yo Acad, Seio 81s 
635, 1959 o 

Singer, Mo P., Esppel, Lo Ao, and Hilaee, So JoS Oligonucleotides 
aa prlsers for polynucleotide phosphorylaseo Jo Biol, CluWo, in 

prasao 

SiasBSTe >1° '09 Hila»»9 to J«i, mA Eappalo to AoS fte pal yia i rt s a . 
tiea of giwaiiMinfmn diptes^taa ftr pel|nael«^ite piiMylweylaMo 

Jo Bi©!., GfMB.8 toJBBJg&o 



ras»HiH 

Indi^iduAl Project Raport 
Caisndar Yaar 1939 



Serial HOo MIAMD^ »i 



lo BlochoBlsti^ & KetabollsB 

2o Eiusynes & Cellular Biocheaictry 

3o Bathesda 



Part Ac 



Project Titles Studies on the Structures Biosyntheals aad Iater<= 
mftdlary Itotabolian of Huclelc Acids and Saall 
Nucleotides c 

Principal Zanreatigator: Dr. Leoa Ao Heppel 

Other In^aatigatorss Uto Sussell Jp Hilaoe 
I$r, Marie Lip sett 

Cooperating Qaits; DTo Audrey Stevens 

DTc Eo Pc Anderson (lationel Cancer Institute) H^i-'jllh 

Man Years (cal^i^ar year 1959): 
Totals 3 years 7 nooths 
Professionals 2 3A years 
Others 10 sumths 

Project Oaseriptioas 

The (^Ject of this project is to discover pattoays 
for She biosynthesis and breakdowa of nucleic acids 

and smller polynucleotides aad to dl8ca>»er features of the structure 

of IHA that are iaportant for its function. 

Methods Baglo yed; Biosynthesis and degradation of SNA and other 

polynucleotides is studied^ using ensynes purified 
froBi bacterial, plant end aaiaal oourceso The reactions are followed 
by veans of paper and coliam ehroaatography, dieadcal analysis and 
isotope tracer tgaathodso Also, purified ensyaes are used wlb specific 
analytical reagents for study of polynucleotide structure. Physical 
■ethods for study of Bacrenolecular structure include spectrophoto° 
metric ■easeraawKie, ultra centrifugation, optical rotation and infra°= 
red spectroscopy o 

In the field of nucleic acidSg, e^er<»increaslng 
importance la being attached to the isatter of 
secondary structures vhich gbmbis the secondary aggregation of 



Part B included X Tea ___ Sto 



Pa&« 2 



Itejiog gladinR», coat. 



individual polynucleotide chains. The forcaa holding thcM chain* 
togathav are hydrogen bonds betwaan certain pairs of bases. This 
is hov the double helix of DMA and the double helix consisting of 
poly A -f poly asre fonoedo Specific hydrogan bonding also as» 
plains ansyaic replication of SNA, and probably of BNAo Several 
aignif leant observations in this field were osade during the current 



The m»8t striking result, by B?o Upset t, vas the f< 
of a regular, triple^stranded structure between the polyaer, poly«- 
adeaylic acid and a uridine containing tetrasnicleotide« At a 
slower rate, interaction vam observed even ifith a trinucleotide <> 
The reverse interaction, between polyuridylic acid and the adenosine 
containing tetranucleotide, pApApApA, has also been observed » These 
interactions proceed to the saaa extent esad have all of the features 
of hydrogen beading between two large polyoere except that they fall 
apart or "nelt <mt" at less elevated tear^eratures o The question of 
how large a nolecule aaiat be before a double helix can be foz»ed is 
eoneidered to be a crucial onoo 



In othar work <Lo A. B^pel) it was found that the eaxyM 
polynucleotide phosphorylase is subject to powerful and highly 
specific Inhibitory effects. Any nucleoside diphosphate can be 
polynarisad in the pres^ace of any polyaer with the following 
exeeptiseas (!> Polyadeaylic acid specifically inhibits W^ and 
IDP polynerisatlon. (2) Polyuridylic add inhibits only AO? poly- 
■Mrisation. (3) Polycytidylic acid Inhibits only ID? polyaMrisa° 
tion. (4) folylnoslnlc acid inhibits CSP and AI»P, The exchange 
of radioactive phosphate %ritk nucleoside diphosphate is also 8ub° 
jact to the saste specific inhibitory effects o Only certain cos^ina^ 
tlons are involved, and they represent these, pairs of bases which 

~ strong evidence that 

of this sasyne is not a purely randoa process, but sub» 
Ject to real restrictions «hich could govern synthesis of a 

.c 8M« 

A new mielease was disc<3(v«red in Axotobacter aitil&s extraetiS: 

investigated <St«veaa and 



purified and its aacbanism of action investigated <i 
Bllnoe) c The ensyna is proving very useful in studies of SoiKA 
structure, A phoephodiesterasa was purified froa leukesia cell 
extracts and found to eichibit a new fom of specificity^ for it 
hydrolyses nothing snaller than a dinueleotlde. Other phospho° 
dissterases hydrolyse sia^le eaters of nucleotides as well. 



Page 3 

Slyitf icance t^ MXAMD Rasearch s The present studies help to 

clarify oechanisaiB for KMA 
•yntheeis and utilization. They als® give nev inforuiation on 
the eit^sAcAl aad the sMtcroooleculer structure of polynucleo» 
tides « Geasequ«ntly they are o£ significance for problems 
of hereditary laeehanisiss, certain taetabolie diseases, and 
plant and animal viru&eso Governing principles coning out 
of this vork ^Iso apply, U» socne extent, for ISSA synthesiSo 
Finally p the \i&sk per&lnent to SoMA is Inpmrtant in protein 
biosynthesis o 

Further studies lon secondary 
structure of interacting polyiaers 
and of BHAs are eenteiaplated. Ths specificities of poly- 
nucleotide phosphorylase will continue to be eaqploredo Studiei 
en the structure of S«RHA will be pursued, making particular 
use of specific nucleases and phosphodiesterases as analytical 
reagents o 



PBS-NXH 

IndlviduAl Project Report 

Calendar Year 1959 



MI AMD-21 
Page 



Part B. 



Honors ; Appointment to the Board of Editors , Journal of 
Biological Cheaistry, 

Publicatione durini^ 1 959; 

Strooinger, Jo L„ » Heppel, L» A., and Majsuell, Eo S.: Nucleoside 
aonophoephate kinases lo Transphosphorylation between adenosine 
triphosphate and nuclooside oonophosphates. BiochiiOo et Biophyso 
Acta 32: 412, 1959, 

Heppel. Lo Ao , Strominger, Jc Lo , and Maswell, Bo S,: Nucleoside 
sonopbosphate kinases IX o Transphosphorylation between adenosine 
monophosphate and nucleoside triphosphates . Biochiac et BiophySo 
Acta 32: 422. 1959 . 

Shuster, L« , Shorana, Ho 6,, and Heppel, L, A.: The node of action 
of ryegrass ribonuelease. Biochiaio et BiophySo Acta 33: 452, 1959 o 

Heppel, Lo A., Singer, H„ Fo , and Hilmoe, Ro J,: Mechanisa of 
action of polynucleotide phosphorylasso Proe, of the New York 
Academy of Sciences 81: 635, 1959o 

Hilooe, Ro Jo: The effect of endgroups and the initial site of 
attack on polynucleotides by polynucleotide phosphorylase and 
certain phosphodiesterases o ProCo of the Hew York Acadeay of 
Sciences ^: 660, 1959, 

Singer, M, F,, Hiliaoee Ro Jo, and Heppel, Lo Ao: The polyaieriza^ 
tion of guanosioe diphosphate by polynuelaotide phosphorylaseo 
J. Biolo Cheao, to appear March 1960o 

Singer, M, Fo, Heppel, Lo Ao , and Hilooe, R, Jo: 01igoQucleo-° 
tides as priiaers for polynucleotide phosphorylaseo Jo Biolo Cheso , 
to appear March 1960o 

Bappel, Lo Ao: 5* '-Nucleotidase <, In Boyer, Po Do, Lardy, Ho, 
and H^back, So (edso): The Enzyaes o New York, Academic Press, 
Inco, 1960, Volo XII, in presso 



Individual Project Report 
Calendar Year 1959 

Serial H©. Nliao- 22 

1. Biochemistry & Metabolism 

2. Enzyaes & Cellular Biocheaistry 

3. Bethesda 

Part A . 

Project Title: Gsne«Sazyns Selationshlps in Hlstidine Bi<!>aynthesiSo 

Principal lavestig&ter: Br. Srues No henBts 

Other Investigators: Barbara Garry 

Han Tears (calendar year 1959): 
Total: I l/a years 
Professional : I year 
Other: 1/2 year 

Objectives: 

^ S*laoBella and huai fciKid tbsy are all in a cluster on the 
Salttoriella ebroaoaoiBe. We hsve set up enzyise assays for the 
different enssyntes of the pathway so a@ to determine vhich cutants 
are missing which enzymes and to see i£ there is any correlation 
between the fle<;isence8 of the gomes on the chroopsorae and the 
sequence of the ensymes they control in the biossmtbetie patbv 
way, 

^^ ^^ 2« Beprassiea of the hiatidine bloaynthetic enzymes by 
histidine . We h»ve in««s8tigated the repression by histidine of 
the synthesis of the ensymes of histidine biosynthesis. Two 



points ware under investigation: a) Q^es the histidine ?«•> 
pression of enzyme synthesis affect each of the ensytaes of the 
pathway to the em& esstent. b) la there assy influence of the 
eoneentratictai of the enzyme substrates on the enzyme synthesis 
control mechanic. 

Methods B»loY «d: Mk have used vari<ou8 histidine mutants of 

Sala^nc^ f isolated by !^. Po S, Bartmsn and 
modified the fiissaye for the enzymes of histidine biosynthesis 
we have previously described in K a ur^spor a» 



Part B included fee ^ No 



NliUNDo 



Majffr Ftadlnpsi 



I. The bi@ehe»lcal ^suslysis of Ha7t3aan''s mutants has 
indicated that, in general, «aeh genetic class of nutants can 
be aesociated with the loss of a particular biosynthetie enzyise, 
of thsa histidine genes on the chroaaosoae linkage 
to tha sequence of the enzymes they control in 
the biosynthetie pathvsy. £n addition^ certain of the mutants 
itieally as if they vere odssing a section of 
all o£ ths histidine genes have been 
to be nissing all four of the biosynthetie enzymes that 
were tested for. 



2. It has been paMsiblep by lo«»ering the histidine pool 
in SalBBoaella t& raise the level of the histidine biosynthetie 
35-fold over the level of these ensyiaes in 

growing on aininal sasdiuao One «ay of doing this 

to grow a histidi^ieorequiring mutant on a derivative of 
histidine as a soesree of histidine so that the grovth rate is 
liaiited by the aaeount of histidine available to the organism. 
Sy using autants blocked at different points in the pathuay «e 
have been able to show that histidine alone controls the rate of 
synthesis of the various ensymes of the p&tisaAy, that the 
histidine repression is independent of ths ^^uantlty of each 

bstrate present ia the cells. Depending en the omtant, 
can be in the presence of substrate or no substrate 
without influencing the repression of its synthesis by histidine. 

The aajor finding of this study of repress iosa was that 
histidine affects the synthesis of each of the enzymes of the 
pathway to the so&s estsnt. flftis pheneiBeueia has been called 
coordinate repressi@a. Several other enzyaaas (glutaiBie de» 
bgfdroganase and histidine activating ensyss) have been shown 
not to he influenced by the size of the histidine pool in the 
organisa. Many hypotheses would be c^^sistent with the finding 
that histidine represses the synthesis of all of the histidine 
biosynthetie ensyiaes together. Om attractive possibility which 
is suggested is that, as the histidine genes are closely linked 
on the chronosoBS, this feedback aeehaaisia stay work at the gene 
level and histidine (or s htstidine-nueleie acid repressor) has 
a specific affinity f®r t\m histidine section of the 
and can "turn off" these g^es vban the internal histidine 
eeatrstion rises. 



NXAfiD" 22 
Pages 



Significance t© MIAMB Research: The etm&sol w&ehmiem of tb® eell 

are of fundmsental Imporeanceo ftte 
prebloK of the ger^t£« control of mee&tolism is o»e of tha central 
ones of biologs' and has ioi^lie&tiens for all of otsdical seienee. An 
understanding o£ hma gsnes are "turned «n and off" may be the key t@ 
th® probieia of differentiation. 

Proposed, QmxBB of Prgjegt; In te^eg^ and yeast it hss been 

sh^ssm tbst the hietidlne genes are 
scattered on the ehrea^sosns and it ^11 be of interest to see if 
repression is jpossible in these organissss. Ftirther \msk ie planned 
to try and characterise tle^e repressor in Sali Boaella end t© study 
its BBode of SLCtixm^ 



FBS-NlH 

Individual Frojeet asp®;£ 
ealendar ¥e£? &9S9 



Serial K«. NIAMP«23 

lo Blechaaietry & Mstabolism 



2. Eaz}^ota6 & Cellular Biocheasistry 

3. Beet^sda 



iM^' 



Fff©Jeet Titla: "Sse tol® o£ PeiyaiBines in efee Heutraiizatim ©f 

Bae£@ri®£>lifise l^&.' 

Qth^T Inv@s£iga£®?s: Pr. Sisasld T„ ^bin 

Sfeffl ¥®®rs Ce©le«sd©r year 1959): 
T®£al: i year 
frsfeasional: I y@s? 
Otiser: 



Ob^estiyes ; It ws previously ra^reed ehat baeeetriophfige T4 

KaiBfid the j^lyaisisies ptstreseine. fi3H.(CH,).Ha, 
spensddine, m,iGBJiJmimJiJSSL» in fiaasunta aufflelaSt^to i 
ize abeut half of fhl viraf mf The putreseina and spersaidina in 
the phage vere feund to he derived froea the large aisount of 
pelyaaiines norsBslly present in the bast baeteriwa, S| 
e»li« Xt was alse ehoen that these eations vere tkiT 
cesBpounds in phage T2 rep«9rted by Hsrshsy t® be injected into the 
bacteria almg ^th the viral SSSAo 



These findings gm polyssiiiee in ^M^ raised s@versl 
qisestisna: 

1) Is the rsle of tha polymlms in phags that of apeeifie or 
tBm»speeifie cations for neutralising the negatively charged 
phosptote groups in the Mkl 

2) ^@ ths ffiwunts and kinds ®£ poiy^^^ts in fm ptege det@miffi@d 
by the pha^ or by the bacterial pool of cations? 

3) Gan 3toichi<metry bet^en eati@^ ia the phage and th@ ptosp^ts 
of the mk be deaenstreted? 



4) What is the distrilmtls© @f p©ly^^s iis vlrose®? 
Part B included X Yes Is 



i^ll 



Methede ga elgyed; Varlaws visnsses were ^aaa and pur&fled and then 
sesojred for polyesaioes ast6 pfaosphASe, 



and to||l 



: Tiae c££&ens of T4 phags ha»7e been 

baianee teas l»sen o&t&iaed bet^en total eat|^s 

,$ess. "Site eati«os p^strreseioe s epestoldine , 
aeutraiize the Dim of tha T4 &aet«?i^liffige obtaiasd frora 



E. egU srs«m in lainiml stsdiusa. ?laa satisas in f4 ptog@ hsve 
beeaT^mm to b« a funetiosa of both the ooiBiiositioa of the pool 
of eatiame in ths host b&eteri«sa at the tisoe of phage AseenSilf 
«ad the affinity of e&eh species of cation for the phage nuclele 
acid. Viable 74 ph&gs have be^ (Dbtained viith vasious cations as 
the S^A«setitralizing ag@at; the role of the polyaoiines in phage 

to be that ®£ & s®n-speeific cation for mA neutralizstiSB 
stabilisation. 



fhe absence of polyataioss in esrteia Eo coli «ad 

^_ _ iaages «as eerselated ^th their persEeability to 

i; it appears as if the polyasiiQes are displaced by other 
cations dtsring purification of the phage. Sdlyssaines are not 
present in fM^, Cucumber ?inss, Tonat® Bushy Stunt Virus » or 
folio Virus o 

Sianigieanee t& 1£AI®, leseareht The present studies help t® 

•'' ^^ " ^' — — Claris the role of the polysnines 

as !%SA neutralising agsnts. Censequently they ore of signifiesnee 
for probless of hereditary issehanisias and the structure of viruses, 

gr^sed Ceurse ®f Project.' mt ^jaeti^s of the study have been 

realised asid m& further «ork is 
pla^nad at this tlae. 



iBdiv&dufil Psojeet Itep®rt 
Calendar Year 19S9 



NZAMD* 23 



!8, B. H. And Oobia, D. t.t 1 

%tT&ll3Atijoa of baeteriopbage 



sole of polyesa&nes 
J. Biolo 



in the 



Xadividual Projeet Seport 
Calendar teas 19ii9 



1. Bioehemlstxy & Hetalieiisn 

2. Bnzyaes & Cellu&aff Bi@ehaaistcy 

3. BethesdA 



Part A» 

Fr«ject Title: Sazy&atie Utilizatieo of Hodel 
Prinei!»al Xonmstigators UUiifiiB B. Jakol^ 



Cooperating Units: Sr» B. «;. Yaitiada, Fall«» o£ the Jane Coffin 

Ghilds Menerial Fund fo? Medieal Research 

Ctr. M. Nirenberg, Fellot; of tbs 
Society 

Sr. Wj^ae Albers, HINDB: HA>NCo8 



Sian leaxo (eaiendar y@ar 1959): 
Total: 3 

Professional: 2 1/2 
Other: l/I 

Project Baseription: 



invastigatioets were concerned ^th the reaeti-^Tit^ of 
vasi&&a cheseleal groupings in ensyoe catalyzed reacti^&So T^ 
ccmpounds studied noet intensively have been fsm&s^kmtyrte acid 
and aeetylenesiesocarlioj^lie aeido 

Sfeth^ amlg yed; By use ®f the enrieh^at culture technique » 

fl^eroorgaaisas vers obtained vith the ability 
to gxow on various aodel cos^ounda as solo carboa source > Inzy^es 
free these orgaaimas vere investigated by the usual techniques o 

The biosynthesis of ^«eiBinobutyrate froa pyrroli« 
dine end putrescine and the subeetpient utilization 
of this eesiiiound have been studied at the enzyise level. Each ®£ 
the enzyse reaeti«&8 which are denoted by solid lines have been 
purified and characterized and evidence for each of the intenaedi^ 
atas listed has m& been obtainedo 



Part B included I Tes 



^^ 



Major Piodinga^ coat. 



pyrrolidine putrescine 7<>hydroxybutsTat@ 



i 



' I 

1 ' • 

^ •pyrrolioa — ^ 7>-amio^«styr aldehyde — ^ 7»ai&inQbuCyrate — ^ euceilnic senlaldebyde 



i 



succinate 

Of particular interest has been the study of the kinetics 
of one of these reactions » tl» transsaainseion of 7«>aiBinobutyrate 
and yketoglutarate resulting in the fonoation of succinic seatto 
aldehyde find glutassateo The dsta h«d suggested that tronsaminA^ 
tioa occurred by i^y of a series of binary cosiplexes of enzyoe 
and esch substrate » Further support for this concept has been 
obt£iaed by the dissection of the transamination into two 
reactions. 



Conti£m£ng studies on the mseb«nisi9 of anzynie catalyzed 
aldehyde osidation have resulted in inforeiatioa concerning the 
sites of substrate binding to the proteiRo Eaploying techniques 
of ensysae digestion and eo^etitive inhibition it has been con» 
eluded that aldehyde substrates are bound to closely juxtaposed 
SH groups of the e^nsyme, vhere&s pyriditie nud.eotides ere bound 
&t sites other than sulfhydryl groups. In & study of a novel aide*' 
hydfi dehydrogenase oxidising msloaie semialdehyde» both DI>N and Ck»^ 
were fo«md to be involved, resulting in the direct fortaation of 
cArbon dioside and aeetyloGoA. 

Siipif icanee. U» nimi Besearch; Each of the compounds studied 

\ is^ingee oa an area of vital 
interest in the fields of biology and is^dicine. A particularly 
clear esaaple of bsisic research leading to practiesl applieati^tn 
aay be cited frost th@ above»noted tmrk on y»aminobutyrie jscid 
nstaboliem which ims led to m estreoastly sensitive and specific 
nethod f^ the detertain@titcm of this coopound in brain where it 
appears t© play «i role in both narvewas m.d isetabolie activity, 

Pro^sed CoMsse of ggojact; It is enacted that the msehanisa 

of aldehyde oasidation by enzyi&es 
will be further investigated. A study @f the oetaboliea of 
other fi»del eQss^eund8« e.g., g^^^^^^s ^^^ ^°^ erythrltol, will 



Sadlvidual Project 
Calendar Year 19S9 



^.ji 



fart B, 

Jt I'l Tinir M inff I ■^NTffir.MiiTJiriffr 



Scott, S. M, aad Jalwby, W. B«; Soluble 7»aBiinobut;if<ic-gIuta.aio 
transaainase froa Iggudtegoas ^^l^gM. J. Biel. Shea, 2^: 
9329 1959a 

Jakeby, Vo B, and Seott» Sc K,: Alddiayde oKidatioa r.lo Suecinie 
semialdehyde del^difogenase. J. Biol. Chea. 234: 93? « 1959. 

Jakoby, W. Bo and ^redericke, JoS Fyr^olldlne and pu^^esdne 

saetabolisBU ^o^ainsibutyraldeSssfde debydaogenase, J. iS'^l« Cheeo 
234 ? 2141, 1.959 o 



I, Sc Wo and Jak®5»y. W. B,: Snayaatic utUisatloa of 
aeetylenie <ai»^ounda IX. Aeetyleneffi^oearbos^lie acid '3;ydraae» 
Jo Biole Ghsm, 234; 941 » 1959, 

Jak@by, W, S. and Harrod, S. A„: Aldehyde oisidation £V, An 
aldehyde b«af£er f<BS g]io%?th studies » Jo Bact, 77: 410, 1)59 o 



Jakobyc, Mo Bo sni iBsmd&p E. H. : 0iree4 enxymic eonversim of 
aal^iic s^tialdehyde to aeetyl'^eoenzyms Ao Bioehiato Biopliys. 
Acta 34t 276, 1959. 

Nirenbcirg, Ho and Jakoby, H. B.: Sazymatic utilisation of 
^»tqrdr@s7b4ityrie acido J. Biolo Cfaes.» te^^^yress. 

Jak^ty, H. B.: Snxysatic fonaation and utilization of yaniitofcutyric 
«ctd in PeeudoBsooaSo Sn Soborts, B, <edo); 
nervous Systeai and y^Aai^butjriy ,Aeid, in,Br|gao 



Albere, Wo K. and Jakoby, W. BoS transaminatioa and the isotttpie 
labelling of glutamate in brain. In Boberta, S» <edo): Inhihitioc 
in the ^rvou|» Syaten and y^Aaiapbutyrfc Acid, in,jr^8o 



Jakoby, Wo B,: Enzyoes of y*aniinobutyrate aetabolism, bacterial a 
In Colo«ick. So and Kaplan, K. 0. <eda,)f Mf thod. in Enay«olo^, 
Msv York. Acadesiie frees, ¥olo IV, ps. -ff ^gga » 



PabUeaticaa d«rtnj^ 1959, e<ynt:. 

Yameda. S. H. aad J®k<3by, «. B.: Aldehyde «xldati<m V. Direct 

cemretsioxi of ssaldsic s^aldehyde to aeetylccwnsyate Ac J. Bi<sl<, 

Bayai8hi» 0., Slaa^eer, C, and Jakoby, Wo B.: 3»Bydresy b&le 
aeid dahydrogeoaae froa Bacharichta fremidli. J. Bact,, to press. 



Jakoby, W. Bo : Qssaiate deearbaacylat&oa. Sa G«louiek. S» and 
faplaa. No 0. (edso); Sfethoda in lte«y»»lo)|y. Mev Y^k, Academe 
Press, V©1. r?, JagB Pes i B . 



Albars, V. &o and Jake&y, Wo BoS Ssehange reaetieas catalysed by, 
7-siBinoS»tat:yriG»giu£sisie detsydrogenaseo Bioehlrao BiophySo A@&s, 

in .y;g«8s. ■ ■ ■■ 



Serial No. NIAt/iD-25 
1. Biochemistry S, 
2o Intermediary Me. 
3, Bethesda 



PHS-NIH 
Individual Project Report 
Calendar Year 1959 



Part A= 



Project Titles The Biosynthesis of Inositol 

Principal Investigator; Dr. Frank Eisenberg, Jr. 

Other Investigator: Dr, Yoh Imai 

Cooperating Unitss None 

Man Years (calendar year 1959) s 
Totals 2-1/2 
Professionals 2 
Other; l/2 

Project Descriptions 

Objectives ~ To elucidate the mode of biosynthesis of inositol 
in the mammals 

Methods Employed - Various carbon-labeled sugars and sugar derivatives 
were administered intraperitoneally to ratSo Three hours later the 
animals were killed and myo-inositol was isolated from the whole animals 
and assayed for the amount of isotope.. In some experiments the inositol 
was partially degraded by three different methods to allocate the isotope 
to one or at most two carbon atoms as follows; 

(1) Oxidation by A, suboxydans to myo~inosose-2 followed by 
periodate oxidation to yield C0_ (C-2). 

(2) Oxidation by HNO_ to D ,L -epi -inoso5e-2 followed by periodate 
oxidation to yield C0„ (C-4,6). 

(3) Oxidation by a rat kidney supernatant to D~glucuronic acid 
followed by acid decarboxylation to yield CO^ (C-l). 

Major Findings - Of the various labeled six-carbon and smaller 
compounds administered to the rats glucose and galactose were found tc 
be the best precursors of inositol. The extent. of synthesis of iso- 
topic inositol from both of these sugars was independent of the location 
of the label, suggesting that a six-carbon unit is the immediate 
precursor of inositol in the rat. This unit, however, is not glucuronic 
acid, since although glucuronic acid-U-C^^ was incorporated, glucuronic 
acid-6-C^'^ was not. 

These results indicate that the mode of biosynthesis of inositol 
in the rat is different from that observed in yeast where a two-carbon 
and four-carbon unit combine to form inositol. Furthermore, the cleavage 
of inositol to glucuronic acid observed in rat kidney extracts is not 
sufficiently reversible to account for inositol biosynthesis in the 
whole animal. 



Serial NOo NI^'^ID- ■; 
Page 2 

Partial degradation studies have supported the six-carbon cyclization 
mechanism. Glucos^jl-C gives rise predominaijitly to C-4,6 labelec ino- 
sitol; glucose-2-C" to C-3,5; and glucose-6-C" to C-1. 

Significance to MIAMD Research - The presence of inositol in 
animal tissues both in the free state and in combination with lipide 
materials makes the study of its biosynthesis of interest and importance 
to a complete understanding of carbohydrate metabolism and its possible 
link to fat metabolism. Without an understanding of the normal course of 
these reactions a derangement in one or more of them might not be 
recognized. 

Proposed Course of Project - The definitive conclusion that inositol 
is derived from a six-carbon unit must await the complete degradation of 
the compound. Chemical methods to achieve this end are now being tasted 
and will then be applied to the biosynthetic labeled inositol. 



Part B included? Yes 



PHS-NIH 
Individual Project Report 
C.cilendar Year 1959 

Part B . Honors, Awards, and* Publications 

Publications other than abstracts from this project; 

Hisenberg, Fc, Jr., Dayton, P, G. and Burns, J. J. Studies on 
the glucuronic acid pathway of glucose metabolisni» J. Biol. 
Chem. ^4, 250-253 (1959).: 

Eisenberg, F., Jr. and Leder, I. G. An imp3:«ved scanner for 
radioactive paper strips. Anal. Chem. 31, 627-628 {1959).. 

Dayton, P. G. 5 Eisenberg, F., Jr. and Burns, J, J. Metabolism 
of C-'- '^-labeled ascorbic, dehydroascorbic and diketogulonic acids 
in guinea pigs. Arch. Biochera. Biophys. 81, 111-118 (1959). 



i. Biochemistry & iVieta.' 

2. Intermediary Metabo: 

3, Bethesda 



.' PHS-NIH 
Individual Project Report 
Calendar Year 1959 



Part A. 



Proj/ict Titles Studies on the Degradation of Insulin and Insulin 
Derivatives by F^ilammalian Tissues 

Principal Investigators: Dr. Frank Tietze and Dr.. Glenn E. Mortimore 

ether Investigator; Dr. DeWitt Stetten,' Jr, 

Cooperating Unitss Clinical Endocrinology Branch- NIA^ 150C 

Man Years (calendar year 1959); 
Totals 2-1/2 
Professionals 2 
Others l/2 

Project Descriptions 

Objectives - Previous studies in this project on the fate of 
insulln-I^"^-*- in intact, perfused rat liver have shown that this organ 
can carry out the extensive degradation of the labeled protein. Such 
deijradation is presumably catalyzed by an enzyme system, termed insulin- 
as*>, which has been found by other workers in soluble extracts of liver 
anil other mammalian organs. More recent work with intact liver has been 
concerned with the specificity of the enzyme(s) comprising this degradative 
sysitem and with the role of the cell membrane in the capture and degradatios 
of insulin-I-'-'^-'- within the intact cell, 

Methods Employed - Intact rat liver is cyclically perfused, at 37° 
or 0°, with oxygenated whole rat blood containing trace amounts of 
insulin-ll3l. At intervals of time, aliquots of the perfusing medium 
are sampled for total radioactivity, TCA-solubie radioactivity (a measure 
of dggraded insulin), and TCA-insoluble radioactivity (a measure of 
undegraded insulin). Such perfusions have been carried out with native 
and denatured insulin-I^^^ either alone or in the presence of possible 
competing substrates. Paper chromatographic methods have been employed 
in d number of experiments to determine the raonoiodotyrosine (MIT) and 
diiodotyrosine (DIT) contents of undegraded insulin prior to and 
following perfusions. 

Major Findings - Determination of the MIT and DIT contents of 
various preparations of insulin-I-'-'^-'- has indicated that the fraction 
of iodoinsulin resistant to perfused rat liver increases with increasing 
initial content of DIT in the labeled substrate. Furthermore, the DITs 
MIT ratio of the resistant fraction of labeled protein is significantly 
higher than that of the substrate prior to perfusion. 



Serial No, NIAMD- 26 
Page 2 

The results of a number of experiments heve suggested that the 
degradation of innulin-I^^^ by intact liver may proceed by a sequence 
of steps which, in the simplest case, may consist of the following 
events: l) Binding of insulin by the cell membrane; 2) transport of 
insulin to the site of insulinase activity; 3) degradation of insulino 
Thus, for example, when liver is perfused with insulin~I - at a 
substantial uptake of the label is observed without, however, a con- 
comitant appearance of TCA-soluble products; in contrast a soluble enzyme 
preparation obtained from rat liver possessed considerable insulinase 
activity when measured at 0° « 

Evidence for the role of the cell membrane as a determinant of the 
specificity of insulin degradation by intact ret liver has been furnished 
by a number of observations^ Thus, whereas soluble insulinase prepara- ^^^ 
tions are. capable of extensive degradation of alkali-denatured insulin-I 
intact liver preparations are essentially inert with respect to the altered 
protein. Furthermore, whereas ACTH behaves as a potent competitive sub- 
strate of insulin-I^'^^ degradation by the soluble enzyme preparation, the 
same protein is without effect on the degradation of the labeled substrate 
by the intact livero 

Significance to MIAMD Research - Although the liver does not appear 
to constitute a primary target organ for the action of insulin the' 
substantial- binding of the hormone which has been observed under condi- 
tions not complicated by degradation, i.e,, at 0°, may bear a significant 
relation to the uptake of the protein by frank target organs, e.g,, 
muscle. In particular, the location of such binding sites on or within 
the liver cell would be of value in the further understanding of the 
mechanism of action of this hormone. 

Proposed Course of Project - It is proposed to couple insulin with 
a fluorescent dye, such as fluorescein, and to incubate the conjugate with 
a suspension of intact liver cells. It is then hoped to localize the 
site of binding of the hormone by microscopic examination. Preliminary 
bioassays will be necessary to determine the effect of the coupling 
procedure on the biological activity of the hormone. 



Part B included; Yes 



Serial No, NIMID- 26 
Page 3 



PHS-NIH 
Individual Project Report 
Calendar Year 1959 



Part B , Honors, Awards, and Publications 

Publications other than abstracts from this projects 

Mortimore, G. E. , Tietze, F» and Stetten, D=, Jr. Metabolism of 
insulin-ll31. Studies in isolated, perfused rat liver and hind 
limb preparations. Diabetes 8, 307-314 (1959). 

131 
Mortimore, Go E. and Tietze, F, Studies on the fate of insulin-I 

in the perfused rat livero Metabolism 8, 479-480 (1959) . 

Mortimore, Go E, and Tietze, E* Studies on the mechanism of capture 
and degradation of insulin-I-'-^ by the cyclically perfused rat 
liverc Ann. N. Y, Acad. Sci. ^, 329-337 (1959). 

Tietze, P. Release of amino acids from the carboxyl terminus of 
native and modified egg-white lysozyme. Arch. Biochem. Biophys. 
In press. 



Honors and Awards relating to this projects Nonec 



Serial No. NIAMD- 2 7 ,^ 

1, Biochemistry 8. Metabol: 

2. Intermediary Metabolisr 
3.. Bethesda 



PHS~NIH 
Individual Project Report 
Calendar Year 1959 



Part A » 

Project Titles The Mechanism of Action of Hormones 

Principal Investigators; Dr. Yale J. Topper and Dr. Elizabeth S» ?i!axwell 

Other Investigators; Dr. T. David Elder and Dr. Stanton Segal 

Cooperating Unitss Section on Metabolic Enzymes- NIMS) 32 

Clinical Endocrinology Branch- KIMD 143C 

Man Years (calendar year 1959); 
Total; 3 

Professional; 2-1/2 
Other; 1/2 

Project Descriptions 

Objectives - The immediate objectives of this project are l) to 
determine the mechanism(s) by which progesterone, testosterone and 
androsterone stimulate the oxidation of D-galactose by certain mammalian 
tissues in vitro and 2) to determine the mechanism by which progesterone 
enables galactoseraic subjects to metabolize galactose. 

14 
Methods Employed ~ l) Determination of C" O2 production from 
galactose-1-Cl^. 2) Analysis of pyridine nucleotide levels in tissue 
preparations incubated with and without steroids, 3) Determination of 
certain enzymic activities as influenced by steroids. 

Major Findings - Since the effect of' steroids on D-galactose 
metabolism was reported last year the following observations have been 
made, l) The site of action of progesterone on galactose metabolism has 
been localized at the level of the UDPGal-4-epimerase reaction, 2) One 
mechanism by which progesterone stimulates the epimerase reaction in liver 
relates to the fact that the hormone lowers the level of DPNH by inhibit- 
ing aldehyde dehydrogenase reactions, 3) Kidney aldehyde dehydrogenase 
is also inhibited by progesterone. It has been deduced that one of the 
reasons galactose metabolism in kidney is normally not influenced by 
progesterone relates to the virtual absence of alcohol dehydrogenase 
from this tissue. 4) Menthol simulates progesterone in its effects on 
galactose metabolism in vitro and in galactosemic subjects. 



Serial Uo. NIMID- 27 
Page 2 



Significance to NIAMD Research - Studies on hormonal control of 
D-galactose catabolism might be expected to shed light not only on 
galactosemia, but on other metabolic diseases as well. 

Proposed Course of Project - More information relating to the 
in vitro mechanism of action of progesterone and menthol will be sought. 
In addition, the mechanism, of action of progesterone and menthol in 
galactosemia will be further investigated. 



Part B included? Yes 



Serial No„ NIMD- 27 
Page 3 



PHS-NIH 
Individual Project Report 
Calendar Year 1959 



Part B e Honors, Awards and Publications 

Publications other than abstracts from this project; 

Simon, E, R. , Pesch, Lo A. and Topper, Y. J. Localization 
of the steroid hormone effect on galactose metabolism. 
Biochem^ Biophys. Resc» Commo 1^, 6-8 (1959) » 

Pesch, Lo A,, Segal, S. and Topper, Y. J. Progesterone effects 
on galactose metabolism in pre-pubertal patients with congenital 
galactosemia and in rats maintained on high galactose diets ^ 
Jo Clin, Investo In press. 

Topper, Y. J. Isomerization reactions, in "The Enzymes", 
Second Edition, Vol. III. Academic Press, Inc., New York. 
In press. 

Topper, Yo J. Aldose-ketose transformations, in "The Enzymes", 
Second Edition, Vol. Ill, Academic Press, Inc., New York. 
In press. 

Topper, Y. J., Maxwell, E. S. and Pesch, L. A. On the mechanism 
by which progesterone stimulates galactose metabolism. Biochim. 
et Biophys. Acta. In press. 



Honors and Awards relating to this projects None. 



Serial No, NIAMD- 28 ,„ 

1» Biochemistry 8, Metabcj;!. 
2o Intermediary Metabolism 
3. Bethesda 



PHS-NIH 
Individual Project Report 
Calendar Year 1959 



Part Ao 

Project Titles Studies on Oligosaccharides and Polysaccharides 

Principal Investigator; Dr, Marjorie R.. Stetten 

Other Investigators? Dr, DeWitt Stetten, Jr, & Mr» Howard Mo Katzen 

Cooperating Units: None 

Man Years (calendar year 1959) s 
Totals 3 

Professionals 2-1/2 
Others l/2 

Project Description; 

Objectives - To gain insight into the normal structure, synthesis 
and metabolism of certain polysaccharides and oligosaccharideso 

Methods Employed - Glycogen is isolated from animals by acidic 
methods, purified and used in studies of the nature of the changes 
which occur on treatment with alkali under various conditions. Light 
scattering methods are used in following the decline in molecular weight 
and chemical and chromatographic methods are used for identification of 
the products produced. 

Usual methods for the isolation, purification, characterization and 
kinetic studies of enzymes are used. Radioactive glucose-°i-PO^ and 
maltose are prepared from radioactive starch^. Samples synthesized and 
isolated are assayed for radioactivity. 

Major Fin dings - The rate of degradation of glycogen by alkali 
under various conditions has been studied. Among the principal products 
of such degradations a number of mono- and polysaccharinic acids have 
been found. The most abundant of the monosaccharinic acids has tentatively 
been identified as isosaccharinic acid. 

The mechanism of action of a rat liver transglucosylase has been 
studied and the existence of a glucosyl enzyme intermediate proposed. 



Serial No. NIAMD- 28 

Page 2 

Significance to NIAMD Research -Alterations and defects in the 
way the body metabolizes various carbohydrates have been found to be 
characteristic of certain nutritional states, drug actions and 
metabolic diseases.. Any additional knowledge as to how carbohydrates 
are normally handled may be expected to contribute to a better under- 
standing of the nature of these conditions and diseases. 

Proposed Course of Pro.iect - Characterization of the saccharinic 
acids produced by the action of alkali on glycogen will be pursued. 

The purification and properties of the mammalian transglucosylation 
enzyme will be further studied. Evidence will be sought for the transitory 
existence of glycosyl enzyme complexes with transglycosylating and phos- 
phorylating enzymes and attempts to isolate such complexes will be 
undertaken. 

In a separate program with guest worker. Dr. Nancy Cummings, a 
study has been undertaken of the in vitro metabolism and respiration 
of brain tissue derived from normal and uremic rats. 



Part B includeds Yes 



Serial No. mmp- ?.8 

1. Biochemistry &. Meiaboli 

2. Intermediary Metabolisni 

3. Bethesda 



PHS-NIH 
Individual Project Report 
Calendar Year 1959 



Part B o Honors, Awards, and Publications 

Publications other than abstracts from this projects 

Stetten, M. R. Transglucosylation by a mammalian liver enzyme. 
J. Am. Chem= See. 81, 1437-1441 (1959). 

Stetten, D., Jr. Gout. Perspectives Biol, Med, 2, 185-196 (1959). 

Stetten, D., Jr. Symmetry, asymmetry and meso-symrnetry (Editorial), 
Am, J. Med, 26, 161-164 (1959), 

Stetten, D,, Jr. Introduction to deficiency diseases, in "Textbook 
of Medicine" (R, L. Cecil and R, F, Loeb, Eds,), W. B. Saunders Co,, 
Philadelphia, pp, 527-532 (1959), 

Stetten, D,, Jr, A current view of metabolic errors. Am, J. Medo 
26, 659-661 (l959), 

Stetten, D,, Jr, Hormone regulation. Rev, Mod. Phys, 31, 563-568 
(1959), 

Stetten, D,, Jr, and Hearon, J. Z. Intellectual level measured by 
Army classification battery and serum uric acid concentration. 
Science 129, 1737 (1959), 

Stetten, D., Jr. Comments on the fate of and responses to insulin 
in the liver. Metabolism 8, 559-564 (1959), 



Honors and Awards relating to this projects None, 



Serial No, NIMD- 29 

1. Biochemistry & Metaboli 

2. Intermediary Metabolism 

3. Bethesda 

PHS-NIH 
Individual Project Report 
Calendar Year 1959 

Part A . 

Project Title: The Biosynthesis of Thiamine. 

Principal Investigators Dr. Irwin G. Leder 

Other Investigators: None 

Cooperating Units; None 

Man Years (calendar year 1959): 
Totals 2 
Professional; 1 
Others 1 

Project Descriptions 

Objectives - To study the mechanism of synthesis of the vitamin 
thiamine. 

Methods Employed - The enzymes which catalyze sequential steps in 
the synthesis of thiamine will be isolated from crude extracts of bakers' 
yeast by classical fractionation procedures and by column adsorption and 
elution techniques. The enzymatically synthesized vitamin and precursor 
compounds will be tested biologically with various mutants of neurospora 
and Eo coli. Intermediates formed by purified enzyme systems will be 
isolated and studied by chromatographic, electrophoretic and spectro- 
photometric techniques. 

Major Findings - The synthesis of thiamine from its two constituent 
moieties, 2-methyl 4-amino 5-hydroxymethyl pyrimidine and 4-methyl 
5-(i3 hydroxyethyl) thiazole, involves the initial formation of thiamine 
monophosphate rather than the free vitamin. The synthesis requires three 
enzymatic steps; The phosphorylation of the pyrimidine to the correspond- 
ing pyrimidine pyrophosphate; the phosphorylation of the thiazole to 
thiazole monophosphate; the condensation of these derivatives to form 
thiamine monophosphate with the elimination of pyrophosphoric acid. The 
phosphoryiated substrates have been synthesized and the "condensing" 
enzyme purified approximately 100"foldo 

Significance to NIAMD Research - Cyclic compounds containing sulfur 
are represented by such diverse compounds as biotin, penicillin and 
thiamine. It is hoped that this study will contribute to our understand- 
ing of the synthesis and metabolism of compounds of nutritional and 
medicinal importance in man and in microorganisms. 



Serial No« NIAMD- gg . 
Page 2 

Propos ed Course of Pro.iect - The properties of the "condensing" 
enzyme will be studied. With the aid of the separate enzymes and the 
phosphorylated substrates, extracts of mutant microorganisms and 
mammalian tissues will be examined to establish the locus of the genetic 
defect in thiamine synthesis. These enzymes and substrates will also 
provide a sensitive technique for studying thiazole ring synthesis = 



Part B included; Yes 



Serial NOo NIAM_D_".2i_ 
Page 3 



PHS-NIH 
Individual Project Report 
Calendar Year 1959 



Part B , Honors, Awards, and Publications 

Publications other than abstracts from this projects 

Leder, lo Go The enzymatic synthesis of thiamine 
monophosphate.. Biochemo Biophyso ResCo Commo 1., 
63-66 (1959) » 



Honors, and Awards relating to this project; None 



Serial No= NI.AMp». 30 
1, Biochemistry & Metabolism 
2„ Intermediary Metabolism 
3. Bethesda 



PHS=NIH 
Individual Project Report 
Calendar Year 1959 



Part A. 



Project Titles Metabolic Fate of Intracellularly Generated Reduced 
01= and Tri~phosphopyridine Nucleotides o 

Principal Investigator: Dr, Ben Bloom 

Other Investigators; None 

Cooperating Units s None 

Man Years (calendar year 1959); 
Totals 1 
Professional; 1 
Others 

Project Descriptions 

Objectives - The object of this project is to gain information 
concerning the metabolic fate of "various" reduced diphosphopyridine 
nucleotides (DPNH) , and "various" reduced triphosphopyridine nucleotides 
(TPNH) generated intracellularlyo 

Methods Employed - l) Those generally used for the specific tritium 
labeling of substrates or when needed, the development of sameo 2) Standard 
techniques as in current use for in vitro studies involving tissue sliceSo 
3) Analysis of various metabolic products isolated from the in vitro 
incubations for tritium contento 

Major Findings - The results obtained from this project, last year, 
suggested the possibility of using intracellularly generated reduced 
diphosphopyridine nucleotids-4=T for evaluating the hydroxysteroid augmented 
transhydrogenase concept in a cellular system. To this end the influence 
of several hydroxy steroids were tested for their ability to catalyze the 
approach toward equilibrium of the DPN=DPNH/TPN-TPNH couple« No evidence 
came forth suggesting that in liver cells, hydroxysteroid dehydrogenase 
functioned in a transhydrogenase capacityo 

Significance to NIAMD Research - The concept applied in obtaining 
the findings recorded above can easily be used as a generalized technique 
for study of alterations in the DPN-DPNH/TPN-TPNH equilibrium state in 
both normal and pathological cellular systems.. Thus an increase has been 
effected in the techniques available for the furtherance of our appreciation 
of those diseases which comprise the category of metabolic disorderSo 



Serial Mo. NIAMD- 30 

Page 2 



Proposed Course of Project - The application of the above described 
concept to other cellular systems wherein a hydroxysteroid augmented 
transhydrogenase reaction might likely be found. 



Part B included: Yes 



Serial No. NIMD- 30 
Page 3 

PHS-NIH 
Individual Project Report 
Calendar Year 1959 

Part B o Honors, Awards, and Publications 

Publications other than abstracts from this project: 

Bloom, Bo The intracellular occurrence of reduced 
diphosphopyridine nucleotide-coupled reaccions in liver and 
kidneyo Jo Biolo Chemo 234, 2158-2160 (1959) o 

Bloom, B= The hazard of orally pipetting tritium oxideo 
Jo Labo Clin= Medo In presso 

Bloom, Bo An evaluation of hormonal augmented transhydrogenase 
activity in rat liver cellso Jo Biolo Chemo In presso 



Honors and Awards relating to this projects None 



Serial Noo NIAMD- 31 

lo Biochemisfcry & Mefeabolism 

2, Metabolic Enzymes 

3o BeEhesda 



FHS-NIH 

Individual Project Report 

Calendar Year 1959 

PART Ac 

Project Titles- 

Ao Studies on Steroid Reduction 

'lo Hechanism of Steroid Hydroxy lation 

do Mechamism of Action of Steroid Hormones 

D Studies on Oxidation of Cyclic Secondary Alcohols 

E^ MetabolisiB of Steroids by Microorganisms 

Principal Investigator? Gordon Mo Tomkins 

Other Invt-fcigatorss Joseph So. McGutre 
K. Lo Yielding 
Vincent Hoi lis 
Jean Curran 
Sllzabeth So MaKwell 
Marshall I^irenberg 
Richard F, Bakemeier 
Glovanna FerrcLuzzi 

Cooperating Unitss Ac Do Merrittj Dental 
Leonard Garren, Cancer 
Virginia Evsns, Cancer lid' IZ^ C 

Man Years (Calendar ye.',r 1959) s 
Totals 6 
Professionals 5 
Others 1 

Project Description 

Project A 



OBJg C TIVES - Previous work has established that there are 
a nisaber of enzymes In maaanalisn liver which 
saturate the 4=5 double bond of hormanely active steroids » Enzymes 
in the soluble fractions of cell had been shown to be highly specific 



Part B included Yes 



Page 2 



and to produce the 5p isomer of the reduced compounde Microsomes 
were found to have enzymes saturating the double bond to yield the 
58! isomer,, These were also TPNH°°specificc Questions under investi'^ 
gat ion during the past year have beens 

lo What is the mechanism of the reduction? 

2a What are the characteristics of the enzymes considered 
as proteins? 

3o What is the physiological role of these enzymes? 

4o What are the genetic implications of this series of proteins? 

MEfH OpS^D_MA JOR FINDINSS - 

gart_la^ Hi^crosomal EnjBymes = A n»re rapid and sensitive 
spectrophotometric assay has been developed for the mirrosoraal 
K steroid reductases based on the oxidation of TPNH<, Most of the 
experiments, however, were done with an optical methTd based on 
substrate disappearance o It has been found that fl'.vine analogs 
or inhibitors do not interfere with steroid reduct'.on, and isotope 
studies have indicated that a proton is added to fitie A» but not 
the 3"position in the course of Che reaction, wh^.ch would be predicted 
if there were a direct transfer of a hydride ior from TPMH to the 
substrate without the intermediate action of a .t'lavineo This is an 
unusual mechanism for a double bond reactioa fud provides direct 
evidence for the participation of a hydride fon and a proton in this 
(as well S3 other) pyridine nucleotide^ linked reactlonso HitherfeOj 
the evidence has merely been for a direct t^'ansfer of hydrogen 
(without specifying the electronic form) fvom the pyridine nucleo= 
tide to the substrate o 

In addition,, it has been found that £here are a number of 
microsomal SCI reductases; possibly » eaca one specific for its own 
substrate, in th? same way as the soluble 5g series » Of considerable 
interest was the finding that one stevoid could interfere with the 
reduction of another by interacting T/ith its enzyme^ This inhibition 
was dependent on raolecular size, ioCo^ the inhibitor had to be of a 
smaller size then she substrate whoae reduction it was inhibitingo 
This indicated thai; the active sit^ of the enzyme had the same contour 
as the substrate and interacted with it at saany points, and also that 
this interference of the metaboiifsn of one hormone by another may be 
important physiologicallyc The administration of small steroid 
hormones such as the androgens could interfere with the metabolism of 
larger molecules such as the adrenal cortical hormonesj, and overlapping 
effects of the hormones might be produced in that wayo The microsomal 
enzymes appear to be "inducible" under certain circisnstanceso Other 
investigators have reported that laale ra'fis have less enzyme than 
female ratSo We have confirmed these observations and, in addition- 



Page 3^ 

found Chafe the sex difference I3 independent of either gonads or 
adrenals since it persists in adrenalectotalzedj gonadectomized 
ratso Ihis may be an Indication that the genetic information for 
the enzyme resides on the X chromosome o Other explanations for 
it are presently being investigated^, Furthermore, thyroxin was 
found to cause an increase in the level of the enzyme o More 
recently, drugs which are known to increase the levels of other 
microsomal enzymes, such as phenobarbital, have been found to 
elevate the levels of the steroid reductases o The mechanistic 
and physiological implications of these findings are presently 
being investigated o 

£BE. t, -? «, -Splo bl,® Enzymes ■= Using the methods previously 
developed, baaed on substrate disappearance $ the cofactor require°° 
ment for the soluble ensymes has been elarifiedc It was reported 
previously that some of the substrates could be reduced by DPNH 
as well as TPMo This finding has now been shown to be artlfactuaT 
due to the finding of small amounts of TFS to various reductases s> 
but not to all of themo It appears, therefore, that the soluble 
5^ reductases are TPM specific as are their 5C3 counterparts » i 
new purification scheme for these enzymes j using DEAE cellulo? , 
has proved highly effective and produced evidence for more df terete 
5P reductases o In addition^ other enzym® sources besides re liver 
have been investigated^ for example, pig^ calf, horse, guJ .ea pig 
and hisaan livero In the first case, pig liver, definite -vidence 
has been obtained that there are multiple SP reductases :md these 
enzymes are being purified from that source by means o^ ansnonium 
sulfate fractionation and ion exchange chromatography. 

Part, 3^, Biological Consi derations = There is ' aeries of Si 
reductases in the microsome, each of which is specific for its 
substrate in requiring XPM, and a similar serie-^ of 50 reductases 
in the soluble fraction of the cellc These findings have raised 
two indspefvdent questions. Is there a relafclor between the & enzyme 
corresponding to a given substrate and the p etzyme, for examples 
is the & enzyme a precursor of a given ^ enzyriej, or are they derived 
from a coasaon precursor? The second questlor of interest is^ in 
view of the multiplicity of the steroid reductases one might consider 
alternates to the proposition that all live;, cells make each of the 
reductases and that there might, in fact, 'jb "mlcroheterogeniety" 
among liver cells, where one cell would n-ake only a limited number 
of these enzymes, by analogy with the Br "net concept of antibody 
formation in which only certain cells o\aks certain antibodies o The 
answer to the first question., the relecion, if any, between a^ and p 
enzymes, could be obtained only if tbs Of enzyme, on solubilization, 
were converted to the P enzyme 9 or Jf a genetic experiment were 
possible in which independent deletion of genes for the Q. and P 
enzyme could be obtainedo to date, although many attempts have been 
ajade, the ot enzymes have not been solubilized,. Genetic experiments 
with maimnalian liver are, of course, impossible^ One approach has 
been to examine hepatomas which contain both a and ^ enzymes to see 



Serial ^ Oj:-._NIAMD,-, 31 
Page 4 



whether deletions of an @ enzyce results in loss of the correspond^ 
ing P enzyme In one case no relation between missing 'S2 and missing 
enzymes was found, which sug{;ests that at least precursor product 
relation between the two does uiot holdo In answer to the second 
question, whether all cells are. able to make all the enzymes, two 
approaches have been made, Tht; first of these is to develop 
specific raicromethods for the determination of the enzymes in a 
single cello Progress has bee?; made toward developing methods to 
determine either the oxidised rjyridine nucleotide produced, or 
substrate disappearance based on fluorescence o The second approach 
has been Co examine tissue cultures derived from a single cell to 
see what their enzyme complement iso In various cases this has been 
done and, in fact, a limited nuiabsr of enzymes has been found in 
tissue cultures derived from single cello This approach has not 
been exploited to the fullest yat and experiments are to be 
continued on it in the future. 

Project Bo 

OBJECTIVES •= The method by vhich moiscular oxygen is cleaved 
and one of the atoms inserted into the steroid 
nucleus to produce the hydroxylat Ion reaction is unknown, although 
this reaction is the primary bior^nthetic reaction in the synthesis 
of the steroid hormones as well as many other important biological 
compounds o 

METHODS MP FIND INGS •= Additional studies with mansaalian 

systems has progressed although those 
studies using isicrobial systems have been dropped due to extrene 
lability of the enzyr:ies involvedo Mananalian experiments have 
confirmed the fact that three enzymes and TPHH are involved as well 
as the heat stable co factor,. Some indication of the nature of this 
cof actor has been obtained reccmtly. It seems to be a O'-rbohydrate, 
possibly a phosphorylated hexoseo One of the enzymes involved in 
hydroxylation may be able to convert gIucose°6°phosphate to the 
CO factor. In the presence of large amounts of boiled liver extract, 
one of the enzymes can be eli&iinated from the reaction mixture. 
However, with G"6-P as thg prt'.cursor of the cofactorg this enzyme 
must be present. 



OBJECTIVES - The steroid hormones are potent biological reagents 
and an understanding of their action at a molecular 
level is essential for an understanding of physiological control 
mechanisms in the cell. To date little specific information is 
available and theoretical controversies rage over even such mechanisms 
.».?. have been presented. 



'»ir_l - => 

Page 5o 

.HAJQR FIMDIHGS - We ave found that numerous steroid 
hoE! ones inhibit DPNH cy£ochrome c 
reductase from many sources, both nsanitalian and microbial, as well as 
from neoplastic tissues » The site of £his inhibition has been 
localized to a step between the fla'«.njrot;ein and cyCochifoma bj 
the same step affected by amytolo l^s inhibition is competitively 
reversed by 'S- tocopherol and other lipidso In some tissues a 
steroid insensitive pathway of eleeoon transport has been 
discovered where electrons are tran: erred directly from flavo" 
protein to cytochrome c, circumvent:. ig cytochroiae bo Methods 
involved in this study have been pr^tarily spec tropho tome trie 
assays of reduced pyridine u«cleoti«. oxidation or cytochrome 
oxidation reduction o In addition, s .nventioaal Warburg manosaetry 
and respirometric measurements with tlae Clark oxygen electrode 
have been usedo Since this reactios as the main pathway of 
electron transport beyond the subatEiie level » interference of 
it by steroid hormones is of obvious physiological importance o 
However, the inhSbition is so generei, uot only in terms of 
tissues affected, but also in terms >f what steroids are effective 
that it is difficult at the present ;i!se to see how the specific 
effect attributed to steroid hormottu can be as a result of this 
inhibitioHo Studies have- been under taken, however, to determine 
whether, in facts specific effects ccti be observed on the basis 
of inhibition of this sortc, One of ihs forma of steroid 
inhibition of electron transport the!, might be reflected in the 
physiological effect is the carcinos attc effect of steroids o To 
this end steroid^sensitive and sterc;,d=resis£ant C^Jior lines have 
been developed and the amount and se-isifcivifcy of the DPHH cyto= 
chrome c reductase enzyme has been diterminedo Other studies have 
been continued on the use of steroid;; as 'fPM oxidiaiag agents in 
intracellular economy. As previously reported, various steroids 
which can provide XPN through double bond red«c£ion» stimulate the 
oxidation of glucose^d^phosphate in this way^ Het accumulation of 
TPN in the presence of different steioid horsaonea has been shown 
using isolated intact liver cells so that £his metabolic effect 
may, in fact, play a role in steroid iuetabolism« 

Project Do 

OgJgCTI.VlSS = Previously it has been shown that crystalline 

horse liver alcohol dehydrogenase could oxidize 
cyclic secondary alcohol,. Since fcheisa compounds are structurally 
related to steroids, a study of the interaction of the enzyme with 
these compounds was undertakeoc Thit has been pursued somewhat 
during the course of the past year^ 

J ^Sl'J?>g_4KP J!.I^B];gGS. - Findings of further interest have been 

that the axial hydroxyl is preferentially 
oxidised by WB with alcohol dehydrogenase. This conclusion was 
derived from the fact thac cis 4 tertiary butyl cyclohesanol is 



Serial Wo. NlilMD :- 
Page 60 



oxidized by the ensyae in DPN, but the trans isomer is not. Since 
the tertiary butyl group is sufficiently bulky to be fi:xed in the 
equatorial conformation, the els hydroxyl group id necessarily 
axial and the trans necessarily equatorial o Xha cis hydroxy 
compound is oxldizedo this Is an interesting situation in view 
of the fact that the liver Xi hydroxysteroid dehydrogenase 
preferentially oxidises the equatorial hydroxyl as one might 
expect, since it is the more unhindered project alcohol group o 

Project Eo 

OBJECTIVES - Microorganisms are &hlf. to metabolize steroids 
often in a dramatic w^y, although the role of 
steroids in the metabolism of mlcroorg anisras is thoroughly 
unknowno Recently, as described in r^K>ther project report by 
Sc S.} Maxwell, we S^^ave isolated a mntant yeast which Is resistant 
to the antibiotic effect of steroirlso Some of the metabolic 
transformations of the steroids by the yeast have been investi'^ 
gated o 

METHODS AIjDJlMQ^ glNDIM^ - Frra both mutant and wild type 

3e ^charomyce s^ fragllis , a 
metabolite of 4-androattene=3,17"dione w.'s recovered which was con» 
slderably less polar by paper chromatography, which was rendered 
more polar by alkaline hydrolysis o Chromic acid oxidation of the 
hydrolyzed compound yielded the substrate^ 4'--androstene=3,17=dionf <. 
These facts can be reconciled with tho formulation that A-andros^ene 
3,17-dione is first reduced to testosierone following which the 
testosterone is conjugated to form an acetylated compound at 
17 testosterone acetate » ^his comprund has never before beer 
found in natural sources and it is therefore of considerablr 
interest. The Identity of the isolated conjugate has been further 
confirmed by first hydrolysis and activation of the acetrce moiety 
with acetokinase, following which the hydroxamic acid w.s chromato= 
graphed,. Infrared studies of the intact conjugate alsj Indicated 
that it was testosterone acetatec 



Part ^ Be HoiJ^^i-, r->*ard3, and Pub 1 teat ioru 
Publications other than absttract;; from thi* project- 



Xotaklns, G= SCudles on the Mechanism of Steroid Hydroxylationo 
Colioquim on Oxygenating Enzyraea, 4th Into Congre.ia ot 
Biochemistry, Vienna ^ 

McGuire, Joseph S. and Xomklnj, Gordon M. The Efjfecti; of Xhyroxlr> 
Administration on the Enzyraic Reduction of A -'3'=keto.3.tGrcidj 
Jo Biol.. Chem. , 234, 791 (1959) o 

TonikinJi Gordon M^ Enzyraatlc Metabolism of Cortlcosfeeroidso An?!, 
Hew York Acad, of Sci„, 82, 336 (1959). 

Merrlttj A, Donald and Tomkins, Gordon Mo Reversible Oxidation ct 
Cyclic Secondary Alcohols by Liver Alcohol Dehydrogenases » 
Jo Slol. Chsssso, 234, 2778 (1959). 

Yielding, Ko L«.mone and Torakina, Gotdon M„ Inhibition of Ensvalc 
Oxida':ion of DPNH by Steroid Bonaone^t, Proc, feto Acad„ 
Scl, (In pce33)< 

Yielding, K, Lemcne and Tomt'.ina, Gordon Mo An Effect, ct £nxyiT!ic 
Redocfeioi! of Steroids on Triphosphopyridine Kuclec«:ide= 
Dependent Glucosa-o^phosphate Oxidation. Biochlr-., et BiopV.ys , 
Acta {in press).. 

Tomkins, Gordon .Ho and McGwire, Joseph So Ihe gfiect of Thyroid 
Hormones on <ldtsnai SEaroid Metabolism,, N, Y. Aond of Sci,, 
(in pEe;;i).; 

McGuire, Joseph Sc- an^ tomkiosj Cordon M.. the MultipliciCy and 
Specificity of A"*- 3 -ke Co steroid Hydr.ogenasea (»)„ ArcL,, 
Biochem„ & Blophys, 32, 477 {1959% 

ToEjkins, Gordon M. and irJcGuire, Jasepli. So The Adrenogenital Syndic. 
in J» Be, Staoburyj, Jo B^, Wy» gaarden and Do So Fredcrickgon 
(Editor 3> |;he M^^t^bolic Basis iter Ijaherited Disoases, McGtiv/ 
Hill, New York Cifl'prejs),, 

McGuire, J„ S.. and Ton,ikias„ Go Mo The Heterogeneity of .cT-i-kfS!.)- 
steroid raductas^ea (5'J).. J, Biol, ilhtas.,. iirk presij)^ 



Serial No.: NLAB«> - 

io Biochemistry & neu&DOiism 
2o Metabolic Enzymes 
3o Bethesda 



PHS-NIH 

Individual Project Report 

Calendar Year 1959 

PART A. 

Project Sitie; 

Ip Galactose Metabolism 

Ao UDPgalactose=>4"epiiaerase from yeast » 

B. A Steroid Sensitive Aldehyde Dehydrogenase 

Go An Assay for Galactose-^l'^P in Husian Erythrocytes 

£Io The Antibiotic Effect of Steroid Hormones on Yeast 
and the Isolation of a Resistant Mutant 

Principal Invesfeigatorg Eo Maxwell 

Other Invesfeigatorss Gordon TotakinSj, Joseph McGuire, Leroy Pesch, 
Yale Topper, Ho Ho Kirkman 

Cooperating Units g 

Man Years (Calendar Year 1959) 
Totals 3 
Professionals 2 
Others 1 

Project Descriptions 

Id Galactose Metabolism 

OBJECXIVES ° 

Ao Previous studies carried out in collaboration with 
Dtr, H^ Mo Kalckar and Dr= Huguette Szulaiajster demonstrated that 
UDPgalactose»4"epliEerase purified from yeast differs in several 
respects frota the same ensyme from calf liver o The enzyme from 
liver requires exogenous DPN and is inhibited by DPNH,, In contrast;; 
the enzyme from galactose-adapted yeast is fully active without the 
addition of DfW and is not inhibited by DPNH The enzyme from 
yeast contains a tightly bound substance which fluoresces with the 
characteristics of DPNHo Treatment with p=chloromercuribenzoate 
brings about a disappearance of fluorescence and a loss of enzymic 
activity o Activity but not fluorescence can be restored wich DPK 

Pare B included Yes 



Serial So, MIAMD- 32 

?a^.,e 2 

plus cysteine but not with cysteine alone« These findings 
suggested that the yeast enzyme contains protein=bound pyridine 
nucleotidec Studies were, therefore, undertaken to identify the 
protein^bound material and to investigate further the mechanism 
of the enzymic interconversion of UDFgalactose and UDPglucoseo 

Bo Previous studies carried out by Dr^ Yale Topper, 
Dr, Leroy Pesch and Dr, Ernest SicEon desaonstrated that the 
oxidation of galactose by the soluble fraction of rabbit liver, 
as measured by the liberation of C<=1 as COga is stimulated by 
certain steroid hormones and that the iaterconversion of UDP 
galactose and UDPg'ncose is a limiting step in the reaction 
sequence o In collaboration with Dr^ Topper and Dro Pesch, the 
mechanism of the steroid stimulation of galactose metabolism has 
been investigated^ 

Co In collaboration with Dro Neil Kirkman, the development 
of a method for determining galact09e=i°phosphate in red blood 
cells uas under taken » 

MAJOR FINDINGS AITO PROPOSED COURSE 

Ao An improved method for the purification of UDPgalactose=4" 
epimerase from galactose^adapted yeast was devised^ The purified 
enzyme was shown by three independent methods to contain protein-^ 
bound DFHo About Oo3 jjsnole of DPH per 100 mg<, protein was present 
in the most purified preparation^ Using SEAE cellulose chromatography 
a correlation was desaonstrated between bound DFNs enzymic activity 
and fluorescence at 450 mpo VJhether or not the fluorescence is due 
to bound DPN is not yet certain, but it seems clear that the 
mechanism of the yeast enzyme is similar to that of the same enzyme 
from livero The difference in response to exogenous DPN can be 
explained by the presence of tightly bound DPN in the purified enzyme 
from yeas to The yeast enzyme, like the ensyme from liver, failed 
to incorporate tritium into the hexose nucleotide fro® either 
tritiafeed water or DPNH labeled with tritium in the para positiouc 
The detailed mechanism of the reaction and the role of DPN thus 
remains to be determined o 

Be In collaboration with Dr, Pesch and Dro Topper, it was 
found that progesterone and certain other steroid hormones bring 
about a decrease in the rate of reduction of DPN in systems 
previously employed by these investigators for studies on the 
stimulation of galactose oxidation by steroids » Since UDPgalactose-A" 
epimerase in liver requires DPN and is inhibited by BPNH, a 
decreased rate of reduction of DPN would be expected to result in 
stimulation of the conversion of UDFgalactose to UDPglucose, a 
step previously shown to be limiting in the reaction sequence leading 
to CO, formation from C=l of galactose » The mechanism by which 



Serial ISOo MIAMD- 32 

Page 3 

progesterone decreases the rate of DPN reduction has been 
investigated and is now at least partially understoodo The 
40~60 per cent saturated CKH,)2S0, precipitate from the soluble 
fraction of rabbit liver contains DPN^specific aldehyde dehydro- 
genase activity which is 40-75 per cent inhibited by iO"^ M 
progesterone o The system is active with a number of aldehydes 
inciudingj acetaldehyde, propxonaldehydej glycolaldehyde, 
succinic semialdehyde and glyceraldehydco When coupled with 
alcohol dehydrogenase J the reduction of DPN by alcohols, such 
as propylene glycol, can also be showi to be inhibited by 
progesterone o The steroid is not acting stoichiosaetrioally as 
an electron acceptor since the difference in DPM concentration 
in systems with and without added steroid is as much as 60 times 
the concentration of steroid presento The 0°40 per cent saturated 
CNH,)2S0, precipitate also contains aldehyde dehydrogenase 
activity but this activity is unaffected by progesteroneo Aldehyde 
dehydrogenase purified from calf liver according to the method of 
Racker is similarly unaffected o 

The steroid'sensltive aldehyde dehydrogenase is being purified 
from rabbit liver o ^e properties of the purified enzyme, or 
enzymeSs will be investigated in detail c Aldehyde and steroid 
specificity^ as well as tissue and species dlstributioaj will be 
investigated and attempts will be made to evaluate the physiological 
significance o£ the reac;!:ioEic 

Co In collaboration with Dro Neil Kirkman, a sensitive, highly 
specific and comparatively simple assay for galactose=l=P in 
erythrocytes has been devised^ Such an assay applied to galactosemic 
patients should be of value to physicians in determining the 
efficiency of galactose^free diets or for detecting divergence 
from such prescribed diets o 

IIo The Effect of Steroid Hormones on the Growth of Yeastc 

OBJECTIVES = Studies in other laboratories indicate that the 
growth of a nimber of Gram- positive bacteria is inhibited by a 
variety of steroid hormoneSo Although certain organisms, including 
yeast, ate capable of synthesizing steroids from acetate and of 
metabolizing steroids by reactions similar to those occurring in 
mammalian steroid hormone biosynthesis, the physiological significance 
of steroids in microorganism is not knowno The present studies 
were undertaken in collaboration with Dro Joseph McGuire and 
Drc Gordon Tomkins in an attempt to gain some information about the 
role of steroids in Saceharomyces fragiliso 

MAJOR FltaPINGS = The growth of wild<=type So fras^ills is almost 
completely inhibited by 0»13 mg/mlo of 4-andro3tene=3,l7'=diones 
l-androstene'=33l7"dione, androstane=3,17=dione, deoxycorticosterone 
and progesteroneo Other closely related steroids either had no 



Serial NOo KIAMD ^ 
Page 4 



effect on growth or Inhibited to a much less extent o One of the 
inhibitory steroids, 4«'andro3tene"3,17'=dione, was shown to be 
lethal to growing cells of this species of yeast<. Resting cells 
were much more resistant to the steroid. 

Several mutant strains of .S,o fragilis were isolated which 
were relatively resistant to steroids, Attennpts were made to 
discover the basis of their resistanceo No qualitative difference 
in the metabolism of 4»androstene"=-3,17-dione was observed in the 
wild- type and resistant strains. Whole cells of both strains 
convert the added steroid to a previously undescribed metabolite 
which has been identified by Drc McGuire as testosterone acetate c 
The uptake of C^^ labeled 4-androstene»3,i7"dione into growing sells 
of the resistant mutant was about half as fast during the log phase 
of growth as was that at the same phase into wild-type cells. In 
both cases the rate of uptake was slow during rapid growth. As 
the stationary phase approached, the rate of uptake increased and 
became the same in resistant and sensitive strains. Whether or 
not the slower rate of steroid uptake into mutant cells is sufficient 
to account for their resistance is not yet known. Further studies 
will be instigated to delineate the mechanism of steroid resistance 
in the mutants. 



Serial No. WIAMD « %o 



FHS-KIH 

Individual project Report 

Calendar Year 1959 



Pa rt B ^ Honors, Awards, and Publications 
Publications other than abstracts from this projects 

Anderson, Eo Po, Maxwell, Z„ So and Burton, Bo Mo The Enaymatic 
Synthesis of C^'^ labeled UDP glucose, UDP galactose and 
Galacto3e-l°phosphatea J., Mxo Chemo Soco (in press) c 

Maxvell, £« So, antl Szulmajstcr, Ho de Robichono The Purification 
if UDPgalactose^^-epiiaerase front Yeast and the Identification of 
Protein^bound Diphosphopyrldine Hucleotideo Jo Biolo Chenic 

(in press) o 

Maxwell, Eo So Enzymic Epimerizationo Volo III The Enzymes 
(in press) o 

Kirkman, Eo !^o and Maxwell, Eo So Bnsymatic Estimation of 
Erythrocytic Galactose-l-phosphata,, Jo Labo and Clino Med, 
(in press) o 

Maxwell, E= So, McGuire, J„ So and Tomkins, Go Mo The Antibiotic 
Effect of Steroids on S accharoiayces fragllis^ and the Isolation 
of a Resistant MuEanto Jo Bacto (in pre33)= 

Topper, Yo Jo, MaKwellj Eo So and Pesch, Lo, On the Mechanism by which 
Progesterone Stimulates Galactose Metabolismo Bioch^jSo et Biophyso 
Acta (in press) o 



serial HOo HMMP°_.33.._^ 

2o Metabolic Enz^ea 

3. Bethesda 



PHS^NIH 

Iiiidividtssjil Peojecte Repot fe 

CalesMlar Year 1959 

PART Ao 

Project titles 

lo 2?HiydroxybMtyric Acid Ca&abolism 

a... The pathway of I'^hydroxybuteyric acid metaboliam 

bo Shared genetic information ° A test: case 

Co The xoechanism of succinic semlaldehyde oxidation 

2c Hie role of the inducer in penicillinase indttction 

Principal Investigators Marshall Nirenberg 

Other Investigators t 

Project lo a,, b^ e„ Dro Willi«3in Jakoby 
Project 2o HVo Gordon Mo Tonkins 

Cooperating Units e None 

Man Years ((Calendar year 1959) 



Totals 


1=-1, 


Professionals 


1 


Others 


1/2 



Project Descriptions 

OBJECTIVES <= Iqo To determine the enzymatic steps involved 
in the oxidation of j^^hydroxybutyric acid by Pseudoatonas fla orescgnso 

Ibo To determine whether one cystron contains 
the information necessary for the synthesis of a protein suboait which 
may be an integral part of two or more ensymesc The j^hydroxybujtyric 
and P^hydroxypropionate dehydrogenase systems will be investigatedo 

Icc To obtain information about the sites 
of substrate attachment of TPN-^succinic semialdehyde dehydrogenase 
in order to devise an approximation of the mechanism of aldehyde 
oxidationo 

*'asrt B included Yes 



Serial NOc NIAMD .- ?.•> 
Page 2 



METHODS AKD MAJO R FINSINGS 



la = 5'-Hydroxybutyric acid dehydrogenase and two different 
succinic semialdehyde dehydrogenases have been purified from extracts 
of Psaudomoaas^ species and the properties of these enzymes have been 
described^ j^BydroxybuEyric acid dehydrogenase is specific for 
y»hydroxybuterate oxidation and forms succinic aldehyde as the 
product, Diphosphopyridine nucleotide is required for the reaction. 
Both succinic semialdehyde dehydrogenases are specific for succinic 
semialdehyde oxidation and form succinic acid as the producto One 
enzyme has a high affinity for diphosphopyridine nucleotidei the 
other for triphosphopyridine nucleotideo The differences between 
the enzymes as veil as their possible relationships have been 
investigatedo 

Ibo The induction of the enzymes in the pathway of 
y»hydroxybutyric acid aetabolisra was investigatedo A g^hydroxy- 
propionic acid dehydrogenase was formed by these microorganisms 
when they were grown upon p-hydroxyprop ionic acid as the sole 
carbon source.. The appearance of both 7=hydroxyfautyric acid dehydro^ 
genase and ^»hydroxyprop ionic acid dehydrogenase were dependent 
upon the growth phase of the culture.. ^'''Hydroxy butyric acid was 
found to induce the formation of y^hydroxybutyric acid dehydrogenase 
at low inducer concentrations „ Higher concentrations of /-hydroxys 
butyric acid were highly effective inducers not only of 
y°>bydroxybutyric acid dehydrogenase but also of ^»hydroxyprop ionic 
acid dehydrogenase o The relationships between the inductions of 
similar enzymes in different metabolic pathways by the same inducer 
were investigated further o No evidence of shared genetic information 
bec;ween the two closely related enzymes was foundo 

ICo The effect of trypsin upon the TPN succinic semi° 
aldehyde dehydrogenase was investigated.. When TPN combined with the 
enzyme, an intramolecular rearrangement of the enzyme occurred 
which exposed a bond labile to trjrpsin activity. Enzymatic activity 
could then be rapidly destroyed by trypsins This phenomenon was 
utilized to study the half reactions involved in succinic semialdehyde 
oxidation^ The enzyme was inhibited by arsenite which suggested 
that two closely juxtaposed sulfhydryl groups were present. The 
pyridine nucleotide cofactor did not combine with the sulfhydryl 
groups, instead competition between arsenite and the aldehyde 
substrate for at least one of the two closely juxtaposed sulfhydryl 
groups occurred. On the basis of these findings a mechanism of 
aldehyde oxidation was proposed, 

OBJECTIVES ■= 2 - To investigate various parameters of Pollack's 
penicillinase system with the hope of gaining some knowledge of 
the role of the inducer during enzyme induction. 



Serial No.. NlAMB_j:;„Jl, 
3 



METaODS MP MAJOFx FINDINGS 

Possible roles of the inducer are now being studied in 
penicillinase induction in B. cereus <, Induction of penicillinase 
is being studied in protoplasts and protoplast lysatesc Highly 
sensitive methods of assaying penicillinase have been developedo 



SSRIAI, NOo NIAMD « M 



Pare Bo Hoaorso Awards sstd pyblicaeions 



Hirenb^rgg Mo A Bloch^nlcal ChaxacterisCie of Ascites Tumors o 
Jo Biolo Chaaio (in press)o 

Nirenbergj M<, atsd Jakobys, Wc The eraaymatic iJEiliisafeion of 
7<=fiydroKyb«tyrie Acido Jo Biolo ChsEo Ci® press) o 

Niretibergs Mc Wo and Jakoby, Wo Bo On the Sites of Attachment 
and Reactioi^ of Aldehyde Dehydrogesmses o Proco Nato Acado 
Sciq {in press) o 



Serial No, liIA^ffl :^ 

1„ Biochemistry & Metabolism 
2o Metabolic Enzymes 
3o Bethesda 



PHS»NIH 

Individual Project Repore 

Calendar Year 1959 

PART Ac 

Project titles 

Structural 3£udy of nucleotides, polynucleotides, sxsd 
nucleic acids by njeans of infrared spectra in TiJ} 
solutiono 

Principal Investigators Ho Todd Miles 

Other InvestlgaSorss None 

Cooperating Units? None 

Man Years {Calendar year 1959) 
Total? 1 
Professionals 1 
Others 

Project Description 

QB^CTT^ S - This project has been primarily concerned with 

a study of the Eautoaeric forms of the n^acleotide 
components of nucleic acids and vith the application of the 
information obtained to the structures of nucleic acids o 

I g^ QBS EMFLOYEB = The infrared spectra of nucleotides, poly= 
nucleotidesj, and nucleic acids have beesj 
observed in D»0 solution^, A number of model compounds have been 
synthesized to permit the spectra to be Interpreted in structural 
terms o 

MAJOR FIHDINGS => During the past year it has been found that 
polyinosinic acid definitely exists in the 
keto form in aqueoas solution and polycytidylic acid probably in 
the amino form, and that these tautomeric structures are maintained 
in the helical interaction product formed by mixing the polyn^rso 
In addition it has been proposed that the changes which occur in 
the spectra upon mixing the polynucleotides may be explained largely 
by the reduction in dielectric constant caused when the close'^packed 
helices are formed with consequent exclusion of water from the surfaces 
of the heterocyclic rings* The changes in spectra of DEJ^ upon 
denaturation obtained by other workers may very well have the same 
explanationc 



Part B included Yes, 



serial Ko« ^um » 34 
Page 2 



The helical lat:erac£lo» products of £he polynucleotides 
have been found £o have essentially the same stability ia D2O 
as in H„b solution, denKJnstratii'sg the applicability of the 
results obtained in the former solvent to the latter as well,, 

SIOIIFXGAMCE TO BKMEDICAL RESEARCH - The question of tauto^ 

merle forms of the 
nucleotides is fundameatal to the structures of the nucleic acids 
since the hydrogen bonding schemes that hold the nucleic acid 
helices together are deteraiined by this structural feature of 
the component nucleotides » A related point of biological interest 
is the proposal of Watson and Crick that the ch^ical mechanissa 
of mutation involves formation of the less stable tautomeric fona 
in a polynucleotide chain with consequent pairing with the "wrong" 
base in a DM molecule o 



Serial No, NIAMD - 3^ 



Part B o Honors, Awards arid Publications 



Miles, Ho T« A Proposed Interpretation of Infrared Spectral 

Changes Occurring upon tbe Interaction of Polynucleotides » 
Mature 183, 1814 (1959) o 

Miles, H» To Infrared Spectra and Tautomeric Structure of 
Folyiaosinic and Folycytidylic Acids in D^O SolutioBo 
Biochinso et Biophys, Acta 34, 274 (1959) » 

Miles, Ho Toddg Smyrniotis, Fo Zc, and Stadtmanj Eo Ro Bacterial 
Degradation Frod^scts of Riboflavino IIIo Isolation, Structure 

Determination and Biological Transformations of l='Rlbityl'=2s3=> 
diketo-l,2,354=teferahydro='6,7»di!aathylquinoxaline, Jo Anio Gheaio 
Soco, 81, 1946 (1959) o 



Serial No. NIAMD- 35 

].. CaieriD.stiy 
2. Ifetabolites 
3 „ Bethesda 



PHS-NIH 

Individual Project Report 

Calendar Year 1959 



Part A. 



Project Title: BfetalDolism and Biosynthesis of Catechol Aiaines 

Principal Investigator (at NIAMD): Bemhard Witkop 

Other Investigators: Siro Senoh (left 3/17/59);. John Daly, Yc Kanaol^. 
(VoSo arrived 8/31/59) 

Cooperating Units: So IHenfilend and Co Ro Creveling, Ml, Serial 

HOo HHI'-216 Dr. Sydney Archer, Sterling-Winthrop 
Research Institute 

Ifen Years (calendar year 1959): 
Total: 2/3 
Professional : 2/3 
Other: 

Project Description: 

Sjmthesis of Novel Jfetaholites of Dopamine, Norepinephrine, 
Adrenaline and Other Catechols of Physiological Impoirfcance o Clarifi- 
cation of the Biosynthesis of Norepinephrine. 

Oh jectives : To establish metabolic parameters for iriportant 
endogenous hormones, to characterize new catechol metabolites, to find 
labile transformation products of dopam5.ne and precursors of norepi" 
nephrine by elucidating the mechanism of its formation. 

tfethods Employed ; Cross labeling of dopamine vith' tritiiim and 
C^^ was used to follow the chemical and enzymatic transfoz^oations in« 
clviding the conversion to noradrenaline » 

Major Findings : The addition of nucleophilie reagents such as 
water or methanol to (N-acylated) dopsuninequinones produced noradren" 
aline and 6-hyidroxydopecnine (2,ifr,5-'fc37ihydroxyphenylethylamine) in a 
ratio of 10,000:1, 2,i|-,5-Trihytlroxyphenylethylamine is easily formed 
by autoxidation from solutions of dopamine on standing, with boiled 
tissue or in the ascorbic acid-versene system. This new autoxidation 
product of dopamine is chromatographically indistinguishable from, nor- 
adrenaline. Its discovery was made possible only by cross -labeling 
technique whereby it was found that the "noradrenaline" fraction had 



Serial MOo NIAMD-35 
Page 2 

not lost any significant tritium activityo The same technique showed 
that tissvie from selected regions of the "brain, such as hypothalaznus 
and caudate nucleus, convert dopamine to authentic noradrenaline in 
yields up to k'^. 

A number of new aminochroxoes and tetrasubstituted indoles have 
been prepared from derivatives of dopsminequinone and the mechanism 
of these transformations has "been followed "by tritium labeling. It 
remains to be seen "whether these new aminochromes or some of their 
derivatives are centrally active. Such activity has been claimed 
without sufficient support for adrenochrome and adrenolutin. 

Since it has recently been postulated that dopamine may be a 
new hormone in certain tissues, it is of interest to note the excre- 
tion of 6"hyiroxydopamlne after administration of dopamine to animals. 

Collaborative efforts with Sterling-Winthrop aim at the synthesis 
of amino acid precursors capable of penetrating the blood-brain 
barrier and of the release of active amines such as adrenaline, nor- 
metanephrine etc, in the brain. It has been noted in the National 
Institute of Jfental Health that these biogenic amines when labeled 
and administered to animELLs did not reach the brain. 

Significan ce to bio-medical research and the program of the 
Institute ; The metabolic fate of peripherally and centrally active 
biogenic amines is a key problem in modem neurochemistry and psycho- 
pharmacology. 

Proposed Course of Project ; In analogy to the foimation of 6- 
hydroxydopamine (2,lj-, 5-trihydroxyphej3ylethylamine ) one may expect 
the occurrence of 6-hydroxy-(nor)epinephrine by a similar mechanism. 
Judging from previous experience with 6-hydro:£ydopamlne, such a 
hyd3X>xyad3?ena3.ine analog would have to be synthesized first and known 
in all its properties before attenrpts could be made to prove its 
presence as a metabolite. Such synthetic sttsdies are planned. 



Part B included: Yes 



Serial No. HIAMD- 35 
Page 3 



V&Tt B o Honors, Awards, and Publications 



Dro Witkop received the I958 SLllfebrand Award of the Washington 
Section of the Atoerican Chemical Society for outstanding contribu- 
tions to the structure and oxidation mechanisms of natural products 
and intermediary metabolites.. 

Publications other than abstracts from this project: 

Senohp S., Witkop, B», Creveling, C. R. and Uienfriend, So: Oxida- 
tion Mechanisms of Catecholamines and the Biogenesis of Nor- 
adrenaline o Fourth International Congress of Biochemistry 13^ 
176-188, 1959 o 

Senoh, So, Witkop, Bo: Formation and Reararangements of Aminochromes 
from a New Jfetebolite of Dopamine and Seme of its Derivatives, 
Jo itoo Chem. Sooo 81, 6231-6233 s 1959- 

Senoh, So, Witkop, Boi Non-Enzymatic Conversions of Dopamine to IK>r- 
epinephrine ^adTrJ-hydroxyphenethylaaasineSo Jo Amo Chem. Soco 8lp 
6222=6231, 1959 o 

Senoh, S«, Creveling, Co Ro, Udenfriend, So ami Witkop, B. : Chanical, 
Eozymatic aiai Metabolic Studies on the ffechaa3.!3!B of Oxidation of 
ropcmineo Jo Amo Chemo Soc„ 81, 6236-62l«-0, 1959 • 

Khy, Ho and VJitkop, Bo : Chemical and Baajnaatic Studies of the labile 
!fe-oabolite lf.(5H)-Imidaaolone-5-aGetic Acido J= Am. Chemo Soco 
62i^5"625l5 1959. 



HIS-WIH 

Individual Project Report 

Calendar Year I959 



2o Metabolites 
3o Betheada 



Part A. 



Project Title; Selective Cleavages of Peptide Bonds by Chemical .Agents 

Principal Investigator: Bemhard ii?itkop 

Other Investigators; W, B.. Lawson^ Srhard Gross, Lo K. Ramadb.andran 

Cooperating Units; To Vis^/aaatha, LET, L-. C. Craig, The Rockefeller 
Institute for Medical Research 

Man Years (calendar year 1959): 
Total; 2 
Professional: 2 
Other: 

Project Description; 

Objectives ; l) To investigate and apply usethods for the selec- 
tive cleavage of peptide bonds « 2) To selectively modify or cleave 
peptides ; protedjas and enzymes in oai^er to correlate structural 
elements with physiological or enzytaatic activity., 

Methods Employed ; Special organic oxidizing agents, such as H-= 
bromosucclaimide, M-bromoacetamide, sodium periodate and others, are 
capable of selectively attacking, eogo, tryptophan residues in pep= 
tides and proteins. In situ observation of the changes in absorption 
by differential ultraviolet spectrophotoEsetry in a self-rccordi33g 
instruHient is used to follow this reactiono DISP and Stein and Moore 
techniques, paper chrosiatosraphy and electrophoresis serve for the 
identification of cleavage products. 

K^jor Findings ; The well"kno^«i photo-oxidation of tryptophan- 
containing proteins is accompanied by characteristic shifts in the 
xiltraviolet spectrum to shorter TjavelengthSo Similar shifts are also 
produced by the controlled oxidation of, £ogc , H-Cbz-tryptophan, poly™ 
tryptophan, gramicidin, lysozyme, chymotrypslD., etc., with selective 
oxidants, such as W-bromosuccinimide in v;atero The reaction which 
was followed in situ by differential U¥- spectrophotometry paroceeded 
smoothly In a^IO-^ molar solution « After the rapid consumption of 
approximately 1,5 mlo of W^ per mole of tryptophan the Indole chromo^ 
phor had disappearedo Volhard titration of the reaction mlxtiire 
showed the presence of only ru80^ bromide ion pointing to nuclear 
brominatlon accompanying oxidation of the indole ring to derivatives 
of oxindoleo Model studies with skatol, indole -p-propionlc acid and 



Serial No, .MIA>ffl- 36 



Page 2 



N-'benzoyl-tiyptophan showed that substitution, oxidation and group 
participation merged in the bromination of indoles. 

The structure of the dihromoskatole , resulting from the action 
of fj-hromophthaljjmide on skatole in benzene, has been proved to be 
2,6-dibromoskatole by acid hydrolysis to 6-bromo»3-niethyloxindole ; 
an isomer of the bromination product of 3"=Jnethyloxindole, and by 
oxidative degradation of 2~acet8ajino-4-broinobenzoic acid. Electro- 
philic substitution of indoles in the 6-position has been shovm, in 
the case of S-phenylskatole^ to proceed via an unstable yellow per- 
bromide intermediate, rearranging rapidly to the 6-bromo compound. 
In aqueous media, intraiBOlecular participation of "the carboxyl group 
of indole -3 -propionic acid, possibly by displacement on a bromonitmi 
intermediate, has led to (5-bromo)dioxindolespirolactones which have 
been hydrogenolyzed to oxindole~3~propionic acid. 

This neighboring group effect of a potentially nucleophilie 
amide imidol group in an indole p-side chain was utilized for the 
cleavage of the W-peptide bond adjacent to tryptophan, VJhereas I'JES 
treatment of K-Cbz-tryptophyl-glycine gave free glycine, the iso- 
meric K-Gbz-glycyltryptophan under these conditions did not liberate 
an amino acid. The general usefulness of the method was demonstrates 
with glucagon, the crystalline hyperglycemic-glycogenolytic peptide 
from pancreas, containing only one tryptophan among 29 amino acids, 
N-Bromosuccinimide leads to the liberation of a major new ni.nhydrin- 
positive peptide, giving positive platinic chloride reaction for 
methionine and negative reactions for histidine and arginine. Its 
hydrolysis yielded aspartic acid, threonine, methionine and leucine. 
This tetrapeptide , which arises from the C-tenninal seq.uence TRY- 
LEU-Ml?r-ASP-THR, has been obtained by the action of chymotrypsin and 
trypsin on glucagon. However, the cleavage of gJ.ucagon by W-bromo- 
succinimide is more rapid (<1 min. ) and more selective than that by 
aay known peptj-dase. 

The reactions of trypsin, tarypsinogen, acetyltrypsinogen, ajid 
an enzymatically active fragment of trypsinogen with W-bromosuccini~ 
mide have been explored. Under the conditions usedj the reagent 
selectively oxidized the tryptophan residues without significant 
cleavage of tryptophyl peptide bonds. The marked difference in re- 
activity of tryptophan in trypsin and trypsinogen is ascribed to 
differences in their secondary or tertiary structure. Enzymatic 
inactivation (trypsin) or loss of activatability (trypsinogen) was 
studied as a function of the oxidative modification of tryptophan. 
Such partially inactivated enzyme preparations still had their DFP 
phosphorylation sites intact. At least one tryptophan residue may 
be needed for activity. This demonstrates that an intact phosphory" 
lation site per se is not svifficient for enzynKitic activity. 



Seriax m. i>iIA^iD- 36 



Page 3 



The applicat5.on of the JM-bromosuccinimide (MS) cleavage to 
proteins under specified conditions releases new M- terminal residues o 
Bond cleavages generally average 20-ifO^ and the number of new K- 
terminals formed corresponds to the number of tryptophans in the 
molecule o The results indicate the presence of Tyry-Ljs arid T3'y-ALa 
bonds in tobacco mosaic virus (TM7) protein^ of a Try-Ala bond in 
the I=peptide from TMV protein,, of a Try- Ala bond in human sej^um 
albumin, and of Try-Gly and Try-Ser bonds in bovine serum alUimino 
Lysozyme which contains seven tryptophans is cleaved by the reagent 
with niuch lower yields. 

Significance to blo-medical research etnd the program of the 
Institute ; There is a great need for mild and selective loethods for 
the controlled and systematic degradation of proteins =, These chemi- 
cal "peptidases" in xcany ways promise to be superior to all knovm 
enzymes customarily used for breakdown and structiiral investigation 
of proteins o 

Proposed Course of Project ; jJto Gross has spent some time in 
DTo Lo Co Craig's laboratory at the Rockefeller Institute to find 
conditions for the cleavage of a number of cyclic antibiotic pep- 
tides. Active work is directed toward elucidation of gramicidin Ac 
Hew cleavage reagents and conditions are being investigated. 



Part B included; Yes 



Serial So. BIAM)- 36 



le k 



part Bo Honors^ Awards, and Publications 

Publications other thaji abstracts froni tills project: 

Bamachatidran, Lo Ko ,, and Witkop, B. : Selective Cleavage of G- 
TiTptopJayl Peptide Bonds in Pl-oteins aad Peptides, J. .Aia« Cbemc 
Soco 81: kQ2d-h032, 1959= 



lo Chemistrj' 
2o ^fetaboliteB 
3 o Bethesda 



EHS-KIH 

Individiial Project Repoart 

Caa.endar Year 1959 



Rirt Ao 



Project Title: Studies on Substrates and Inhibitors of Cholic- 
esterase and on the CJhemistry of Neuro-^Sascula.r 
Blocking Agents 

Principal Investigator (at WIH): Berahard Witkop 

Other Investigators: H= I<hy (Left MIH ll/?./59)f J» W<= Daly 

Cooperating IJiits: So Friess, R, C. Durante Haval Ifedical Center 

Mmi Years (Calendar year 1959): 
Total: 1/3 
Professional: 1/3 
Other: 

Project Description: 

Objectives : To esteblish a role and possibly a -use for deriva- 
tives of those mono- and diaiainohexitols that occur as building stone? 
of antibiotics; to explore the steric lisnltations and reqirljreji'eats 
for (polyfuQctional ) substrates of cholinesterase; to correlate neuro- 
muscular blocking activity with the structure of cyclic analogs of 
dimethylaminoe-aianol » 

!fejor Findings ; In oilier to investigate the labilization of ester 
bonds in the acetates of tertiary and quaternary 2-deo3{y-2-d3jiiethyl' 
eanino-myo- and scyllo- inositol and of 0-acetates in the strept-amine 
series^ the synthesis of analogous conrpouads. in the NjN-tetraa^tJiyl- 
deoxystreptamine series was carried out. An extremely labile tri-0' 
acetate was obtained ;, the hydrolytic activity of which approached 
that of esters as labile as £-ni.trophenyl acetates o. Two diacetates 
were obtained, one of which was qtiite labile while the other was 
fairly unreactiveo Kuclear magnetic resocaace Investigation suggested, 
that the reacti-'.'^ diacetate -i-ras the symmetrical ^,6-0-diacetyl deriva- 
tive. The quaternary salts of these esters were prepared and are 
being investigated.. It will be of great interest if -the labilisation 
of the esters in these nsodel compounds can be correlated with the 
overall conformation of the molecule » 

The Ettxticholinesterase activity of synthetic Dj,L"muscarine and 
9 further derivatives ira,6 determined with a highly~purified enzyme 



Seria:. 

page 2 

preparation from electric eel tissue "by a constant pH titsration 
teclmiq,ue used previotisly (S, L<> Friess, A. A. Patchett and B= Wit?' 
Jo Am. C!hem„ Soc,.79: 4p9, 1957) o A greater activity than by imisc-. 
was e:^iMted lay trans -4 , 3 -dehydromuscariae , allo- muscarone and 0- 
acetylmuscarine » !Diese enzymatic results parallel the pharmacologic a 
findings » 

Significance to "bio-medical research and the program of the 
Institute; Two major aspects of the cholinesterase problem are of 
special interest o l) The mode of hydrolysis and natvire of ester 
labilization. 2) The correlation of structure and activity in natura 
aod synthetic inhibitors o 

Proposed Course of Project : Dr, Friess is presently occupiec 
pharmacological and isolated nerve studies in/olvlng inhibitors of 
cholinesterase. It vill be of special importance to construct inhio-. 
tors capable of penetrating the blood-brain barrier and of acting on 
brain cholinesterase directly- 



Part B incltided: Yes 



Serial No, MXAMD- 37 
Page 3 

Beurt B. Honors, Awards, and Publications 

Publications other than abstracts from this project: 

Witkop, Bo, Durant, R, Co and Friess, S. L, : Acetylcholinestejrase 
Inhihitory Activities of I^iscarine and. Muscarone Derivatives » 
Experientia 15 t 3OO-3OI, 19590 

Friess, So Lc^ Standaert, F. G.;, Witkop^ B,, Durant., Ro C. and 

Reher^ L„ J, t Son^ Toxicologic Properties of a New Series of Aryl 
Ethers Derived from Trans"2-Aminocyclohexaaolo Toxicol o Applied 
Pharmacol „ 1, 609-617, 1959 » 



Serial Ko. tj IAMD- 3 8 

lo caaemistry 

2. Jfetabolites 

3 . Bethesd.a 



EHS-KIH 

laclividxial Project Eeport 

Calendar Year 1959 



P©,rt A. 



Project a?itle.' iBhibitors of the Biosya-Uiesis aod Ei^eMoim of 5'» 

Biydroxytiyptainine and Other Centrally Active Biogenic 
Aniiaes 

PrincipEQ. Investigator (at HIAMD): Bernhard Witkop 

Ottier Investigators: M. OzeM 

Cooperating ISaits: S. Udenfriend, H. Weisebach ard Bo Redfield, IBEj 
Serial Ho. HHI- 216 

J&B. Years (CaleMar year 1959): 
Total: 1 

ErofessiOEial: 1 
Other: 

Project Description: 

Project ; Synthesis of conrpouods which itihihit 5"hydro:£yc;:cjn:'to - 
phan-, dopa-decarhoasylase and monaaiine oxidase (M0)o 

Ob,1ectives ; The inhibition of the enzyme which decarbojq/lates 
5-OH-tryptophan or dihydro:}Qrphen3'-lsaanine \TovCLd. prevent the foi'matio;.;. 
of serotonin or dopamine and otb.er catecholamines and have practical 
applicability in diseases characterized by overproduction of raerotoicj. 
and catecholatBines such as liver carcinoid syndronje phaeochromocytonr. 
tumor and chronic ij^rpertension. The inhibition of MO in vivo anil in 
central locations is known to result in marked (soicQtibBes psychic) 
effects. The control of both of these processes is highly desirabls^. 

Methods Employed J.; 'Bxe technique for assaying MO oxidase aetL.. 
is described in the p'ublication by Ereter, ¥eissbach et al. Similar 
techniques are being used or investigated by Dr. M. Ozaki working in 
Dr. uaenfrierd-'s laboratoiy for the decarboxylases acting on dopa, 
tryptophan and 5-hydroxytryptophan, as well as on cat-echol-O-insthyl- 
transferase. 

Ifejor Findings : In the laboratoiy of Dr. ISenfriendj, Dr, Ozald. 
has been screening over 80 conipoux^s for ijahibition of rnonasiine ozidr 
MDst tests were performed in vitro following the disappearance of 
serotonin, and in some cases dopamine. Some very active inhibitors 



Serial No,JiIMD- 
2 



have been studied in vivo in rats, and serotonin bi^in levels have 
been deteiTnined in a number of cases. Other studies concerned the 
activity of monanine oxidase in various organs as a function of 
the species. For instance, determinations were rsBde in brain, 
liver and kidney of jnice, rabbits, dogs, rats, cats, guinea pigs, 
hamsters and toads. OSae effect of various monamine oxidase, inhibi- 
tors was not the same in all aniiasLLs; it was foimd that Marsilid 
does not act in toad liver, whereas asnphetaMae hydrazine (JB 516) 
is active. 

Over 30 compo\jnds were tested for activity as inhibitors of 
5»hyd2X>xyfcryptophan decarboxylase. It was found, that meta-O-methyl- 
dopa was a better competitive, inhibitor than dopa itself, JIB 51^ 
was also active. 

A conipletely new approach to the prolongation of the phaiTDaco- 
logical activity of catecholamines was made by the study of com- 
pounds competing with catechol-0-iasthylt3:Bnsf erase , It was first 
established that methyl-deficient aninals showed no difference in 
their response to norepinephrine with regard to nox^ual control 
aninfflls. No activity was shown by JB 516, cysteine, methionine and 
ethionine. However, striking effects were displayed by glycocyemiae, 
nicotinamide, arterenone and adrenolone. All these compounds acted 
as competitive methyl acceptors. Hie fate of norepinephrine was 
followed in vivo in a study involving over 300 mice by detemiining 
accurately the levels of 1) norepinephrine, 2) normetanephrine, 3) 
dihydroxymajadelic acid, all in the presence or absence of inhibitors 
of 0~methyltransf erase as ^-rell as monamine oxidase. !Biese st\Klies 
for the first time yielded accurate physiological half "life times 
of norepinephrine, namely 20-25 minutes noimally, and l^O-yo minutes 
in the presence of inhibitors. 

Significance to bio-medical research aM the program of the 
Institute ; The field of monamine oxidase ii^ibitors has acquired 
much importance. The only exact methods for assaying MAD inhibitors 
are being used in the laboratory of Dr. IJdenfriend. -In order to 
support these important investigations Dr. Ozaki of this laboratory 
has been delegated to participate in a pxt)gram of developing and 
screening iJihibitorB in this axea. Many pharmaceutical con^janies 
supply comrpounds for this program. Biey also adopt these ensymatic 
screejiing methods in their research progism. 

Proposed Course of Project ; Attempts will be made to develop 
harnala alkaloids which will be more readily absorbed from the gastx-xs- 
intestinal tract, Beversible inhibitors of MO and various decarboxy- 
lases will be looked for. Die program of clinical cooperation will be 
ejcpanded. 

Part B included: Yes 



Serial No, NIAMD- 38 



Part B . Honors, Awards, and Publications 

Publications other than abstiucts from this pro^ject: 

Udenfriend, S«, Creveling, C» R., (Ml), Ozeiki, M., Daly, J.Wo and 
Witkop^ B. (KIA>D): Inhihitors of Norepinephrine Mstahollsm in 
vivo. Arch, Biochem. Biophys, &)-, 2ij-9-251, 1959 ■> 



Sexlal Ho. JHAMD- 39 



1, Claerslstry 
2o Metabolites 
3 o Bathesda 



EHS-ETH 

IndividiEal Project Beporb 

Calendar Year 1959 



Part A. 



PitJject Title: Assay of Msnoamine Oxidase 

Principal Investigator (at HIAIffl): Berahard Witkop 

Other Investigators: Jolm Daly 

Cooperatir^g Tjrdts: Herbert Weissbach, lEhoiaas E. Smith and Jo B. 
G3X)utj, MI;, Se3:*ial Ho. MHI- 203 

Man Years (calendar year 1959): 
Total: 1/3 
Professional : 1/3 
Other: 

Project Description: 

Objectives ; Jfe,jor progress was made in the purification of 
monamine oxidase when Weisshach succeeded in obtaining preparations 
devoid of particulate matter. S^ich soluble enzyme preparations 
have been enriched 10-20 fold. This and similar work necessitated 
a reliable and rapid method, for the quick assay of monamine oxidase. 

Methods Employed ; The underlying idea for the selection of a 
staiteble stibstrate was the introduction of a reacts. ve ortho siibstitueat 
such as a primary amino group into a suitably sifljstituted prismry 
amine in which the initially formed imine or aldehyde would undei^o 
self -condensation to a stable product. 

Ifejor Fiadings ; All these requirements were met in kynuramine, 
for which a new synthesis was developed in the oaonolysis of K-carho- 
benzoxytryptamine . Ksitureimiae was found by Dr. Weissbach to be a 
good substiute for monamine oxidase. Its enzyimtic disappearance 
can be followed spectraiast3rf.cally by the disaHPearsnca of the absorp- 
tion peak at 36O m^. The product formed in this reaction is is-hydroxy- 
quinoline, which no longer absorbs at 36O mn, but at 329 aM 315 ^H^' 
Thus, by measuring the decrease in absorption at 36O mn one has a 
simple and rapid assay for monamine oxidase. 



Serial No. Jf^l®; 
Bags 2 



Si gnificance to Taio-medical research and the program of the 
Institute ; "Biis new rapid assay should prorve useful in the ex- 
panding progi^an on the purification of sionoamine oxidase and similar 
enzymes and on the screening of compounG-s affecting them. 

Proposed Course of Project ; Fur-bher purification of the enzyme 
is being contemplated. 



PEirt B included: No 



Serial HOo ULA!-®- 40 



lo Chemistry 

2. Metabolites 

3. Bethesda 

IHS-JJIH 

Individual Project Report 

Calendar Year I959 

Fart A . 

Project Title: Histochemical Studies on Monoamine Oxidase 

Principal Investigator (at KIAMD): Bemhard Witkop 

Other Investigators: Dr. Yuichi Kanaoka (V.S., arrived Augo 31^ 1959) 

Cooperating Units: Herbert Weissbach and Betty Redfield, MI^ 

Serial Mo. HEI- 208 and Br. G. Glenner, LEE, IIIAMD 

Man Years: 

Total: 1/3 

Professional: i/3 

Other: 

Project Inscription: 

Project ; Synthesis of substrates of sionoamiJie oxidase and of 
D-amino acid oxidase which ^ vivo might be converted to histochemical 
stains o 

Objectives ; Earlier studies by Glenner, Weissbach and Redfield 
have shown that during the oxidation of tryptajnine by monoamine oxi- 
dase a concurrent reduction of added diiodonitrotetrazolium chloride 
took place J, the aldehyde from the amine acting as the reducing agent. 
It has now been attempted to have the f eatiires of a histochesiieal 
stain built into a possible substrate, such as a primary amine or an 
a-amino acid for monoamine oxidase or D~amino acid oxidase. 

Methods Employed ; Derivatives of o» uj-amlnoacetophenone and 
phenylglycine with amino groups in the ortho position of the phenyl 
ring have been synthesized or are in the process of synthesis o 
Spectrophotometrlc assay in situ has been used to follow the disap- 
pearance of the substrate or the appearance of oxidation or conden- 
sation products, such as indigo. Investigation will proceed to in 
vivo systems, and histochemical staining phenomena are being looked 
for. 

Major Findings ; So far only o-co-diasiinoacetophenone has been 
subjected to the action of monoamine oxidase and found to be a much 
poorer substrate than the homologous kynuxamine, in the same way 
that adrenalone is more slowly oxidized by MAO than noradrenaline. 
The preparation of dihydro-o-cj-diaminoacetophenone is under IR'" 
vestigation. 



Serial I^^o-^j.^.u-:->.-:.. 
page 



SlgnifieanGe to bio-medicel research and the program of the 
Institute; The localization of the enzymes involved in the hreaic- 
down of biogenic aajines and amino acids in vajrious tissues and 
organs has been a matter of considerable interest to histochemists. 
The approach chosen in this project develops the stain from enzyxcatic 
transformation of the substrate rather than froas an interaction wd.th 
an extraneous compoundo Such an approach is much more direct and 
should^ if successful, lead to topographic maps of iarportaxit cata» 
bolic enzymes. 

Proposed Course of Project ; Limitations are imposed upon 
these synthetic substrates by the specificity of the enzymes. A 
systematic study on a larger body of compounds will be required to 
reconcile minimum useful rates of enzymatic oxidation with optisial 
staining properties of the condensation products thus produced- 



Part B included; Mo 



SerisJ. Ho. EIMm-hl 



1. Chemistry 
So Metabolites 
3. Bethesda 



PBB-KIH 

Individual Project Report 

Calendar Year 1959 



Part A. 



Project Title: Selective Cleavage of the Hy!iros.yproline Peptide Bond 
in Model Peptides and in Collagen. 

EriBcipal Investigator: Bexiiiiard witkop 

Other Investigators: John Francis, A. ¥» Rohertson (V.S.^ aixived 
Aug, 26, 1959) 

Cooperating Units: K, A. ?iez, EUMs Serial .No.___ , 



Man Years (calendar year 19,59): 
Total: 1-1/3 
Professional: 1-1/3 
Other: 

Project Bescription: 

Objectives; To find a selective chemical method for the cleavage 
of peptide hcrnds next to hydroxyamlno acids, especially hydroxjiprolins 
with a view of applying such cleavage methods to the structural eluci- 
dation of co3J-agen and gelatin. 

Methods Etaployed; It had been observed previously in this lahorc, 
tory that suitable O~tosyl hydroxy-^^proltae derivatives^ in the 
presence of proton acceptors, undergo an inte3:T2al e2.iiQination reactior 
with the formation of allo-hj'dre'oxy-i-proline lactones. The principal 
of this reaction is now being axjplied to suitable hj-d-roxyproline pep= 
tide derivatives. 

Major FindJAgg; Although the carhoxylate anion readily displaces 
a trans-O-tosyi. group in the natural, hydroxyproline series with eon- 
cosaitant lactoniaation^ no such participation occurs in IJ-caji-bobensy- 
lo2sy~0»"tosyl"hydroxyprolylglycine. 

Significance to 'biO"medieal research and the program of the 
^stitute; Collagen, the quantitatively raost import£uat protein in 
ms^JEnals^ is characterized by its high content of hydroxj-prolrlne, aa 
amino acid occurring more or less exclusively therein. A special 
method for 'bhe selective cleavage of the hydroxyproline peptide bonds 
is highly desirable, since snost enz;yBJes fail to cleave this bond. 
Such a method would Kake it easier to find, as has heen reported, the 



Serial Ifo. MIPMX^-Ul 



Page 2 



areaa of high h]/6xoxypTol±ae content in the peptide strajods of coi].a=. 
gen /Grassujaxm), ajad ^ro-old facilitate the analysis of urinsjry hydroxy- 
proline peptides obsea-ved in some patients i-Jlth metabolic distorloanees 
(Br. F. Ii'reverre)^ 

groijosed Course of Pr oject; The reasons f02* this lack of re- 
activity are heijig im-estigated. K-Car'bohenByloxy-Ss^'-'iehydroproline. 
its i^ptide vith glycine and other derivatives vill he subjected to 
i'-hromosucciniEiide and the asoimt of lactonization or peptide sleavage 
detenained. 

At the sajce time proaisang leads on partially selective aleavage 
of U"( hydroxy )prolin.e peptide bonds hy sodamide in liquid aEsaonia 
will he followed up. 



Part B included: Eo 



Bethesds 



:iiidiri-?.ual P.roject Report 

j>:,3nd:-r Year I059 



!?art 



Project 'Title: Oxidative Cleavage of T^/rosyl-Peptide Bonds: 
& specific RhensicaJ. peptidase 

;i?riDCipal Inv8£itigato:fs Lcuis A>, Cohen 

Other- Investigators? G» Lo Schiair, J. 0= Wilson (VoSo^ arrived 
Hovo 12, 1959) 

Cooperating Units: iJone 

M's.a Years: 

Total: i 

Professional: 1 

Other: 

Project Description: 

A study of the cheaaical fragnsentation of palypci>tides and pro- 
teins at tyrosvlL -peptide bonds by use of oxidizing agents sucli as 
bromine and W-bro3io3ucclniiaide<. 

Objectivea: To effect the splitting of eomplei: peptides by 
the addition of a chemical reagent which attatjks tys'osine exclusively 
and Iabi3.i2es the adjacent peptide bondo To study tiie use of 
phl.oretic acid as a unique and highly specific aaiine blocking group 
in peptide synthesis. 

Me thods Employed; Eapid recordiJ3ig ixLt-raviolet spectroscopy is 
used to foilov tJie course and extent of cleavage inactions o Infrared 
spectroscopy is ueed go elucidate the st:ructui:-e of reaction products c 
paper and colu.ran chroi»tography and high-voltage electrophoresis are 
\ised to separs.te and purify polypeptide fragjaentSo 

Major Findings; i^Jumerous peptides of tyrosine have been cleaved 
selectively at the adjacent peptide bond jjavolving the earboxyl group 
of tyros ine» The octapoptide hormone hypertens3_n has been cleaved 
exclusively at its tyrosyl -valine bond. 

Signific ance to blo-'iaedical reGearch and the program of the 
Institute ; The ability to S}iLit coisp2.ex peptides (enzymes and pro- 
teiri5y~at specific bomis can contribute g:5featly to the determination 



Pe-ge 2 



of aoaino acid seq.uences, to the jaodification of protejjas without 
deiaaturation and to the isolation of active fragments of enzymes - 
By these techniques proteins can be split vinder very mild coi:dition 
(neutral pH, aqueous 8o3.ution) by a rapid controllable reaction at 
se3.ected positions » 

Proposed Course of Project; To extend the study of the selective 
cleavage of the tyrosyl peptide bond to complex systesas such as 
polypeptide hormones and proteins « 



Part B included; Ho 



ftta-boi-it 



Individual Proved. Hapox^t 
Ci^-leadar Year 1959 



Part A. 



Project Title; Stu<l:les on a Bovxi.d Forja of the Mevirotropic 7- 
■butjT-ic Acid in Braiu 

Principal Investigator (5.n EIPMD)i Louis A, Cohen 

Other Investig&tors; VU:Lli6,n! M„ Jones 

Cooperating Units 1 Jeaa Do Wilson ajid John Jo Pisano, iffij% S^~2'icd 
NOo MX" 220 

Msxi. Yeto-s (caJ.eada.r ,i^ar 1959)2 
Total r 1-1/3 

Professional: l/3 
Other: 1 

P3ro,ject BescriptioB.; 

In the course of isolation of Coenzyme A from "brain a fraction 
was foujad by Jo J.. Plsaxio which, upon hydrolysis, yielded y^^inlno" 
butyric acido 

Ob^ctivess This occurrence of a bourd foinoa of 7 -afflinobutyric 
acid in. brain, raised the question of its eheioical straoturco 

jMethods Employed; Furification on chaj;coaj.. and I)ovex-50 colurani 
followed bj- paper electrophoresis sex-ved as ffietbc«5.s for conceutratiO' 
i-nd isolation of the bound forxa of /•"aajinobutj'ric acid,, whose conceiri 
trati.on varies from 300-5,000 i.igo par kiIogi.«eiD brea-Q ixf dogj pig ezS. 
beef. 

^_jor_S^lridin{|8 ;; The new cojiipoaQd is a peptide h;jn:'h»alyzable i-vo 
hiBtidine~ajtid'7"Su2liG,oLu.t;)'ric acido 7-MiiaobutyryJ.-|;".'iiotlcli;iae_, 
obtained by synthesis, had all the properties of the a'itureU.,y 
occun*i.f^3 compcnoiido 

^jjSfiM.^g^^^-^-. ^""-^ bio-"m edicaI. re see.2.'eh fxnsi the pro^y.r'ari of the 
Institutes a^ie"Tite7.a'ture on the signif icaace "of the neurotropic 7- 
QiBinobu-tyric acid in brain has been growing rapidly du;.-ing recent 
years o The occurrence of this amino acid, la a botind ptrotide iforia wi 
histidine as a hcsnolog of carnoalne raises Taaciy interestir^ question: 



bCX'lLftJ. DsO 



such as penetration probleias, mode of ■bj.os;iiil"liesis„ active axil 
taactive transport foriDS, etCo 

Proposed Course of Proje ct; It wi3,l be teKpt:lQg to sj/ntaesize 
iXirther analogs and iiomologs of this kixid and to subject theu! to 
phsiiTnacological and xieurologlcal studies^ 



Park B included s fio 



Individual Erojact Report 
naleadar Year I959 



k^ Metaboiriej 
3. Bethesda 



P&r-t Ac 



Rx5ject Titles Selective Oxidation of Free and Bound Hi£tid:'u;ie and • 
Other Iiaidasole derivatives 

Principal Inve0tigator; Louis A« Cohen 

Other Investigators: Go L., SoMair 

Cooperating Units: None 

Man Years (calendar year 1959); 
Total: 1 

Pifofessional.; i 
Other: 

Project Description; 

A study of the ox5vdati%'"e dega-adation of hiBtidine deiivatives 
by use of reagents anah as N-bi'vaffiOSUCciD.imideo 

Ob^eoW^j"'- ^° achieve the oxidative remov.?!. of ijoidazole rings 
in histidins peptides with a vlev of possibly effectlr;g the cleavage 
of peptide bonds adjacent to histidine by an intraaiolecuiar rea(;l;ion 
involving the disintegrating imidasole ring of histidlae; to study the 
use of a-rylsulfonj'l ip-oupa at; protecting groups for tlis ijtildazole rinj/; 
in protej-n degrade.tiOAi and in peptide syntiiesisj; to correlt.ite aiet^al 
binding and histidine destruction jji proteins-. 

Methooi.s Employed; Recording ultraviolet sixs-etroscopy to foJJLcw 
reactions of sulfcnylated jjDiidaaoles^, Infrared spectroscopy to de^ 
terxaine the stractures of reaction products » Paper cliroinatogra-phj 
and paper electrophoresis to help in structure elucidation azs-d puri-- 
fication of hisxicJjae derivatives o 

Major Findin££; '^i'he isiidasole ring has been rapidly oxidizefi b}/ 
W-broi2osuccinimide'\mder mild, conditions to jrield a keto aldehyde, 
aaaaonia and formic acidc Ring-niti'ogen substituents such as ^-tolue.r-j 
sulfonyl protect the ring agajjist o:iidative degradation and provide a 
new route to the synt.b.esis of histidine peptides o 

Si gnificance to bio-.rcedic al research azid the prog;rajc3 of the 
Institute ; The general objectives of specific chiemicsj. cdea'/ages of 
peptide bonds lie in the area of protein sequence F.tudie&, .■Bodifica^ 



tions and preparation of active fragments o These aims^ partiaiJLy 
achieved by the previously observed cleavage of tyrosyl aad tr^/pto- 
phyl peptide bonds., vfill be greatly furthered if other peptide bond:: 
such as those iavolving histidine, can be al.so selectively cleaved » 

P roposed Course of Project; The study of the action of variouf 
oxidizing agents on histidine peptides aoad to investigate tiio use oi 
tiie tosyl group in specific modification of the histid:Lne residues 
in proteins o 



Part B included? Wo 



i'is-fcaooiites 
Eethesda 



PHS-KIH 

Individual Project Report 

Celcndsx Year 1959 



Part A» 



Project Title; Ivfechaaism and Maaipulation of the Biosynthesis of 
Hjn±ro5£7proliae aod of ColLlagen 

Prtocipal Investigator (at JSI.'M)): Bemhgjrd V?itJiop 

Other Investigate- "s; Au Vo Robertson (VoS=, arrived Aug. 26 , 1959) 

Coopers-tiag Units ^ K, Gibson, So Udenfriend, mi, Serial Mo. MI". 211 
A.o :8erger (VoS. ,ln Di-o Anfinsen's Laboratory) 

I4sxi Years (calendar year 1959): 
Total: 1-1/3 
Professional: 1/3 
Other: 1 

Project Deecription: 

Ob jectlvea ; At the outoet of this e^jpaasive investigaticii the 
folJ.oi<r±ag limited objectives will be pursued: 1} The synthesis and 
x-esolution of 3; ^"<ie^^J^opro.l;LQe, 2) The reductive tritiation of 
dehydroprolxne to Si'^-H^-L-proline, 3) The synthesis of other spa- 
cifJ.c (dias,tereo}isoi!jer8 of 3-.» ^^-3 aJid 3,4-tritiat^d proliiies qx>& 
hydroxyprolijaesc h) Sj-n thesis of 3 , 4"epcjxy-Jt»prolix-.e as a key iaiter- 
E>sdiate for further fractional dsrivativea o? proline having £ OH 
groups, ilu.oro groupSj etc, 5) Synthesia of polj'dehyd.ro-L-p.Tcline as 
a further model for the existence or nonexistence of t,v-o rotational 
isomers analogous to polyprolinet. 

Me thod s Employed ; Special reduction of pyrrole-S-carboxaaide 
following So Fischer's method yielded 3,'^"-'3-shydro-g;L»i!roliiie and lis 
amide whose striacturae were proven by reduction to proline and prol." 
araide and by IMi spectroscopy" Resolution of the s^jnide was acbievrr 
by chemical and euzyiaatic methods c Tritiation is in progrej 



■JCK: 



tfejor Findiaga; The nev emiao acid :jd. two-dijnensional paper 
chroiriatograjas is vqty close to proline, gj.ves a ye.llov7 ninhydrin spec 
but .is not identical with anj^- unknovm spots of this color from marine 
or eoaimal coll.agen« The rotatory contributi.on of the new double bond 
is high and njakes for a totaJ. {(i)-q « -209*'' » 

Signifi cance to bio-gjediceil resea rer.'. and the progr-aEi of the 
Institute ; ICo Gibaon :ln the labora1>oiy ol' So Udenfriend will coiitln- 



Page 2 



the 'biosynthetiG studies on collagen atai'ted by Cb<. MitOffia. The 
jaecbanism of hjdroxyiation of prol;tiie (free or bo\md) will "be studied 
vith m laaay proline derivatives tritiated in selected positions as 
possible. The coji^etitive inhibition of dehyiroproline, Ji-fluoro- 
proline etCo wll3„ be studied » 



Par'G B included; No 



2. Metabolibfes 
3» Bstiiesda 

lodividufoa. Project; Report 
Calendar Year 19 59 

Part A , 

Project Title; Studies vith the 'Knz-yma, O-Mathyltraasf erase 

PrincipsJ. laveatigatoi' (at iSIAJffi); Bernliard Witkop 

Other Investigator b: Joim ftily, Si.ro Senoh (left 3/17/59) 

CooperatiJag Units: S, Udeiifriend and aaeociates^ Mil, Serial KOc JHX- 
J. i\xelroci;, KIMH? Serial flo. M-CS-Fa~3 

Mem Years ( ealendarr year 1959): 
Total: 2/3 
Professional: 2/3 
Other; 

I^oject Description: 

Q^4^gJj:X.Qg.' -^^s shoim ty Aselrod and coworkers., tJae enzyiBe ■ 
metbyltrans.ferase is primarily responsible for the metabolisiD of 
epinephrine and norepinephrine , Because of the great interest in 
catechols and in their metabolism a thorougli study of the action 
of O-ntethyl transferase on a variety of substrates has been initi- 
ated.. 

Methods Employed; The enzymatic 0-aiethylation of various 
catecholB has been stedied in vivo and in vitrOj, and the products 
investigatedc Feriodate oxids-tion of msta- atod paraaephrine deriva- 
tives has led to vaniLi.iae and isovanilline whose separation is 
rapid and quantitative o 

i«jajor Findings; In vitro studies using O-methyltransferaBe 
and various catecJhols e.s substrates has led to the interesting 
finding that ncton].y does 0-aiethylation occur meta to the side ehaiiji 
as reported in the literature, but that a sj^^gnif leant amount of 
^ara-0-nsethylation also occurs. The eaaount of this ^ara-O-metliyla- 
tion varies acco3"ding to the nature of the side eha:Ln, For compounds 
containiD^g electx^on -withdrawing groups in their side chains such as 
aoetovanlLlone J arterenone and adrenalone., the pfara isomer totals 
^"■56^ of the metAhylation product^ while vith cojupainds conteining 
saturated, side chains such as 3,4~dihydroxyphenylineti3ylcarb:in.ol, 
dopamJjQe, epinephriiie and norepinephrine, the pM'a isoEer is forced 
only to t-he extent of iO-15^o The occxu'rence of ^-O-raethylation in 
vivo is of great interest, and. with acetovanlllone^ arterenone, and 
adrenalone, p-0-Eiethylation has been deTuonstrated in the intact ra.t 



Seriail .No, j;JlAi!®-"46 



page 2 



although to a lesser extent than in vita^Oo h possible exijlaoation 
of this was found for aestovaniUone when it "was sho^m that the gara- 
and meta O-methylated derivatives of acetovanil3..one uMei'go a novel 
interconvarsion in '''ivo;, with the para ooiapoixQd heing xnost labile to 
conversion. Studies -vrere undertairen to demonstrate the forsmtibn of 
jj^ra-O-ssethylated spiuephrine (pararsephrine) in vivo tut the results^ 
in contrast to the ^a vitro studies^ indicate no foSnation of psira- 
nephrine , 

Stiodies on the half -life tiae of norepinephrine admiaistered to 
mice showed no effect ifith monoamine oxidase inhibitors while various 
0-methyltransferase inhibitors aimost doubled the half -life time of 
norepinephrine;, an ijnportaat phaisnacological finding » 

3?^b5'='2?rihydroxyphenethylamine, shown to be an inhibitor of O." 
methyl transferase in. these studies, is also of toterest as tri= 
desmethyl jnescalineo Studies have been initiated on the methylation 
of this compound and also on the enayjaatic demethylation of laescaline;, 
and the products are being investigated. 

Signif icsmce to biO'-ffiedi cal research and th e pr ogram of the 
Institute ; The great iasportance of catecholamines in regard to 
central and peripheral neurochesaistry lends great interest to the 
fun.ction of enzymes such as O-methyltransferase which effect their 
metabolism.. 

Proposed C ours e of Project; The biochemistry and metabolisia of 
mescaline, and tridesmethylmescalj^e in reference to the enzyiaes 0- 
methyltransferase end O-demetbylase will be investigated further.. 
The possible fos'siation of Coenzyme Q, recently reported as an isipor- 
tant oxidation-reduction coenzyiae, from a tetrahydroxybensene deriva- 
tive through the action of O-Eiethyltransf erase , will be investigated.. 



Part B included; Yes 



Serial m„ HIA?0 -'4$ 
Page 3 

Bart B . Honors, Aw;!5Tci.s, aad Publications 

Biblications ether than abstracts from this project: 

Senoh, S« ;, Daly, JaVr, , JiXelrodj, J<, and Witkop, B. ; Enzymatic £-0- 
Methylation by Catechol O-Msthyl Transferase, Je #jii. Chein« Soc, 
81^ 6?hO-62h5, 1959 . 



Serial Ko» a iA2«g)- 47 
1» Chemistry 

2, Metabolites 

3. Bethesda 



PES-KIE 

Individual IToject Heport 

CaleacLar Year I959 



Fart Ao 



S^ojeet Title; Ragulation of Grovrth of Miiaal and Plant Cei^.3 by 
Barj-vatives of He-tural. Bydx-oxyajsino Aaids 

Principal lJive£-tigator; Bern,bar6. ¥itkop 

Other Investigators: Ao ¥» Bobertson (VcSo., arrived Aig. 26, 1959) 

CooperatiBg Uaits; K. Slbson, MI, Serial Ho» H ai- 217 

BTo Fo Co Steward^ Ccmell University 

Ite'o S^ Archer;, Stsrling-VJintbrop Kesearch Institute 

;)fen Years (calendar year 1959): 
Totals . 2/3 
Professional: 2/3 
Other: 

Project Bsscriptioa: 

I^iject: To detarsilne the influence of hydroxyaaino acids and 
their" an£;logs as Tjossxhle regulators or inhibitors of celli.ilar gro^-rbho 

Objectives; To acccrsplisb a sore di2.*ect control of protein syn- 
thesis, tissue regensDration, formation of collagenous zees tissue by 
direct and local application of eyco-active agents than vas hitherto 
possible by remote and hoxmonaj. control o 

Methods ISxgplo yed; A plant tissue culture system has been used to 
detect the gro'^roh :;xhil)itory effect of a number of nitrogenous com" 
pcuads and to detsruiinej, where possible^ the siataboli'i site at vrhicb 
the substance in qu.estion may acto The tissue culture system consiste-; 
of explants froHi cairot root stiJisjlated to grow by cell division* Thif 
•was sug^jested because the cells %rhich groi^ ixx this way synthesiae a 
protein in which proline is incorporated aad which is unusually rich 
in hydroxyproline for a plant protelno 

At the EOBjent the act3jioiayGin producing strain of streptos^j'-ces 
is the only microorganism known to incorporate unusual exiS. foreign 
amino acids such as ketoproline , pipecolic acidj aaetidinecarboxyllc 
acid into the peptide pai-t of the antibiotic -which it elaborates « Eto 
Katz is the only expert laao^m or available at the present time who 
masters the technique of following the incorporation of these foreign 



Serial HOc JSIAMD- 4*/ 



Page 2 



amino acids and of the eoialysis of the resulting modified actinoEiycino 

>fe.jor Findings ; l) In plant tissue: see puTalication by Stewsx^i, 
Pol].ard;, Patchstt and Witkop; "The Effects of Selected Kitrogen Cam- 
pounds on the Grovrth of Plant Tissue Cxiltures," Biochimica et Bio» 
physica Acta, 28: 308, 1958c 2} In chicken ernhx-yos ketoproline, an 
analog of hydroxjrproline, causes a prolonged elevation of f2*ee 
hydroxyproliaeo The Hsschanism for this elevation has teen estah].ished 
to he due to inhihition of hydroxyproliue cataoolism hy ketoproline 
and by enzj-ffiatic corversion of ketoproline to hydroxyprolineo The 
enzyBie for the latter reaction 3 s found in the supernatant fi*action 
of rat kidney and liver, and requires reduced pyridine nucleotide. 
Some of these findings have been siinajarized at the 4th Interns.tional 
Congress of Biochemistry, Abstracts, Section 12, Ko. 27, p. I52. 

Significance to bio-medical research and the program of the 
Institute ; The existence of enzymes capable of reducing ketoproline 
to hydrcxyproline raises the question of a reverse transformation 
which, on the level of collagen, could lead, to the formation of cross- 
linkages . Whether the hardening of collagen with age may be due to 
such a sequence of reactions remains to be seen. 

Proposed Course of :i?roject ; la'So Gibson and Udenfriend will 
Bttidy the effect of prolonged administration of ketoproline on 
animals and will try to purify the enzyme involved in the conversion 
of ketoproline to hydroxyprolineo The synthesis of S-iiydroxy" , 
3,4-dihydroxy, 3- or 4-fluoroprolines will be attenrptedo 



Part B Included; Ito 



Serial lio. MMD- 48 
lo Chemistry 
2o ^Setaboli■tes 
3« Betbeadf'. 



EHS-KIH 

Individual Project Report 

Calendar Year I959 



part Ao 



Project Title; The Chemistry and Metabolic Fate of Tryptsmine 

Principal Investigator (at KIAMD): Bemhard Witkop 

Other Investigators; John Daly 

Cooperating Units: So Udenfriend and H.. ^Jeissbach, MI^ Serial Ho»_gHL:;2i 
Regis Cheanical COo, Chicago (custom sjiithesis of 
certain hydroxyindoles ) 

ffen Years (calendar yesx 1959): 
Total: l-=2/3 
Professional: 2/3 
Other: 1 

Project Description: 

Objectives ; The discovery of Dr« Udenfriend that tryptamine 
arises from tryptophan by the action of a new decarboxylase, and that 
it occurs in significant amoiints in the brain, focuses attention on 
this new biogenic amine and its transforusationso 

M ethods Employed ; Hon-enz2/matic and enzpaatic oxidations of cold 
and radioactive tryptaaine lead to a number of new compounds which are 
imder investigation <> Kuclear magnetic resonance spectrophotometry xp 
utilized to gain infonration on the possible existence of unstable tri 
cyclic tautomers of tryptamine » 

^^ajor Fi ndings: The appearance of tryptamine in the brain has 
raised the question of its possible transformation to serotonlno Such 
a conversion, however, has now been ruled out, since radioactive tryp- 
tamine in vivo does not lead to radioactive serotonin » The nuclear 
magnetic resonance spectra of tryptamine derivatives leave no room for 
the assiaaption of a sma3J. aaiount of a labile tricyclic tautomer= Oxi- 
dation of tryptamine by a microsomal enzyme system has been shown to 
lead to a new hjndroxytryptaaine not identical with serotonlno In 
analogy to some Japanese findings this new metabolite has been con- 
sidered as 7-hydroxytryptamine= However, recent resvats have proved 
the structure of a 6°hydroxytryptaminec Through Sajidoz Phaarmaceuti- 
cala, Basle, Switzerland and the Regis Chemical COo, a number of 6- 
and 7-hydroxyindoles have become accessibleo 



Serial Ho. MliSM)- 28 



Page 



Significance to 'bio-'iBedical research and the program of the 
Institute ; The recent finding that the Mexican jEushrooias used for 
producing central effects in religious rituals contain as their 
active ingi-edient psylocybia^ J:°£°^ "^^® 0-j^osphate of 4-hydroxy- 
N^N-dimethyltryptamine^ injparts special interest to the novel 
hjr-droxytryptaaiae metaholites mentioned ahoveo 

P roposed Course of Project ; The chemistry and psychopharma- 
cology of 6- and 7"hydroxyindoleethylamines will be inves'bigatedo 



Part B included: Yes 



SsriaJ. Ho. HIAMD- 48 

Page 3 

Part B o Honox'S, Avard-S, acd Publications 

Publications other than abstracts froia this pro^iect: 

Udenfriend, So, Creveling^ C» Ro ^ Posner, H= , Sedfield, Bo Gc ^ Daly> 
J^ and. Witlsop, B. ; On the Inability of Tryptainine to Ssrve as a 
Precursor of Serotonino Archo Biocheja.. BiophySo 83; 5OI-507, 1959< 



Serial W3',M£^L&3^ 
3. B&t^@Ma 



Cals^ar Year 3-959 



^&^' 



pEt>j®Gt SI tie; Ea#K©r°Cferb9a Sasgss^^ Mh^^^xo Sis^s^g ^nin© as^^rs^ 



Ofels®r Ime&tlm^^i lea® BslMsfe ^©r (?.S. frm 9/&/39)s 

mjsssm^r J, aarlsoa (IT. So tatil 11/13/59)^, 
feass ¥. Imtt (mtil V20/59)is Bigo He Sesfetoa 
(?cSo fs^a 9/1/59) f l^ma«iel Elssis^ Jolm 1?o 



>: 
ftolals i) 

l^f®ss±®a®l; 3 2/3 
©«ter: S 1/3 

Ob^leetiyes; ®3 erolv® g@a@mlisati«»® ^©latiHs to -fcbs sfcyaieal 

^^1^0^, ^^qy^ ^ WQ£,^^^^' ^ coatinmtioia of ©iir 
eaaadaatlcaa of tfe® M^r-ca^lKKi s^^^^ ia tfe© ©imeaao ©j^ ^dm 
species tj© Mv© s&osm tSsat th® sass© oettuos® ocenra in Ijotb of 
pleat ^^ri®ls sad hm® e8tal>lisli@& its stsKsture as B» 



glycearo-S^agmBO-oetalog® '^ dsg:reS®tioa smd ^y cjrasiQh^fteia syathssis 
froB D«glycea?o°B°Baa'fflO''l8SFtog'S - '^ Mv@ isolate frc® •^® ©'voesdo 
"the fli'S't lssso?fflffi ^fes3£@ll3r occisir^ing octilxsl k:^ tev® ptov©3. i'fcs 
istffuctur® to b® !^KSgy1a3go-^^^ga lagto- octitol| we have fomsd ©^Idsnc© 
also foz* the proMbl® pseaencs of D-^i]^"b©p?^alos® in W.® m^oo^Oo 



^ fesnre isolated ^-aiMoSi^ptitol (S>-glye@yo°D'"^mso°h@ptitol ) fs-osa 
Sedmai this ia ttos first rgporfc^ occ-aETsae® of iMs si:@©r s3.cohol 

ia 13S,tU?@« 



■•xitea to iSm prspamtioa of startles amterials s^l iates^ 

Bropoaaa Counae og^^oigct ; Osatimstioa ©f ttes® @M closely 

TSlGftsd tropics e 



Birt B iaclTsaM: Yes 



serial ». ^^zM. 
Bag© 3 



gfegt 3o IBSss^rs, AaaMs, and Pablicatiesjs 
Puiblleaticms otisss* tSaea absti^cts fseoaa this isro^ectj 

Chsylsoa, A. J. ©sd M-chi^pgr, 1. S« : Isolatioa ef 

1959. 



Serial »o.K^iJ@. 
3. istlassaa 



OaleiiSar Yesr 1959 



^^A,' 



Project ISLtls: Ste;fii®s <^ iitm Syataaeeis of OesSsohydiat® Beriva- 

tif^ for l^acal 



Other InTOStiga-feorsi l. ]^istee (^.S. frs® 8/28/59), 1. W» Bi^hl;, 



!i»i years (cal®i^.©2' y^&S' 1959) s 
»ytjal: 7 1/3 
EtofoBsional: 5 2/3 
0«h®rs 1 2/3 

itoj9ct Bsseriptlcmi 



Bo 20 BSke difficultly accessible css^xSais^Lmts d^ri-mtlrm 
emtil^le to BieAieal rs8S8TOh®£® eitSasr -^roia^ dlv&st 
giffe or 1&a?ough pablicati^i of dixisetiems for tSs® ssrepaza- 
tlon tbds^sof > 

C. 2b eacfeas^ ki^wledge of -aie ehssDaical psogerties of Mo» 

Specific Ca>,1^ti^a; ®5jt^ the ^'^'^fj^ f ®**S^' ^% 

6yBtli@sisiii@ 3ubstaac®0 ^lich Mvs bsen d^sonstx^ted 
to be (o? sus^cted of beisjg) iatejsi^iated ia cea%o» 
bydrsts n^taibolissiQ 



Serial m.mmj" 50 



Eroggess dmlc^ 19^^: 



for s«latiw3ly IsjRge-sGalfi preductioa of tMs sugar 

has "be^Ei @volvedl, 

B. A Jsst^Qd h^ be@ii devised fo? the syathssis of S» 
d®OQ^rxmcI®>sld®s . 

G. tS^ "bmmylMi&a. B-sl^^l Mfl L-a»abiaosyl £luorid@s 

(a) & txsasfoximtiQa fxnm tbe D-riliopyxaiaose to -^3@ 

D-ffiboftoanose se^d-es, (fe) a ffecil® coswgs^ion fs^ 
ttm a^bisiose to the iribose eeries ^sd (c) % ?^y s<sut® 
to ribofisssaaose 8Sid ribosiyis&siose derivs.ti'V'ss st&ssti'^'t^ 

"by cJiOTlc©! iBsam for tt® first tin®. 



£. His z^^sfitiOEi b8t«?@@SL vsislous l-'iMoaMosd 6jss±r&t±w®& 
Qsod cspfsdis £@n>^ ss@t@J. s&Lts has 'b^si Sl30{{& to ;^o?id@ 
a zastr ^^^^stic pa^nsay to logi&stitat^ aldose ^rit^" 
tiws. 

Fo !Ei© pefflgBsati<m of S-deosgr-D-gliseoa© aad 3-deo:^-B" 
mmDOB® fsma 2-djeos;y^!>>7il}oa@ baa l3®ea acMe7ed» 

Sj^fjcaaee of the pgo^eet to the ^gt^am of the Bi8tit«fe®: 

She ^atbetic assthods ead -^^ imtexlals produced ia tl^ 
couTse of this project h@3?e 1>@e& aod ^11 "be of utility to 
^msiam seseauKh gxm^ in lilAM). 



ggopoBed Oourae of Sao^ect; 



<I!he area descrilMSd uaier "S^peciflc Objectives" tsill be piisrsued 
during 1959o 



®B3rt B inclM<sd: Yes 



berial Ko. JIAMD- 50 
Page 3 

Part B . Honors, Awards, anfl. Publications 

Publications other than abstracts from -ttiis project: 

JfecDonald, D. L. and Fletcher, Ho 6., Jr.: 2-Deoxy-D-rihose . II. 
The Synthesis of 2-Deoxy-D-ribose 5-Ebiosphate. Jo Am. Chem, Soco, 
81, 3719-3722, 1959 « 

Ness, Ro K» and Fletcher, H. G., Jr.: Synthesis of the Two Anomeilc 
9-(2--Deoxy-D-Hihofuranosyl)-Adeniveu. J. Am« Chem.. Soco, 8l, ij-752; 
1959. 



Serial BOo HIAMfi^Jl,. 
Ic Caeiaistry 
2o Analgesics 

3 o Bethesda 



PHS--MIH 
Lndividual Project Report 
OalenclftT' Ti-?«5^ 1959 



Part Ai 



Project titles Gh^smical structm's ^md action of 
iiaorpA:xine=like analgesics o 

Principal Investigators Sath^an Bo Mdjo 

Other investigators s Hone 



Professional; 066 
Othey ; 2 066 

Project descriptions 

Some thirty odd coiBpomids h&¥e been made tkls year 
in tha Section" s laltoratory,, notafely foenaoaorphaiia aiid 
the new series of sininoacetates aixd amino biityrates o 
In addition well o"?er one hundred compounds ha^e be§i?x 
received fi'om laborato^fies in the United States aii.d in 
Emropeo In addition to quantitative comparisons .of the 
series of coffipo-unds feeing built up \^ oiir o^n chemists ^ 
a large group of cofflpounds--?8late!i' to pethidine and 
another group related to le^orphanol are being evaluated ^ 
partly f 03? the accuia.ulation of data and structure^aetion 
relationship and partly in an ssffort at fiirther separa- 
tion of useful analgesic aetionj, froai addiction 
li&Mlity and side action incidence (see last 5^ear"s 
report) o Our major contribution in this efforts 
ph@nasocin@p is now ready for isarket after trials in 
thousands of patients The GoverniEent has alloi^ed 
foreign rights in thi.s cosip'ound to lapse end its intro- 
duction abroad by private industry is anticipated o 

The few patients in the Clinical Center which have 
eome to oth' attention this year on accour).t of their 
pain problems c> hav€j been treated with oral doses of 
phanassocineo Good relief has been obtained with as 
little as 2o5mgo£. ^hen the patif-mt has had littl© previoBs 
narcotic experience o If the patient, \sb.b tolerant tO' a 
previously used opiate a larger dose was required biit it 
is oiiif impression that cross- tolerane© is in«o.iaplet&^c 
Mo side effects hava been enKomintfired 'sslth adequate 
9-naIgesic doses o 



Serial Koo MIAMD^_|L 
Page 2 



Individual Project Keport 
Calendar Year 1959 



A good start has been jaade with the Coded Informetion 
Center^ supported in part by KIMHo Several thousand 
dociaments have been coded and keysort cards p STiithor ijidex 
eardSg etCo,, ha¥e been made for well over a tho^Asand of 
thes€o We ax*© beginning vjith_ current mid very rseent 
literatiire and the aecijaiialation is already proving a very 
useful sotirce of information for 0urssl'*res and others o 

As a part of our consujl^tative se3?vice to other 
Governiaent agencies a significant contribution was sade 
to the legislative prograia for IwpTovement Xn the national 
narcoti'Ss control regimenc Conferene-ss viith the Addiction 
Research Center ©f NIMHj, the B^areau of Marcoticsjj the 
lational Research Comisilg and the sponsor of the legis- 
lation^ have rasiiJ^ted in amendment of HR=529» to be kiioyn 
as the Has'cotic Act of 1959s providing for- flexibility 
in raarcoties eontrolg modification of control iri eithsr 
direction as e:4;perience warrants and technical advice tc 
establish a degree of eontrol commensm'ate with the riaic. 
Lie healtho 



Part B Included s Yeso 



Serial ...-. .-lAli P,^!!. 
Page 3 



PHS»MIH 

Individual Project Report 

Calendar Yeax- 1959 



Part Bs 

Publieations 



The analgesic equi "faience- to morphine and relative 
side action liability of oaymorphone ClV=^hydro.xy-= 
dihydromorphinone) 9 hj Mthan Bo Bddy and Lyndon 
Eo Lee. Jro Jo Phariaaeolo (1959) X25) 



Tfee rats of development of physical dependence ®nd 
tolerance to analgesic ds'ugs in patients with 
chronic pain^ lo Comparison of morphine p oxyiaor- 
phone and anileridinej, by Nathan Bo Eddy^ Lyndon 
So Leep Jro J, and Carl Ao Harris o B'ollo NarCo (1959^ 

The s?ate of development of physical dependence anci 
tolerance to analgesic driigs in patients with chr-oi;; :.;. - 
pain* lo Comparison of MoyphJ-ne^ oxymo:i'phon0 and 
anileridine .. Condensation in French o Hathaii Bo .^Idy, 
lyndon Eo 'Lee^ J^o^ and Carl Ao Harris « Bullo- Vfid 
Hlth Orgo (1959) m» 12>H-5o 

Structures related to morphine » XII o Sjiithesis of 
2 " =■ hydroxy-- '? » 9=diEiethyl=2-=phenethyl=6 p ^^hanzosaor-piyAii. 
CMIH 7519) by Eo Lo May and Mathan Bo Bddyo Jc Orgo 
Chemo ^ (October 1959) » 

A new potent synthetic analgesic by Nathan Bo Eddy 
with E?erette Lo Mayo Jo Orgo Chemo (1959) ^s 29-5 o 

Addiction Liability & Mrcoticjs Control by Nathan 
Bo Eddy and Harris Isbello Public Health Reports 

(1959) 2^. Hoo 9, 755o 

Chemical structixra and action of laorphine-^like 
analgesics & x'elated substances s, toy Nathan Bo Eddy. 
Chemistry d Industry (Ekigland) (1959) 21 lo v ember o 
Moo h?^ ppo 1^^^2-=li^9o 



Page h 



PHS^NIH 

Individual Project Report 
Calendar Year 19^9 



Part B Csontinusd) 

Eonovs sad Awards s 



Secretasya Coimaittae on Drw^ Addietion and 
narcotics £, Matlorxal Research Goiancil <■ 



Osto 19"2^p participated in 10th Session ^ Expert 
Cojasmittee on Addiction=prod-ucing JyrugSg World 
Health Organ! sation^ Geneva^. Switseriando 

Octo 1 - I4.ster Memorial Leettirerj, Edinburgh^ 
Scotland o 

Octo 2 =• Member of Paiielp Sysjposixiisi on Analgesies» 
MlnhUTghs Scotland o 

Octo 1 to Octo 31 visited laboratories working on 

analgesic problems o - Edinburgh^, Scotland | Beerssj, 
Belgi\2a| Louvaiiis Belgimiig Ingelheiiaj, Qermaiiy; 
Baseli,. Switaerlaadf Copenhagerij Denmark? Stockholm ?/ . 

Swedeiio 

Octo 6 "- Addressed postgradiiate classesp .Louvaln 
University^ LomraiOp Belgiujn on "Methods for 
Determinisig Addiction Liabilityo" 

Oeto 30 = Addressed Drug Control Unitp Karolinska 
Institute J, Stockholfflj, Sweden on "Addiction liability 
and narcotics control" o 



Serial MOo HIM1I >JS2_ 
lo Chemistry 
2o Aa 



PHS^-NIH 
Individual Project Report. 



I xne erreat or -sin 
thyroxin on the recovery of M-demethylase a,fter ab:rupt 
withdrawal of narcotic dru^So 

Principal Investigators s Joseph Coohin and Louis 
Sokoloff CNIMB')o 

Other investigators! Mone 



Man years 5 Total s 1/3 
Professionals 1/3 
Other s 

Project descriptions 

Objeoti^es =■ To -determine i-^hether the adralnistrs." 
tion of thyroxin before and during abrupt withdrawal 
of morphine affects th<a resynthesis of the M-demetbyiase 
diminished d"uring the period of chronic morphine 
adiaini s t ra t i on o 

Methods and results s 

The administration of 90 micrograms of 2-tlyroxin 
to rats for periods of 7 days doss not affect th 
■I'=d©methylas^8 fe'ttt tliree days after withdrai'^al of 7"day 
hyperthyroid aniaala that ha^e also been given Kiorphina 
chronically J, M^dejaethylase activity is about half of 
that of aniflials treated with morphine alone^ Thus there 
sescQS to foe a real potentiation of the effect of aorphi!!'; 
hy thyroxin 6 Fourteen day treatment u'ith thyroxin alone 
depresses aoti^^lty of the enzjme moderateljp but the 
combination of morphine plus thyroxin results in a most 
profound depression of demethylase acti'^ity far greater 
than that follo^'ing narcotic driigs alone o The TQCoveTj 
of li-demethylase activity after ^^ithdravjal is delayed 
significantly hj siaking the animals h^yper thyroid pr-ior 
to withdraMalo 



Serial MOo I^IAIJD » »x 
Page 2 



PES-MIH 

Individiial Project Report 

Calendar Year 1959 



Significsnce to the program of the Institutes 

Contribution to the imdarstanding ot the effect of 
tbyroxin on microsomal ensyme systems and a possible 
lead toward imderstaading the mode of action of the 
morphine effect on N=-demethylationo 

Proposed course of the projects 

It is hoped to continue the project insllQ. fey 
varying tiiae intervals of tiijroid administration and 
by using thyroidectomized animals and thyroid blocking 
drugs o It is also planned to carry out extensive 
lg,°'^itro experiments to see whether this effect can be 
reproduced by addition of thyroxin to tolerant non^ 
tolerant liver preparations o Attempts will be aiade to 
simplify the in- vitro system in order to loealisje the 
site of action and in'«'estigate the laechanisai of this 
effect o 



Part B included s 2'Io< 



Serial MOo MIMD ~ 
lo Cheraistrj'' 
2o Asiaigesics 



PHS-IIH 

Indii?idiial Project Report 

Gal end ar fear 1959 



Part A 



Project t.itl€!S Biocliemical aiid phars.acologieal changes 
after ehronic ©Jjainl strati on of narcotic driagSo 

Principal Invsstdgstors Joseph Gochinc 

Other investigators s Julius Axelrod (l 

crating Units MIMH ■« gi-GS"Fl»3 

jearss Total t I/3 

Professionals I/3 
Other s 

Project descriptions 



M=dealkylation of narcotic cli-iig siibstrates hj ^at-liver- 
rjoiaogenates after cteonic adiainistratioii of narootie 
driags parallels diminution of ia-vivo responses and 
whether changes in the enajaies ini'olved can series as a 
mod el ^ for the .in°j? lTO changes uitii tolerancao 

b) To investigate the nature of the reduction in 
ensyjaatic actl¥itjo 

Methods aM Res\0.tss 

a) CD In an attejapt to correlate the in-'Mtgo 
S-dealkjlation of a series of compoTOids of tie sorphinaxi 
series vith thair relsti'^e analgesits potency ia-v lgO g 
efforts were made to de'-ifelop methods of eztraetion and 
separation of the ensysaticallv- dealkjlated nor Gompoiind 
from the parent H-s-abstituted raorphiaan in order to' 
determine the rate of dealkjlationo W"s ha¥e had only 
partial success in these attaispts and a clean separation 
of closely/ related jsorphinans is not jet possibleo 

2) Contiii"aation of the studies of parailelisia of 
the l,n°fltro and In-gitfo changes accompanying tolerance 
repealed that the stereospeeifieitj hei'etofore considered 
a fmiction of analgesic potency » that iSj, that the isomer 
's?h.1.(C!,h %,'j?.s the more potent analgesic was also th© one 



Serial Ifoo MlAlil) «■ ,53 
Page 2 



PHS-MIH 
Individual Project Report 
Calendar Year 1959 



was dealkylated more readiiy» ¥as true oxiij In 
a partiailar speciesj, and that in another speeiesj, 
the rebel's e .might well be tinieo 

W It was fouud that the dealkylation reaction 
as it md been described heretofore iias not sat^l^&tect 
with respect to TPM and that increasing T'PM 10-2vO fold 
and adding glucose 6='P0if boosts the rate of dealk-fla- 
tion fi¥Q to eight times <, However j the ratio of 
activity of normal .and tolerant livefs remains about the 
ssaiej, indi eating that the defect in the tolerant , animals 
is probatolj not one of cp-faetoi' defieieiicy bv.t rather 
a tr^e destruction or blocking of &nzfm® acti^^itjo 
Attempts to piarifj and isolate the N-demethyias© ha^e 
not h&sn successful th-as fsXo 

Signifieance to the progrmn of th© Institutes s 

W© believe this to be a contrihiitioji to the 'under- 



Proposed com'se of the projects 

We hope to contlmie these studies of the relation- 
ship of the Tate of M-dealkylation and an&lgesie 
effisiencj axid/or dependence liaMlitjo W© also propos'a 
to continue attempts to p-urifyj, to some extent at lea^.t^ 
the crisde Bicrosomal ensijme we are at present \ioTklng wil 



Part B included 5 Yes< 



Serial Koo MIAI®^ 
[e 3 



PHS-NIH 
Individual Project Report 

Calendar Year 1959 



Part B: 



Jo and Axelrodj, Jo s Biochemical 
pharmacological changes in the rat folloving chronic 
administration of morphine p nalorphine and normorphini: 
Jo Pharmo & Expero Therap^j, 125s 105» 1959 o 



Honors and awards s 

Q&'we seminar on "Bioehemical and pharmacological 
changes aecompanying tolerance at NIMH Addiction 
Research Centers Lexington^ Kjog January 1959? at 
Depto of Pharjsacologyj) George Washington Universityp 
May 1959; at DaptoOf Pharmacology j, Bkiory Universityj, 
Atlantaj, Georgiaj, Septo 1959° 



1 Hoo IliklSIK^ 
3 o Bethesda 



PHS=.IIH 

ladividiiai Project Report 

Calendar Year 1959 



Part Ao 

Project titles 



1) Chemistry and neuroptmrmacologie atiidj of Qc>Ei.-- 
po-onds derived frora 3j^-='<iil^<i2'o-7='sa®thory I'ClH)- 
naphthal enon e c 

2} Stereochemical direction of addition to the 
cartoonyl groyp of 2''=BiethcuQr"2s5=--<ii2iethyl-9"«^3!;o- 
69 7"beBigOffiorpha3i ( OK^codone and ©.xymorphone 



3) Preparatisn of miscellaneo'as bensomorphans. 

Principal Investigator? B^erette Lo May 

GtHer investigators s Hiroshi Kugita & Jo Harrison Ager 

Cooperating imitss. Siaith^, Kllsy? & French liUboratori es ,,. 
Department of Pharmacology 3 University of Michigaiij, 
and Addiction Research Center^ Lexingtons Kyo AXsiOj, 
eoffipleaientary to pharmacQlogisal investigations in 
this Seetioiio 

Msn fears s Total s 2o5 

Professionals 2o5 
Other t OoO 

Prft^ect descriptions 



Objectives s To add to our basic kno\«fledge of 0J:?gsin: 
chemistry and of chemical atx^uctiu-e - aeia-ophas-B-iP- 
cologic behavior implications | to synth^^siss© s>ui, 
medicinal agents o 

Methods employed s The standard arid the siore „r -, ;.. 
techitiiqiaes of organic cheiaistr-y incjlndirig' spectral 
methods of analysis's as well as. pharmacologic 



Serial MOo HIAMD«_54_ 

9 



PHS=.NIH 

Individual Project Report 

Calendar Year 1959 



Major findings 5 1} A more versatil«g approaeh to 
the phariaacoiogically and chemically interesting 
bensoaaorphan (I) faiaily of ciompounds de-¥elop©d hj 
oxjT lateoratorj was soiiglit and fowiido TMs approaoh 




involves 3 !)^-«3.il^(^ro=7=iBetiio^'=2ClH)'=nEplithalenone 
as a starting aiaterial &rA 2"=-inethoxy-2"s5'^<ii{E©thyl« 
9-=oxo=6g7=-ben:soiBorphan ClI) (Tp R2~R2"Meg R^sOMe» 
Rl^g^oso) as an interesting intermediate 6 In the 
synthesis of the latter a pyrolysis reaction on the 
methiodide was necessaryo This pyrolysis conducted 
by dry distillation yielded aiEinly tar but aJ.so a 
small amoimt of an apB-tmsatiarated' ketone resulting 
from elimination of HI and nitrogen ring opening 
CHofmann degradation),. The structiiire of this eoBi- 
poTand%i?as readily proved by standard methods o The 
desired comporaid li was 'finally obtained in satis- 
factory yield along with varying anaoiantf; of the 
Hofmann product (depending upon the solvent used) bj 
condMC ting the pyrolysis in hexanolp heptanol or 
octaaoli octanol was ' optimalo Surprisingly p in tl:u-' 
presmit® of acids compound 1.1 formed yers' stable 
hydrates (or alcoholates) at the .sarbonyl group as 
shown by analysis and infs*a red study ofBeverai or 
its salts o Regeneration of the free car bonyl gro'up 
as shown by analysis and infra red study of s©¥02'al 
of 'its saltSo Regeneration of the free carbonyl 
group with base -^^as instantaneous o Finally » II %7as 
converted in 8 sjteps to 2^-Jf»^droxy-5'^!aethyl'='2-'ioheia- 
8thyl-6p7"beusoaorphan (Rj^^CHgCHgPhp Hg^GH^p R>OH^. 



Serial lloo KIAt'IDj;^-^.-^ 

Page 3 

PHS-WIH 
Individual Projeet Report 

Calendar Year 1959 

R5 sH^^H) the 9-demethyl homolog of the clinically 
promising phenazocineo This 9=denethyl coffipoimd^ 
although only about half as potent as phenaaoGisi© 
in analgesic activity.) was at least h times as 
active as morphine in the laouseo 

2) Addition of H to II or CHv-=H by means of 
organometallic reagents (GH^LSg CH^Mgl) was found 
to be stereochemically controllablio With II as 
the methiodide (positively charged N) one dia- 
stereoisoiaeric 9=carMnol C"fe?ith apparently the 
l^droxyl cis to the laiinoethano systemi, equatorial 
for the hydro aromatic ring as indieated by spectral 
data and degradative experiments) is formed to the 
easiclusion of the other o This addition can be almost 
completely reversed to give the opposite configura- 
tion at Cg if one starts -with the free base 
(negatively charged M) of 11= The resulting carbi- 
nols (III) CI» R^sCH^, ^'2''^^3* ^^"^^ °^ '^^^^y ^i^^'^<^ 
Bi-sN or CK^) may be looked upon as analC'gs of oay- 
ccjdone and'^oxymorphone (the diastereoisomeric foriag 
of which are unknown) clinically useful drugs of the 
morphine series ^ and are feeing evalxiated pharmaco- 
logically o Some show interesting properties o 

3) The synthesiSj, optical resolutiong and evalua- 
tion of bsnsoiiiorph&ns more closely related to 
phenazocine CSIH 7519) have continuedo While 
analgesic activity is practically nil in the N-etl'isrlj 
propyl and butyl derivatives corresponding to phena- 
zocinep the H-asayl homolog. is equivalcjnt to moA-pklns 
and shows low physical dependence capacity in th® 
monkey o Purthermorsp it has been .possible by optical 
resolution to effect a separation of neuroptorHaeologi\5 
action and ad'^rerse side actions o For exaaplej, levo" 
ratatory 2 " -hydroxy- 2 » 6 » 9-= triiaethyl-6 ^ '/--benzoiaor pli®n 
(Is Rs sR2"R5^CH3i, R3sOHa Rkj."!!) coaiparatols to morpMne 
is ti^ice as pofe'snt as the racematej, has a much loi^er 
acute toxicity and is rated vej"g Mm. i^a physical 
dependence capacity in the monkey as compared' with 

lo¥ for theracemateo Finally j, l§vo-=2''-'iQethax:f-59 9~ 
dimet.hyl- 2=phenethyl-6 » 7-benaojaorphan ( I » R s.-GHpCH^G^H,- 



Serial NOo KIAMD- 54 

Page h 

PHS=NIH 

Individual Project Report 
Calendar Year 1959 

R2sR^CH^)sCR3sOCH»a ^\-il) is as potent in miee as 
morphine \^ith no pnysieal dspendenee capacity in 
the monkey o 

Significance of the program to the Institutes 

Research in the field of neurophanaaeologic agents 
has pertinence in the area of pain and anxiety 
states associated with Arthritis & Metabolic diseases < 

Proposed course of projects 

Present plans are to continns along lines suggested 
by the major findings above o 



Part B included s Yeso 



Serial NOo NIAMD- 54 
Page 5 



PHS=KIH 
Individual Project Report 
Calendar Year 1959 



Part Bs 

Publications % 



Eo Lo May and No Bo ikidjc A new potent synthetic 
analgesic o Jo Orgo CheiBoa ^g 29^ (1959) o 

Eo Mo Fry and Eo Lo Mays Mannich derivatives of 
analgesic agents o Jo Orgo Cheuios, ^» li^ (1959)° 

Bo Lo May and Jo Ho Agers Structures related to 
morphineo XI „ Some analogs and a diastereolsomer 
of 2''=hydroxy=2j, 5i3 9"triiaethyl=6g7'=bensomorphano 
Jo Orgo Chemos 2^ (October 1959)° 

Eo Lo May and Ho Bo EddyJ Structures related to 
morphineo XII o Synthesis of 2'''=hydroxy=5»9=' 
dimethyl- 2= phene thy 1=6 g7-benzoinorphan (MIH 7519) ^ 
Jo Orgo Chemoj ^ (October 1959) = 

Eo Lo Mays Chapter, on Analgesics in Burger "s 
Medicinal Chemistry » 2nd Edo (in press) CInt@r= 
science) o 



Honors and awards? 

Received "Alumnus of lear" award from Bridgewater 
Colleges Virginias in May 1959° 

Presented lecture "Synthetic analgesics" to 
Hoffmann=I^ Roche Laboratories in Kutleyj, New 
Jersey in February 1959° 

Presented lecture on "Analgesics" to members of 
the Philadelphia Section of the AoCoSo^ as part of 
a series of 10 lectures in Medicinal Chemistry in 
April 1959o 



Serial Hoo 11^11)^^15^ 
lo Chemistry 
2o Analgesics 
3 o Bethesda 



PHS=NIH 

Indii'idual Projeet Report 

Calendar Year 1959 

Part As 

Project titles Synthetic Analgesics 
Principal Investigators Jaiaes Go Murphy o 
Other investigators? Noneo 
Cooperating Units? Hone 
Man Years s 



Total s 
Professionals 
Other I 



IcO 
loO 

OoO 



Project descriptions 



Analogs of Acetylcholine o = Because of the possible 
role of acetylcholine in sensory ner^e transmission^ a 
gromp ©f analogs. (I) has been prepared in which by 




progressi'^e substitution of methyl for hydrogen in 
groups Rjp Rp and R^ a graded steric hindrance at the 
ester linkagi is produced with the view of attaining a 
competitive inhibitor for acetylcholinesteraseo Also 
prepared p ha^e been analogs in i^hieh one or both 
H-=fiiethyl CR^^^ R^) has hB&a. replaced by phenethyl^ a 
substitiaent which has been shown to prodnse aiagaenta^ 
tion of pharmacological response not only in analgesics 
but also in other classes of medicinalso 



Serial Noo HIA14 D_^_g5__ 
Page 2 

PHS^IIH 

l2idi¥idual Project Report 

Calendar Year 1959 

Thus far no response has been sho%m for the 
meiabers which are disubstituted T^fith methyl groups 
on nitrogen CI^sHwsCH^=)j, nor- for membei's bearing 

a single phenethyl group CR|^°CH^=| R^^ // v^'CE^CE^'') ^ 

but definite neurotropic activity (analgesic action) 
has appeared ^hen the nitrogen is doubly substituted 
by the phenethyl group o 

As a byproduct of this ^ork seven substances have 
been released for cancer screening o 



Part B included; Yes 



Serial Noo NIAMD.^^iS_ 
Page 3 



PHS=NIH 

Individual Project Report 

Calendar Year 1959 



Part Bs 



Publications s Synthesis of aainoi^drophenanthrene 

analogs of mor-phineo James Go Murphy g 
Jo Orgo Cheso In press o 



Honors & A^iardss 

PliDo = Gsorgetowi University » June 1959' 



Serial No„ HIAM)^-^^ 

2o Analgesies 
3o Bethesda 



PHS'-MIH 
lBdificiiia.1 Project Reps 
C«,leM&r Year 1959 



Project titles Mro Perrine has been on loan to ' 
Rocky MoBntain liaboratory^ Hamilton ^ Montana foj? , 
two years o A brief sumciary of the ifork done there 
follows s 

Principle Investigators Tiieodore 'Dc Perrine 

Other investigators s lone 

fcit! MAID 



Years s Total ; loO 

Professional; ,loO 
Other J OoO 



Projeet description! 



Siteri tides .andotoxin studies a .Endotoxin prepara= 
tions were treated with some 90 organic liqiiidSg, aiisj th-j 
aiixtares then examined for gross solvent astion? and 
-subsequent to evaporation of the liquid j, for effect on 

the; endC'tsxin and (in some cases) antlgenie po'tencjo 
ASMJUt 10 goC'd solvents were foimdj, aiid the rvile fo^r-sau- 
lated that strcagly foasie asiineSij and reagents which 
liberate strong ssirieral acids oie formic aeid on laild 
hydrolysis J, Will ha^e a delete^eous effect on the endo- 
toxino Report© on the effect on antigenicity are not 
yet availafeleo 

TB Cell Vlallso A production jsodel .px'ess was design? 
and constructed 8 which appears to be applicable to the 
preparation of all types of fc-acterial ceil walls o This 
has be^in OTitten rip sj.id should be published shortljo 

Vi AsitigQiio Cooperation on this project Mth DPo 
Jarvis iijas more or less terminated hj Sssecutive Ordero 
However J, t'&o ptj-blieations '*fd.ll probably r©sv.Ito 



Serial Noo MIAMD - 56 
Page 2 



PHS=HIH 

individual Projeet Report 

Calendar Year 1959 



Synthetic antigeno 'rfork was aainlj concerned •fejith 
the preparation of vinjl gliicosideo This substance 
liasg so farj) eluded us but ife think lie are ^ell on the 
%jay to a suecessfui synthesis <> 

Considerable .experience was gainsd in the techniques 
of preparing synthetic high-poljmarso 



Ciirrentlj returned to the Seetion on Analgesics and 
resusaiiig u'ox'k on the syntheses in the pethidine series,, 
more parttsuiarl^T in the preparation of quinuslidine 



t B inclMedo yeso 



Serial lOo 1IIAMD-. ^&_, 



PHS-MIH 
liidividrial Project Beport 
Calendar Yesj* 1959 



Part Bi 



Publications s The woi'k at Rockj Mountain LaboratD,?j 
has resulted in fche following publications and is 
exp©eted to yield .fi¥e additional publications?---- 

Magnetically stirred separatory flannel 9 hj Eo Oertii 
aM To Bo Ferr5.neo Sent to Aiage^^o GhsiSo 7/29/59 



5zie and antigenic fractions from the cell wall 
of So aiteritidis methods for seperation and soiae 
biologic activities by Eo Bibi^ Ko Co Milnei% and 
To Do Perrineo Jo IfflMiJiiolop Sg^ 75 (1959) o 

Use of a pressTsire cell for the preparation of cell walls 
of layeobacteriaj, by Ec Ribig To Do Perrineg, Ro Listj, 
Bo Brown and Go Goodeo FroGo Soco Esptio Biolo Medoj, 



Physical and c hemlcal analysis of endotoxin from So 
enteritidiSp by Eo Ribig Bo Eojqt^ lie C. Milnerj) To Do 
Perrine and Co Larson c. Jo laimimoio In press o 

Method for attaching glass. %iater aspirators- to water 
lines o Uiipi3Misliedo by To Do Perrineo 
Jo CheiSo Sdoj, Oetol'959o. 



Honors and aiiardss Mone< 



Serial NOo NIAMD_^S7_ 
lo Chemistry 



2 c. Analgesics 
3o Bethssda- 



PHS^NIH 

Individual Project Report 

Calendar Year 1959 



Part A. 



ect titles (a) The chemical transformation of 

certain opiusa alkaloids (or their 
derivatives) into novels medicinal2=y 
useful drugs o 

Cb) The structure of the S0f=called 
"Hydroxy cod sine" o 

Principal Investigators Levfis Jo Sargent 

Other Investigators; None 

Cooperating Units Vo WeisSp LFBj, HIAMD^ Serial Moc.,U9___., 

Man years s Total s 1 
Professionals 1 
Otiiei' s 

Prdleot dgseriptjon s 

Ca=-1) To determine the effect on analgesic activity 
of altering the point of attachment of the nitrogen ring 
from carb0n=>9 to carbon-7 in the morphine system (using 
dihydrocodeinone) « 

(a-2) In view of the highly encouraging pharmacolo- 
gical results obtained with the recently synthesized 
KIH 7519 p an attempt was made to convert ^'ysS-desoi^- 
codeine into an analog of the above in which the h-^^^ 
oxygen bridge remained intact o This should afford 
pertinent pharmacological data relevant to the importance 
of the dihydrofuran cycle in such systems c 

(b) Clarification of the presumed bimoleeular 
structure of "hydroxycodeine"o 



Serial Noo NIAMD^ 
Page 2 



Methods employed; 



Ca=l) The initial sequence of reastions leading to 
this new class of morphine derivatives o in yhich the 
nitrogen terminus ®f the ethanamine ring was shifted 
from earbon='9 to carbon=7£) was described in the praced= 
ing reporto Infrared analyses havej, in the interimn 
necessitated altering oiar conception of the stmetiirs 
of the new isomer o The absence of hydroxyl absorption 
led to the conclusion that no ^s5=oxyg^ fission occiirfed 
during the decomposition of the quaternary methobromide. 
and that the product must in fact be a new isomer of 
dihydrocodeinoneo This is being investigatedo 

Ca^2) In pursuing the ides of transforming a 
derivative of a naturally occurring opium alkaloid to an 
analog of the potent synthetic analgesic KIH 75l9p an 
unfortunate impasse ttarned up in the attempted lead tetra= 
acetate cleavage of 7=hydroxycodeine C6a7=glycol)o The 
intermediate aminodialdehyde proved to be exceedingly 
sensitive and polymerized to intractable products bef j'^j t^ 
it could be reduced with lithium aluminum hydrideo This 
difficiilty was eventually circumvented by operating on 
the neutral N-phenaeyl derivative of the glycol o How- 
evers because of the greater accessibility of the 
corresponding 7»8=glycol this new approach was pursued 
as follows? ^ =7»8-desoxyeodelne was converted to the 
nor-cyano derivative and hydroxylated with osmium 
tetroxideo Acid hydrolysis afforded the nor- glycol 
which was selectively N-phenacylated and then cleared 
with lead tetraaeetateo The intermediate H-asyl dialdehyde 
(which appeared now to be stable) was reduced with lithium 
aluminum' hydride whereupon the following) 
N=phenethyl dicarbinol was presumably formed; 



^-C'd^Cli^ 





HgOK 
pH^OH 



Serial No, MIAMP. 5? 
Page 3 

Cb) Fiirtiier evidence in support of the Mmoleculsr 
natiare of "hydroxycodeine'" was obtained throiagh prepapa= 
tian of the hitherto unknown monoxime and intermediate 
dihydro derivative j, thus completing this projecto 

or findings? 

The successful transformation of two opiuai alkaloid 
derivatives into the novel ring systems described under 
Ca=l} and Ca-2) should lead to clinically promising 

analgesics o 

Significance to the program of the Institutes 

The search for new drugs (whether synthetic or 
chemically-Biodified naturally occurring ones) capable 
of controlling severe clinical pain^ with a minimum of 
undesirable sid@=effectSj, properly falls within the 
purview of the Institute programo 

Proposed cotirse of project? 

Ca=l) Attempts will be madep in this area^, to 
hydrolyze the S-aethosyl function as well as to replace 
K-methyl by the M^phenethyl group o 

Ca=2) 'Abolition of the two alcoholic groups (via 
lithi.um aluminum hydride reduction of the di-^tosyl 
derivatives) followed by hydrolysis of the S^-siethoasyl 
function should s it is hoped » lead to the desired 



Part Bz les 



Serial Noo 
P&ge h 



PHS=MIH 
Individual i^rojsct Report 
Caieiidar Year 1959 



Part Bi 



Nog- symmetrical Biffloleeialar Reductions Structure of 
the so=called "Hvdrosycodeine"^ hy Lewis Jo Sargent 

and. Ulricfe ialeissc Jo Orgc Chemoj, In Press o 



MIAMD Mo^_M^ 



lo AsialgesiQs 
2o Chemistry 
PHS='HIH 3o Bethesd® 



PHS=JIIH 
Indlvidiial Project Report 
Calendar Year 1959 



Part Ao 



Project titles Synthesis and use of dihydropjridine 

derivatives » 

Principal Investigators Edward Mo Fry 

Other investigators s Hone 

Man years s Total s loG 
Professionals loO 
Total OoO 

Project descriptions 

Objectives? To develop a method of alkylating the 
2 and 3 positions of N=alkyl dihydropyridines o The 
extreme instability of the lansubstituted dihydro- 
pyridines has thus far thvarted the attainment of 
this endo However „ results are promising enough to 
continue the investigationo A favorable end result 
would provide a new and versatile synthesis of the 
morphihan-type analgesics o 

Methods s - The routine chemical agents and physical 

instrument So 



Part Bo Yes 



Serial Noo KIAMD_^_jsa- 
Page 2 



PHS=NIH 

Individual Project Report 

Calendar Year 1959 



Part Bs 

Publications s 



Hannieh Derivatives of Analgesic Agents by Eo Mc 
Fry and Everette Lo Mayo J., Orgo Ghemo 2^? 
116 (1959)0 



Ssrial Koo KIAIdE L^ga, 



1.. CheiBistry 
Analgesics 
Bethesda 



PHS=MIH 

Individual Project Report 

Calendar Year 1959 



Project titles Comparison of the de'/elopment and loss 
of toleranee to the effect of morphine on an analgesic 
Chot=plate} response and a general behavorial response 
{swimming) in the rato 

Principal Investigators Joseph Coehino 

Other Investigators Conan Kopnetskjo 

Cooperating Units s IIMHg Depto of Pharmacology j, Boston 
Uni^yersity School of Medieinej, Boston^, Masso 

Man years s Total s 
Professionals 



iectlvess 



1/: 



To determine whether the rate of tolerance develop- 
ment on the one hand,, and loss, on the other hands is 
different for two different effeets of naipcotic ds'WgSo 

Methods and Results s 

The response of the rat after a test dose of 
morphixie to the analgesie effect is measured by ^ising 
the hot plates and at the same time the effect on spead 
of swimming a circBlar alley is also measiired„ The 
observations were aade before p during and after a 
seventy day period of ciironi© mox'phine admini strati on « 
The effQOt of thi.s same test -dose on the speed of swimm- 
ing a circular alley were measured before^ during and 
after a seventy day period of chronic morphine adminis- 
ti-ationo It was found last year that tolerance to the 
analgesic effect de'^slops more rapidly than loss of 
sensiti'S'ity to the sffeet of the test-dose on speed of 
swimming o " Continuing this study through 19599 '^^ noted 
that 262 days after abrupt %?ithdra\*>al of narcotics j, 
sensiti%"ity to the effect of a 20 mg/kg dose of morphine 
on shimming speed had retui'ned to initial control values, 



Serial Mo. MIAMr )._^J9, 
Page 2 



PHS-^NIH 

Individaal Project Report 

Calendar Year 1959 



font the analgesic; response to this test dose is but 
^5^ of the initial control values one year after 
abrupt *^ithdrawalo 

B@oai2.se of the possibility that the repeated 
admlni strati on of the test dose might be "priming" 
tolerance and the non-refc-arn to initial control drug 
seiisiti%"itj m&,f tee due to thlsj, the experiment was 
redesigned so that this "pfiming effect" can toe studied 
and its importaiiee ®¥alTisat©do This part of the study 
has Jiast gotten vmd&r ¥sy here and will be mider way 
shortly at Boston Uoi'b'ersity t&'here Dro Kornetsky is' 
pi-esently located o 

Significance to the prograja of the Institutes s 

V/e believe this to be a ccotribution to the under- 
standing of the mechanism of the loss of tolerance to 
narcotic driagSo 

Proposed course of the projects 

We hope that this newly designed experiment v^hieh 
will isolate and study the "priiaiing effect" of repeated 
test doses as well as the effect of age and weight on 
driig response and will answer the question as to whether 
OT not narcotie d.rag sesnsitivity is really diminished 
for such long periods of time after withdrawalo QTo 
Kornetsky also plans to do some psychological testing of 
rats dUji^ing the periods of addiction and withdrawal 
which may also throisj some light on the probleni of 
tolerancso 



Part B included s Ho< 



Serial Hoc 

Ic Gbendstry 
2o Steroids 



FHS-MIH 

Individual Ps^Jeet Ssport 

Csleaday fear 1959 



Part A. 

Proj|®ct Title? Study of tbe Steroidal Alkaloids aed Sapogsains 

Principal Investigators loshio Sato 

Other lavestigatorss Kobuo Ikefcawa aad Erich Mosettig 

Cooperating Ojaits: None 

Han leaxB 

Totals 2 2/3 
Pawfessionalt 2 

their I 2/3 

Frojeet Description s 

0]JigSl^Q|:— In order to find nsws rare and friaitfTjl sources for 
the production of biologically aetiv® stsroids, th® ottidy of th« 
degrsdativs possibilities of various steroidal alksloids end 
eapogenins (particularly solasodine) bsve continued o 

Msthods E^?^loY<^(^o°— ths OjK-diae@tyl derivativss of solasodine 
and tox^tidin® are Isosierised by trestsu^mt with acids (acetic, 
I^Tidia© iigrdro<3hlorid®s etoo) to A^^^^aj unsaturated pseudo 
derivatives s osidiffied with chromic acid and hydrolyaed with 
acetic acid to ^-ae«tosy-5sl6-pregnadi8n-20~oa© and ^-acetosgr- 
5a9l6-pragnea-2C>-one respectivelyo A nsw tetrahydrosolasodine 
has been obtainsd froja the aluffiiaum trichloride catalysed litMxjsa 
altunlnuffi i^dride reduction of solasodine o This has been con™ 
vorted into the hitherto unknoan G".22 isoiaeric solsaidan-3-on® bgr 
oxid&tion and subs@quent reduction. This latest addition cosipl®t®e 
the set of the foijr poseibl® C22» C25 isoiseric solanidaaoneso 

Ma.'f ff>y,.|',JLp,^jt?'?P °~~ Cl) Convsrsion of solasodin® and toiaatidine to 
ths acetates of pregnadienolone and pregnenoloao in excalleat 
yields » 



Serial No. .Ji^„.s»|0. 
Paga 2 



PHS-HIH 
iBdividml PTOj©Qt Rsport 

Calendar lesr 1959 



(2) Hev@latioa of feha iatei-^sting ciaemisti^ of th& 
spiroafflinokstal system present in tb® steroidal alkaloids. 

§i^SX^M^''-^ "^^ d©fradatioB of eolasodia® to prsgriadlenoloa® 
in good yields (65-70^1 makes pessibl® th@ atiliaatioa of solgm^jis 
plaats as a costaisrclal ist@roid.al sob^c©^ Ib fact Eussia has 
laimsbed on a large scale cisltivatioB of solaiiiiim sp@ci@3 £md 
industrial eoav©s?sion of solasodin® to biologically activ® steroids = 
ffeo kHOKledg© of the manifold ch®iaical intsrr^lationsMp of tbe 
gpirosffiiaokatal aystem is a definite coatribiition in the steroidal 
alteloid field o 



Pas-t B incl-udad I®s 



Serial Koo .JJ^L^:l_§IL„, 
Pag® 3 



lEifiividual Pj'oject Raport. 
Calendar I©ay 1959 



^jCL^o Soaorsg Awas-daj asad Publications 

this projeets 



Sato 3 loj Ik@ka«as Mo mid Mossttigj 1;>3 ^''Impfovsmsi&t iu th^ 
Preparstioa of ^~AQeto3ty-5asi6-pr©ga©ii~20fon© and ^-Acetoxj- 
59l6-"p2'©gnadieB-2Q"Oa@ fsrom th® Steroiclsl AlakaloidSs Toiaa-fcidiae 
and Sol^eoain@o" J. Oyg. Chesioj 24s ^93 (1959) = 

Sffitoj lo amd Ikskawa, M,^ "Prsparatiori of Chossodeosychoiic Acidc 
Jo Org, Chsnias ^ 



SatOj loa IkekEwaj H. and Mossttig^ So, "Tfee Cssejaistsy of tiie 
Spiroamiaoketal Sid© Chain of Solasodia® aad Toiaatidia®. lo Ibi» 
pycved Prspaifation of ^~Ac«to:^"'5jl6-pr©gna<3iea~2O-oa0 asid ^" 

Ac®to2y-5asl6-pr©gnen""S0-»on© from Solasod3.n© and Tofflatidine<,** 
Jo Orgo Gfeeaioa aceeptsd for publieatioBo 

SatOg lo and Iteka%s®s M»j "Th® Cheiaistry of th® Spiroajsiaoketel 
Side Chain of SolasodiB® md Toffietidia®» II <. Ghemistiy of 
^ s X^-dise@to3qr-2C5-C 2 " -^'^•'•~H"ac©tyl-5 "-jasthyl-tetx'sfeydi'op^X'idyl) « 
5^p3?9^©K®o" Je Or-go Ghsfflos accepted for publicatioao 

Sato J lo asad Ikeksswaj Scj "Th© Cteaistay of the Spiroassisoke'tal 
Sid® Cbain of Solasodins and Toatatidiaeo III:- The Esaation oS 
OjS-diaeetylsolasodiia© ia Aeidie I'fediao*' Jo Orgo Cfeamoj accepted 
for jniblieatioao 



Serial IS&c MIAM)_~ 61 
Xt Claeaiatry 
2. St©3?oids 
3o 



iBdi-vidTisl Project Keport 
Calendar Year 1959 



Part ^» 

Project Title: Stvily of Fscal Steroids 

Principal InveBtigator: Eridi Heftjaatm 

Other Investigators: Slskebard Weiss, Harold K. Miller and 
Erich Mosettig 

Cooperating Uaite: lone 



ToUlt 1 l/3 
Profession®! : 1 
Other; l/3 

Project i>@scri|>tioa: 

Otoj^scti^o -=- So identify stsroids in fsces.. 

Msthods ^?loy9d.-~ AiSsorptioii and partition chrojsatography^ 
prepar&tioa of d©rivetiv»e and infrared spectroscopy o 

Major FiMiog8o"«- In addition to tbe steroids referred to in tb© 
^jS repbrtj^e hsvs isolated ss&ll aeoxsnts of cryctallina siib- 
ffitsnces, tiio of ^^ch Kay b© hydrosylated fatty acids and osi® 
T^ich is probably a nsw sterols 

Slgpifieapce; 'Bie nature of the fscei steroid fraction may d®p©nd 
not only on tJh« di®t and iatesstiiml flora. Changffs in various 
disease states and la agiaing are quite likely and dejsarve further 
investigation. 



Part B includ<5d Y®a 



Serial No. JJULs^^ji, 
Pag© 2 



Calendar Year 1959 



gasl^. Hoaorss Awards ^ and 

PwblicaMoBs othar t-laaa abstracts froHi this projects 

HsftinaaHs ^^j Wsiss, E.. j Millers Eo So a aad Mosssttdgj E-jj 
"Isolatixjii of Soms Bil® Acids and Sterols from tfe© Fsees of 
Healthy Man, " Arch, Biocls^mo & Biopbyso ^<, 324-^1 (1959). 



1. ChQiaistxy 

a» Steroifisi 
3« 



ms-wm 

Individual Project Report 
Calendar Year 1959 



Part Ao 

Project Title: Idsntificatica of Acrasin 

Principsd Investigator: Srich Heftaana, 

Other Investigators; None 

Cooperating Units: Laboratory of Cellular Hiysiolo©-' and 
Itetabolism^ National Heart Institute 

Man Years 
Total: 2/3 

Professiosml: l/3 
Oth^r: 1/3 

Project Description: 

Ob jsctive^ -»••»■ To identify the aggregation honsons in th© slims 



l.^thQd3 ^ EBgloyed. -^ The ?ESthodology developed in ths study of 
fecal steroids was applied to the isolation of the hor^sne froa 
Dict^osteiiiJS digicoide\3^. 

IJis-ioT Findinga^-^ The identity of ecrssin -sath 22-3tigaasten"33" 
ol was est^fclisbsd hy cheisical isesiis. Othar sterols,, includizig 
ergosterol wers foimd to have eci'ssin activity- 

Sigaif icBace^ '°- This is the first desoastration that sterols aay 
ha^'e hortiozssl effects o It is also the first insteacs in which 
aa organizer has b©sB identified. Stai'ols sjay be of gsasral 
iiaportanc® in cgllular differssatiatioc 



Part E iacludad Ysa 



Serial lio. MIMm - 62 
Pag® 2 



Ta&lvi&ml Project B@port 
Calendar Y®sr 1959 



?art^.> Honors,, Aifards^j SBd Puciicatioas 

Ftiblicatlons otb©r tfeeu abstracts fvom this projsct: 

Heftisaaa^ E., Weight,. B^ Ecj. aM. Lidd©!^ G. Ue^ "IdeatifieatiOR 
of a Sterol v5-tii Acrasia Activity in t!ss Slise Ifeld," Jo ^c 
Che®. Soc, (seceptM for publieatios). 



Serial HOo MMm - 63 



1» Cbesiistry 
2» Steroids 



Individital Project S©»ort 
Ca"..asdar Year 1959 



Part Ao 

Project fitie: Biogenesis of Sapogeaias 

Principal Investigator: Erich HaftBana 

Other Investigators J Hob® 

Coo^arating Units: Division of Biology, California lastitut® of 
Sechaology, Passdes®, California 



Sotal: 1/3 
Professional: l/3 
Other; - 

Project Description: 

Qb^eetivQo "" Tt> dstcrsjin® the ^chaaiss vhei-ebj stsrslas ©3?«5 



^thoda Eaplogf ed. "^ Dioscox^a tiabex-s are sitter sliced or 
hoiiogeriized and iacubated 'ijith radioactlv® prscrorisarso Ife® 
labalefi prod-aets ar<s isolated aad idsatifiado 

Ma,jor Findixjt^s^"-^ A sjethod for the isolation of sapogsjaiaa has 
baea adapted and agpli^ to two giogco^a specisso D. floribgada 
has been selected on t'ais basis for the biosyatheais^Wperijssnts 
initiated at California Institute of Tschaolos/. Sioscorea slices 
iacubatod with !a®%>'alo!Jsic ©cid convert th© latt©r into 4 "radioactive 
pro3.ucts^ mem.® of 'i^Msh is identical with diosgenin.. 2ti®ir identifi' 
cation is in progress.. 



Part 3 iaclufied Y<sn 



serial ^o-_P^M.:L^ 

Pag© 2 



FHS-SffH 

Individual P2fo4@ct H®port 

Caleadar Year 1959 



P art__B o tenors, Avas>dSy and ftablicatlosia 
F&blic&tloxm otSvsr timn abstescts fro^ this pr©J®ct: 



Reinhold PublisMxig Ccspasgfj, Ifew Yorko (accepted for piifelicatioa) 



Serial BOo HIAMD «» &4 
lo Cli@Hi3taty 
2= Steroida 
3» B«tliesds 



Individual Project Report 
Calandsr Year 1959 



Part Ao 



Projsct Title: lo D^tersainatioo of IMivldtial IT-ifetosteroids 
by Gradi^it Slution Chrcsatograpby 

2o AdrsK^cortical HorEones in Rat Adrenal 'Itoor 
Tissue 

Principal Investigator: Devid F<. Johnson 

Other IiiTeatigatore : Daniel Francois and Erich B^ftsiaaa 

Cooperating Units: Hatlonal Cancer Inistitut® ♦» fi»or tissue 

Man Yeara 
atotal: 2 2/3 
Professional; 2 
Others 2/3 

Project Description: 

Objective t-"" lo Developsssat of & qTsantitativ® asBthod tor tte 
d«teralnation of iadivldtisl 17«»kato»st@roids in Kistursso 2= leo- 
iatioa of adrenocortical horEJoaes fro® sssplss of tr®jaspiant®d rat 
adrenal t;mor tissusc 

Methods Baplt^gdo '«' In tiKs first project a method is being dsveiopsd 
for the (jpaantitstive deteraination of iadividxtal l?*ketosteroids by 
a KOdification of the gradient ©Itttion t@cha5.qu® with silicic acid 
colusasy devolopsd in tlais laboratory for adrenocortical horsoneso 
The individual fsractions are saaJ^zed by ^eaas of the Ziaaaexisaa 



Tte second project is being investigated by th« saethod for 
odresocortical horsscsie detensiaationji ice« gradient elution irlth 

petrolecm ethsr containing increasing suacante of dichiorcasethene on 
vator impregnated silicic acid coli^asi- 2fee fractions obtsiasjd aire 
analysed by ultrs.Yiol®t absorptitm and reduction of blu® tstrazoliijjji.. 
Furbher identification ia achieved by pap<er chroisatography . 



Serial Ho. ,.MAM) _» 64 
Page 2 



PHS-NXH 

ladividBal Project Report 

Calendar Yesr 1959 



Major Fiadlngs ^-^ ExperiEienta tht^s far isdicat© that alteration 
of tl^ amount of wat®r# as the stetioaary ph&B&p on silicic acid 
Goluims will persait the separation of tb® laa^or IT-ketostspoids 
encoimtarsd in biological fluids o Coaplete seperatioa end 
quantitative estissstion of dehydroepiandrosterone^ endrosteronea 
etiocholanalosej ll«-k!etoaE!droBtsron8j, and ll«iiyfiroxyetiocl\©l®a&lon© 
has been schisvsdo Difficulty ha© be®n eacount®red in th® c(K3- 
pl«t« separatitai of ll«k©to®tiocholeQalone and ll-^'hydrasyandroster" 
on® Ijut ©xperiffisnts iadicat© that tMs caa be achieved with the 
propsr coaditioaoc 

Th« s«coad project ia belcg carried out in coo|J©rati©a ^jlth 
Dxc etherise SneXl of HClo Shs tissor tissue being investigated 
is a transplant frosg an original s^Msnteneous adpsnal t&soT in 
ratoo Biological observations indicate that this tat^r say be 
producing adrsnocortieal hozs«i»s« ft?eliiainary fractionations 
are being carried owt to attempt to identify thss® compounds o 
Farther iavestigatioa of the ®aay^^ syst®®s of this tissue with 
radioactive tracers is plam^do 



Part B includ«d Yes 



Serial EOo M^^^r 64 
Pa^ 3" 



PHS-SIH 

ladividual Project Report- 

Calenday Year 1959 



Parfc B» Boaors, Asjsrda, and ?ubliestions 

Pablicatioa coacemiag raport- for caXenfiar year 195^.; ^^t ^^'^ ^'®-- 
porfced there: 

Jotmaouy D» Fo^ Frascois^ D<. aad Heitaaim^ Eo, "DeterisaiaatioB of 
iMivld-ual Mreaocortical St®5.-oids in Urine of Pregjaant Woissn?" 
Acts Saaocrinolo, J2j, 8-l6, 1959u 



Serial EOo 
2p Steroids 

3e 



PBS-HIE 

Individual Pnjject Report 
Caleadar Year 1959 



Paart Ac 



Project Title t The Structure of tbo PsKsduet Obtained tycm tb® 
Lithi«a M,«siBS3 ^drifi© Reduetiea ©f 22^,26^ 
G2ido='A3-?C20j„2hol©sti^3®-'3&s22»dioX-l6-*oaeo 

Principal Ia-r@8tigator: l&leols J. Bio^paon 

Other Investigator: Erich Mosettig 

Cooperating Ifcita: Hone 

Man Years 
Total: 1 2/3 
Professional t 1 
Others 2/3 

Project Desci-iptions 

OWsctiveo "=> Litiiitist sJlusainua hydride risductioa of 22;26«oxido» 
^^it^)«'CiioleEtena"3&? 22=diol-i6-one had been reported to yi©M 
Al7C20)»22~isoallospiro3t@ia«'33'°ol. Shis spirosten© wo\ild have 
■becjn an impoirfcasat link ia tlie elucidation of the structure of 
peanogsrdrio 

^ttods_^^i^j[^^*« Ultraviolet asd infrared ©jaaiysis of th® 
OKidative product of the supposedly spirost^ne aloag -id-th 
analytical data of th© origiaal product siad derivatives l®d to 
the elucidetioa of 'the stractar® of^ the .lithium aliiisiinuffi hydrid© 
reduction product of 22^26-a3d.dO'-A2-'?^^'^^»ehole3tea®-'3^*22-.dio:i-l6-'02i.: 

tfei^or F i^Uaga c. -^ It 'S'qs shown that tSie lithiGie altisairi^JEt hydride 
rediciiofflT'of &, 26-oxidO''^iTC20 .Ucholesteas'-SPf SS-diol-lS-oja® ;v ields 
a 22y26-oxido^TC20)-cbol©stene"3^?l^ f --diol arsd not the A^TCSO)- 
22«ieoiallo®pirostsa-»3^-ol ®s for?s®rly b®liev©d« i^aslogou® r©sults 
were obtained with the A^-ssrieso 



Serial 






Individiaal Project E«|>ort 
Caleadar Ysar 1955 



Signlficancgc -"^ Catalytic reduction of ths 22^2&"imi&Q-^'^^^^* • 
c]f^iesf«sae-53» 3.6 f-diol has yieid©d the hitherto ^jsilosowi 17c- 
cholestane sids -chaiao RessoTml of f^aaetionsl groiapss givsss fhB 
hydrocarboaj!, 17«ia©cfeoleeta»So This cosspciand would be o;? great 
val^«! as a r®fer®i7.e@ coaapoaad where eesafiguratioiial ®r3raag®issat 
at C«»17 is guestiOEsbleo 



Part B Included Yes 



Serial ®0» _IXAMj_ 
Page 3 



Inaivid-otal Projact Report 
Calendar Ysar 1959 



Part B « Bojaor8/> AvarfiSp and Publications 

pablicatioas coneeraiag report fox- calendar year 195S, "but aot re* 
ported there: 

Thtmpaotis hU Jog Sch©®rp 1= sad Mogsttigj, Eo, "Th® ChoX©g®aias<. I^. 
X6,22-Ep02ycoprostai®«3«j26,S7-triol and its ^ms-idsatity Td.t]i Di* 
hydrochoXeg^jia, " Jo ite« Chaaj^ Soc.>, ^^ 5225-5230 (1959 1 » 

'E&tmgsoat H<> Jo^ Scb©®r, !<, asd Mosettigj Ec..i, "Sb© Chol®g«ninso XIo 
Stractjsr© of Cholsg'Saia Isocboleg@ais and Dihy<23fcciiolegeai% " Jo 
Cheso Socop 8l,> 5222-522^ 



Publieatioa coaceraijag raport for calsadar y®ar 1957 ^«it not r©|»r&ed 



von Bread, fo« McMabOBg Po» Joimsoa, Jo, 5SiOiapsonj> Mo Jo and Mossttig,, Ec 
"Cheesj-cal Ccmpoeltica of th® Culture Fovm of SrypssoEOsa Cr^isiy" 
Escp. Perasitelo 8, 171-lSl (1959)- 



Serial, Mo. JJI^„;l.J£_„ 

1. Chemistry 

2. Steroids 

3. Bethssds 



Individual Project Report 
Calendar Year 1959 



Project Titles Th® Unsaponifiable Lipids of T^galt %msdMstQ,^^^. 
and MggijiJj, §^. 

Principal- 'lBV©"Stigat6Ss Hslcolm. J. Thompson 

Other lavestigators Erich Mosettig 

Goopsrating Units? Ssctios of Physiology (Dr. Th®odor von Brand};, 
Laborstoi^ of Tropical Diseases s HHID ~ 4| 

J'Jan Tears 
Total; 2/3 
Professional s 1/3 
Oth©r: 1/3 

Project Dascriptions 

Objective."- It app«sar«d desirabl© to r©inv©stigate more sxteMsively 
the nonaaponifiable fractions of tapaworms to search carefully for 
products accompanying chol«st®rol and finffilly to characterise all 
tfe® purified compounds that ware isolated. 

Mett^ods Employed.""- The noasaponifiabl® lipids from tapsworffis 
ware purified by chromatography. lafrarsd, ultraviolet and 
specific rotation analysis vrors performed on sll compounds isolated. 
Further identification was based on direct com|5arison» 

IMsaL^iSJdJjsga^"- I^ ^^s sboun that in Tj^^jt ^mmMismkS. «att in 
MSiaiSSM Sa» cholsstsrol is by far the most prevalent uasaponif-iable 
sabistanGQs 98 and 85,^ x-aspactively^ Th© fact that a search for 
frisdelin in Tamla. t j^ni aatoxmJka we® nagative strengthens th© 
asstuaption of Cmelik and Bartl that the friedelin found in J^^ais 
S,lgl^saM <51d originate from cork stoppers* 

S.igal£ift imft SP,. --' Th® finding of o?ily cholasterol in the tsp^sworais 
©stablishess bsyond doubt that th© uasaponifiabl© lipid fraction 



Serial Ho. .^I&M-^, 
Fsge 2 



Individual Project Report 
Calendar Year 1959 



of perasitic worms ar® not sa diversifiad as ia soia© inv®?- 
t©bi'®te pbyla, such as molluscs or spoa^^s. 



Part B iacludsd Yes 



Serial Ho, .J^^^^Ml-. 
Pag® 3 



PKS-NIH 

Individual Project Report 

Calendar Year 1959 



^X^3.-> Honors, Awards 3 and Publicatioas 

Publications other than abstracts from this projects 

Thoinpsonj M, J.j Mosettig, S. and von Brands T.j "Th© Unsapoai-^ 
f iabl® Lipids of T^SSM i^m^sM^ aad l^mSMiM. Ifi" " 
Exptl. Parasitol.j acc®pt®d for publication. 




PH3-EXH 

IadivlduQ.1 P3roj®ct Report 

Calendar I©ar 1959 

Part_A« 

Frojgct Title: Study oa Hydro^l(st®d Antlirssteroids 

Principal Investigator: Os^m fssa^a sad J. A. Steele 

Qfehsr laYSsti^tors: S.ri«^ ^bssttig 

Cooperating Usalts: Sos© 

I^a Ysars (eaieEdar year 1959) 
atotal: 2 a/3 
Professiojial: 2 
Otfear: 3/3 

Project .'Dascripticia: 

Ob^e^^j^o-^ Shis sttidy tsss ;xM®rtak©a to obts^la the h^dre3Q^lal-,ed 

!l5is classical stnictur® .and tl:^ K®el3aBiss of the rearraag®" 
asBt are "being invsstigat(Sd» 

Me-^ods S aploye do"^ B^hj&rosrgosteryl aestate aad 3^•»'®ce&o^f■«• 
i65* 7» 9Cllj»cJiQiestatri®ne were treated with j>'toIi2©nesulfOBie acid 
in CHCl^ at rooss •tesaperatur® for I5 hours o Ha© product •^as jniri^ 
fi@d by chrtaaatograpisy Knd frsctioaal recs^stsillisatioa as ds.?iva- 
tives (2 aad o-clilorobeasoates hessibydrobaasoiat©)* 

In order to elucidate t3i© f ©actios ffiscbanis^, ps-sparatloa of 

aahytoidi© adduet has bean undertafesnc 

Ma^or glndiCigSa'*^ (a) T's?© iso5ssric isydrosysBthras'i^s^ids w©r® iso- 
lated ia 6rgost®roI asd cholsss'tecrol sarieso It ^-as foisM that 
hezahydr^sazoates ef both isosssre forsed a molecular cospoisad in 
th® case of anthracfcolesterol and dibyclroaiithrasrgostSjfGlc. 



Serial l^-^JEBRj:^--' 
Pags 2 



ladiAd-diiai Project Report 
Calendar Year 1959 



(h) A OX'S' stalling fet© essgouad iras obtsissd fs'©^ oa© 
of the iscsa©rs of <3ifeydro©sithra©rgostsrol by cstaljtic ^@«- 
b;^d2?og©jmti(ao IB si^ U? opisctra sho^red this ke'feo gresp ¥-®s n©t 
ia eonjugatioa xd.tb the areaatic ringo 

¥■&© ctetsiasd in a good yisldo 



SigEif iesBceo •=« Ib contrast to tbs ^s-Sfesettlg eatferastsroid 

hydr©:!cyl groups tha ae? rearraiigffi^jat ?d.th ^toiusasmilfoalc acid 
gav® two Iscs^rs o? aatlufastsztJid "vfit^ a Isydrsiyl groisp'o 

S-ies® co®poiJ3ids ©ee^ to te ^sor® iater©stiag ia tha |sIi73ie--= 
logical pr©gerti®s coaipared vl-th tfes eorr®spoBding st«iroids.l 



mis foiHstioa of an «neon^.?.gat©d bato cospouM strcagly 
staggestsd that tfes bj^dro:^! group ©sd.i?ts in rlag A.. 



Parfc B isael^aded Yea 



Serial lo. S3 



PHS-IIE 

ladividisal Project Report 

CalSGdar Year 1959 



Part B« HeaorB, Awards, aM Poblicaticsas 



Riblicatioas concsmijog report for calendar year 1959? ^^t isot r@-» 
ported tter®: 

Burgstahlery Ao Wo and Mosettig^ Soj, "Th^ !Dotal Sgrsthesis of dl-C-I? 
Oxygenated Anthrasteroids, " J, Aa. Cbea. Sos., 8lj 36'97-3Y01 {1959)" 



Serial 'Mo^^^MMSd 

2. Steroids 
3" B®th©sda 



InilvidTasl Project Esposi; 
CffileEsdar Y©sr 1959 



Part Ao 



Project 5?itle; Thm Partial Syatfeesis of Sulfur-saalogs (Poai- 
tic^iS 11 sM/oT 9) of B'y<iro3^1at©d Cosrfcicoids 
aad Othsr Steroidal Soreajsag 

l^'incipai IsiTOstigstorss ToshiQ Kawoeki (laft ME B/lh/$9) 

Other 3iiveatigators: .Irich J&settig ei-d Yo Ueda 

CooperatiBg Units; Csacer Chesotherapy Ifetlonal Service Ceatrr^ 
ECl - 

Man Year C calendar yaer 1959) 
9?otal: 1 2/3 

Professional; 1 X/3 
Othsr: l/3 

Project Descriptions 

Ub^ectivBo ««- !EM8 attidy ha© been ccssaseacod. vith the object! vss of 
finding sntiiaetsRbolitess of corticoids sad steroidal e&s. horsseaes^ 
Shvsii aatitEstabolitss say shed light upon the sechaaies of actios 
of antirheiasetoid and cencffirchtSKOtherepQutic staroiosl ®g©ats> 

Msfflods i^Bplogeda '-"^ ^ghe isBthods «sapl^©d coESist aaialy in -fchig 
opening oc? th© 9^,X1^» or 9q» llOfr^epoxide rizjg with b5'(?jrog®K taic--- 
cyanide, and ia the liydrolysis of the rssultisag thiocyano groups c 

Ito^ipr Fladlia^eo "°" In contiauation of th® search for s©s hoi-feoas 
antiEotaboliteSf i!r''«androatea®-9<&'thi©c7a23,o»3.s 13.^ IT'-trioas has 
been coavarted to the coiTosponding 9(3-thiocsrhsside and 9t>-thio'i. 
(9c&-jn®rca|>to adrar!ost0roae)r> Similarly., 9othiocyeaocortlgoji!S 
has l>3on ojnverfced to th® correis'posiSing 9othlocarba©idSo A new 
route to lip-aercas^to corticoidg has bson opened throu^ ths 
syntheeis of S^ 9Q^'■®poay'-Il^-thioey^aO"5^■•■■prag35SIK8'-3^* l?Oy Sl^tslQl- 
20-oEts 21=ac«!tateo 



Part B included Yes 



Pag© 2 



PHS^-NIH 

Individual Project Reporfc 

Calendar Year 1959 



P a£fe^ .» Honors 9 Awards j and Publicatioas 

Publication conceraiBg report for oaXendar year 1958^ hat not 
reported there; 

Mosettigs Ecj Biochemistry of Steroids c A Report on S^posium r/. 
Vole IV of the Proceedings of the FoxisTth latematioaal Congress 
of Biochsmistxys Vieimas S^posiiMa IVj Pergaiaon Press 1959» pp- 
283«296o 



Sarial Nco .^^M^s^M. 

2c StsTOids 

3o Betfeesda 



PHS-KIB 

iKdi^dual Project H©port 

Csl©adar leas' 1959 



l^LA" 



Project Title; StrucUup® aad S-fc©r®och@Bd.stry of Sts^iol asd 
Isoatsvioi 

Principal Investigator; Peter Qtiitt (loft HIE 9/30/59) 

Other Investigators Erich Hoasttig 

Cooperati»g Units; Hone 

Man I©ars 

Total? 1 2/3 
Profsesion®l5 1 1/3 
Others 1/3 

Project Description; 

0^3act,1.veo"" To elucidate th@ atructur© of th® aglycon® of 

StoVi03iS®o 

^^PX yi^nfjing^o-"" Ste^iol ssath^laster aoi^etol-allogibfe®ric acid 
and iaostaviol-gibberie acid h&ve superisipoaabl© RcDo curves^ 
This ©stablishea tba stes'eochefflistry of tfee six- aad fi^s- 
ffifsaabered (C/D) ring Jimctur® of steviol and isost®vioIo Th© t%so 
©piffi©2lo difeydrosteviols ob'feaiaed 'by catalytic reduction fi-om 
steviol and st^vioaide «nder different and specific coEditions 
Her® cojyrsrtad in an ©igfet-stsp degradation process to (-"ia- 
dih^rokawreae and C-l^-^dihydrokaureneo 

§lgal£ii3fflg$t° °-° Ifflportaat to know t&e chsiaical stanaoturts of 

sts"yioaid3 which is a natural sweetening agent = 



Part B included les 



serial l!o.., .Mm^:^t= 
Pag® 2 



Individiisal Project Report 
Calendas- Tear 1959 



Part Bo HoKorss A^rdSs and Publicatioas 

Doldayj, Fog Liehtig Hog Mosettigg So aad Qnlttg '^^s '"^® Struetnr© 
anS St®?«oefe®iaistiy of St®Tiol asid Isoste^iolj*' Jo Mo CteKo Socog 
aceeptecl for £%iblicatlosto 



Sesial fee Mm^s„^^ 






Project Titl®8 Iafra?®a Spseta^scopie St«^es 
Prineipal Iwestigatoys Ha?old Ko Mill®? 

Coopsi^tlsig Ifaaitsa liJoa® 

Han Jb&vb 

Totals 2 
?Fof0salomls 1 
Otheyg 1 

lalas All iafrax^d faeiliti®s op@s?ati»g P®jfkia=El2ier fe4©l 
21 infrared spactroptoli^MMtei^e ia tfee Laboretosry of Cfeeaiistry 
vo7« eons®li(S«t®d ia r@csi SB^S, Bldgo 4$ Svi^ng Septsinber 
1959o TMs report iacltMes all isafrareci actl'Bl-feies fos? th® 
caieaidap y^ar 1959a ^®a? tte® g®slods befosre aa^ siac® 
consoli.da'&loB o 

Project Sescslistiona 

AjKPS'OJsiffi^tftly 11©0 inf s?a2f©d spswitra wsre mad® t© ma|spos^ 
iBEyestlg^tioES is tfe© Labesmtoiry of Chsadstsgro Sh® po?*®? of 
■fe&a iaf rajped matfeod was illwSmted In tb© M|iia ideatlficatioa 
of the «at©5Elffll "acspaaiss* as iS^^st;i©aagt®si-^<=©la ■yfeieh ws 
GQBfismed th?i9i^ spoicts^ ag»@m®Qt %dth amtltoiatic imtesds^ 

Losramtaias ftoctioas t© ej'sst&esis© tlss iiQffmj?©^ absas^ioa 
©awolop® is beiag lOTrestigated In coop®s?ati©a witt Bro Jo Hsyesy 
LTDo Th© 1JC»=15€0 oEo"^^ s?@gi0a of tins spectj-a of eboleslaESs, 
asadyostsfiSg aud ergostsaes as sf®porfe«d is So Doferia©s:'''e 
®Ijaf3?ai?ed AfefsoTgiieffli Sp®<st?a of Stssoids - &si AtlaSg" hmte 
be®a aaalyaed foy the emalop® asalysi® t®«l2ffiiqu®a aad essrtela 



Pag© 2 



PHS-IIH 
ladividtaal Project Report 
Calendar Year 1959 



Lorentaias coinpoaeEts appear to form eorrsXatable s®ts» 
Th®s® spectra wer© smd® ^th cslciusn fluoride i^ssoiutioxio 
TMs iavQstigatioa will b© continued using the rssoliation 
of s prisja-gratisg spectrophotometer @ad other Isjdro- 
carbons in &n ©ff ort to gaia fuz^ter knowl©dg® coneeraiag 
tfeQ structure of tfe© steroid moleciile and its side chains. 
1b attempt to corr®late sbsorptloas for morpain© typ® 
compouads is contimAiag. 



i*f3rt B incX'iided 



Serial ^ _„„,„^ 
2o lM®pss2i@nt USait 



.■jml Protest B&'psx'iii 
(M^" Y^s" 1958 



Rroject Title t ilsalytical Ssrrlces Islso2@.tosy 
Briseipal Is'/sstigators Willie® S, Alfosa (ts? li/1,59) 



Hisabstfe Ho Ifeta^f i^frca Bssx-j, Ji^-l© Fiper 
1959) 



T&i^li 3 1/8 

% lA? 



of Healthy ss^ t© a liELtM earfcsati, fer ps)T&on& of othsr @Oi^vf®i?ja»" 
E^nt®! @g^jci®3« Es® 8G<y^ of 'SMs ^70Tk is £4^iBsmris®a ©@ fOllOs^St 

1^ ^gg^oaliKtely 10,000 ©Iss^atalj, fteetioaal gBS^^, ss:^ 
iastsf«;as3atal ssmlyses \-5©rs perfossa^ toilssg ttes past ^rem-o 1fess®j, 
wit& "to® aK^roslBa-fc® aisbsr of ©acJi iacl\sae: smstoa (2175 ), 

(2100), 2>^uclng siigai? (^^), lmlo®®ss (8%0)j, sulfur (100 )j, ptosjiiaj^^y^. 
(90)j. fimctlosml g,TOt^Bp swch ®s &e©tyl, :ss'te32?yl;, ©tSso^^vlj) 'b®ss©ylj 
caiijosyl^ actii?© Js^toogea {85 )j ^sight losg;, sssistiist*, solvate© j^ ashj> 
etc. (350); i?d.e5fO"3©i^l^gs (90) j s^tals, swcfe as sodi^, copper., 
hBXlmif sine I clis^Bsiiass^ HEaroury, cobalt, iroa^ eatlisojoyj. etc» (65)., 
optical s^tatiosa (75) p iafssr^ spectra (11CK3), selesslsa (5B5)^ 
ffiiscellaaeous (250)0 Besipieats of tM.e sex^ric® iEClisae abom; 125 
r®&^sxih sorters of tke MM S'teff « 2q ©Mitioa, saalyses a3?e per^* 
fosassd for gofverm'^sa.tal ©g@3aci©s outside SI2H, ^s;^ stscii service can 
l3@ gi-^iga "sritboiit iatsrfsadag ~td,-& the pyogress of ME 2'©s®src!i, 
D^HlBg tte past ysar such s@s'^c€5 •®a0 i^s-foHs©! BBialy t 



serial ^..JX^bli^ 
f&^ 2 

2. Duriag i^e j^^ the staff has coESlmieA gsr'/ieas of a 
speciaO. saatm^ to ■U^ sss€;®rch tesa of Dr. Klesss SslEJars (mi^m) 
Ismslviisg a sttsSy of tlss dista^ importance of seleai'iaa. C^r 
or^-balf 12ie f«3j.'-tli^ attsa-bioa of oae Esasfesr of tb® staff 'iias 
rajuiE^. la gdditloa a eoasMas:^!© asassssat of '^S2*k bss beea 
S®3?fosiEea f&r Dr. Co H. (Is«^ai, G^os^stosm Ifeivsrsityo Ds-. Gs^asi, 
althou^ ^ii^loyed t^^ llCl is p3^^s'ij3g c^(bo?3^s oa s coatrset 
basis fbr C^saesr Ghi^theanpy. 

3. Coaside^tbl® tls^ is ^seat ia ccmsul1a.ti<aa ^^th s^s^^Jh 
^so^srs eone©2iiisg prol3i@35S ^i(^ Idsey i?ish Iks handle to their 
oan laboxs,to2l©s. Ifethods of ©sp^ssach age discuased^ ©tvlce is 
giv@!i} ssSl in 3^^ cs3@s ©(^'uljssss.'t i@ ps'ovi^sda 

^o Sllfi2ig l^v® "^en foHsal&t^ to eat up a ehs^iK^t^gss]^ 
ssr^ce lQ3^iratoxy. ^ ©oaayst has tsesa obtain^ foi* this work, 
@S3& this s®a?^c® ^11 "bs offes^ t^fe^n sssce is s^milsble. 

5. Sa® iBfz®.-£^ sarvlce labosmtosy is a ^opt of t!3S 
tosilytical Ser^ces ISsit. Bstslls of tfesir ^ork are disscri'te^ 

iSl & 6€tp££%tS ?@]|SOX^<> 



VsLSt B incited I Ko 



Gaieiida? Yeas? 1959 



Serial Kbo ilA^-?2 

2. Histocb»ist3£f 
3e Betiliesda 



PslQcipal I^r^sbigato:^: E« D« LHHe 

00€>p©ffati8ig UJaltss Fons 

!fen Teass (Calendar Year 19S9) 

fotali 3 

B?of@ssio23alg 1 

Otfe®2?s 2 

P2?©ject 3tese3?iptioag 

Work on tha Msfcochcsdcal si^aetioas of to© eas^isoid tusioxs of the 

©f sma X50 titles re-ffiewed ejsitical3j md tbs s^pos^ haa fossa accepted 
foa? pKtoHcation in the Msa^tsm Joimial of Payiolo^. 

Woxk on th@ identification of the &n^Toei^ows££ija stibstsnce h£!S 
continasd, JJa view ©f the r©po3?ted isolation of ^l3g?idj?03gftxyptasdm 
in the Is'psi'toaie stxcrose ^ntrifagatc, the sia^pose tecisiie mas adapted 
to histologic iK3©j bT2t it did not pro-ws possible to d^aoastrate iadol© 
reactions in a^terochrozoaffin cellSo 

Detailed stiadsr of the aao coiisplisag reactioK of esaterodmssnaffiKt has 
b®®a Kade "Kith a coasidsr^ajl© range of st^ls md f^sfe diasotateSe 
revi^^iabls ©sedation blockade of the szo reaction has hBsn deEonstratede 
Hhe psrediaet of ■^he chEosaffin reaction of eatss^oehsroaoaffin is probably 
a caiti02^iG acido This tarald appesp to demand sing clea-^agej, bis& 
ftothes* Btts^ is ifeqaissd oa this point* I&t^ chelation of tlae sntes©« 
dn^ossafflB siSsetancs has be@n desicnsts^tedo ^i© in-tei^s^tatios of this 
i''®aetioR is Mot jet established, though aa orbho diphsssol or assiaophoaol 
sssesi to te iadicated. K«3d®l preparations of 2ij, selectsd 03?gaaie 
cojj^iHide ha^e been snade in serjjmj Hlth foKKald®!^de fiasstion* Th®£s© 
haf« been sia)j©cted to the viaslo^as s?eactions i2sed for d^aonstration of 
©atsrochi^oajaffis ^Jd the data are beizjg ^U£li^» 






Par-i Bo. Horo^s, km^rds^ and FiJblicataons 

Peblica-Saoas o-'d^ss? than abefcs&cts toasi tlds psrojectg 

S, S. Sploer aad B« D« Lillies Sapoaification as a mms of aeHe&bx^&lj 

v&VBH^^ tSsa s8tl^i?la,tloa bloekad® of tiss^ basot3hilia„ J^ Eistoelissu 
(^feodJaa. 78 123-1253 1959, 

Go G« Olsimsg- and Ee D, Ulli®? Pepsin raleass of guinea pig ent^o- 



H« So Lill3.eg FT@S®T^ed ecsmon names, lormAaBg coloto? aM©s: references 
asid i^ynoqpiis of stable diasoniisia s^ts tised in histochesaistay, J^ 
mstodssBJ. C^ochesw 7i28l«281t5 3559« 

6» G. Glenaer aM E, D« lollies C&S8i?vatioas on tlis diasot:Lsa-&ion 
coigjliag ^aetioa fos? th© iastodhsiaAcal dessoasts'slaon of i^sosinej 
3©tal dJslstion and fo^jssan ^ai^sjtSo J^ ELstoebsa^ Qf1joeb@2;;» 

G« G, Glesn^ sfid Eo S, I.illiei Ojsarvations cia tfes dissotisaticR- 
Qov^lism mB&tion for the kistodse®ical deKosste'ataou of -l^oBlmn 
Sfetal chelation and farsimm vaslaatSo Jo Estocbaafu ^tocteia^ 
(35a P:pess) 

R«. Do ttHie and Jo G« Keasoas An et-iec^t at ilesosstration of m 
IMolie st&stanee in ent©:^dB«3a3affi33 e@lls by ibs of l:ro©^"^05uc 
sasspos© solutio!io J, Hlstochem* C^»iodi©2i<, (353 Press) 



Honors aisd A^a^s relating to this projectj 

DTo Re Do Lillie wias ©lected P^sidssit of ■S'b.s Biological Stain 



Gslendaa* I©ar 3,959 







Fsv^ect Titles I^tochesleal desK»n3t:?ation of ozidati^ 
and pTOteoljtic ^issp::^ in nos^sil snd 
patbiologic tissus 

Principal Investigator: Dr. @. Q. Qienner 
Other iavestigators: jfone 

Ccoperating Diidtss . Hiaiffl«LC8 DTo L, A, Gohea l«XAI®-44 

ISiaiSMJEs Dr<, J, B, Field SI^»»145G 

TCI«Es DffSo Ho Hertz & Do KerLogg i^ti-<(03c 

HXDB^ISPg DTe K, Sc Busstoiie SIDE-® 

Mis'LCBj Dy* H, Eo Wsis^ada HHI-2il 

Uni-roxsi-Jgr of Pennssrlvaniaj Hemsr I*hij^ Dasfcitntss 

ISP* A« Daanefeea^j jx-a 

Man Xeasas (Galeader Year 1S$9) 

Totals 2*3/2 

Frofosslonsa.s 1 
OtSters 1=1/2 

Project Dese3?iptioR8 

Estmmire stisdles on S37e£>aL o^dative eo^pses ha,'^ been m^er^^ 
tak@a in an att®:^ to evaXtiate kncnn i^t^olic changes in tlu3 iiiitacb 
atiiasal and bicch^cal fitsdisngs on the basis of indi^dx^ cell 
foncbion. Using tlis inf ozmsition obtained fsN^m tiie biocheesdcal 
e:^0ZlBBSts doscssJbed by fills® and Talalsy xsslating to the preseae© 
of SQ C0t3pogen«smisitive trsnsl^d^ogenas© sjstfsia in plaeaatal tismsBg 
tbs fiffsfc MstodsejBicaL desaonstyation of both a.DPW and TP!?;=.link®d 
17 p-aQStradiol dsSg^drogeaaee ia placental tlssHs ■&ajs ^adSe On tbs 
basis of tba diffes^nce in localisation of tbea© two rmeleotide« 
linl^d ^stKss it tjas possible to absolve tbs <3onts'o'7ers3?" ss to tbs 
esistsnce of specific 1? p«»estrediol DPIf essd TPT? liakod ejessnaes and 
to iiidioat© the possibility tte.t a fbixd eru^pBj a tsr-ansI^Mx^gsnases 
t-ias present ift a singular ktstolegic oite isa tbs placeatao 

BaOTd on the oyiglasl description hj Biue'^joh© of aas a»xviop33t2.'jciSCr3 
like ©BOTsae ia tiiie invasive fsront of epitbaliaX ttsr/©rss an ensSjssBti^s 
stiBJ^ of this enssgrffi© isa the stroisa of nor?cal^ s^goKeratiwg aad 
Booplaa-fcie Samais tismie tas sadSo IMs straS^ s^tAsaled -^at jJ5 oa"iy 

cestaia tmsor tj'pes wm tte ejsjgpse evident iKsi'shiiii tifflor cellis proparo 



Serial Wo,>jij^^B©"13_ 
Page 2o 

The majority of epithelial timers revaaled high enayaiatic activi-Jy 
in the invaded sstsroina only, Awinopeptidase actimty in the stsfoma 
adjacent to invasive ttunors was di2?eet exridence of proteol3rsis aiid 
was tmselated to inflaiamatoay or fibroblastic reaction in ttanor 
stroma, taerelay indicating that a pK>iHinent ssoaehanism of tmsor 
invasion is by the proteolytic destruction of %h& s-troaial cosHpartment. 
Ho^rever, this did not iu^ly that all iavasxT© ttasors ^oiisgd increased 
stromal aminopeptidase activiigr. It is indicated, rather, that 
different tumor ii^pes (notably sarcomas) invade tisSBs by a reechanism 
other than tliat demonstrated by this histocbeiaisad teehniqae. It ¥as 
also evident from the above study that increased stroaial ajninopeptidase 
activii^ was probably related either to the activation of the enzysss 
by a metabolic product of the invasive ttroor or by sn iiramme 
response. 

Further studies on the presence of other entsyass in the stroxna 
of invasive tumors is being «nderfcaken in an atteinpt to determine 
•whether specific tiimors invade on the basis of tursor sjpecifie 
proteolytic or Ij^aluroridase^-lik© ensyaatic solution of adjacent 
stroma, m this regard a histochesdcal substrate specific (in a 
bioehemical sfy^em) for trypsin was ^Tithesised, Shoiagh the 
enzysne l^rolysing this substrate ^sjas not evident in tumor stroEia, 
it Kas present specifically in the mast cells of eerta3.n species. 
This initial demonst3:«.tion of a speci@s«limi-feed tsypsin^la^ 
en33?^B in mast cells gives concrete definrbion to an ensjnae 
capable of producing intrac^oplasraic proteolysis. An enayme 
of 'this type had been previously implicated by tJngas' in tli release 
of intrac^oplasBiic granule substance (histaraine and heparin) from 
the raast cell follomng chsEiical injmy, 

A. contimiation of woric on the localisation of specific amino 
acids by new histochemieal technj-qoes has revealed the ps?esenee 
of high conceitrations of protein^bound tyrosine localized almost 
esjciusively in the alpha cells of the anterior Ijypophysis. 
Evalaatioia.of this histologic find3.ng as it relates to pituitaxy 
hoEaonal production is in its preliminaiy stages. 



Part; B included 



Serial Wo, i?^j)~73 



Part B. Hoxiors, Awards, and Rxblications 

Piablieations other than abstracts from this projects 

G. G. Glenner, M, S. Buratone and D, B. Meyers A stBdy of airainopep'&idase 
activity in the stroma of neoplastic tissue mth a comparison of 
bistotdieiaical techniques. J. ffet. Cancer Inst, 23? 857«8?lij 1959. 

G. 0. Olenner and R. D, Lillie: Pepsin release of gmnea pig entero«» 
chromaffin substance. J. Histochem. C5?-tochem. 7$ ZOhs, 1909. 

G. G, Glerafier and R, D, lilliej Observations on the diaaotiaation« 
compling reaction for the histoobemical demonstration of tyrosines 
metal chelation snd forsiasan variants. J. SLstochem, Qrtochem. 
7$ iil6»U22, 1959. 

G. 6. Glennerj A nitrosophenol reaction for tyrosine end related 
compounds in tissnie sections, J, HistocheBi. C^ocbsm, 7i U?3=*h2l!.s 
1959.. 

G. Gt Glenner and L. A, Cohens The bistoeh®i!d.cal dejeoRstration of a 

species^-specifie ensycie tsypsin'=>like in roast cells, T(5'at'm?e (In Press). 

Q^ G„ Glenner and H. E. Bagdoyan: Tyz^sine localissation in ]l:^npopI'3yseal 
alpha cells. J., Hiatocheai, GytochsK, (m Ftssb) 

S, D. Lillie and G. G. Glenneri The histochefflical reactions of the 
carcinoid tumors of the gastrointes'tinal tract. Am, J, Path, 

(In TTQSi 



6. G. Glenner J H. Weissbaeh and B. EedJield; The histoeheiaical 
demonstration of enaymatic acti'eitgr by a nonenajnaatie redox 
reaction. Reduction of tetrasolitjm salts by indolyl«.3«acet.2lde!^de. 
J. ELstochem, Q^tcchem, (xn Press) 



loKors aid Awards relating to this projects ?tone. 






Sasdsa Kb, HI&MD-74 

2e Histochesdstsy 
3, B«?&hesda 



Pas* A 



Psjoject Titles 



Sii3di.@@ on the MstoeJssmeal' s^&etion of 

Wt0Op3fO'ti®3il'$S 



Pi'ixieipal BsTOStigatoss S. S« Spicsr 



Ocoperating t^tss 



C LPs 
A LPTs 
G LPHTt 

G tPg 



Eg", Ss H, Wolffian A/C/>fofc 



^// 



Tofeals 2»V2 

Pxwfessionaltl 
Otfesrs: 2^3/2 

Project dss^ptioBs 

Tli^ pjrtoaatgr iirfjex®s1i diJSlHg the lest yeas' coacstmed Mstochssacal 
is^t^o^ of locsHsSXigf differentiating ssul S^ss-i^v ehax?aeb@S7i&ing 
rodent aaicopolysacchsffiides. Basically, this pswjaot iiafrolTfid GcmpBH^ 
ison of resralts yielded l^ (airi'CKat staajsing piffo«s8(te«s i^th rasi2lts 
obt$|&@d ^ resdioss^graphic loealisatioa of sulf a|ed nnxcins mtli 
JSag^^O^^.The aatoracJiog^^jhic sttidies ^Ic^idng S^^ wes® done in eo21ab« 
ositioQ tdth TSTo B« Eo S^Km of the IfaMonal GaQcer J]:iiStitut@« I^ tMs 
B@Qne it tiss sho^ -bhat a En^iB£}<32' of alcian bliss ^acti'^ aM se'Tss'al 
sset&elisoinatic aeid sozcopolysacel^aTideis l&ek sulfate astexSo Charaoi^£^» 
isfcieaJjy a laild fflfft%lation iwoceda^e estraifies the easSsosgrls in sioe« 
sislfatsd acid smcins bloddisg their basophiliae Anotlsa? fe&t'ose of 
these polyB®rs is their cs^acil^ to eoBSsin® iH3.th tMassin© «^e3 at pH 3«0 
bizfe not at pH lp5» A finsCl ebaracteristic ^&icfe diffesoatiatss Ron.i=> 
3u3-fated acid saicins from those tQ-^i salfate esters li- tbs weaker 
sffini^* of th® fosmv for basic dy^es» Thiss id-yx a i^fcaiising proced'urs 
3Eplcg?is?g tw3 basic d^es siush as aldisi bltsa acd alde^de fuehsia ia 
seqt2SHc©a sislfates as© colored by the first and tlm carbo:^! grotips of 
nofisnafsted said nse^ias bj the seeond c^e, 2)n col3^iborat-ion tsith Br. 
Ls, Hars^n of «rar JSsatittit®,) sams (iml not all) of the noKSulfated 
acid aKicins hs«© bssn iderrbifisd as sialoasacins l^" ths loss of basophilia 
follo^iag spscific d:lges-fcioQ of tisstss secliloiss taith pslfiM bactsria!. 
3ialid©s8 ssnd 1^ spscif ie eoloiliristffiic qu^ititatiTO &sm^ of the si^ss^ 
natssit flrdd acd digested section for sia3Lie aelde 5f tMs ss^&hod which, 



SerislTTo. m&sm-'i^ 

for the fiz^t time identifies sialoiimcins histologicallyj sialic 
acid containing mucins of Hie mouse have been localized in the 
sublingual glands, laxyngotracheal glands, tl:^roid <^st contents ^ 
and vaginal surface epithelixmi during pregnancsr. The basophilia 
of sialoinucins in the rat resists digestion by the available 
sialidase pseparationsj but the nstaehromasia of the gmnea pig 
sublingual glands succoaibs to such treatment. The metachromasia 
of the follicular fluid in certain sialic acid rich thys^id cancers 
in the ^at, esaisained in collaboration tath Dr. S.H. Wollsisn of the 
T^ational Cancer Institute, also disappea2*s follotang sialidase 
digestion. 

As shosjn hj congparison of alcian blue s^baj-ning mth S^-' radio« 
autographs and assure A staining at low pH, xnany highly sulfated 
imicins, including those of mast cells and carti.lage lack alcian blue 
reactivity. However, mast cells in some areas in iiace (like certain 
epithelial sulfated mucins) stain with alcian blue and show relatively 
weak nsetachromasia indicating functional depletion of sulfated im,xco« 
polysaccharides. Investigation into the distribution of alcian blue 
reactive mast cells . haa sho-sm that th^ are usually intincately 
associated mth nmnerous phagocytes laden with stainable iron and 
U-poftiscin, These raast cells and phagocytes increase concurrently 
mth age in association mtli the appear^ice of intracj'toplasinic 
iron positive granules and/of lipofusein bodies in certain epithelial 
cells. The significance of the latter observations is under invest^ 
igation currently from the point of view that mast cell Eiucins ja^ 
play a role in the phagoi^e disposal of wsar and tear pigments and 
products of cellular degeneration. 

An additional project currently under investigation relates 
to the chemical nature of certain hematosylin stained bodies observed in the 
adrenal gland hj Dr. Thelma Dunn of the National Cancer Institute, 
Ij3 eollaJooration mth Dr. Dunn it has been found that these bodies 
consist of a reducing substance related to, but not identical wildi 
the catechol asdnes as demonstrated by the chswinaffin reaction. 
The presence of these bodies in adrenal tumors and of histocheraically 
siaiilar granules in cortical cells in cases of Gushing 's eyndrouje 
has been demonstrated. 



Part B included 



Serial ^To. M, 



Part Bo FIonor«5 A'wards, and Publications 

Publicationa other than abstracts fTom ■ihXs projects 

•3. S, Spicer & R. D. Lilliej Saponification as a means of selectively 
revsrsiriig the metbylation blockade of tissue basophilia. J, Fli^ochem, 
Qrtochem. 7slS3°125, 1959 » 

S. S. Spieen A coyrelative st'udy of the hiffbocheajxcal properJiiies 
of st'dent acid imicopolsrsaccbasldes* J, Histochem. Qytochein. 

(m psess). 

S. S. Spicer^ Helen J^ Btartner and R, L. Snarinj Coiapayisors of 

basopMlia mth S^^ label in s^ormal ^ad methylated snucopolysaecbaridsSo 



S. S. Spice? and Lb Warrani The feistochenastsy of sialic acid eontajjfjing 
ffiucoproteins, J„ Eistochem. Q^ocheva, (m Press) 

S, S. Spicer ssnd D, B. Meyers Histoehemical Biffex^ntiation of acid 
muccpolysaeeharides by combined aldel^de fachsin alcian bltse stainirsgc 
(Am. J. din. Path. Si Pz^se) 



Honors sBd A^-jas'ds relating to this projects 
Wone 



Bidividiial Project lieporfc 



Gelendaj? Year 19$9 



2, ■ 

3c Bethesda 



Pagfc A 

Pso^ect Titles Stisdies on xettsS. s:b^a&tm?B and fjsnstion 

Otijsr Jjarosfcigatop? M. b. Ifeiss (Student ScieKUst) 



Ito T®a2» (Galasdar Teaa? 1$$9) 

Totals 2=3/2 

Bfcfsi^ioaals 1 
Otbss"? l<al/2 

Psoject de^ripfcioas 

a) Stijdles of the ccant8r<«H?rsat vascular- btasdlss fomtd. in tbs 
B»a«lla of sJSSBBJasliaii IsMn^s bm@ been coatinosdj, ^th the aid of tijs 
elect^n mcsoseope. She fine straetiore of t&e afferent snd effe^-^irfc 
capillaries has been obsei'vsd. The afferent vesssle (branchss of the 
?f^. ®^°-^®^ °^ *^® d^ssfca^Kficitaia^ glomeruli) reveal a thick 
eafiotheliiaa aa ¥hich the cells o^rlie one another esfeeasi^2y. These 
are the C831s ^sMch in tlm rat have previousls?- been fonad to possess 
strong eg\>erase aetiTitsro The bassE^t la^abrane is cmspl^Q. end on3y 
occasional psi-lc^jillaisr inrostiag cells are seen, Sisoilar capina^s 
jave been desczabed in easxiiac md skeletal seubcI®, in l«ng, in elastic 
tasso®, a-ad in the nerroiss ostein. The efferent vessels are lined W 
a delaeat® fenestrated endothelim siciilar to that alreac^y described 
in g3«Bffiralarjfed interttjbislar c^inaiies in the aasasalisn kidnsr„ These 
cagJOaxaes also possess a continuotss basosient ssCTsbranei iiwesUap- 
cells have not been seen, Bettjsea the two igrpes of capillaries 
s»derate intersfeitial spaces occur. The struetm-al details seen in this 
sic^ raxse the qasstion ^Meh has alrea^ been esked in pre^otis 
Matoc^eaieal stra2iess ^rather it is nofe liksly that these vasctJlar 
etructoes do not have important functions in addition to peraitting 
omm&o egialabration between the tsso opposii^ streams of blood thev 
ea.'i^e As atwdios coatimie eloes to this problea w^ b© foi2nd« 

. b) •nm distnbiafcion of glntaiainase I in the Mdn^ of the rat 
has be^ deteiaoned, B haa been oondl-aded that -a® xcai^ si*es of 
rena3. ^istaadaeae I acUvil^- in the rat es'o in the cenvoliited part 






of tiie ps?oxiTaal tiibTsle and ia tbe stsmglxb eegBavit of ths distal 
tt:S}%!l9o I& has h@®n f^aowi that the amotsat of glo&amtiasa I 
acS^-fltsr in tha renal papilla; of tbe rat is iosofficient to 
accoisit Sov ths obser^sd 2?^t8s of sssKtois ^josTStioQ^ snd it 
is tl^^efore indieated that this ^mzyms is not tlis direct sgent 
of s^lsase of isssinasf airsooniao / , 

0) Tb& haadliag of tbe d^® cia.oyophesol ff@d by the pro^jaal 
t'libtale of ite rat kMngnf h&s b@@£i in'vsstigated. m ^'ro, or at 
low p®s::^jsion prigssiafej 02* iraisr las'iBasy stop flotj eonditioQS^ d;y© cotio 
eenr^tse ia tbs conrolo&ed seg!!:^at« la vX-^o ths dye eoneentr%t@s 
in the m«^ight ^@i^nt. fhis diff@ren^ e&mel&t&s Hith Insb^ar 
flo^ of -wi^SiBf ssd the 2©si2lts shoi? that tt^mlssr eiros^tion of ths 
c^ taikeB plaee oiOy iB the con^olufc^ sogs^uft^ and -yiat pax't of tbs 
i^x^eted (^ is acti^^ly 3?ssSs3os?b€d ia i^m stx'alght segsosnto fMs is 
ths iirelt di3?3j»b di^soiistration of a fimstional diff@:^»ce in tl^se 
tm ssgmesito of the ssajsaalian TCKal tiibisle. 



Pa5.% B iHcl^edo 



Serial lD« m^im-15 



Pai't Bo Eon&SBi, Aisasds, asid Fubli.ca'tions 

PsbUcaiions otbsr thaa sObstrasta l^mt ^Ms px'ojects 

J« B« L©agl^9 Wo Go Banfieldj and Do Co Bslsidl^o Sfcractssre of the 
s^te ■Mxabll® in ths kidnsgr of tise ?at sa si^n uith the elsetroa 
B)ic^3seop8o Jo Biopl^o Biocheffio (3ft Pross). 

M. Bo "W^ss and Jo 3o Looglq^s Benal gltslissiiiia;^ I disti^ibiition mid 
airssonia esces^stion in tlis rat, ilai» J, I%;^elo (m ?sess) 



Honoz® and At^jj?ds selating to this projects 

ISPo J, Bo Jjotigl&y ifjas elected Trusts-© of tJse Biological Staia 
Coaaslssion in Apill l^^S'e 

RPo Jo B» Longlegr w^s appoiated Society Hsp^es^'fcati'^e to the 
Blologieal Stain Cozsmssion by %hB ELstoch^cal Society at its 
aamsal insatisag in Atlantis City in llS$9o 



Dridimcitial ?5?oject Heporb 
Galendaa? Tear 1959 



Serial .!^o»__j3lA^_° 76 

2. Hi.stocbeH!istS3r 

3. Betfeesda 



Part A 



Prefect Titles Histologic stiidies of getnes tmd ohrosROSOEes 
in nossial and pathologic eonditions« 



^ators Dr. J, H. Tjio 

O^sr JJj-ssstigatorsj !Tone 

Coopeffafcing Unitsi Fone 

Man Tears (Calendar Tear 1959) Dr. Tjio anared on October 15, 19p9< 

Project descriptions 

Studies on the kajcyotsrpe of laan in relation to 1) the localization 
of specific humsn genes and chromososial dsens^tions in subjects mth 
heraditasgr defects^ 2) the identification of chroiaosomal ses 
constittation of indixadaals m-Mi vss^ng degrees of clinical hermu!- 
phroditismj 3) the nature of malignancg^, A study of the sex 
chromososies dtsrlng snale sseiosis (spe^!iatog©n,@sis) in tssn and on the 
nature of malignant from a chromosal point of ^ew are also in progresSc 

Ifo Part E. etc«. 

Wo Honors and Atjards relating to -yais project. 



mdixrid-asl Project Report 
Calendar Year 1?59 

Serial "No, EIAM )^ ° 77 ^_ ^ 

1, PaSEologTlJtl-fiS^ocReinistsv: 

2, HiBtocheniietiy 

3, Bethssda 

Parb A 

Pyoject Title: KiBtodaendcal studies on phoaphossrlating ensyinea 

Pslucipsl JJavestigators T» Ts&euehi 

Cooperatiag Units: TTone 

Man Tears: Dr, TakBuchl asriired as a Visiting Scientist on 
October 30. 1959. 



Jiidimdual Project Reporb 
Calendar Year 1959 

Sejfial Wo. 
1. 



2» Histocheinist27 
3. 



Part A 



P^jeet Titles Histocheiidcal studies of neTaroEelanins 

Psancipal lEi"?estigatorj Dr. H. Yamada 

Oiik&r Jiivestigators IJTone 

CkJopes-ating Units: ?fone 

Man Yeass (Calenda? Year 1959) 

Totals 2 

Professionals 1 
Others 1 

Ppojeet Descriptions 

Mi eiraaaiQation of the hisbodieid.eal stsining of tieuroiBSlanins 
in the locos caeruleus and substsntia nigra, of htsnan and nx)n&:^ b.?5lns 
was siad®. !Ekis s^e^aled that netsros^lsnins fail to e^iibit a reacti'^ 
sulfate or stilfonic acid grouping as seen in cutaneous melsnin. The 
presence of eosinophilic graniiles in the nerve cells of the locus 
caaruleos are much more highly developed than those of the substantia 
nigra. These eosinophilic grantdesj ti&ich probably z^present phenolic 
protein substances $ decrease mth age and appear to have a relationship 
to the formation of melanin pigs^nts in these sites. 

Part B. Honors^ Axfards, and Publications: ITone 



Serial No. «JS^^J1„__, 
1. Pathology & Histochesla 



2. Pathologie Anatesig" 

3. BethQsda 



PHS=4JIH 

Indi-g^idaisl Projeet Report 
Calendar Yssr 1959 



iM^J.- 



Pr@Jeet Title: Pathalegy ©f ehelin© deficient in gers^free rsts^ 
3^§l?^S®Mg. j^iM gaUi ^ iaf 8eti®a ia Kiee and 
allej^gie @ne@p^litis ia guinea pigSo 

Priaeipal Sx^estigaters L. L. Ashb?a?H 

Othar In'^estigat®2t8! David L. Beaver, Ernest G. McSaniel, Floyd B. 
D®ftj Steals Leveassn, George Hettl© and 
Ln^T Re@r^n» 

C@@p©rstiag Units: Um^imn D@partseat ©f Gersa-fi^es Ressareh^fflAIi 
DBS LPP=HIMD 

Man Years (ealeadar year 1959) 
T®tal: 2 
Professional: 1 
Other: 1 

Project Deseriptisas 

Prelisicai^ studies ia gena»fre8 rats indicate that cheliRe 
def icieney develepa in these animals as readily as in tsoa^ 
ventiei^ snin^s. Fat aceum«:dLatQ8 in aj^rois-iate lecatioas 
ifithin liver lebales, ceroid is tomed (osd liver oil is diist} 
and phago^osed, liver cells degenerate, l®b©les beeeme di8-= 
torted and eonneetive tissue is giTeatly increased in asssumt, 
appearing ia trabsenslae separating r^sants @f lobbies ©r 
largely replacing parenchyma in ssain lobes n^p portal hiat^is 
and the papillary lobes. This 8t«i<^ will be extesided and 
inelssde an e^alastioa ®f factors (protein adeqwa^, dietary- 
supplements) influencing the localisation of fat within the 
liver lobul®. 

Gontintised studies of allergic encephalitis in gadnea pigs 
have ai!!^ at detexmining the earliest age at which the 
"allargin" appmrs in the rabbit brain. Sssch infoz^iistion 
was needed in connection with an attea^t C cooperative atwjy ° 
DBS) t@ produce a rabies vaccine from rabbit brain that will 
not produce allergic encephalitis wh^ tested in guinea pigs 
using the usml adjuvant. The data trm& these studies show 
that the "allergin" is 'imriabSy present in brain tissue ®f 



Page 2 



rabbits ? t© 9 (Sejrs @ld bat not ia those wad^' 7 dsjsj also 
that lsaf©©ti0E: t-fith rabies d©9s aot alter the tiiae %fliea the 
allergia appsersj gad that a -^-sseise say be msda from ssaeh 
^©BHg rabbits, Stusfy ^ili eoatiBas ©n ths Istte? poiato 

la seareh fcs> ^ liw asthod of ©faltsstiag agents to^@ fsr 
Tyiefe0iBQBQ3 ia.gigilM a strain has fessa fs«ad CLatoi?at®s^ ef 
Psrotosoolo^y KIAID) tbat p^Kesd ss^sra les£©as in si©s 
■f©ll©wisg iKt-rsp@rit©si^l iiaj9@tioH« Orgasisss p8?olif©?®ts 
her®, iadia©© isifi.aKsati©a is the ©mantsss seseat^^s sad 
sreslap tissue about the kiise^j pan@£'©ssj, aafi portal Mattes = 
Swasioa of the li^er Q®&3^a frmi the lattor l©©ati©a as well 
as SS^egtlj th3?®5agfe ©spsiileo Ones the li^er is ia^©l^®d» tha 
oyganisBs rapidly ia(^©sse ia nisabs^j nofe fora^ard on a brosd 
front, leaviEg bshiad oislj Ese^oti© tissue. Ths protosoa 
forsiiag this "lao^iag wall" sr© filled with gljigogsn (red %fitb 
P.A.S, teehniqus) sad s?3^est a "red tide". Li^s? sella 
iraaediatelj ahead of the psraait© ^all show littls ehsagSo 
Studies will be imde ia aa attsmpt to detsraia© hms the 
trishosQEads destroy the liirea* eellso 



Part B iaeliided; Moc 



Serial Ko. vs-amm ^ ao 

1. Patfe©logy & Kisto®he^st5j 
2o Pathologi© AoatoE^ 
3. Bsthesda 



PHS-MH 

ladi'^'M^aal Pffojeet Report 

Galeisdar lesr 1959 



EieOo 



?3PoJe(gt fitlQs StM^ies on dietasy fees©sM©^©siSp ©a ssreoidosisi 

mid on pffl@ffi9s^3ti9 iafeetioa ia staEc Ekpes-iiaeatel 
studies MtSi pa©uBi!9@|^stis ia rats aad mareterial 
psisQsiag is eats* 

Prissipal te^estigstors Gsrt L. Laquawr 

Other In'^sstigator s s Laoa JaeobSj L. T, Km-landj 0, Miekolseia 

Cooperating ¥aits; UTD-gMD E-HEIBB L-!!S~MBi® 

Msn Iea?3 (©aleadas- jear 1959) s 
Totalj 2 
Professionals 1 
Other; 1 

Rr®je@t BesgE-iptiea; 

The stm^ of the s-elatiw© iaeidenee ©f degeKsrati'??© ©ardio- 
■^ssettla? diseases aaossg Japsaaso ©f Hig^shiB^ has beea eosple^ed 
ia its essentiel sspsets ead 'rfill be psablJ-ahsd aftsr asri'^al of 
esrtaiQ outstandiKg data flrom Ja|®a» 

Pathologie esaaiKatioa of surgieal aad asatopsj SMtra^ials feoa 
the PHS Eadiasi Hospitals and ees-taiia Fed^al lastitutioas fess 
been eosatinasd sad ssves-al studies ha^ro beea iaitiated from 
this laateriel. isasng thssa, the problem of disterj hessosidsrosis 
aaossg ladiaas is of iEterest ia "yie© ©f the relative fpsquesssj 
with tshieh severe fori^ of ©bse^re li-^or dJ,ssasss perhaps 
distas^ ia sat^s-a a?© seea ia this pasrtieulas" ssaterialo 

Cases ®f sareoidosis &jmng fall-bloodsd ladisas iReMdiag a 
folloM-wp ©oyrespoadsnee ^ith the respeetiT® B^iaa Hospitals 
wa?© re^ieyedo This has beea doae p^'sparatory to partieipatioia 
ia aa Ihtersatiosal Coafereae© on sas-soidosia eeirt; years Mong 
this liasj prelSalKary asrangeaeats ha^e beea ®ad8 to participate 
in an epidesdologie study of sareoidosis asoag the Oklahonia 
Indians o 



Serial No , wTAun - an 

Page 2 

An outbreak of a pulmonaxy Infection with high mortalitj 
occurred In a Korean orphanage early in 1959? Material 
sent her© was diagnosad as plasiaoeyfcie interstitial pnemonia 
sssoeiated with overwhelaiag infections with pnemtacjBtls 
earinii. A smasarj of the rsznj problems in this disease was 
reeently sMtaitted, and it ms pointed out that experimental 
stiadies had been initiated designed to investigate siaeh phases 
as taxonoi^, i^thogsaesis, antigenicity, and responses t© 
GhesiDtherapy aad/or antibiotics » 

The first objestiv© was to pro'^ide large mmbers of lining 
orgaaisffls. Fresh bxm&n aaterial being difficralt te obtain, 
the attempt was Bade to utilize the rat as a sonjrce beeaws© 
pneranoeysta have been fomid occasionally in various rat 
colonies. By using cortisone and antibiotics, large nrabers 
of organism ^ere found after several months. Havisg established 
a sosarce for pnenmocysts, the second objective was to obtain 
M 2i*^ grovth of the organisms. These experiments are 
presently in progress in collaboration with" Br. Jacobs of KIAIDc 

The outbreak of a newologic disorder @f ama, cats, and birds 
with a. high mortality was observed in a Japanese fishing village 
0a I'linaaiata Bay. The disease according to reports results froev 
consHsdng fish ca«ight ia this bay into which & factory ©apties 
its refase. Organle mereiisiy eoaponands have been implicated as 
the agent responsible for the developaeat of th© disease. A 
coUaborstive study with the EpideEiiology Branch of the NINDB 
and the LHE of MAMD is under way. Pathologic studies on cg.ts 
fed seafood from the Japanese bay thtes far have been inconclusive. 
In those which ^?are affected, severe organic alterations were 
fo«nd in the cerebellian and basal ganglia. Experiments are still 
in progress and incMde for coiaparison studies on cats fed ©rpanic 
aercmy compoKnds, "^ 



Part B included J No 



Ssyial Mo. „ Jg am?-8I_ 

2o Pathologle IsatQay 
3o Bsthesda 



PHS-MIE 

ledi-^idml Pp©J9et Report 

Caleadar lesx- 1959 



iMLA' 



Pspsjest Titles St«!iss oh hi^ altittad©, ^sperlmQatal J^a^stisa 

aad ©tfeer @@s^ia© lesioiMj, art^pisselos^slg s, fea@t©^ial 
9®d©@as"ditis afii gleae^aloaephyitiSs eatadsolaHiaeSp 
adE-eaeopgie and gssaglioai© bloeSsing ©geats, MOBoaffliao 
©sialase iahibit@?Sj aad fat iK>biliaati©n asd d©|^siti©r: 
iB th© livor. 

Priacipal Sivestigators Beajaiain Higkaaa 

Oth8? le^Qstigatos's: Po Do i0.tlaad, B, B.. Brodiej ¥, M, Sutler » 
?. H, Coha, Jr=,p Ho M. Kaliags Jo Roshep 
©Ed Eo Co Thosiipsoao 

Cooperating Uaits: LP3-HIMD (Altlaad aad Thox^soa) H1AI3?-104 

L0P-ini (FMiag, B^sdi©, Biatlss?, ©ad Cote) MI»202 

Maa lears (©alsBdar jsar 1959) s 
Totals 1 
Ps^f®ssioaals 1 
Ot-h^"? Koae 

Projeet Deseriptiea: 

Stwdies id.tb Drso Altlaad aad Roahs ©a the ©ff©@ts oS scepeatsd 
daily egcposares 1;o 30,000 fast ©a aorssal doga aad oa dogs %?itls. 
■9>al^tslar defoEniities wsrs @oMpl©tedo la addition to '^al'^ialar 
thiekealBg and ■^egstatiosiSp resaal iafaretisB aad hssaosidsTQsis^ 
aad oitheoc @haEges ps-sviowslj desa?ib8d ia rats ^spssed to 
2§,0OT feat 5 th© dogs oftea developed aoalipid aytsrioseleyoti© 
iatliaal plaques ijs^ol'^iisg th© aos'ta smd ©eeasioaallj th© ©©s-oassy 
fflftoiPles; these lesioas er® ©tts'itated t® hg^xiao This asperi- 
Biental sceproduetion of lasioas reseaibliag hmm& aj?t©ri®s@l®p©sis 
aaay pa:>o^e rasafsal 1m elueidatiag the geassis ®f ©s'tsrioselerosis 

Aaothsd? st'J^" with Ifeso AltMad md R®@h© ^sas eosapleted oa ths 
treatmeat gf ©xpsyfeeatal stapS^loeoe^l eadoearditiso Thia %fas 
parodueed bj aa iat^'airsaoiss iajeetioa of a etsltiia?© of StaphTlogpeeus 
aureus in do^ pead^red highly suseaptible l^ sjsrgieally-induesd 
aoptle insuffieieaey. Endoeayditis eoBld be p-weated toy a 



Serial Ko. KTAtan-ni 



Page 2 



single iatr©v@E@Tias injeetisa @f peaieillin given wltMa 8 hsisrs) 
after isJQugtiisg the bact@rte= If treste^t was daMjed 24 hsisrs 
©r l@sg©rp the s^^ptesss ©ft@a racm'red after esssstiea ®f tr©at- 
Eieat. The ©Bdeearditis v&s arrested bgr treatmant of r@]Lsps«?Sy 
biat th© "mh^ss often b@eas58 thickeasd and ief®iia©d aad soa© 
®s5s^ls died frma &&&t@ hm^t fssiltar® fe® t® ^pal^^sla? issmfficf ; 
A diffm© ps'Qlifsrat-i"^© gl©©gri!sl@E@pteiti8, whieh ds^(^@p®d is 
ae@3?lj ©11 d@g@ giv®B dalsjred tr^taseatg p«rsist©d despit® tfe®?®!?-, 
Tfe«r® i^s s^idsE©© ttet ® ©teoaie gl®ffi«ri!il©siepbritis i!®2r b© S3 
sgqsael t© this pir©lifer®ti-^© glsseri^oaepbritis. Thes® fiBdiag© 
sgapfessis© the iiapsrteaes ®f prepbjlaetic trgstaeat ia ssssegptlbl© 
iadi'^idisals and of esrlj gsd adegimt® trsatsesi; of hmmn eHd©=> 
esrditiso Th®^ s^pp®rt the thesis, reeeatlj q'?s©sti©a@ds tbat ® 
ebroai© gloasnsloa^pbritis is ® s€q®©l t@ aa &&&t@ aephritiso 
Ow espsrliasEtal method sboiald ^&W9 ® taasf^ tool is stsi^iag 
stsph^loeeal iafeetioBs resistsnt to aotibiotie th@ra^ ®33d ia 
th® sttadj of gloiasnaloBQpliritis. 

Sttadi@3 i^ith Br. Mtlaad sa th® ©ffeets ®f high ®ltit?ad9 ©a 
eli©l@st®r@l'»f®d rabbits ar@ aesritsg eompletioa. Stiadiss w®?© 
beg^em with Dr. Htlsisd ®a sbsiagas ia bl©«d eh@Jaistrj iii&©©d 
i3^ ©xposis-^ of dogs t® lj%h altit^d©,"oia possibl® ehaages ia 
iasa^mity sad aatigeaie respoBS® ia rata imposed t@ hi^ sltitirad®^ 
sad @a the ©ffeet of -m^lQ-m dsngs @n th© smeepti&ility to 
eadoes^ditia of norml rats sad rats i^icposed t@ high sltit^ideo 

In stiadies with Dr. Ifeling, it vas fomd that Mrge dosas ©f 
cateehoS^iaiaes produe® a laarksd elevation in seram lactie 
defe^drogesaaa® ia additioa to ths pa's^ioisaslf reported Barked 
H^eardisl fatty ehaages aisd ©lerotioas ia s^^sa transamiEsses 
and alkalis© phssplmtss©. Ssieh ehsages, eseeptiag the ris® ia 
saareaa a21saliae phosi^atas®, ®r@ reduced or |sr@^@at®d h^ the 
adreatirgie blodciag ageatj ph@a@^b@assMia@. Eowwer, phesoKj- 
beasamia© does aot prgf^eat a sfeilffir rise ia serwa ®Bg|«@ Is^^s 
thst ®<g@W8 aftor s^es^dial iafar@ti©a ia dogs following 
ooroasry lig®ti@a»- aor doss it ^mm% the fattj eh®ag@s ia th® 
i^®©ardi®a borderiag ths infaret. 

A @t^ wss md© to test th@ leoaespt of Hars'is aad Bisteni thst 
th@ ventri0ialar taehycardis r®giialtiHg fro® s^esrdisl iafss^tioa 
ia d®e, at least ia |srt, t© ©piaephriae aad aos^pia^hriaej 
whieb @r® libf^ated f^oa th® asigFotie s^e«rdi^ ®nd i^ii@h tmj 
a@t ^poa th© ftoetioasl e@lls borderiag th® iaf®r@t. The ffig?®° 
osirdiiffla was depleted of sjorepiaepteiae by ®^aiaist®riag reserpiae 
to dogs before mT©nwj ligation. This did ©ot pr©y®nt th® 
veatrieiidar t®e%©ardfe, th© fatty ehsagss sromd th® iafaret, 
aad th® rise ia senam easya® Iwels. Th@s@ fissdings do aot 
stapport the o©a©apt of Harris and Bieteaio 



Serial N©. JBTAmvoi. 



'&g& 3 



A stiffidy with BiEtlar^ MaliBg, aad Brodi© is©nc©rns an increase 
iffii n©iH.tr®l fat ©r triglycerid© ooBteat of the liv©r ®f rats 
isdueedl ly ©arbon t@traehl©rid© (1.5 scAg s.g.)^ etlsaa©! 
(6 gffli/kg ©ralljls or ©thioaiae (750 fflg/kg i.p. ). R ^^^as f®aad 
% ehemicsl aad histologie a©tli©ds (stainiigg f®r fat with ©11 
red O) ttat th@ iacreasQ in fat im@ largely |rw@Et®d by pri®r 
a<Saiaistrsti@B @f adpes©rgie blsdcisg sgents. This projest 
sSieiHld iaersass @nr TrnderstaadiHg ®f th@ pr®e©@s@s isrol^sd 
5js triglyeerid® msbilisstiea ®ad cbp®siti@a sad msif giv@ ssss 
eMes t@ the smt^sr© ®f h©p®ti© eirrhesls ^d sth®i'@3©l®r®sis<, 

Aaothra* stt^dy with Bra. I&liag asd Spg@t®r eoaesras th© 
atoisiistrstisn of =^®ri®^as isss&sajiE® ®s:idas® iafcibit©rs t® d@gSj, 
0®ts, rabbits, and sqaiETsl s»Ek^s. Sob® @f these ©sapmsEds 
prodwea marked changes in belm^i'or @Bd marked fism^olegiig 
^raptoms. These fiadiEgs sre ©asrelated ^^ith etenges ia tfe® 
Iwels @f s@r@t®saiffl sad TOS-epiaepteia© M "^ariets^ ps^ta ©f ths 
ffi€®^@i!as g^steas ©ffid %jith |mthol©gi© ©teBges. This sl'&^j smj 
eMcidst® tto lEsed© ®f action aad pessibl© tasieitj @f so^es @f 
thss® e€®p@TOds, Tliis stsady is iaportant bse®»® s®a@ @f th@s@ 
eoEsp^taads ar® t^ic'aly tased bgr psychiatrists -and ia tfe© treatsasat 
®f l^'p®rt©Ksi®a. 



Serial Hoo KiAKn-fti 



Cril®adaff Jew 1959 

Rm.%,3.' Hoaorsj, Ma^Ss^ aad Publieatic-as 

P.srtieipat€d in the Sya^sijaa ©a "a© Cateeliolaaiaes is Cardio^sseular 
Pattelog^r" %-iihiGh ws held at the J!n±mTB±tf of YerwHtj, CDllege ©f 
Msdieiaa, Aiig^t 23-»26j X959<. 

Fartieipated in Meetiug of Joial; C«aitt©e Oa Aviatios Pathology whieh ' 
was fe®M at ths ^SrEed Fo^ees Ihstitttts of Pathology Wasfeiagtony D, C„ p 
oa Kwsaber 6, 1959» 

Ps3.bli<sst5.oas ©thas' thsa sbstraets fr©a this pris Jests 

HebstsTj, S= H.J Rie©, M. S.g Higteaa, B.y aai Stoblmaj, E, Rei fh© 
ToKi©©log|' of Potassissm aai Ssdi^aa lodates II» Sisteeate Toxlsitj of 
Potassiisa lodat© ia Mi©0 aad Guia@a Pigs, fosiieolo^ aad Applied 
PtoM^eologj Is a7~96.» 1959 <. 

Higbssaa^ 3o, Jfelisag, Ho M»j aad Thoaspsoa, E, G<,; Ssrraa Transaaijaase 
aad Alkalia© Phosptetiage Lsvsls aftsr Large Doses of Norepinepteia© and 
EpinspM'ia© 5ja BogSo ia. J. Phjsiol, ]^% 436-440j 1959. 

S=Jali2sgj> H. M. sad Highaanj BcS High Altit?jd® Tolca-aa©© of Kcsisal Dogs 
aad Bogs Hith !-^esrdial Bifareta, M. J. P%siolo ^s 50^-511j 1959. 

IfeliBgs Ho Mc, CohBj ¥. H.J Jx'cj aM Highisaai, B.s Th© Effegts of 

Cos'Qffiffi'j OaclusiOQ la Dogs a»0a1;®d with Reserpiae asd ia Dogs Ideated 
Hith Phsno2^b®as«iB©. J. Pfeajsaeol. & Efepea?. f&srap, l^s 229-2J5j 1959o 

AltlQBdj P. B.p HigteaSj B»j ajsd Eo^b©j, J.s Bff@@ts of AltitMa ©a Eogs 
witlj fal^xlsE" HssFt Diseas©. A. M* A. Affgfe. Patla. ®s 4'^5<-4S65 1959o 

Hig:te!aHs B.j, Altlaadj P» D.^ OEid RoBh^^ J.s Stapfejloeo®©®! Endo@®rfitis 
and Gloaenalonepbritis ia Bogs, Effeeta of Trsataaat with PeaieiUia m.d 
StreptoiiB^ino OlrtMlatioa S©s®ar@bi 7: 982-9S7 , 1959 » 

l--hliMgs H. M.J HlgkaaHj B., md aoKpsou, E„ C»2 S&m Sisailar Effaets 
Aft®r Largs Doses of Catsefeolffldaea arod ifyoeardial Iafar@tioB is Bogs. 
Paper pe-esented at S^poaiws om "Th© Cmteeholasalnss in C^dio^aseialsr 
Pathol©^" held at BOTliag^oa, Vatp^ist, Angmt 23--26s 1959 sad psper 
ssataitt®a at request fog- p^lieatiosi la the MB^lmn S&mml ©f Gs^diologjo 

AltMiady Pc D.J md iigtaaSy B.s Eff©©ts of Sigh Altitiads @a ttoste^el 
Fe4 Rabbits o Produetiosa of Swara PtxlsaeMry Ath0r@s@l®s«La)sis with 
&l©ifi©eti©a<. A.M. A. tosho Pat-ho Im ppess. 



1. Patlioio^'& Histocheiaistry 

2. Pathologic Anatonigr 

3. Bethosda 

PHS°!3KI 

Sidividml Project Report. 

G©ls2adsr Year 19.59 

Part A. 

Pr®jQet Titles St^d^ of jasebaRisaas involved in iafaetiotas diseases. 

Prjjaeipsl Bss'estigators Edwin M, L&tn&^ II 

Other Siwstigat©rss L©sa Sokoloff, Robert R. Hilliaissj Ksart J. 
Blochj, Joseph J. Bwaimj Robert T, Hsbssiisaap 
Shsldoa ik-ay 

C©©p®?ffitiag fcitss I-ra-IIBJffl-109Aai-lIfil© - MB.4SIS LMIIilB- 55 

125C 
J&n Ie@rs CealsMar ysar 1959) s 
■ T@talj 2 

PTOfessiomls 1 

Othars 1 

Projeet Deseriptioas 

The pathogenssis of experisseatally iadteeod artbritis isa rats 
l^ infestieli %fith St^jgt g^nAHM. rosuqil^^pra^g tas been d©f iaaJ 
in detail. Tho Issions fesrs been sh.a'JSi to develop as a pritsarf 
osteoiE^elitiSj with sQSOKdmy axtsnaion into tha periartisialar 
soft tissues, or into the Joint f3?om either of th® two pressding 
foci. Th© slMlarity of this infectioiis process t© other so^ 
called iafeetious arthritidas has been described. Bacteriologici'- 
and iaanamologie®! st^ies hsva been eorrelstsd with a detailed 
time Btndff, of thia proeess. 

Tlie iafeetivitj of the cyrresatl^ e^loyed straia has bs«a 
explored in Mee %M.dh had been raised so &b to be free of 
astm-al infection with Streptob ^e^Up gi or Hitfe pl@?^ropa©®B9sia- 
like organisms, fhe IneideRce @f gross ^oint lesioss tes fe®6fs 
as great in aieo &b was c-bserysd in rstg, ®nd th© gross ©^dosie® 
of iiaflsiMation persisted for KUeh long@r periods of time in 
mice. Th© laieroorganisia vaa found t© be letlml for aie© aftar 
iatravencms, intraperitoneal, or a«ibe8st®n®©?as Sajeetions with 
deeressing rat® of Esortslity in that order ©f routes of ija©e?s= 
lation. Gross Joint lesions appeared in mice as esrij as 24 
hours after injection, ®nd have persisted for ma long ss thr®® 
months. 



Serial No. niAm-B2 

Page 2 



The serologieal rsaetioas for rhewmatoid artteitls ia the 
iafeeted rats, namely j the sejjaitiaed sheep ©ell hemagglutinatis® 
reaetioa aad the bentoaite floeeulatiea test, hsT© been showa 
to be an jjsauaologie raspoKse to aatigeaie stismlatioas indepea- 
deat of seti'^8 isafeetioa or joiat isflaasaationo Rabbits isaaimisad 
with foffnsalia-killed asstigeiss pa?epared from S, maailifogmia 
developed high beatoaite floeaulatioa test titres, raiagiBg «p 
to Is 1CS24, Attempts to ebaraeteriaa the ps^teia f^aetiea 
iBRrolved ia this ismme respoEss ia rabbits iiadieated that, tka 
scespoasiblo faetoy ia rabbit senaaa differed f^oa that ia hsM&s. 
rheumatoid arthritis serws l^ altraeeatrifugatioa behaiPios' aaS 
by agar gel preeipitatioa tests. The rabbit faetor had the 
ehgraeteristies of a SHsall jaolee^© by Kltr®@aatrifagatioaj, th'as 
differiag trasa. the hraaai^ rheumatoid fa©t©r» A^r gel pz'egipitia 
experimeats indicated that the imesmized rabbit sertssi coataiasd 
aa aatibody to himan gsMna globulia. Although the sieroorgaairsm 
was routinely eiilti'V'ated ia medium eontaiaiag feyiman ascitic fluids 
this substaaee had been fouad iaaeti-ye ia coatrol expsrimsats 
involviag both these serologieal reaetioaso Further exploratioa 
of the role of h?jaaa ascitis fluid imeludod adaptatios of th© 
Eiieroorgaaisia to growth ia ssni-synthetie laadiim, aad f iadisg 
that sueh cultures were aoa-isaiuaogenic ia the tests in^oliredo 
Ewian aseitie fluid aloae, or in eombiaatioa ^ith bsateaite 
particles or with S, laoailifonais grotsa ia the abssace of 
ascitic fluid ^las aoa-stlsmlatoryo This iadicated that the 
Bjia?o©rganisa either ©oaceatrated or aodif ied himaa gwm& 
globulia preseat ia the HsediiSB so that it besaiaa aat^^-eMcj, 
aad the sSiailarity of this ppeeass with the elafeoratioa ©f 
haptsisogeaie saatoriafe from ©ultwr© Ksdia ^ ©thsr id.®poorga.aisKs 
has baea defined. 



Pas-t B iiE@li«deds fes 



Serial No. .msMa, -,«g 



PHS-JJIH 

Ifcdividml Project Repsrt ■ 
Calendar Year 1959 

Part B i Hon®rSj, Awards, and Pnablieatieas 

Publications @th@r than, abstracts frsm this pr@J®ets 

Lernerj, ESwin M. II, and S®&©l@ff , hmnt Th® Pattogess@@ie ®f B®a© and 
J©iat Ihfe©ti@n Prsdiaeed in Rats b^ gts^pMatHhM l^alAMSzMff.. A.M.Ac 
Ar©h. Path. ^, 36»4-372j 1959. 

Lerner, Edwin M„ II, Bloeh, Kssrt J., and WniSjias, Robert R., Jr,s 
"Rli@!jaiat®id" Ser@l®gicsl Reaetions in Ehcpariiamtal Anlswls» II, Beatssni-t. 
, Fl©ee^jl®ti@a Test in Rats with Experlmeatsal Arthritis. Arthritis and 
Rheraaatiam. In Press, 

Lemer, Edwin M. II, Bl®sh, Kwt J., and Milliaias, Robert E., Jr,s 
"Rhewiatoid" Serological Reactiens in Ebtperisaentsl AniSiEsls=— ftoe 
Ssnsitissd Sheop CaU H©sagglmtiMti®n Reaction rad Beat®ait® Floeeis- 
lati®ffi Teat in R®ts -^ith Experissaent®! Arthritis. See®ad Psn-lmsricaa 
Congress @n Rheto^tic Diseases, Jim®, 1959. 

Lerner, Edwin M. lis Arthritis QmaB& bgr S^j^^^iltt^ meiMf^^.g. 
and Pl©!ar@pa®®ffisaia<»likQ Organi^as in Saall R®d@nts. C®nf®r@n©® ©a th© 
Gsiapsrffltive Patholsg^ ®f ikrthritis and RSi®imatismo I®b@rat@2y In¥®sti--=- 
gation. In Press. 

Lerner, E^in M. lis Pattolegj ©f Ac®t® and Qjr©aie Rrac©ll@sis in 
Expsriiaentally Infected Gainea Pigs. CJoafereac® on the CsmparatiVQ 
^ Pathology of Arthritis and Rheiim®tisa. Laboratory Investigation. 2a Pr©r 

I-arnsr, Edwin M. II: Morphol©®' and Clessifieation of the Flmxs'&mmm&ix, . 
like OrgsnisKs. Swwar^r of Session CStiaii'Ean. H&is Y@rk Acad'cssy of Scieas^a 
Conferenee ®n Biel®^ of the Plem'^pneraaoniallke Orgajaisas. AnBals ®f 
lew York Acad«3ai^- @f Scienees. In Proas, 



Serial Ho, HIAHD » 83 



1. Pathology & Histocheiaisti'y 
2o Pathologic Anatoi^ 
3o Bethesda 



PHS-Nm 

ifedi-^-idsxal Proje@t Haport 

CaleMar Year 1959 



ParJLA, 



Projaet Title: Mor|&ologie aad histoehosiieal 'fas'latious ia the 
■ p2»Qputial glaedsj, oadoerin© s^stessp and iat^Mil 
gQaitalia ©f the @owfenti.oml and g9s?3a-fe'8Q ratj, 
as infiueneed fey hoMKjaeSj Titsaissj baeteria, 
and tissue fisation^ 

Priaeipal Isyyestigatox's Da-yid L, Bearer' 

Other Swestigatof s Eraest Q. MeDaaiel 

Gooperatiag Unit: ME-KMMD - 3 

Itos leas?® {©alendar jea? 1959): 
Totals 1 
PffofessioEsals 1 
Others None 

Projeet Descriptioa: 

It has been shown that the rat preputial glaad is a "di«srine" 
organ and exi^ histocheiaical or endocrinological studj smst 
take this into consideration. A method has been developed 

for staining the two seerstorj products simultaneouslj and 
is now being iised, along with -various histoeheaieal jstssedwresj, 
to assess the effect of various honaones on the glando Coa- 
©oEsnitaatlj ^itasain A deficient is being studied ia the eon- 
'9'entional rat fSt>m aa endoerinologie point of irie^p and ia the 
ges'sa-fjceQ rat in order to deterjaine the importance of infeetios 
ia the production of sqaoEous sietaplasiao Isi addition, the ^ 
relatioaship of normal bacterial flora t© the aeutrophilie 
•paginal axtadate of the !aflM»ine estroMs ^©le is being e-^alaatedo 
It is hoped that the infos:TOation obtained from these studies 
Mil lead to a better understanding of expsris^atally induced 
tissue changes o 

I aa also collaboratiag ■with others ia an escperiaent eonceiised 
with the role of vitajirfja B12 aad choline on the production of 
dietary cirrhosis in the germ-fc*ee rat (see report bi^ Bro L, L. 
Ashburn) e 

Part B ineludeds leso 



PHS-IIH 

ladi^idml Protest Repsr'^; 

Galeadar Imr 1959 

^2^^: H@M@rs, Awards, and Publieatiosss 

Psiblicatioas other than abstraeta from this project: 

Bearer, Bsvid L.s "Der Einfl«ass vorschiedener Fixienangsnittel aiaf 
das Stndctm'bild der PripiiatialdrSssn der Ratt©": T@ b© published ia 
Zeitsehrift ftr Zellf@rsch?iag tmd MSki^akopigohe Aaat©iaie. 



Serial Noo wyAag>.. g4 

lo Patholo^ & Histosheiaisti^ 
2o Pethologie AaatoBjf 
• 3« Bethesda 



PHS-NSI 
^di-dldaal Protest Report 

CalsMsr Year 1959 



f^t A„ 



P?®Je©t Titles Prepaeatioia of staiaed tissue ssetioa for 
iH^estigatioa and dlagaostie pt2s?poseo 

Prineipal Investigator? I^o Roy Read - Head, tissue ireparation 

laboratorjo 

Otheff' fewest igators Noae 

Cooperating Uaits leas 

Ilea Years (ealeBdar ^©ai- 1959) j 
Totali 7« 
ProfessioBials 
Othsri 7 

Projest Sesc^iptioas 

^e^ statistieal report of this wait is sh8%?a below. la addition 
to prepariBg nsaterial for projects eoisdueted ia the laboratory of 
Pathology and HistodietEsist^jr? tms^ ©thstr ias'sstigatos's s-eesiifsd 
ad^©Q and ssryie©. Those iiseludos DrSo Brods^, Hesseltmghp 
siad Rowe - Lafeoratosy of Iflfeetious Diseases j Bra, Boiyitt aed 
Reardoa - Laboratory of Tropieal Diseases j Brso Freimd, MeJ&s-iex'Sj. 
affid Tobie - Laboratory ©f IssaMsology, Tissues :fe©ffi a sa&ll 
anntbar .of animals were ^spsjped for eight other isrs'sstigaters . 

Polio ^aeeine stsjdiesj eosts?©! aad eacperlaeatal aspecsts soatiaKsdj 
tissue t^eim 2pl52 moasieys were proeeesed fcjir histopittologi® stm{. 



^ In additioa oae teefeaieiaa furaished fey UBS for polio vaeeiae ■uo^kg 
approxisaately oae-hslf a lEaa year. 



Serial NOo jSiA^sM-^^. 
Peg© 2 



Aalmsls 
Siffgicsls 



Spsciiaens 

2,193 

103 



StalQed_^8lMes. 

Eoatine Sgacial 

ll,04Sl "^ 

4.j298| 18,254.1 

h^ J 

16,555 



SlMes Totql 



49,181 



'^Soiirffie of as?i^al imterials 

DBS 3,34-6 

Emm 1,137 

Othsi' laslixtutQs 1,001 



i.rt B iKcladads !IOo 



Sei'iai Ho. NIAf^D-_85_ 



1. Pathology & Histochemi 

2. Hematology 

3. Bethesda 
PHS-NIH 

Individual Project Report 
Caleadar I©ar 1959 

Part A. 

Projeet Titles Regulation of heaopoiesis. 

Principal Iir,f0stigatorss Frederick StoMaianj Jr,? and George Breeher. 

Other Investigator: Archie A. MaeKinaeyj Jr. 

Cooperating Units t Medical Department? Brookha^en National Laboratory. 

Man Years 
Total: 5.S 
Professionals 1.2 
Other 4.6 

Projeet Beseriptioas 

Objectives: Stttdy of red cell and white cell turnover with partica- 
lar esaphaais on the determinants of rates of production and the 
meehanisa of action of these regulants. Attention is also 
devoted to the physiology and clinical identification of dis- 
orders of eiythropoiesis. 

Methods eaployediQ Rates of red cell production are estimated by 
Esans of Fe incorporationj reticulocyte appearance and bone 
marrow morphology? destruction utilizing Cr j modification of 
rates of erythropoiesis is achieved l)y varying pO„ concen- 
trations of inspired air, hyper transfusionj blood lossj iri=sdi~ 
ation and the administration of erytbropoietine. 

Cellular proliferation in marrow and peripheral blood is being 
studied by in vitro labeling} using tritiated th^idine and 
autoradiographs. 

Major findings: 

1. Bone marrow eellularity influences the plasaa level of 
erytbropoietine s presumably through utilization, 

2. Short tern in vitro marrow culture teighnics cannot be 
adapted for the assay of erytbropoietine. Presumably this 
reflects the fact that these primarily are maturing rather 
than dividing systems. It also may be attributed to the fact 
that erytbropoietine may act primarily at the stem cell level 
and there are inadequate mmibers of these cells available to 
permit detection of an effect. 



Serial Ko. ICTflMD ::;85^^ 
Page 2 



3. E^idenee has bsea ad"^anc8d to support the coneept tlnafc 
"popolation pressisre^ is not of iisjportaiies in the early 
release of red sells under maxiaiEs stiraidatioB, 

4= A s^Jor-fcened stem cell to emergsnee t5Me has been desioa- 
strated following the adiainistration of srjfebropoietine in 
aniiaals in which erythropoiesis %m3 suppressed hy hypsr- 
transfusioaj iaereassd pO„j and irradiation. Fwrthsr sbor'ien- 
ing Mas been observed tmdar continixed erythropoietins sd~ 
ministration^ sjjggssting that the action of erythropoietine 
is not confined to stimulation of ©arly pree«rsor cells, 

5o In steadies on eell'Ular proliferation it has heen dason- 
strated that an inereasa in red ©all production is aebis'^ed 
throijgh an Increase in the niishsr of di'^iding progsnitor 
eslls together with a deereass in the ntsiber of nomaoMasts 
norsially lost 1by attrition in the isarroij. 

6. Studies to date indieat© that the sesond regulant of 
erythropoiesi© whieh we ha-^e postulatedj is related to tts 
number of cirewlating red cells. However attempts to dosraaent 
that a postulated feedback fi'<sa the deatt of senescent cells 
is the sole factor has not been possible. In fact bone marrow 
suppression has been induced by young red cells in the presence 
of no3fmal oxygen deli-^ery. 

"7. Is 2ME2 studies on thymidine indicate that at the time 
of withdrawal fTcss the body a certain proportion of cells are 
in DNA ^nthesis and continue to ^nthesisse DNR up to 4 hom-s 
after withdrawal. Howe-s-er these cells do not enter mitosis 
nor do other cells enter DNA ^nthesis after r^o^^al frara the 
body. 

Significance to the program of the Insstitutej Anssnia is a eosMon 
complication of arthritis and in snarly instances may be con- 
sidered to be a metabolic disorder. Understanding of the 
mechaniaas of red cell regulation are not only of basis 
interest but should eventually result in iiaprovsd therapy. 

Proposed course of projects Studies on the site of production of 
erythropoietine and its meehanisn of action, ftlso further 
studies directed at elucidating the nature of the second 
regulant of erythropoiesisj the latter to be pursued in both 
experimental and clinical studies. Patients with anemia of 
rheumatoid arthritis, polycythemia vera and thalassemia will 
be the subjects for the clinical investigation. 



Serial No. NIAMD 2_g§^ 
Page 3 



Gooper-ati'^e work with Gheaotherapy Sers'lesj General Medicine 
Braachj KCIj is bsing conducted to explore the determinants 
responsible for thymidine iiieorporation into DNA in sitro 
and the effect of eheaotherapy agents thereon. 



Parib B incltsded I©s 



Serial No. KIAMDt;^.§___ 
PHS-NIH 
Individual Project Report 

Calendar Year 1959 

Part_B: Honorsj Awardsj and Pablieations 

Publications otlier than abstracts from t&is project: 

Stolilmaiis F. Jr, s Observations on tfes physiology of exjthropoistine and 
its role in the. rsgwlation of red cell prodiictioa. Anoals of the New 
lork Academy of Science. 77 j 710-7245 1959. 

Breclier J G. and Stolalmanj F, Jr. : Hxsaoral factors in erythropoiesis. In 
"Progress in Heanatolo^", 2iit2 ^^ol., (Toesatiagy L. M,^ ed.)j New York and 
Londoa, Gruns and Stratton^s 1959? pp. 110=-132„ 

Stohlffiaaj F. Jr. and Breeherj G. t H'i;^oral regtalation of erythropoiesis, 
V. Relationship of boae marrow aetivity to plagaa erythropoletine le^'^sl. 
Pro©. Soe. Exp. Biol. & Med. 100s 4.O-435 1959» 

Stohlmanj F, Jr. and Braeber, G, : Effeet of bone saarrow aeti'^ity on 
ersrthropoietine utilisation. la "Proceedings of the 7tli Congress of 
International Society of Hematology" 5 Rcaaej Italy, II Penaiero Seientifieo.- 
1959. 



i3j E, D.J Loehtej H, L. Jr. and Stoblmanj P. Jr. s 
develop an in "^itro systeia for the as^y of erytliropoietin. J. Lab, a: 
Glin. Med. In press. 

Stoblmaa, F. Jr. s Erythropoletine, Pediatrics 2|s 835-936, 1959. 

Stohlasan, P. Jr. s Preface. In "Tlie Kinetics of Cellular Proliferation^^ 
(Stohlmanj F. Jr. 5 ed.)j Net^ York, Grune and Strattonj 1959. 



"ilmans, P. Jr. s Observations on the kinetics of red cell proliferation. 
In "Kinetics of Cellular Proliferation^ (Stohlajan, F. Jr.? ed.)j New York 
Grune and Stratton, 1959? pp. 318-324. 

Stohtoan, F. Jr., editor "The Kinetics of Cellidar Proliferation" 5 New 
York, Grune and Stratton, 1959. 



Schmid, R., Breeher, G. and Clsmensj T. i Familial hemolytic anemia 
erythrocyte inclusion bodies and a defect in pigment metaboli^j. Blood 
Ms 991-1007s 1959. 

Brecher, G. j von Foerster, H. and Cronkitej E. P.? Produktioag Ausreifang 
und Lebensdauer der Leukoajrten. In "Fhysiologie und Pbysiopathologie der 
Welssen Blutaellen (Braunsteiner, H., ed.)s Stuttgart, Germany, Georg ThieES 
ferlags 19599 PP« 188-214. 



Serial No. NIfiMD'^;_85^ 
Page 2 



^sLBs 



Breefeerj G. ^ Smitlij ¥. W, and Gronkitej E. P. s Strahlenschiita disrsli 
GrantiloayteB, In "Physiologie nnd Pbysiopathologle der Isfeissea 
Bltttsellen (Bratznstelaerj H,j eS.), Stuttgartj G-ei^aayj Georg Thieaa 
¥erlags 1959, PP. 215-226, 

Bondj ¥, P,j Pliednsrs f, M., Cronkite, E. P.j Rufeinij J. R,j Breclierj (1. 
and Sehorkj P. K, : Proliferati'^© potentials of bone marrow/ and blocnj 
sells studied by in -i^itro uptake of H""*-tb^idine. Aeta Haemat, 21s 1-15$, 
1959. 

Crorfcite, E. P., Fliedner, T. M., Bonds f. P., RuMMj J. R.g Brecher, C% 
and (^stlsr^ H. % I^namics of hemopoietic proliferation in 2»an and mice 
studied hy H -th^ldine incorporation into BNA, Annals of t&e New fork 
Acadaaiy of Sciences. 77s 303-82O5 1959. 



Serial No. IfXSiMD ::i,S£,..____„, 

1. Pathology & Hls'ioeliGjaist: 

2. Hematology 

3. Bethesda 
PHS-HH 

Indi"^iclual Projoet Report 
Calendar lear 1959 



Part A.. 



Project Titles PatSiogsnesis of IB^perimental Arthritis and Pathology 
of Rha'jmatisa. 

Priasipal Is^ejstigators Leon Sokoloff, M, D. 

Cooperating Unitss NCIj Dr. George E. Saj, Jr. 

Man Years 
Totals 3.2 
Professioiiais 1 
Others 2.2 

Project Iteseriptions 

Objeeti"^es: Investigation of factors infl^sneing develocsasnt of 
degensrati'-fe joint disease in anall laboratori" animals. 

Methods (snplojeds The role of genetic factors in osteoarthritis 
is Iseing stiidied fey aaatomieal PI and F2 bybridsj backerosses 
and reeiproeals of certain mice are being made. 

Major findings? In addition to papers listed below, 6 aan'JLScripts 
eo'^ering the work of tbe last report j ha'5?e bean or shortly are 
to be subasittsd for publication. 

In the genetics studies; upwards of 2500 laiee have been pedigree-: 
and are at present 8-13 saonths old? to be harvested at 16 



A previously imdeseribed pel'S'ie inflaimaatory disease has been 
recognised in males of one strain. It apparently is secondary 
to occlusion of the urethra t^ a proteinaceous pltJg presussed to 
be of seminal origin. Genetic factors apparently affect itj 
scsse crosses being more susceptible than others. The lesion is 
of interest because it is lethal to the artferitis^prone strain? 
because it has interfered with biochemical analysis of ^8riae in 
mice; and because it may prove to be a deleterious effect of 
celibacy. 



Serial No. NIAMD^M, 
Page 2 



Proposed so^srge of t&e projects the genetics ssperiiaents ssill 
reqisirs a ysar for eoispletioa. The genesis of tlie goii. 
dissase will te stedied further bj eastratioay breediagj 
east examination for struetm'al anoiaaliesj seareb for a 
neural sausSj possibly related to a gastro-h^iatopoistic 
defeet. 



Part B Ineladed Iss 



Serial Ho. 
PHS-NIE 
Individual Pro.lset. Report 
CaleMar Year 1959 

Fart B; Hoaors, Avjards? and Piiblieatinns 

PsiblicatioKS otSisr tfeaa abstraets frcsi this project; 

LernsFj E. M, II and Sokoloffj L. s The pathogenesis of feoae and joiat 
infaetion prodiaeed In rats by Streptebaeillus monilifonais, 
A.M.Ao Arssh, Path, 67s 3€4."372s 1959. 



Sokoloffj L»5 Lilliej R. B, and Andersons F. 0« 2 k papain digestion 
apparatus. A.M<,a, kveh. Path. la press. 

Sokoloffj L. s The ccxriparatii?© pathology of artlsritis. In "Advanees 
in feterixsary Science" (Brandlyj Gc A. and Jiiagfe®rz*s 1, L.^ eds, 
Rcadeaie Prsssj, N. 1. In press, 

Sokoloffj L, s Osteoarthritis is laboratory aniisalso Labo I^B'ssto 
In press. 

Sokoloff 5 L, 5 Gtirrent Cosaasnts In praise of folly. Artli, FJieiaa. 
In press. 



Serial No. IlIAJ'iD j^J^ 

1. Pathology & Histosfessiaif 

2. Hematology 

3. Betbesda 
PHS-HHI 

Individ^ial Project Report 
Calendar Year 1959 

Pro.leet Titles Sttxlies on normal and abnox^al hemoglobins. 

Principal Investigators E. k, Itano 

Other IiwestigatorsJ S, J„ Singer 
E, Rolaiusea 

Cooperating Unites Bepartasent of Cfeesaistjyj lale Universiti" (Singer) 

Man fears 

Professionals 1»5 
'3 0.7 



and clinical signifieafjee of th© nonsal and abaorsial hesao- 



Methods s-aployeds Mo'^ing boimdary electrophoresis; speetrophotaaietryi 
:.grap!ij. 



:• fiedingss The fetanaia adiut SO-h^aoglobins dissociate agspsse- 
trically ia acid into unlike sublimits and ree<^bine whea 
nsKtraliaed (195B report), fte submiits are sjssaetrieal pairs 
of th© a- and p-chaias of fc^aogloMn and are designated ttp aad 
p^9 respeeti'sfely. Eesaogloaias S and are abaorsjal ia the 
pSshain and noraal in the a-ebaiaj wheraas hemoglobin I is 
Kormal in the p-eliaia and abno^raal in a-eliain. Acid dissocS.™ 
ation and reccssbinatioa of hemoglobin I witb. either S or C 
rasialted in the formation of normal adiilt hsmoglobia (a) and 
a h^oglobin ecsaposad of two different abnoisaal chains. Appli- 
cation of the Ejethcd to otlier hsmoglobias showed that hemo- 
globins Dj Ej'snd J are abnomal in th© p-chain and that hemo- 
globin Hopkin^-2 CHo-2) is abnos^al ia the a-eljain. Moreo'^ers 
a doubly abnoxiaal sol©erale ec^pos©d of the abnossisl ^-ohain of 
hsffioglobin S and th© abnormal a-ehain of hsmoglobin Ho-S tos 
been d^Eonsjtratsd in the hesaoglobin of an individual do'abiy 
h@t©rosygotis for the respestiv© genes for these hemoglobinso 



Serial No. NI&MD z.„M^ 
Page 2 



Other workers Isa'^e reported on th© basis of farailial studies 
that h^ioglobin S and hsaoglobin Ho-2 ara eontrolLed hj 
different genetis loci. The present resiO-tsj whieh show 
that thsse hasoglobins are ateos^al in different ehaiasj 
signify? that the a- and p~ehains of feeaoglobin ars controlled 
by different loei. 

Significancs to the program of the Institutes farioHs saetabolie 
disorderis in man are known to be assoeiatsd with irsherited 
absence os" deersass of a partiewlar enaysiatie activity^ A 
possible meehaniaia for apparent inhibition of astivity is 
■Jhe syathssis of a structiirallj ateoriaal easyme imder the 
eontrol of a mutant gene- Sinee it is rarely possible to 
obtain ensyses^ espeeially hissan essyaesj in adeq'i^te purity 
and qijantity for eheaieal and physieal efearaeterisationj 
this postidate is diffieixlt to test dix^aetly. Howe^erj 
emrsnt eoncepts coneernlng the genetie eontrol of protsiK 
s^-nthesis are equially applieafcl© to enspsas aad to proteins 
not elassified as ensues. The adiilt hesioglobiss of Eiaaj 
lihieh are obtainable in large qsaantity and in nismerous 
genetically abnojraial formsj thus provide an extr^aaly ussftal 
system in whieh esperiiaentai findings will pro'^'ida generally 
applicable inforaistion on protein ^nthssis and on th© effeei 
of Hiutatione on protein strustisre. 

Proposed eotarse of pro.-Jects H^ioglobin Hopkins-2 will be further 
iswestigatsd both 1^ reeombirsation and hj chsraical studies. 
Other abnoiraal hesioglobins will bs tested by ths teehniqiie of 
aspmetric rseoisbination. Anj forms that prcsaise to yield 
new infosiaation regarding the genatie control of haaoglobin 
synthesis i^rill be purified and analysed chemically. 



Part B included 



Serial No. NISMD ^.,J2„ 
PES-NIH 

Indi'ifidiaal Project Report 
CalaMar Year 1959 



Part B . Eonorsj fttsrardss and Publications 

Pufelieations other than abstraets fr(sa tbis projegts 

Singsr, S. J, and ItarxO, H, A, s On the asjiHsetrieal dissoeiation of 
hman h^ogloMn, Proe. Hatl, Acad. Sei. £^% 174-184, 1959. 

Itaao, H. A., and RoMnsonj E. 5 Fosmation of noraal and doubl-j ahnonsB.l 
feaeuBoglofeins'bj rsecsibination of feasaoglobia I with S and C. 
■ Natiir© l^s 1799=18005 1959. 

Itano, H» Ac s Molecialar disease. Syaposiim entitled "Ens;yiEas in 
Health and Disease^';, edited by D. M. Greenberg. In press. 

Itanoj H. A, 5 Singer j S, J, and Robiason? E. s Chsmical and genetieal 
ijnits of the hsaoglobin moleeide. Ciba Fowid. Symp, H'CSisn Bio- 
ebsisical GenQtics (GhtsreMllj London;, 1959. In press). 

Itaao, H. A, and Robinson, E, t Properties and inheritance of hasrso^ 
globiri "by agpsietric rest^binatioa. Nature. In pres;3. 1959. 



Serial No. mmD-^BB, 



1. Pathology & Histoehemisi 

2. Hematology 
3« Bethesda 



HiS-NIH 
Indi^idtsal Projeet Report 

Calendar Tear 1959 



fe^^- 



Pro;|e©t Titles Swlftoydryl (Mereapto) Groups of Hsmoglobin Studies 
on tfa© Natm'8 of the Mereapto-Mereapto Interaetioa. 

Gcsaparison of Worsnal Mtalt Hmian Hemoglobia with 
H«S!noglo"bin I by "Fingerprinting", 

The Combining Pokier of Norsnal Hissan H^noglobin for 
Nitrosobenaeae , 

Prineipal Investigators Makio Uurajama 

Goopsrating Unitss Medieal Researeh Comieil Unit for Moleetalar 

Biology s Ga'^endish Laboratory, eambridgej England » 

Man Years 
Totals 1 
Professionals 1 
Others 

Project Besssriptions 

Objeetivess To sttidy the function of SH groups of hemoglobin in 

relationship to the process of oxygenation^ 

Methods employed s SH groups of hemoglobins were studied by means 
of hea-^ metal ion binding using the rotating platinwn wire 

electrode as an indicator electrode. The data were analysed 
jnathejnatically and therefrcsn the SH-SH interaction constants 
were derived. 

Major findings s Hemoglobin SH groups interact analogous to the 

well known heme-h^ne interactions. There are two interaetion 
constants J there is the '"toe" and the "shoulder" sigmoid 
coefficientsj respeeti'^elyj of the binding eurre. This find- 
ing suggests schizophrenic character of hesaoglobin molecule. 
The underlying meehanisffii of the mercapto-mercapto (SH-SH) 
interactions seems to be due to the sterie hindrances as in 
the heme-heme interactions. 



Serial Wo„ MI AMD =•■ sa 



Page 2 

Preliminary studies indicate that the aitrosobenzen "wedge" 
decreases the energy harrier due to steric hindrance with 
respeet to SH groups; the "wedge" also influences the SH-SH 
interaction constants. Hr. Max Perutz of GaTendish Laboratory 
foimd hy the x-ray diffraction studies that nitrosoheazene 
acted to ^'open up" the mercury binding site^ (SH groups) of 
hemoglobin aoleeule. 

The @<Mbining power of normal human hemoglobin for nitroso- 
benzene was studied | results indicate '^at the hemoglobin 
binds nitrosobenzea© about 6-10 times more strongly than 
oxygen, ^H of the reaction is about -24 kiloealories per 
mole in contrast to about -10 kiloealories for the process 
of oxygenation, 

A specific eheraical difference between the normal adult human 
hemoglobin and an abaormal hemoglobin I was studied by a 
method Icnowa as "finger printing". Tryptic digest of each 
protein was subjected to electrophoresis on filter paper and 
then followed by chromatography to seiaratQ the peptides. 
The result showed that peptide 23 of hemoglobin I contains 
tryptophan whereas the corresponding one in the normal does not. 

There must be other amino acid in^ol'ysd in this genetic change? 
tryptophan is electrically neutral. Accordingly, the study is 
in progress to find out the other amino acid which is respoasifeie 
for the eleetrophoretic mobility difference of hemoglobin I, 

Proposed course of orojeets Sulfhydryl groups of hemoglobins will 
be studied by means of heavy metal ion binding using the 
platinum wire electrode as an indicator electrode. The data 
thus obtained will be analyzed to find out to what extent the 
sulfhydryl groups interact. It seems ^^^at the sulfhydryl- 
sulfhydryl interaction is extremely important for the under- 
standing of heme-heme interaction aeccsapanying oxygenation. 

It is also proposed that a specific ehesaieal difference between 
the normal adult hisman hemoglobin and an abnormal hemoglobin I 
will be studied. This investigation will be carried out by a 
method known as ** finger printing" techniques a tryptie digest 
of the protein will be madef the hydrolysate is then subjected 
to electrophoresis on filter paper and then followed by 
ehroBiatography to separate the peptides. 

Quantitati'S'o assay of amino acids of the peptide is in progress, 
Spinco-Spakmann apparatus will be used. 



Serial No. NIAMD •» 88 



Page 3 



lastntsaenta-feions pK Stats This is an instrument to maintain 

a constant pH by continuous addition of acid or base during 
the reaction. The instrument was designed around a Leeds 
and Northrup pH meter. It was designed so that a Brown 
servo amplifier "knows" how mush aeid or base Kust be added = 
The instrvenent is used in the study of hesnoglobin peptides. 

Autcmatie foltage Scanners A 10-turn potentioineter is made 
to scan eloekwise 3s600 then stops; and then imiaediately it 
reverses itself 3?600 then shuts itself off. At the end of 
these operations the whole of polarograjihiQ circuit is also 
shut off. The eurreat-=voltage eur^e thus obtained makes 
possible a more eons'enient and more precise Heasurement of 
constants v^ich characterise the cur^e. 



Part B included les 



Serial No. WLmJL »» 



PHS-NIH 
Indi^idiial Project. Report 
Calendar' Year 1959 

Part, Bs Honors, Award Sj and Publieations 

Putelicationg other than abstraets from this projects 

MBrayama, M. and Ingram, ¥. M. j Comparison of nomial adialt h'laBan 

liaemoglobin with haemoglobin I by "fingerprinting". Nature iSjs 
1793, 1959. 

Mtirayama, M, s On the nature of the interaction between binding sites 
of hea^y metals (saereaptoHmercapto interactions) in normal hwsan 
hemoglobin. J. Biol. Chem. 2^s .,.,.5 1959. 

Murayamaj M. s The effect of nitrosobenze on the mercapto-mereapto 

interaction of hOTian hemoglobin. Federation Pros. 18s 496} 1959. 

Murayamsj M. s The combining power of normal hiJinan hemoglobin for nitroso- 

benssena. J. Biol. Ghem. In press. 



{lttach«seat I) 

Serial ^io^^^^^mAm^^^^^^^^^^^^^^^^^ 
le LaboraS^'' ^f Pbaraacology 

2.-> Section oa Pharraacology 
3o Bethesda, Mssylsnd 



?^% A. 



pHs«jm?. 

Ihdividiuffil Project Report 
Cal^sdar lee^ I9S9 



Praject Ti'Kleg Physical cbismieal ©sd Bstsbolle factccps relating Uie 
acljion of pl^ysiological aad phanaac«^of ical agents M 
excitsfole cel2,s svsh. as n&rve ©ud tmsscleo 

Prlaeipsl Imrestigsfcora DTo A^ M» Shsmes 

Otha? liawstig&torsg Srsa Co Paul Blanch.! (Vlaitasg Scientist), No Lo 
Cfei'shfeld, and S„ Winegrad {PHS P'ellow) 

Cooperattag Tfeitsx Hosss 

Man Tears {caXeadar year 1959) s 
Totalj 3 - 1/3 
Profesaimals 3 
Other? 1/3 

Project DescriptioniE 

Ob^QCtlTOSs "SJhe wcspk is continisiBg ©Icag lices ascertaining tha role 
of icmrowKei^ 5.k tbs bloelectrical prapertieis of estcitable tissues tbrcmgh 
stwdies of tb3 aotioa of tretabolie inhibitors, dsvgs and ioas oa nerr^j, 
skeletal and cardiac Biaselei it saSjraces tbs foUowiag projectss 

(a) the eliaeldaticas of iBie role of oalciiam iona to ?aascle coEteaetion 
from the stmsdpotot of its laD-seiEairf^ through aad biKdii^ to eeXltilaa' aspecta 
of striated tississ, sand the erfeeat to 'i^ich dnsgs isill infl^aence thee© 
calcium proceseas 

(b) tfa0 s.7stes5atic study tjf the role af calciim in the eoatractile pro«=' 
tiQSS of ieolatsd ■vertebrate hsert mmcle as affected by pigrsiologicel 

conditicjriSj^ and 



Part B ii}.ali2d®d 



3eri«Jii Ho, 



a««=. 



(c) the study of 'Uie effsc'te of tbsse pbaraacologlcA sgessfes oa saoao^' 
sseleeiilar I'ilms of sifflpls aeleetjles and of cell'slay esta?acts« 

Mi sthods s (a) The rate md qoiaHtity of (Ca*^^) eatiy aad esit in fe'Og 
stri^eT^Scle aad i«R.g«tos& pig ats-ial a,pp®adage ape a:®©8Es«sd ia a©d:la 
^aich ffli?e eitJ^r siodlfled t«5.th :re^>ect to its iosjie coatent or 'bo 'fcMcb 
pharmscologlcai agents affecting contracit4.€a ^rs added | coivpsriaoss Is smds 

betsssea ao&^contrec-ljlz:^ smd coKtraetiag ccsaditions., ffes tjse of a saalX 
sBiotmt of eoealne (2 t^ % -^ IM wg %) elitsijaates spoatasKs^sss -feMi-feehissg i& 
frog striated misele, and the gtdssea pig ateisl appends^ at Toom tssjpsi-'gitiri 
doss not beat spcsit-aaeoijslyo Badioealcitm movBHsn'^ in e®m^eM"?^' tissm 
are followed as modsls for calcitsa blndis^ lis tte iaterstitiel oossKecti-*© 
tiss^ae of naiiscleo 

(b) Left atrial appeadsges frosi small g^ilsea pig feem'tSj sisspeaded by 
specially prcpasi^d clips, ase attached to a sejssitlT© traHSdtscer designed 
to qtmatitati's^ly record chajjges la tha eontractioris of this "biological 
preparatdoBo Tbs sno^ssents ©f rsdiocaleltia ars st-odied on the sas® prspa'- 
rstiem in ^ieh the coatrscti.oi® are reco2"dedo 

(e) Msnola3?BPS ef steasdc acid, ayslin ©xtractSj or pmdfiod coHponeats 
of eKci table cell asi&ranee ®r® spread tm RiagarSg soluti^sa ssd Ifcs Sffl?face 
pE*essur««area releitloa, ^ich is uaiquss tap each 8«rf ace fllK, ia stradlsd ®s 
s fimctioa «f chaagissg conditicsas in the Rissger's sdiitioa substapatSj i e„^ 
ealciisn ecnoeixtration, jS^ asad dftigSo 

(d) The sm=f&ce pK)8a«ifie«"area relaticsae of sxscfws® fUjm of drags oa 
■wwti&aa stifoistmtes ©re stisSied to detea?asi2se the stsrie relatioa© ii^iich esdBt 

b6t^@«a dmgs isfaich bjf?© simiXa^r eheiaieal stm*ctwsi?es biet haw asarkedly dif-> 
fereat phffi?ffiacologie^ prop-ertieSc 

Major Fiadjiggss 1* Effect ^^^of^^ejaffeia e on Ca^ ^ ^ mfy&m nt ix.^^t^^ 
8|gl«^d "''tS ffleafcraBie 

dE^SS^SaSro^iiscreases Ca^ infltse and oiitflUK «?>proal«ateX7 itoee fold? 
the inereasa ia, owibflits: de^felops at a slcPH^r rate thsffii idss i3ac3?ease ia ia= 
fltis, Caffetes iscreasss infltffi: darixig potassitm depolarissatlon. btst has no 
effect tsa. "tei increased infliss: dus -to the iMtisi potssisii;^ depolarisatioa., 
Caffeia© affects calcitsa sites ia the msnbvanB which are distinct from those 
affected hj mr^rwe depolariaatlon, yet both sites aire related to the 
ccsatractile pi'ocesSc 

The increased caLciwa otiUlux dtie to caffeine is only slowly rewrsibX.e 
iipon removal of caffeinei the increased outO-^sx is a3.so dbsenifisd ia the 
absence of external calciiaa @sd in l^e psj^senee of EDTAo 

Caffeine has no effect an Ctr^ t^ta^ce or exit from Achilles tendojio 

2o The int8raeti<M8 ^^,^of ipns^ airi^_ <^ with s pr faee^ filae of _ stearic 
acidg^ A a?e;^BiSEaCl^e"TOX^E8^"°3r«iinSoa^ 

cevadine, TSTace-dne, and imratr^mine isere used to deiaonstrste that phfiinasco- 
logical activity m&j be descra&ed tn texrms of physiOMshemicaQ, pyopartiss of th*Ji 
drugs, Tbs esccitatory alkaloids ^-veratad.disss© and cevadisse » «^r© ehmm to orJ 



Serial Ho, siAMJ-89 
Pag© 3 

horizontally as wall as irsrtically at tie air/watey laterfs^ei ^erata'amlDe and 
tb3 local anesthsties orient only horisontally, and 'ssraceriBe ( a sslati'wsly ixi- 
srt agent) is not surface active and eonseqrasntly sho^rs ao prefegred orieataticsi 
at tlie air/water interface <, The aaanar in ^icb these drugs 03?lent deterairiss, 
in part, the astent to which they interact ^th monolayss's ©f stearic acid.. 
The excitatory alkaloide ;p8nQtrate and interact strongly wi,tb the monol&ysr ■vdiicb 
s-abseqyently leads to tmstable Mxed fil^ss o£ stsarate aod th© drtjgo The local 
saesthotice and veratraalrss siba\s «eak interactions with tte istearats monolsj'es'j 
and verecevine, •^ich is only slightly strfface scti-g© does not interact with the 
ste Pirate filmo 

Slgn^if ieance ^ to^, NXA|@ Risse8ffi;ch| Ow sttsdies in calcium provides a basis 
for cot^aHag"a«Sr*i^"''^mOTm^rMssttS3j' especially in strl&ted, smooth^ and 
cardiac musclss^ where the contractile process has Iseen iagjaired by pathological 
conditions not relatad to inasr^ation bat to the contractile tsschanisni il^elf ,. 
lormal and abnormal mmole can now be characterized in regards to elianges 
calclism distribnttcax, assoclaticsi coastan-tis and tttpao^r rates during activit5»'u 
Caffeine can be i©ed sm a pharraBc61ogieaI egsnt ^lieh Ms a direct effect on the 
cell cBKtorsaB process involved in esBita&ion^coatraetioa eot?>liji^<, 

Alteration in calcitaa n&stsbolism in the patholcgieal conditiosjj of 
©rthritiSj snxssculfflr dystrophies, asd ecatractures can now be exaiaimdo 

The fiadiiigs to date on stirf see films indicate that a means of assessing 
the nature of the physlcaj. chessdcal interaction of drags aad ions 'with cell 
i]£3B&ra»e ctmstitusnts in a model ayet&ai is at hand. Tl^se nay liltimately lead 
to a q^i&ntitatiw deecrlpticsi of phamacological agents in terras of easily 
Ksasured physiocheriical propartiasr The marface film sttdies also provide a 
means for sttsdying ensyssatio processes which occur in the cell msuforanso 

Proposed CoOTse^of Projects Calcium binding in ns(UBcle and tandon of 
aB!phibTan^md''Sfflffll wlSrbeSiaracterised by alteration of pH, ionic enTylormsnt, 
•becperature, and as effected by physiologic&I. wad phari;fsaeologi.csl agentsj 
especial attentiwi will be given to determining the sites of binding In tba 
sarcolemns. that are related to the contractile process. The regulation of 
ca3,ciuia moveiaent as affected by raetabolic Inhibitors (iodoacetic acidj, dinj,tro== 
pbencl, anoxia, etc) and by inhibitors of acti-ro transport aysdh as the cardiac 
glycesldaso 

Tije study of siipface films tdll extend in directions? (a) atisdiee ^iltb 
o'liier dtn^s and ions, the phyaiological aad pharnaacolcglcal effects of which 
In eaccitable cells are well characterized^, to deterndns how general is tbs 
specificity and parallelism of the physico«cheKi.cj!al film interactions « (b) 
St\xdis8 iJith film conposed of Molecules of greater coffipleodtyj e«gc, the in'=-- 
dlTi.dtisl coiBpon.ents of calliwlar lipids and pusrifled erbracts from tissues with 
assnforais38 eadilbitia® different electrocheiaicsl prj^jerties (e^go, chemically 
eaecite-l taeDSjraneg such as occm* at the isyone'arsi jiinetimi as contrasted tJith 
elRctriej.illy excitable oisBisrmies) » 



Serial Ifco^„S» 
Fege h 

Sadividti©!, Project Report 
Galeridsr lesjp 1959 

E^!L2jh Honorsj A-Kards, and Ptsblicatdons 

pTibli,ca.ttons other than abstracts froia this projects 

lo Shaxies, Ac Mo aasd Bianchij C, PoS ThB Distribution and 
Kinetics of Iteloese of Rediocaleitjsi iix Terdon imcl Skeletal yiuBdl&o J. Gen., 

Z-, Shanesj, A, IL ©nd Berioaaj, M„ D„i The Kinetics of Depreesi^jn of 
Fofcasetoa OtitfliEc by Cocaine in Toad Sciatic Nerve <, J„ Pbarmacol^ Ejjpo 

3o Gesfghfeldj, Norsnan L^, &M Sfeanes,, A^ M<,$ Astsgosjism of •'Veratei*.'® bj 
Calaiua Xoa in Moaolaysrs of Steei-ic Acidl Science l|g$U427«lJi28j \9S9o 

ho Qershfeld, Sf^ I.oS Tfce Bsfluaiifte of Stnsctm^ oa Mol®cul8j? 
Orientation at the Air/tfJater Xatsrface<, F«A Studies for Tes-atraa alkaloidBo 
J, PhySo Chemc 1959* Sa ps-osSc 

5« Garshfeldj Uo L^j aad ShKieSj^ A, MoS St^i:iJ.a®r and Labiliae-i' 
Effects sjkJ AEtagonlsBi Dcsoastyebl® with Mc«somoXecul>'S2' Filns. Ja Ph3?3iolo 
iP59e Ssi press.-; 

60 Bimchi, Co P.J, sad She^sjs,, A„ M,s The Effect of the Xosjic 
Milieu im. tba EtsgiygeagBe of Radicealclim t^mx f eadoza and froa SaytaPiiss 
Muscle* J» Pb:?siol» 19^9 3n p?©si3o 



Hrarioirs afid Awards relating to th3.s pyojec^li? Nojh 



2o Section ea Pharsacology 
3o Eetbssdaj Msp?ylaad 



Pfixi; Ao 



ladiTidtial Project Report 
Caleadar loar 195'9 



Project Title J Mechanisa md thsrspy tsf stock asad of delayed 
deaths folle^dsg bum© la hisEjaas 

Prineipai Ija-irastigatoras Drso Nicholas 1» Kef slides, R* C&sl 
MiUlcaa, Kehl J-Iarkley, m, md S» M„ Rosesstbml 

Oldaer Ss'jfes'&igisitsa'as A grotp of Peyjcvtsa doctors., 

CooperatiBig tJsltaj HospltslJs J^oaj'^za., cM Hiao and Dos de Msoro^ 
Liiaaj Pern 

Jto. Tears (calemiar 3?©sr 1959)3 
Totals 9 
ProJfssisionalj 7 
OtSisrss 2 

Project DcKcriptioisj 

Ob;}ectiv98t Cllnioal evaluation of plasmif plmim albusslr^ smd 

lK?g© wItbsqs of "sSlS" soli8tion« 

The basic cschaaisra^ spideraioli^jry aad tr®atB!gEit «f PBetrioa-jsmB 
aEd Stfflphjrlococcue septicemias Vn&t follcw extesjsive bimis ia tatasBs, 

Causes of deal^ed deaths othez* Vim. infection. 

Methods Eti^loffids '.Phe clinical stTxXy in Liasa, Pen?. inrolT»es 
corapayisGR oT"plamaj i^^S ailbmin and Isx'gs toI^jjjbqs of e«li»9 in the 
tbjsrspy of bism shocks 

Balf of the bicmsd patients vee&±v^ large doses of gamma 
globtslin iittjcaas^c^slftrly on adBiissioiai msd dwdtjg the first 10 days after- <. 

On the ®pp®s(r8Bac3 of a pesiti-ro blood ctilttcpe for Pse^Tadot«?aas 
jes^gosaj, a specific antissrixaa is edtaijQletea?ed„ The mitis03?m'''''wsi^"'''""' 
"3©-8is^^3°'in OTJj? Ijstooffi'atoapy (Dif« MUliean) sad prepai^d by Ledesle L^So 



Part B indtuSed Te© 



Serial 



]Ekt8BSi're eheroicsij iia"aunolog5.cal and bacts.riological studies are 
(carr1.ed oixt to datercslae affeeti.'aeaess of therapyo 

M^^OTjlj^iagss Hie sffeetiveH^&'S of oral sallas solufeioKS in "ovrn 
Bhock l-^^b^Tsilssterfcia-fced la an eight ysisr atwdj* Oaly oo© death ll'^om 
i&oek has oectirrsd iii o-ser 90 adtjlts treated with s^lne alone vhile the 
mortelity isn tte plasma g2f©i^ ia adtjlta «a0 12 per ceht^ Eoiwe"S3S'y .to elrildr©K 
tiader 3 ye-as's of agej, sob® added benefit ■usb sho'^m f er plasma ^Ivb sallj^ 
they^y 1x3. ssortsasty fi'Oi® shock (9 ^ ^rsiisB 3$ ^)„ Wb3t'«r tJiis is ;& colloid 
effiBct or an. iKManolcgieal effect reciaiBS to be ©stablish<sd„ 

The adadnislr-ation of gaairaa gldl>vlin prophylacttca!l.lj has had a d©f inite 
effect ia redtjeinji septieeruiaB, While a mora extensive stta^F is seeded for 

fl^ml cosiClKSioaSj the rediietioia has beesi statieticallj sigaificaato 

The ass of Psetsdoaonae antJ^enm has also beea too liadtsd for flaal 
conclujsi<KJS« Hosre-^r tfesre bsvs beers Jf siiryivgl-s oxst of abo'ist 25 casee<, 
In %iB past in over KXi cases ttex^ ha'^ beea ao s'm^'vox's follo^^iog a 
positive disigaosis^ in spltcj cf toteaslv© aatifeiotie ther®p7o 

Prepesed Courgs ©f tSse Praiect? CoHstiJsaed stTidy of Rffichssiisims eed 
therapy of shock md of dela^d deams folloidiig 'bimrBSv, 



(Attachnenb X) 

Serial Ho « JSUm-BO 
Page 3 



PHS-NIH 

Isdividiial. Project Beport 
Calendar Tear 19^9 



P|rt__Be Honors, Awards, aad P^licaticms 

PtSjlicafeions olshav than dbstraets turn tfeis projects 

1„ MasMsy, Ko, Bocorsegra, H.,, Bsaanj A„j, Tcjsple^ R«j, Cbl&ppoi% M„ 
Morsles., GoS GliideA EtSLlxiatitm of Saline Solijt*ioa Thsff^y ia Bnra Stojk 
Ho CoEpsyison of PlaSEsa Ti^rsmj ^tb SalixiQ Soltjfioa Therapyo Jo A, H, / 



Honors sBd Awards reXatiKg to this projects Mobs 



(AttaclusBEit X) 

Ssrial ^o^^^^m^^l 
1« Lsboratory &S Faw^-ia 

axsd Tcjslcoiogy MlkYiD 
2, Section on Phejrmaeolcgj'' 
3o Bethesda, Ilsry2-s:Ad 



FHS»!fIH 
Ii3diTl.dtasl Project Repert 
Calendsr Year 19^9 



Psa"t 1* 



Project Title 2 Sstii!5s.tions ratabollsaa sisd fteetioK cf spepjaias^ 
spes??HidiTis, ead relBt©d pol^Ms^s 

PrJ,nclpsl Bi'^stigatos'ss S,, M^, Rosenthal, D^ D^SslUj. ©sd 



BsB. X©ars (eslendai'' year 1959) s 
Totals h 

'.t 2 - 2/3 
5S 1 - 1/3 

Project De scrip ti.ons 

Obje^l^egs Itifi inportaace of tliese polji^Maas is shoxvB bj-' 
their ^Ide dis'trlbutioa tn -d-rusiSSj, bacteriay plant aad aailmel cellSo 
Studies ai'e conducted to eluci.date thsii' K©ts^olSsm satd tivMitlmip and 
relation to deseasBo 

Meth^sg The ©fB5.r«s arei stwiied by iso'iiiopic l-abeliiti? aad special 
cJiTOffiitograp^cTecbBiqass, «sin.g bacteria ead atiimals as test db^lects,, 

lSstS£.S^I:^2* Eiosyat^iesis of eperBiidine (see s^epoyt 
froaj Section""on Ki^SemfcS. Phartsacology) , 

Asssj?" of px'imasy and Beeoncsiry afsiiies by dinitrofl^ordberiasaa ■ 
derivative 3 c It was fo'^zid that the al:>sorptlo)a spectrtim of primarj amiviss 
had a peak at 350 e^Lj, 'wh52.e the peak of secondary amines Has at 390 Bi.io 
U&irjg the 35o/390 ratio it xm.& possible to dif.ferf.)Btia-o© the po3.j®atoes 



Pert B ijieltidad Yes 



Psge 2 



frora their aeetyl deriTsttiiFes* The method Has farther f«fined te sake 
it se'^ral tisies eioy« seasiti's^s than the one in tase, 

MoaoaeetylptitreselBs mtd t^o 

'acstylsperraidine' is®3?e isolated ■? ^sk ^.,,c^i eslls. 
They ^ere characterized by behavior on ion esKJb^jge s^sins snd paper 
chromatography, by qtiaiititati'^ acetate and andi^ cleterrrliTiations on 
hydrolyzed sati^sleSj and by specific activity of C'^<»labeled GomycmLds., 
E^_coli dees not norn5a3.1y contain sperrsinep but vhsn gs-own iai the 
presence of spenninSj both mono«' and di.acetyl dei'ivati'^s ■mrs isolated 
f2t)m the cells c A similar result was obtairsgd ^th Salmonella, 



^ co^^ate oS aperslEfte Essd sperraidtes mth el'at&tJiionS:, A 
high percentsge of tns glntatSiioae xr lig ^o^i ceils was ro-usd to ©s:i^t as 
s conjtjgsts idth spesisidine ^ This laas ehsracterised by behavioj.' on i&si 
exchange resias ax^ oa papss* chroosstographyj by ideatiAieaticm of 
sj^rraidine, cystSjiej glycine aad gl^asic ssid after hj^Srolysi^j and b;- 
l«ii3ling 'jiith C^Ji japes'iJiidliie smd S35 added to tKhe asedium* That it is s- 
tnss conpoimd was ehossn. by absensse of exshsngs Miaa atteed ^th G^ 
spenaidineo Spei^mine is not aornsaily present in ^^li^ bwt -ahen added 
to the growth niedi^^, s, elnalar eortjtigjate ^th spersdas wss demonstrated 
Jin the cel3.Sa 

Proppeed Coaree^g Ftirther studies of tte astabolisra. fmjctionj, 
sad relSHm" to' " dlses^e of tlie polyaMrieSo 



(AttachsBnt X) 

Serial l^O'^JBSsii.^^ 

Page 3 

Bsdi^dtjaL Project P^sport 
Csl€«dmr Jem 19$9 

Part Bo Heaos'^j, Awsrd®^ ism ?^lieatica:js 

Pi&lics.tions cfcher th®a absfe-aeiits frora 'fchis project? 

Ic Dt±>iaj D« 1\5 Tbs &b®@.j sad C"fca3?aeteriigs.tl<^a ©f A^jies UsiiJg 
g^ii'^DinitrofliK^S'olseasens.^ J^. Blel<, ChBUo 19^9^ la press c 

2o IteiiSf Do T»s STidsaea for GoHjtsgat©© BetHsea Polyaisissss and 
.lysiaes in E„ eoH„ J„ Biol„ Gh.ea<. 195'^" Bi press „ 



Heaors aad ^■'^jards relatiag to t&is projects ifeas 



(AttscfcnsBt X) 

Serial K0„_^jj^»»9|^ 
lo Laboratosy of Fbarmacology 

and Teedoologjj HTAI® 
2„ Sectioa en Pharsj&sology- 
3o BethesdSj Marylssad 



Individ'j^ Ps'ojeet Report 
C@l©n.dsr Xesr 195? 



Part k. 



Project Titles Mechasaism and thas>apy of delaj^d deaths fo^J-Csstng 

Prlacipal liivBstilgatarsj Drs» R* Carl Millican»HicholiiB A„ 

KefalideSj aisd S„ M« Rosepfehal 

Other jirsrestigatca?gs Joha Do Rtsst and S^er't C^ Jaxjsky 
SI 



lim. Tears (caXeadar jresr 19^9) s 
Totals 2 - 1/3 

■si- 1/3 

1 



Ob^eeti^ss To obtsiJa asors potent aatissrum by is3£iU2d.sa.ti»i of 
aairoals ^litlTP^^^mfiSss .ae^'sginoea for treatssieat af esparisjeratal and elfciiie®! 
P^g_ agg.^^to.o ga IsarectienSo 



To sttidy facto3?0 fe the eaisse at delsjed deatl'is fe mice .fol«^' 
lafiftng- hum in^vsry,. 

E-^',alnatioE of gsrasa ^dbtslia ^id aatibiotics In experisissitel 
•infections o 

Me;^ods aaplojed': The prcsduetioK of fatal iareetioas •Kith 
orgmn^sms of'low"'iji^^!3.ee ira saiiaals sads suBceptible by btmj or 
toUTOl.qtjet traiMa or by izjjecting or-gSKistss in rauGia„ 

Assay of protecti^etj titers of vsriows sera agalsist theee infectioasc 

Observe the effects of ©jatibiotic susd other chemotherapy radtiei'ng 
the delayed mortality of bm'aed mice ©i£rid.v1jsg tte ac^e shock period „ 



Pjrrt B isislTided Ho 



Serial Kcj„___siMI?!_«'J2 
Page 2 



Major FXad'iBf^jj Kab'bit asstiser'am. agsiisst PaetdosBssas aeroE-iaosa 
Bas 800 tiBgss 'fflore efxectx-^ thaa feiSEi;^ gamma gldsiuiK agaiJEsst fatal 
motise iKfeetion and ■was ©ffsciti'^ e^en "^Jhen gtven Xl hmwB sthsT ^afectioao 
Antlser-ffli protected against 8 strains isolated from patieats Mtli c'i.ialeal 
PsCT^fflM£__^^^fe^g septicemias o P'orifi cation ©f t^e gaffsaa glcbulia 
c©Bpo358Rt oi* aatiserusa by either DEASU^cellislose cferoiKitcgs'apby or &&=> 
ffiaai"u>.ii si£Lfs!te frac'fcioxiatioa resiilted in a fom'"=^»fGXd iHcraase in pot-ssej 
Gi'Br that of 'l:toe crude antisertsa,, la eollsibor&tion ■id.tt; Ledsrle Labs 
siaa.!! qiiantity of reflried antiserum was prepared fca" elS^Acal 'toial in 
clteicfil septicemias in fena^Bed patients in L^-raSj, Psrtio 

Sevsral. cheaothers.p-3'a.tic! ageats ha^-e bssa desssssts'ated to rja.%«® a 
significaat effect ia rediscij^.g the de^^j^ed Biorts3.ity of bm'fsed ardoe 
6u^TI^5l,■ag tfee sciite shock p9£'iod» The most effect! 'fe of fhe^se sgent-s wm 
ebloraEpbsnicolo Lees ©ffectlT© sgents -sjes© m«asK. ga'sm globialia aad fee 
£jss»?sa of ffiles czsmmLescizig from b?jjk"ia iajtsry^ 3 to 8 ^saeks after injts'jo 
Ho differ©Bce vss noted i« the effectiveness of hmmi gasiissa gldbiilSa aad 
ceairal.eseerst mouse sertimo Tfee coaijined effect of sblos'sjsphsaleol snd 
caa"f^.^geent serxm tterspy «.'^*3 addltlro "s-teK eoe^arsd with each -feerspy 
alOTie„ A variety of othes? .isntiMotics (pol^tnyxlTij ol«sH.doBi5>«ia5 €:Ms3i?*>- 
tstrseyel:|jaej csEy^teseyellsej, ■festracyclissej aad aigasaayeia) irsgre feef-- 
£®cti.v@ la Im&rhxg the delayed mortality after hwut, 

Proposed Cotirse ©f tte Prcdeets E-i^t^ation of Ciatisej?taK. in the 
t?i8fi&E©nt of Fseracmoaas septieeisoLaSo 

lij'^fltigatioR of fsatibiotics to, delff^-ed dt^aths foll,0iidjsg braiss la 
Search for cames of deatei o-fesr tijaa irafsction in Ht&m aiceo 



(AttRCh!(iffitl1.t 1} 

Serial ^Oo^Jl^g;^ 
1„ Lsboratorj of " Phs^iESCology 

sad Tcslcologs'-j KXiaiD 
2e Section on Phsrssicologj? 

IQadiv.lcJtjal Project Raport 
Cal.eadar Tear 1959 

Project Title? Ttoe Biosynthesis of CboXsstsrol 

Pslncipsl Iss-vestigatops Dj?-. Kehl MarKLe^y in 

Othss* inwsstigatorss l-l3?Et. Eltsatscth SiKjUiaaa 

Gooper®tixig Unitsj Hraae 

Itea laars (calender year 19^9) s 
Total! 1-1/2 
Profeseitsaals 1 
Others 1/2 

Project Dsscpiptions 

OfejJecti-e©8s (1) To deliaeate the steps bj which ssYsloatiic ecid (WA) 
13 Qom^^eT""^ sqaaleae and cholesterol by EKusmali«s tissxssSc (2) To 
stttdy tfae stoiciotaetry of eacfe atep^, bsgiiraii^ ^th the first phosphosylatioa 
Btep of M7A„ 

Methodss Sttostratesg D,L 1«C^ M?A and D,L 2»C^ MVA, Bioaesay of 
enz^TOtic^^vit^'"*°Iftsr inactitration of eaayas by heatissg, ti^ 'i?8acm^ 
'S^^^Ts"qiSnlSatl^®ly "tossisfesred to Whatman #1 filter papor and chroEasto^- 
graphed lidth »-butanol«ECC0H«'H20 ijatil front hss wavBd 1$ cau Paper 'J^ies. crtit 
into 6 squares, 2<,1 s 2^1 era each^ and activity msaBisred in each- squarSc 
Square ffl contsJjis the product containiag the MVA'^residBa., E^yroe^^^p^ialfica'blons 
An acetone erfcract ponder of rsbbit Itver is treated with ajmiraSm'^lfate, 
protamitie sulfatej> mid DEfl&:>cell\2loee chroraatographyo ^dea ti ficetdoa^of^ 
prod^ts The product ^ phosphoiEe'mLoalc acid (P^IWA), sepa^teT'^ra pyi^O" 
phosphoase-ral-OTiic acid {PP«MA) hj ehroKatogr^hy in t«birfcanol««HC0CH«4!g0 sol-sant 
systetfio P^MVA separated .^t>ra P^ by chromatograpby iii BKtlianol^HH^-SgOc 
Fhosphste determinations® by EsetSod of Ch&no 

Ma.1or Findiap^s g MeTalonie kinase has been piarified 100 fold f^K an 
acetwie powa(ey"oif" r&bit liveffo This enzym catslysea the foynifttion of 
phosphoiaavfiloalc acid ssid AEtP faron the biologically active iaoaiBy of ssevalonic 
acid and ATP« The ©nayse was free of ATP^'ase and si^ldjiasej- Cys-feaiiie (or to a 
Ijsaser eastont other SHc-con^soxadsi^ phosphate^ said Hg^ or Iln^'*' ax*© reqiiired for 



P.arfe B included - -Yes 



Page 2 

activity, (Msaosine^, urS^dine^ H isioaixm triphospbatas eajsmot replace 
effectively adenosiae triphosphate o Tbs enayss is ii:5liibited by O,,! u-ffioles of 
P-cbloromerc'oribenzoate « 

^S^^S^ '^^S^.^ ^■^^ '^'^^® coatersion of P«lf7A to tSie nest product 
■will be stiKflie^y eazyns pttrlflcaticn fyom j'abbit livsro (2) To steady the 
fate of M?A In L%ct<^_acin.^i>8 aeidopMJLisSo 



HIS-NIH ■ 
^wSividtJiS'l Project Report* 
C&Lend&T Year 19$9 



(AttacbuKsit .1} 

Serieal Ho, SMifrf 3 

Page 3 



P?3blieatioRS oi^er than aSssferaicts tpom this pro^Jects 

Morales^ Gc, ®nd Garrtong A^s Clissic©! E-^uatioa of Selijie SQlwtioa 
Tfeepapy in Btsm Shcck» ITo GoB|)a?:ls©n of Plasa^ 'fhsrapy •Jdt^. Sa3iiv8 
SolwtiK'i flier®py„ J« 1.. M. A. ^il633j, 19S?« 

Honors arad Aisards relating to this project? Moms 



Serial *^0<>.^^J^s9i 



lo Labox'atoyy of Phsrmacoxogy 



2t Section on Phanssocology 
3. Betbest3a, Ste^l&ad 

ladi'fid'i.jsl Project Heport 
Ca1.ends.r Year 195*9 

pyo.iect Titles The chemotherapy of rmm® leprosy 

Prtiieipal Biv^stigatovs Ds"^ I„ To Chsxjg 

OtlKsr Ihvestigatos'sg Hone 

Cooperating Ifedtss The AnBTlean Leprosy Fousdaliiflss, Leosaard Wood 



Man Yefaj?8 (eal.erid8r j&we 1959)* 
Totals 1-1/3 
ProfessdoRSilx 1 



Ps^oject De@cy:lptioaa« 

0b;^Qeti;y38s !Rie evaluation oi' therapeutic effeetin^aess of dirags 
in iaoise Xeprosyo ""^e tiBist!© cultxcre of Intracellular parasites o 

I-Ie-fchods Eiffijlc^df Itttraperitca:js.al isafeGti<m of isdce wi-Si |^^,^^„ 
MCffi jIjac lTiff^"cSKt^^^mac"i^opheige8 in Le^^htoa tifcesa 

Major Fiadiagst DTo BarrySs riedao cosapound, B6635, a dori^rativa 
of phenas^ con^sotmds, showed asapked sT5>p3.*es3iT3 activity xm moisje leprosy^, 
approaching the activity of isoniaaldj wsight fop trelghtj diethyldithiol«>isO'- 
ph-fchalate, roas^kod astl^ty in Isr^ dosesj H. Js^c^bisCdl^alpha^pheaathyl)" 
decai35Sthyl«ftedisRd.T)te (SU u592}p H«(p«diB^thyla8ainociBiRafflyi}doaecylamiae 
(SIT S^ltWiij, orily weak actlTltyi phosphanilic .acids ■bis(p»'ag!iiaophe23yl)phosphimc 
scid^ fcisCdicjBiiiylaniiHophssayljphosphincfUS acid, 3«Bi8thescy»I^=aBdno--if<J«acet,ylajEdao=' 
diphenyl mifonss « deri-watiw of 9, 12«='dik®to»10"0stadecenoic acid (ERL 2hh)t 
leT0«'3«-ssBthaii:y«»lO(3 fi'«di!nethy!l.aid.Bo»29«ffi0thyl"'l''«'pz'opyl)-'phesaothiaairie (Hosissffiit) , 
aad ^'"heptyl-Z-ttiiofca^dQEtoin, ao aati^tgr^, 

Tb.e tsadiasi sxtnriTal time (ST50) of noinsal laice -wias 576 dayB, sad 
that of uatipsated lepyoey control j, 12X days,, fhe S?5o of ai3nJ.B3sae treated tsfitb 
variolas dsongs were as followBt I5DS^ 168 dsysi stareptOKycin^ 18? daysj 
zdeotlnaffiidd, 228 d&;ysj pjv&sismwL^g 256 dej^Bj iscmittsiidj 312 dayisi snd 

Part 3 included Xeis 



Serial JJo, 
Page 2 

hopfK sertunx^ Ha23ks» balanced sslt solistioa, bsef eafei^yio extract ssd f?i.5j3M.3'' 
prepared spleea hoiiK>genates from yovsog mice,, la one expsyiiBsrjt, the Kacre« 
phages were malotalrsed in gi&od eonditioa for 73 ds^^Sj althotigh tte cell 
poptalatiKi decreased to sfoout <me third o At the end of the espe:piaEiatj the 
total number of bacilli increased 6o5 tiires^ and the a-^rage length of bacilli 
increased 2<,6 tisaeSo Therefor©^ the bacillary mass increased a t-otsl of l6o5> 
times ^th only about one third of the nmcrophages lefto Had tiie otfeer two 
thaj-ds of macrophages tlis ssek chance to devslop^ there \fould bs m\Tch higher 
increase of the bacil!!,ary mgss. 

Proposed ^^Cotg^^^^ of ^ Fro:|eet-5 The leprosy studies «r® carried otst in 
coopQr^^i^rv2^rW^'Kmr^^^'^pro8y Famsilatltm. (Drc Chss3g is. on a Fel3j3fe'<= 
ship frora lAsem), The results are applied -to tl^^ir clinical evslaatioa 
studies o 

Continustion of e?a3.uatian of drugs in laoTiffie leprosy^ using both long- 
term and shorfe-'term tecbniqtaeso Goatinaatian in the stiKij of tissa^ culy^re 
af Mp JLepraa cag jian end M^, ttiserculosiae 



Serial So. J 



BidiTidtial Project. Beport 
Cal©ads2> Tear 19^9 



Pa^t Be Hoaors, A-^acdSj sM Prsbliestlone 

Ptiblieations otljgr ttoan absts-aots from tMs projects 

1„ Chaagj Xo Tot Effects sf Kai^aF^sia^ Sts?eptoY@yletec. Parsirsc-iayeinj, 
Horobiocia^ and Ristoeetin oa Mosfirie L&pvosy^ Assro Reve fti>e??» PtsIk^ Di,s<, 

2a Cbiffigj Ta T^s Erolatloa of MiiriEB Leprosy^ Asa-j?, Rev» Ti&ei-o 
Fxsla, Dis„ ?9s8055 



3c Change lo To J More Aboat tns PheRssias Hy^e Intlttfcepciiilosis 
Activity in the Phenastoe SerleSo Leprosy Briefs 2gs37j> 1959» 

I{o Chang J Ye T^ snd Doallj, Jo A„; Mercsptsn Coispeisids in Tii3e3*e\d£S!is 
«Bid Leprosy Leprosy Briefs 10 Jils 19$9^ 



UonoTB and km^ds relating to this projects Sojse 



(Attacbaent I) 

Sei'ial Hoo__J8I^'-95 

md ToBcicology, NI&JID 
2o Section oa Pharsnaeolcgy 
3„ Bethesda, Hary^iHsd 



PHS » lOH 

Individual Pieeject Iteport 

Calfindso- Tear 1959 



Projeefe Titles Tcsdsolc^ic studies of iodates 

Principal Batfestigstors Dr., So H,, Webster 

Othes' Swestigator? Mr^ Eo Fo Stohlmn 

Cooperating IMtsg Dr„ Bejsjassin Righman, KX&MD Pathology S.l 

Man Years ( calendar year 1959) s 
Total? 2 c 2/3 
Professionals 2 » 1/3 
Others? 1/3 

Project Descriptions 

.S^^SMCI^Ii The tOEicology of iodates as a basis for vse in iodate 

Method s Ecroloyed t iJRves-tigatica of analytical nethods for determination 
of iod&lbe '"ffiaS°'l^£(le' "In biological oaterials; study of distribution and es^ 
cretion of KID^ after ^rsrions rontes of adisinistratitm to rodents. 

Ma^or FJJtdiTi^ si Seasitiire HierocheBiioal isethods for idaitifjdng iodate 
end iodide ions in nrins, without the use of ashing, have been devisadc The 
teste used depend upon the liberation of iodine which is s^sequsnia.7 identified 
by maans of the sensitive atarch»iodine reaction^ Certain organic stibstances^ 
i*iich interfere in these tests, wsre present in nearly all urines exarainedi, 
One group of substancesj ^ich tocludes resoreinol, phenols, and ascorbic acid^ 
is capable of uniting or binding iodine j the other groupp -Khieh includes 
thiocysmate, ascoifeic acidj, and asthionii^, is capable of i^apidiy reducing iodate 
in acid soluticaio Ksarly all of ^hese ixtterfering si&tstances can be eliminated 
by « single treatnent tdth activated charcoal „ Minimal detectable amounts of 
KDD^ and KI hafe been found to be 5"17o5y KID. or 150"175y KI per iBi„ of urSneo 

A Biodifieation o^ the above Eethod, to permit quantitati^re evalua'^on 
of iodate in urine, has been developed and is undergoing tests for sensitivitT 
and nsliability„ Tbs most difficult aspect of this matter is the selection of 
a stable standard for this labile material. 

Part B included Tes 



Serial Hoo jiiABg><-95 
Psge 2 



Pr<:^o^d Courae of Projects (X) Testing of a qmntitatiins rasthod for 
the de?e?Sna?!^''oF'lo§StejE°1jriJ38 and biological materials „ 

(2) Study of the diatribtitlon and escx'etioii of ElD-t after various 
routes of administration to rodents^ using itsproved analytical techniques o 



(AttachsieRt X) 

Serial No. jjj^g^^__ 
Page 3 

PHS-NIH 

ladiTidual Projeet Report 

Calendar Year 1959 

Part Bo Honors, Awards, and Pyblieaticsas 

Pi&lications other than abstrscts from this projects 

lo Webster, S„ H., Riee, M. E.j Highcmn, Bo, aad Stohlc^n, E„ FoS 
The TeKicology of Potsssiiaa sad Sodium lodates^ Ho St&acute ToEicity of 
PotassiuH lodste 5ja Iltce and Guinea PigSo ToariLcelogy smd Applied Phsrsaaeolcgjrj 
ls8?"96, 1959. 

Honors and Awards relating to this project! 

Since February 1959 I ^"ws perf orBsed the diiiies of Chairaan of the 
KIAMD Editorial Board o This has involved ■Uie hacdliag of 300 aiantEicriptS; 
panging from abstracts to chapters of books „ Each manijscript required reading 
by ons or more referees© Vfhen no Board jaaober hss q?j®lified the Chairraan sought 
sad receiTred assistance from menSsor'S of other Institutes or occasionally tmm 
specialists oistside the NIH. Chairm^a e-^altiated each rerie^rer's eoismsats, 
partictjlarly ^*sn the saanuseript was not recommended for pt§>licatioa or was 
se-^erely criticizedo A few aant^cripts required reTision befoi® final appro-orf. 
was grentedo The files^ which had not been revised since 1951, 'wes^ coaplctsly 
racandaed and brought v^ to datSo A e^sw card index was set xjp by m&^ect and 
atsthors and a card file sjrBten was stsartedo This esKss it possible to instantly 
ascertain the statijs of any manuscript being processed o About ©ne«qEartsr of 
the Chair2aan»s time is occT?>ied ^th these dotieSo 



lo Laboratory of Phsraacologjr 

md Taaicology, NBl© 
2« Sestioa on Phanaaeology 
3« 



PHS-NIH 

Ijadl-eldual Project iteport 

Calenda? Year 19^9 

Part^ Ao 

Project Titles Patty chsages la mice iadnced by i^ort-tlBte fastis^ 

Principal Imrestigatorg Dr. S, H„ VJebster 

Other Brrestigators MTo Eo Fc Stolilmsn 

Cooperating IMtss DTo Ben^sKln Highamnj WSMD Pathology 81 

Msn Years (calendar ysar 1959)? 
Total: 2»2/3 
Professionals 2'='l/3 
Othsrss 1/3 

Project DescripticEig 

Ob^jectiyeat DeterKinatioa of the dtiraticm of fasting necessary to 
prodtce efoservsble fatty ehsagss in siich orgssss as liver, heart, kidaeys and 
adrenals of cAce atri the tiE» reqaired for reversal of such ehangeso 

Metl iod Emplcgyads Determiaatitm of total fat content and gross asud micros 
seopie eKerdJiat^'oa ' "of above ov^m^ before^ dtsfing and after fasting e Also, 
study of aegm. wsight and body weight changes cormspondlE^ to -Qisse three 
periods o 

MSiJIor Findings s Fasting mice for 7 hotcps was found to produce no 
demoriiw^SjCTTaE^iyehanges in the li"?ey., kidneys, hcsart or adrenals « Hoieewr, 
by increasSiig the fasting tiiae to 16 hours, Karked fatty infiltration was noted 
in li'rcr and kidneys and slight changes ■^ere seen in tesrt and adrenals „ On 
refeeding, tte teart^ adrenals, and kidneys tisijally had a nonasal appearance 
2It hours latere However, fat in the li-«®r persisted for at least another 2k 
hours o 2q addition, the body -Height failed to return to its original valtie within 
2h honrso 

It is known that the raaterial responding to fat staiM in tlie taotjse liver 
is netjtral fat rather than phospholipid or cholesterol o Since dii«ct sisethods 
for the estirsation of netxtral fat were not available, indirect amilyses of other 
lipids isere x^qulredo This made analysis of orgazs of indi^dual ndoe vary 
difficult, if not iupossibleo 

Part B included KO 



Serial Ko o hi 
Page 2 



Propoeed Course of Pgcjeets A new chsadcal nssthod for the dia^ct 
determination of neutral "fat is nm being developed else^^re in the NIH 
and it is eapeeted t!»t tliis esn be adapted to vse in the present studyo 

The effect of fasting oa taice for periods betweea 7 and 16 hoixre 
will be studied in more details Additional data vjill be secxsred ea the 
change of organ 'Height and bod^ ^aeight taking place during fasting and 
refeeding and "Uiis will be correlated with the hi^tole^ic chains taking 
place dtiring tlsse intervals e Such inf<s?taatioa should be of great ralvB 
in oral tosicity sttdies invol^lsg fasting mice sis^e it is often uncertain 
at autopsy whether fatty cher^es are caused by ■rolimtary fasting, iavoluntsry 
fasrfcingj or by action of an administered drr^o 



Serial Hov IIMS>„_,ai» 
lo Phenmcology & Toxicolo^ 

2. 3ioch@sdeal Phanaaeology 

3. B®th®sda 



PHS-NIH 
Caleadas> Year 1959 



iSSLA" 



Project Titles Hetabolisa of Histidine, Eistesaiue end Belated 
Iffiidasoles 

Principal lavestigatorJ Herbert Tiabor 

Other Investigators J (kvia Crowlejr, John Wolffs Alaa Peterkofslcy, 
Eiigo BatwTs Virginia CMlds 

MiA Tears (calendfir year 1959) s 
Totals 4«l/2 
Professional t 3-1/2 
Otbsri 1 

Project Desoriptioas 

Objeetiveas To study the biosynthesis, iaterE^diasy i9@tabolis% 
end phamaeologioal aeti^ltj of these eoB^imds In order to ma&se^ 
sta&d better their physiologieal aad pathological role, 

MaJQr Fiti^^ag Furrier sttadies have been carried o^Jt on the 
folloviag eoayia&tie reaetions iavolved in the metabolisia of 
histidi&eo 

Cl) Histidiae -* xarocaaie acid •<- ^3 

(2) Urotanie acid -* foraiaiilogl^staIaic acid 

(3) ForaiiaiKo-Xr-glataaic acid + tetrahydrofolic acid -^ 
S-formiffiia'otstrahydrofolic acid -}• ^gliatamic acid 

(4) 5~Foraiffldiiot©trahydrofolic acid -* JslO-aetheayltetnai^dro- 
folio acid + NH3 

(5) 5,10-Ifeth®ai|cltatrah3rd£'ofolic acid -* 10-for(saylt@tr«bydrofolie 
acid 

Part B included [X] lea [ ] No 



Serial Noo NIAMD-q? , , 
Page 2 

Cl) Jterther study of raactioa 1 in histidine-adaptsd Paeudosoaas 
liSB rscentlj bson beguce The purification prooedur® has be@n 
revised by tiia iaciusioa of a DBAS-columtt atap. Studiag in progress 
(Dto ?®t&rkotsk^) asp® partioularl^ conessKisd with further purifica- 
tion, elucidation of th® cofactor raquiremeats, sad laschaaigia of 
tho reaetioEt. 

(2) ]feay®® 2 is beiiag purified l^a hog liv^r^ usiisg bQatoait©^ 
USAE, and caloius phoaiMt® 0t@ps« ISie ©nsjsss has been |»3rified 
about lOO-fold* 

(3»4) Hisse two ens^sss have been purified about 7(X>>1000 fold; 
end sepai^tdd from eads o^ero Forffidainotetrabydrofolie aeid has 
been isolated, and obaraoterizede The kinetics and r@quire@®nts 

of th@ tvo ms^sma ha^e l^®n in'?e@tigat@d« 

(5) ^a kinetics of this reversible step ha^rs be^n studi@d| 
botb non-ens^iE^tio and ensi'Batic factors hav© hsen studiedo 
Particularly noteaortl^ Is th© rapid bydrol^sis rata in ■^e pa^sseaoe 
of pihospSiat^o 

Th® reversibility of this step at neutral pH is of particulm** 
ia^rtance since SslO-netlte^yltetxtshydrofolie acid appears to be 
the substrate of the reductase, which results in the li^dro^iEssthyl- 
tetrahydrofolic acido This is the pathway involve in serine and 
methionine bioaynthesiso 

Asa. ensyae has also been porified froia rabbit liver (Dto Qrawley) 
which ^irries out the following reactions 

(6) Igtidasoleacetio acid -^ l-pyrophos;dioryl-5°"Fhosphox7lribose 
i^ iaidasoleaoetic acid ribotide. 

This reaction is of jmrtioular interest since it is the first 
in yitro desaonstration of the possible sedbenissa for the asEOTiSition 
of iaidasoleaeetio acid riboside after administration iaf vivo of 
histamine or itaidaaoleacetic acido Dr. Ho Bmx@r has synthesized 
chcBioally the riboside of both histamine assd of i^ddasoleacetic 
acido 

Procedures have alfflo b©sn developed (Dto Wolff) for idie saalytical 
sepax«.tion of er^othioneine, h^^ynine, and thiohistidine prepax^o 
tory to studies of th®ir biosyathssiso 

SimATi^pmffffi %o ^ll^^JSs^smsf^i Histidine is an essential asaino 
acid, and its products and derivatives enter into mia^ important 
Jietabolic relationships » I3i@ C-2 of the isidazol® ring @nters 



Serial Noc HIMD» a? 
Pag© 3 

into the "one @a2'bo&'' pool^ aod thsia these studies are closely 
s^lated to other studies on the ?ole of folio aeid and iritasiin 
B=>ia eesTied out in this laboratory and elsewhere in NIMD. 

Iffiidaaoleaeetic aeid riboside is of significance in t^t it 
represents a nev kind of jBatural ribosidec It has also been us@d 
else^®j°e as a useful tool for in vi^o trapping agent for riboseo 

jpyopoaad Cpyr^.^ pf ProJeats Further purification and studies 
of the detailed ensysfitic neehanisiss involved in the reactions 
listed, particularly 1$ 2g 5? as veil as on other isidasoles of 
biological significaneee. Where possible, the respective ensjaes 
will b® used as tools for the study of probleas related to enays© 
induction^ cell peraeability, and aechaniss of drug action and drug 
resistaaeeo 



Page4 

PKS-NH 
Ittdividxial Project Import 
Galeadar Year 1959 

PftTt Be Publicatioffis 

Fublicatioijs other tbam abstr&ets fSroia tbia proj^ets 

(1) Tabor^ H.^ and ^dyn^vdme ^•>«s Eha^asatie foraatioa of formimiao- 
tetrahydrofolie acid^ 5$10<»aBa^iai!ijrltetnah|'drofolio aoid» and 
lO-fosfiaiyltstrgfeydrofolio acid ia the sstaboliss of forjaisitto- 
glutaaic scidc Jo Biolo Ghea. 2348 1830=18469 1959. 



1. Pfearsacology & Toxicology 
2« Biochemical Phansacology 
3. 



PHS-IIH 
ladividual Project Report 
Galeiadsr Tear 1959 

Project Title: Mstaboliss of Sislie Acids 

Principal Inveatigatoys Lmmsid Masren 

Other Issvestijgatoyss Gwilia Spaariog .(MoAo C&Mimt® Studaiat) 
Jobtt Goldsb«rry CSaiaisssr Saployee) 



Coopex-ati^ Units; s S. Ha ifoll^iB^ Oancar Pbysiolog^^ Ssstioa^ 
Labovatox^ of Pbysiolog^s National Csaosr 
Isistitut®, #KG2-9C6 (Properties of Ts®iaaplaatabl® 
Tljyi^id Tosors) Mid #MCI-927 (Sialic Acid ia th® 
ISi^'oid Glaad) 

^iaB lea^s (calendar year 1959) ? 
Totals 2 
Professional! 1 
Others 1 

Project DescriptioiaJ 

0,^,1^<ytjLy^a? To utudj th@ iatoraiediax^ atetabolism ^d ohagaie&l 
propiarties of sialic acids in ord@r to imderstand its role in 
pi^ysio logical and imthologieal states » 

1. A new "thioi^rbitTSHdc acid ®©ssy* for sialic acid has b®®& 
dsvdlop®d vhidi is 12 tlsma sot® sensitive than other i^thods and 
is Bjjecific @nou^ to jseasure disreetii' th@ sialic acid content of 
tissu@ac The msthod ie uniqu® in that it @@asur@s onlj fre@ siali<s 
acids a 

The jB@thod has hmn adapted for paper chromatography and can 
detect a@ little a@ 5 Um^ ^^ sialic aoido It can also detoct 
Oo5 iWgJa» of 2-deos^ibos©o 

2o In cooparation tilth Dto S. Spicer of this Institute hi^to- 
chosdcal i^thods Mv& b&m. d®valop@d for th® specific staining of 
sialic acid-oont^ining protsinso llio jsethod depends x^pon ootjpling 

Pert B ittclTjded [X] Yes [ ] Ho 



Ssrial Ho. NIil€^ 98 
Page 2 

th® fr©8 earboi^l group of sialic aeids with tbd basic dyes Amp® A 
or Alciaii blxs® aft@r th@ poriodio acid Schiff reaotioiie Staialjog 
is ollMssated by p:>@tr«atffl@nt of sections witl^ sialidas® tsMdi 
specif ioall^ pefio^iss aialic aeida fros aiusoprotsiaso 13i@ sialic 
acid content of seetioas tr@at@d vith sislidas® is sarksdlj 
decreased sjad there is as equi-mlent increase of sialic a^ids Ib 
the section stipersmta&t floido 

A« !ni'«» histoeh€isd.3ts>j of rodent salivsrj glsnds has been sttidiedo 

Hat sali^aEg? sfficias differ from those of the sous© in that they as*® 
resisteat to sialid&seo 

B» ThQ histochead.stx>y of •wagiaal tissue of the sous® and rat 
have been studied^ ^e ^sialic acid concentration of these tissues 
is subject to horaonal control and increases 5 to lO-fold during 
pregnancy c 

C« la cooperation with 2r» S. 1S« Wollsaa (KGI)^ Dr« Spieer and 
I have found that the sislie acid contents of soTer&l thyroid 
(isaacers are incr@su9@d. ^S^yroid eencera stsin apeoifie&lly for 
ai&lic acid containing aucops-otelns ^ereas normal thyroids do not.o 
The fs*e® sialic acid content of the blood and urine of rats with 
43artain sialic acid rich thyroid eaaeersj is 3 to ^ tisses hi^er 
'^:;han normal « 

3o In collaboration with Miss Gc Spsariag v© ar© purifying 
neuraminidase (sialidase) fros^ the culture fluid of cholera 
(1800 3c purified) and C^9^tyl<3tiiif8^ a@gffclB^g@Ba UO x purified). 
She i@ also isolating K'^acetylneurasinie acid frca hus^n plasmao 

a® 8® are p^elissiaaries to studies ons 

a» The specificiti@B of sialid&ses fsva m&rious sourceso 

bo Tim ffiedainisffl of iahibition of influemse Tirus hemagglutina- 
tion by auooproteino 



4.0 In collaboration ^ith Dr. B. S. Bluisberg of this Institute 
saaples of hxm^ serusa have been freed of virtually all tiieir bound 
siedic acid b^ seans of purified neuraMnidasSo Sets of huisan sera 
had been selected on the basis of their differing genetic ' 
chamcteristics as determined by their patt^sms of binding 
thyroxine^* iron and heiBoglobin« These s®z>a have been ooai^red 
urith their corresponding imtoeated saasples by obseirving migration 
of bands in an electrophoresis apparatus « We bav® observed in. 
sialic-less seras 



Serial Io„ KIM©-,.J£„_ 
Page 3 

A» A B^rksd sloxdisg of these bands soviitsg to the positive pol©.. 

B. A s^golar ofeaage of patt®r3a of fc®fidsc 

C, Qiaages IBj but aot abolition ofg the ability of oerteia 
prot®ias to biad tfeyToxi»®j, iroa and heaioglobiu* 

5. A study oa siglie acids in fish ®ggs ia uow driving to & 

elos®. The fisadiag of layge ajsouats of sislic acids ia troist eggs 
(70 ^ig^®gg), half of which is fp®©, «®s yeportsd last years It 
bias MOW bsea fotjad that bo-Ui K-glycolyl aad l-aeetyl neurssjiai© 
acid ara tha forffis of sialic acid psi-aseiat la th® trout «ggo 

6s, Studies oa tigroid physiologj" hav© b®ea carried out with 

Drs, S« Ho fellEaa aad R, W'u Bates, Th® adainistratioa of pirapjl 
thiovraeil to rats catasss a ioweriag of the sialic acid eoaoeatra- 
tioa of th® thyroid gisad. HoHsvsr the total aaouat of sialic acid 
p@r glaad rig^ias coastaat siac® thertei is a eorrespoadiag iapr@ase 
ia siae of th© glaad.. Oa th® other haadj th® sialic acid coae©M.ra- 
tioa aad total @m>vm of sialic acid ia th© thyroid glaad decs-^sses 
•afeen ToS»H, is giv©R to ohicks» 3ji Br« B&tm^ 1^31 depletioa ass®y 
for T.SeHo Hi® sislio acid eoaeeatratioa in 'tfe© thyroid follows 
the l3-33. depl®tion fairly elosssly. 

Par® bovia® thF^'tjglotalia ©oataias 1^2$ sialic acids (I-acotyl- 
ft©m?5«dMo acid) aad siaee this psfotsia ©oi^jris®© &bo%t 7C^ of th© 
glaad protsin we fe®l that th® soatsm-^aaat of sialic acid ia t&@ 
tfe^froid is a cheisieal ©sti^atioa of its thyroglolsalla eofflt®ato 

7^ Sti2di®3 oa hxm&n uria@a Fotar carbohydrates substeac®@ h&v@ 
be^a det®«3t®d 3^ hismaa ra'ia® by ffiaaas of th® thio barbituric acid 
assay^ Si/itfa th® assistaac© of S&*.» Joha Goldsb®rry (a swaaer 
worker) 60 liters of husaa uriae have been procssssd throis;^ 
d©»ioaiaiag eoltjBjas aad larg© oellulosa ooltsaffls aad oertaia of 
these tjpiaas:^ ©oastitmats have ba®a purified* Tao of th® sub- 
ataaces are aeutral for they do aot go oa to Dowe»-l or Ibwe3c-§0 
resiaso Oae of thes® 1& appareatly a 2-deosysisgar but is aot assy 
of .the coBmoa 2-deosysugars. Hier® is about 0=25-Oeg lag. % of 
this sugar ia hmmn ta?ia®» W® hav® about ^0-60 J8g«So of this 
imterial aljsost pn'Bo A s©«oad aeutral sugar is proS^bly & 
3~d®o-i[;ysugar^ A third substaaee ia a doosysugar i^hieh is pi<&®d 
up by Do«®x-l forss&t® tat e®a b@ eluted vith 0«1 M forasie acido 
a© fourth material has broa fouad to b© sislie acido there is 
approxiaately 3 to fj ag« ^ sialic acid aa huma uriasc 



Fag® 4 



Bv, In conJimetio£i vith Qi><. R« K» JaScob^; o^rar 160 sasplea of 
asjrabTospiisal fluid s&epl®3, bol^ isoraal aad pathologioal, ha^e 
b©«a aaalys®do So strikissg correlatioaa hav© bsea astablishod 
l:«tw®9ia tfe© coiat®£it of fsrse or botsad sialic aeid aed pat&ological 
states » !Hie ttoxti^l l®vmlB of f^ise sialic aoid is 0..4. sg- ^ sad 
tSmt of botmd sisli© acid is lo3 Bg» ^« Thas® vabsas ar® 
considerably loner tb^ja ^on® dstenaiaed by less spscifio aad 
aeaaiti^© astbods* Th® form, of sialic aeid botmd la corsbrospijaal 
fluid is N-ac©tjlaa^!r8®iBie aeid* 

Bimhin^imsLJ^J^l^JBmi^eg^i Sialic acid is £omd Ib 
x^latiTisGly large aiBotmts isi aaajr asJieopol^sacoh&x'iddSe Littls is 
knotm of its fu&etione lli® stud^ of sialic acid aad its flustaboliss^ 
is of sp@cial r®l&vsno@ to aa uMerstamdlBg of rhauasitic piroc®ss®s$ 
cystic filsjpoais of ^® "pmisig^&s, fertiliaatioiaj, aeipv© functions, 
bffi@ts?lal shd riral infections, and mai^ other p:t>o@s3@So 

?gQi>osQ^ .Gfiiagae.. Q.fJEgol@i3teg To coaplet® and extamd iairestiga*- 
tioas listedo 



Serial No, NI&Ml>-18_ 
Page 5 

Isftdividaal Frojscst Keport 
GaXoadas' Tesr 1959 

^^I^J^i PuKLicatioas 

Publications o1^r tbaa abstyaots fJroa this |a^J®ct» 

(1) U'asT«iaj, Lot Th® thioMrbitaric acid assay of sialic scids* 
Jo Molo Chea« 2^8 1971-1975j 1959« 

(2) Warratts Ld IMoterbit'oi^iQ acid spapay ysagaat for deo^ sijgays 
aM sialic acids o Haturs, ia pr@s@« 

(3) Spicer» S., &id ^&Tr®&g LcS Histochaaistrj of sialic acids 
contaiMlsg Mucoprotsinso J, Cjtochea* Hietoch^io, in pr@sse 



U) Warren^ L.: Hiieleotides and nuclecaidsso In dresnberg, D« Mo 
Prass, I960. la pmuBt 



Cedo): Oted^LIa^^aS-fiflJsMiSliSS. Hw York, Aeadeisde 
i, I960. la 



Serial lOo MBM>-^S„ 



3» BsttesrM 



PHS-NIH 
Ggaeoaar lea? 1959 



&ll„iu. 



Project Titl®$ 'Eu&fsm aad Eadoeriias Stedies oa TTjptoptea aj:id 
Niootiaio Aeid Mstabolisja 

Fs'SiiGipal iMYastigatoys Alaa Ho M@hl®r 

Other XuTOstigatos's? G®7d& Asiaden 

Man laars (ealeadar jear 1959) s 
Totals 2/3 

Professioaal? l/3 
Ct&Qrs 1/3 

Projest Qeseriptloas 

oy^^j^jaa* To isolat® tfe® iBdluldtml stsps in th© scq^ae© 
of raacffcioias 2?®s«ltiag is aicotinie acid foimatioia, to study th© 
p3?op@rti®8 of th@ s^jffles Jiwol'^ed, a».d to deserib® th® inters 
aadiat® SBet&bolitss, With tha reactioas amilabl®, to study the 
r^latioa of th®83 ®&&^!ms to altered ^atabolio coMitiosiSo 

IfethodaLlBmljaigadlt Emii^s are obtaiaed frojs -various tsorvces 
and parifiod by tfe® Trapiety of isethods currsatly used ia -i'Ms 
field. Choiaical aad physieal, especially spectsfophotosmtric, 
ffisthoda sr® used to ssasure aasya® actiirity aud to idsiatify 
products o Possible subst^^ites and products lar® syxitiiesised bj 
coti^^jcitiotml orii^ie obsaieal teehniqueso Xsotopic cosapoi^ads 
ara sy]ath®9ia@d and r^dloaotiTity is M«iasm«d to follow th® 
corn's® of reactions l,a»XSsa «tt<5 S^&JSi^SS.' Anisals ara tr©at®d 
to poradsc® altered metabolic sttattss, emd st^iyfflss from such 
aiiiiiials er@ assay^^ Methods hm& b®@n adapted to assay emsyites 
in tissxi® ooltur® and crgaioi oultiar® propsiratioiaso 

|^^Js;g.J^3^jjggs 1!h© smttire of th© iat^iraotioa of endocrijs® 
factors oa the control of the l®v®l of picoiinio carboagrlaffle is 
liver has b&m explored fiixipthor ijs collaboi^tlea with !^« ti[cl5asJ.®lc 
Tim oasysi® l<sv®l is increased throtigh a^i affect of cox*tiso]ae$ 
ami this effect is opposed by th« coeibih«d effects of iatsidin snd 
grovth horaonet^ but iaot by either alo^eo TUm cortisone effect is 
also opposed by thyroxine 

Part B iacltsded [x] Im [ ] No 



Serial Koo HIAMl>-99 
Pag® 2 

3-%diTO2;^ffiBttosaille osldas©p fo«ad in aonsal livers at hi^ 
levels 9 eouid not ba dgit®et©di in s®"v®x®l straias of tiss^a» eulture 
osllse prdsufosid to &@ soiaa® li^e's^r csllsc ^i@ eas^s^ h&s also not 
Soxmd in a solid tvmoT dwlmd from, oia© of the tissis® culttsr® 
li&esy but lae^ foimd in & mm&Q hapatoa^ timt had be@n transplanted 
8Ubeut«B«oti£(ly tor as^^ral yeayso The tissue Gfoltvtr® cells ®nd 
tufflora vera provided bj Or* Yisfiai® Emsm of tSj® Katioisal Caaesp 

IlQ8titl!td« 

Stisdies 0& the di«i@ieal ?e&etiois@ of ths uiQstable product of 
3-bydros3?asith3»gdIie acid oaddatioa hav» sdded support for tb® 
proposed stxisffiturss 2-«aiao"3"ea£°bo:£y~'5~f omyl"-2.1--tr<^s ,. ^ia- 
p^itsaoio Boid. 

Isotop® trapping aspcrisaats with riag-labeled J-hydrossrasathnmilic 
acid should eoiave£>8ioi& to quinolis^de aoid feut not to pioolinic acid 
in non^l rats. 



to W2AMD 



L£iffl5!ag!a-JtQ-JIAI@_BaB^S(agychs f^o lines of inquiry ar® related 
rescercihe One is & study of th@ reactions that influano© 
niacin loatfibolisiffi in order to gain ssons insist into ths biochemistfy 
of this vitaaino Th® ot&«sr is the analysis of th® effect of horKones 
on liver enssyaesj, whids m^ give infonaatioa about th® aatur© of th© 
metabolic lesions in diabetes > 

,pt,^,$sp%t Atteapts will b& continued to find 
systems for dezsonstrsiting the effects of honaones on the 
level of picolinic osTbo3Qrlas®o Tb.® jsjetaboliaa of 3-hydroa:y- 
emthranilie acid will be investigated farther» 



Serial lo, HIMD^-jj^ 
Pag© 3 

PIS-IIH' 

Cal^dar Teas' 1959 

I^eO" Publieatioas 

Fafelioatioas otfeey then abstracts frois. this projects 

CD Mshler, A, H. J Matabolis® of 3-*ydroa;y-aattoaailic acids 1& 
aniEBlso 4tli Iat®5Sjat« Coag. Bioehes. Us 164-171, 1959. 



Seffisl IS©. i^£^aiS-i_lQO, 



3.. 



C«i®Bda? fear 1958 



fr#jee£ Title: %» Si^h^sSxy @f 3»l£u?»e^it^iiDi£i:@ 

ftiaeipal Sawestigfigg®' s Ste®ss K^aek 

Oehs? Isa(v>9«e£s®t@S'Sg Hiss Bl@adel ^»^@@ia, tiSo J^ssass. ^issmxtp &nd 
Wc 3^m Wo fls.®ggss©ao 

less isv@lv«! in £be fi^ntlssfiis of emstieuftaes o£ Uvis® &is;sue» peiPCleii- 



eMlity m e&nv&ft certain sulJhsE-cenesissdf^ e&s^&ai&Aa e@ sev 8uSie«:«2s&e@» 

Ime ^s»» @e»iiie4§ fisS e&£rm:t:<3iri®ai9D It Is f®$ia£ ft® eaasisf: @f ehs«6 sep- 
arable! pr<^»&»isi it&&tim:Ss ^^i^mz^d l^ £1 &&& Xllo In &Mktt&» t@ ml" 

ffosEl^ s«5jK;f.ioaB a a©a"®pe®ltle s-siSacitiea ^f ^s«|gt^s is cffifcalyssji fey 
^h2 tmhtt'M action ®£ 1 m^ tlz 



SI I * SI 

Cli.-S-K ■¥ tFm ~— — ^ CEL-S-S i» SIW 









•¥ S-S-S-K 



•^ eS"SS * 2 asH 



C3) Ihn M«»iE^eh«ai8 @f f®liai&@, 






item inZ9/stm41i&t96 ®i clmlms^&v^l 



3tan^!!.^ @£ eh® q@&w«} ®i M.m&s& mS its tmeti iafesilljiattt 

<SueU#na «£ di8ul£&dae opena p®s&ib£ll«ie@ fe^ 9£u«^ @i such gx^msps sqi§ 

ehei7 ia&«iffeeav«?e&@n!» in p^oettin «@loe«io»9 p?oeessas <&miei<iim^ '&& tew. 



Serial N©o KIAM?j^ J Oo_ 
3 



Pubiie«eieius eehesr ti^n abseraets iv^A this !>fGjeees 



CD Wolff, So C.» aa4 SUek, So.* V&emtiea @£ ehe sie«%lthiol ss^er ©£ 
d-pheaplMglTeerte acid eatalysed by 8lycer*Udeiiy(fc-3-pb®sphfflte elehy- 
Aseh„ Bieehemo and li@fhyso 80» 236 C1959), 



C2) Black, S, Ueehesalstvy <»£ newnr stilf«r>«4meaiai2« mln& aei4e, Iq 



C3) Slack, S, p-Ae^ttyl pbssphsta asd aa|»a?«ie*p-sfisalald^9de. 

in Sa^jneel^y, ¥®lo 6, Aireield 2^, <£» press). 



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46, Cin parens)- 



2. Bl&chssslstTy o£ Asiss Acids 
3o Sa&hesaa 



1959 



0.4 



S»0JGC& !te£«?ipgi«m; 






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^ifllds C -pl»ayalastiae upoa aeid kfSralyslSo ^is eas^pstsad appears t@ hs 
t&tMv clesely ?ela&<5d £e G^fisiieidia Jo ^& a@e ta» Ite identieel ^^th ie« Sm 

Ma el(»e3ly ffela^ed is lad&eated by ehe f«et tUst s&e&t^ acid hTds^lyeis «£ 
eiiOisr esie gives riee «;o phesr^lelsoiae, proliacp valisse (f>^ighias aad leiseis^o 



the iKffiu&aeian sis£ure £e? 6he psr®d»eeiea of Cbie 



aiapU£ie^6 A clear solugicm &i soluble easj^ass plus ^79 end Sfli Sssrii^s 



4UBi&$ aeids, L-C**»ie©l©«K:lo83 I.-C -alsainss l.-C*^««;y3?®sia©B L-C^^-lcBciaSe 
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shange ^slth ATP, is t«sts frnma that the «'nly !>-a)iBine» selSa ^tiveted 
b^ t±« cell free exesa^tt isf B. bgavis i@ B-phGnylAUnice ^Uich is loot 






i<^i£tlf lcfiel<3ii of &h& uzdksm^i coe^OQad ia relation to ©rmjic^idia Jo 
and £® stud^ fus-thffix- ehe cesuHttons of ita f@maelesio 

aieferaei: b^ jmmisniusa sulfate && thet iQ<i«»rps^aei@n ^f C^^-lslt^le^ 
aoiis ia«:@ the 



Serial Noo MMMD^_JLai, 
Page 3 



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3c SeShaskIa, Hetf lsi»3 



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1959 



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%?ith Group C aatiesra ceuplad to 

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gletelia ea^lo4 to fluoraaeeiao 




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fast Ss 



io To Mswastaehso In the isQehAnlssi &i easyiae mtiGUs Cteik?i%«t §]Fti^3)@ifsa 






.1 „ ?n.y8i_ 
3 „ Bethesda 



PHS-NIH 

Individual Project Report 

Calendar Year 1959 



Part Ac 



Project Title: Effects of hypoxia on physiological and 
pathological mechanisms in animals <, 

Principal Investigator: Paul D^ Aitland 

Other Investigators: Edwin C Tliompson, Edna C« Thompson, 
Milton Parker 

Cooperating Units: Dr. Benjamin Highman, Section on Pathologic 
Anatomy J Laboratory of Pathology and Histochemistry o KIikMD=8i 

Man Years 

Total; 4 

Professional: 1 

Other : 3 

Project Description; 

Objectives: (1) To determine the physiologic mechanisms whicn 
influence altitude acciimatiaacion and tolerance; 

(a) Evaluation of the relation of body temperature and rest 
to altitude tolerance in rats. 

(b) Study of influence of aortic and mitral insufficiency or. 
survival to acute and chronic exposures to high altitude 
in dogs« 

(c) To determine che influence of exercise on altitude 
tolerance of rats . 

(d) To discover the nature of altitude tolerance in birds. 

(e) To study changes in serum enzjiEe levels induced by 
altitude exposure in dogs. 

(2) To establish the role of hypoxia in the developraent 
of disease: 

(a) To determine the rata and degree of development of exper ■ 
mental atherosclerosis in rabbits and chickens exposed 
continuously to altitude. 

(b) To determine the influence of hypoxia on the immune 
re ?ponse of animals . 

(c) Hypossia rendiirs rats highly susceptible to endocarditis. 
To study other factors vjhich influence susceptibility to 
endocarditis o 



Methods employed; Altitude exposures conducted in decompression 
chambers. Physiologic » hematologic, and pathologic techniques 
usedo 

Major findings: Rats restrained Imiaedlately before rapid ascent 
to altitude (33,500 ft. at 2,000 fto/min.) die sooner than 
unrestrained rats= Such reduced tolerance is associated with 
an increase in the oxygen requirements as a result of struggle 
to escape restraint. With slow ascent (2-1/2 to 4 hours to reach 
33,500 ft,) the tolerance x^as increased in both restrained and 
unrestrained rats. The body temperature of the rats dropped to 
low levels before reaching the critical altitude thus favoring 
better tolerance. Restraint tends to hasten the fall in body 
temperature commonly associated with an exposure to altitude 
thus providing greater tolerance to altitude. Preaitituda 
induced hypothermia induced by restraint plus exposure to a 
room temperature of 3 to 5 degrees G. £dr 2 hours afforded 
complete protection to exposure to an altitude of 33,500 ft. for 
6 hours. 

Normal dogs and those with surgically induced aortic and 
mitral insufficiency survived 4 hour exposures to 30,000 and 
32,000 ft., whereas at 34,000 and 36,000 the operated dogs 
showed a higher mortality. Prolonged intermittent exposure of 
the operated and unoparated dogs to 30,000 ft. resulted in no 
difference in tolerance or tissue changes. Of particular 
significance was the finding of nonlipid arteriosclerotic plaqr.es 
in the aorta of some of the young dogs exposed to altitude for 
several weeks. The lesions were more severe in character with 
increasing numbers of altitude exposures. The lesions ara 
attributed largely to hypoxia. 

Endocarditis in dogs with aortic insufficiency was inducGc 
by a single in.iect3.on of Staphylococcus aureus . Penicillin 
treatment was completely effective if administered within S h-js. 
after inducing the infection. If treatment was delayed 24 hr . 
symptoms often occurred after cessation of therapy. Prolifer 
glomerulonephritis, which developed in nearly all dogs given 
delayed treatment, persisted despite therapy. 

Significance to NIAJQ) research: Results indicate the importanc . 
the body temperature in altitude tolerance of animals and emp 
the important role that restraint has in influencing the bod^; 
temperature of animals. Findings show that dogs with aortic 
insufficiency and mitral insufficiency have a surprisingly high 
altitude tolerance despite the cardiac disease. The occurrence 
of nonlipid arteriosclerotic plaques in dogs exposed to altitud^i 
suggests that hypoxia may play an important role in the etiology 
of this' disease 

Proposed course of project: To conduct experiments to accomplish 
listed objectives. 



PHS-NIH 

Individual Project. Report 

Calendar Year 1959 



Part B; Honors, Awards, and Publications. 

Publications other than abstracts from this project: 

Higltmanj B.j Maling, H. M.,, and Thompson, S. C: Serum 
trarisaroinase and alkaxiiie phosphafcase levels after large dcsea 
of norephinephrine and epinephrine in doga . Aato Jo Physiol o 
196 J 436-440, 1959 » 

Bartletfc, Jr., S, G., and Altland, P. D.: Effect of restraint 
on altitude tolerance in the rat. J. of Applied Physiol o 14: 
395-396, 1959, 

Bartlett, Jr*, R. G., and Altland, Po D,: Relation of body 
temperature and restraint ts altitude tolerance in the rat» 
Jo of Applied Physiolo 14s 785-788, 1959 » 

Altland, Po Do, Higliman, B., and Roshe, Jo: Effects of altituds 
on dogs with valvular heart disease. A.M.Ao Arch„ of Patho 68: 
475-436, 1959 o 

Hightaan, B., Altland;. P„ Do, and Roshe, Jo: Staphylococcal 
endocarditis and glomerulonephritis in dogs. Effect of treatraer 
with penicillin and ntreptomycin. Circulation Research. 7: 
982-987, 1959. 

Altland, P„ D., and Highman, B„: Effects of High Altitude on 
Cholesterol Fed Rabbits (Production of Serere Pulmonary 
Atherosclerosis with Calcification), Accepted for publication 
by AoM.A. Ai'cho of Pa£h„ 



1, Physical Biology 
2o Physiology 
3 o 3ethesda 

KIS-NIH 

Indlvidiaal Project Report 

Celeudar Year 1959 

Part Ao 

Project Title; 2av«rtebrafc«j Physiology 

Priasipai lavestigetors Joisa B, Beck 

Other lEivestlgatoitsg Lso Leveabook^ Margaret Lo Keietar^ 
Helen D, Parky Clayistyna Eo Mesca^ Vineeat Hollis 

Cooperatiiag Ualts; ^cmi 

Mea Years (calender Year 1959) s 
Total 6 
Profess Icna 1 % 4 
Others 2 

Proj«ct Descrlptioas 

Objectives; The ioag range objectives cas fee defined as the 
research iaterests o£ the four srof^ssioisals in the unit a 
Specifically; (1) Basic tssductioa mechanism la origin of 
reproductive eellB froo: soma cells <> (2) Meehaaissi of p;o» 
t^Ssk syathesis ia ii^sect metaisorphosis o (3) Physical factors 
in respiration o (4) Biological triggers » 

Methods Employed; CarbohydratQ metabolism has be^i followed by 
chroszatographic methods of identiflcatioa aad agftay of various 
t2?pe8 of blood sugars^) cheiaicel isolation of tiosue of glyco" 
gea^ aud respi^'omel:^ of intact orgaaisiss aad of tissues,; 
Enzymatic pathways have been followed by use of radioactive 
tracers o The csrobic-asiaerobic traasitioa Koae is beiag 
studied by meesuriag oaygaa uptake beforej, during eud after 
exposure to vari-ous partial pressures of osygea* The bic^ 
physical espectB of gas tracsfer iavaive dijaeasional study 
of the respiratory ejstwug respirosaetry aad computatioa:, 
Eicutrotaetric ai^thods ^are applied to an intensive iitudy o£ 
neurogsnic initiation of bioli»nineacence in fireflies. 

Major findings; lo During the past year^ 5 cultures cf Hydra 
showing striking 20-'25 day cycles of gonad differentiation 



Serial HOo . KIfeMD°lQ5 
Pas-?. 2 



have been established by Dr, Parko Although the factor or factors 
responsible for this periodic sexuality ia proving very elusive^ 
this and other results definitely eslude CO^s, culture crowding fre- 
quency of feeding^ accianulatioa of material oa culture glassvere^ 
and ©ccumulation of so3.uble metabolites as inducing agents <. 

2o Dr, Levenbook has found high levels of citrate in the blood of 
5 species of insect » These measurements^ together ^ieh sitailar data 
in the literature for 3 insects^ suggest that high blood citrate is 
a biochemical peculiarity of insects. Apparently, however^ the ci» 
trate titer is not aa acciaaulatioa due to blockage of later stages 
in the TCA cycle^ because Dr^ Levenbook has assayed the 10 separate 
enzymes of the cycle plus citrate cleavage enayme, isocitritase and 
malase synthetasSj, and found all but the last t^so present . Further^ 
more^ he has shown that citrate^ alpha ketoglutarate^ tBalate^ 
fumarata and pyruvate are ccKspletely oxidized by the insect's mitO" 
chcndria in vitro „ 

3o DVo Keister has completed a comprehensive study of the relations 
of 0-7 tension and of temperature to respiration of fly larvae and 
pupae and already has much comparable data on the adult stage o Beside 
providing needed basic data on all the developaseatal stages of a single 
specieuj the results have shown that (a) larval respiration is never 
limited by the physical structure or dimensions of the respiratory 
openings (ia contrast to Dro Park's finding that it aay be so limited 
In the pupal stage) ^. (b) larval respiration shous a sharp plateau 
between 10 and 15°, (c) decapitation causes little change in the 
oxygen uptake rates of one day old flies 4a the range 0~45°» 



4. Miss Mecca has cffiupletcd a study of the effect of cuticle punctur 
on respiration of a laboratory moth as a preliminary to isetabolic 
studies requiring injection o£ materials into the pupa. Although the 
literatur.e reports & very amrked stinnilation of respiration after cose 
parable injury to pupae of diapsusiag types of moth_5 no effect was 
found ia Prodenia^ a aon-diapausing species , 

5» Dr„ Buck continued his collaborative w>rk at Woods Hole with 
.Dr» James Case of the University o£ lotsa oa the epxitation of 
bio luminescence in the firefly^ The taajor findings of the suiianer 
include (a) the detection in the photogenic tissue itself of action 
potentials preceding the flash; (b) the fractionafeioa of the overall 

r'jsponse latency into two stepSj, the first of about 50 msec end the 
j3cond of about 15^ the former of which can be by~passed by intense 
stiiaulationj (3) the discovery that a variety of agents^ including 
^serine and veratrin®;,. can disrupt the lantern's coordination mech- 

-aism so that photocytes flash individually and asj^nchronously. 



i'G 



Significance to SIAMD r<a8earchs All the work of this unit can 
be considered as contributing to the basic biology of metab- 
olissn. More specifically, the various projects under%7ay 
Mpinge ons intermediary metaboliSBij, cell differentiation, 
biophysics of gas transfer, endogenous rhythms (biological 
clocks), and biological triggering (biophysics of escitatiou) . 

Proposed course of projsctr Dr. Lefveabook plans to investigate 
Insect organic acid saetabolisa during various stages of de™ 
velopaentp and to initiate a study of amino acid turnover and 
protein synthesis using lysine C '. The other investigators 
expect to carry on in the directions indicated by their pro<= 
gress reports. 

Honors; The appearance of the excellent Vol. 12 of the Pro- 
ceedings of the IVth International Congress of Biochemistry 
(see bibliography below) provides concrete evidence of the 
distinction gained by Dx". Levenbook in being asked to organise 
the symposium on "BiocheiEistry of Insects" and of his- critical 
job of editing the volujB.e„ For his role in the Congress, he 
was presented with a "Service to Science" citation by the 
Minister of Education, Republic of Austria. Dr. Buck was 
appointed to the Editorial Boai-d of the Biological Bulletin, 
and elected to the following postas Board of Trustees, 
Marine Biological Laboratory; Executive Committee, Marine 
Biological Laboratory; Vice President, Society of General 
Physiologists; American Nstioaal Cotamittee of the Inter- 
national Union of Zoological Sciences, Dr. Buck was asked 
to cooperate as a Visiting Lecturer in an NSF supported 
program organized by the Asaerican Institute of Biological 
Sciences "to enable undergraduate and graduate students at 
small liberal art colleges and ualv<£i;aitli5S to meet and 
become acquainted w?'.th leading biologists in the various fieldc 
of life sciences." Re spoke under these auspices at Pennsyl- 
vania State, Lchighj; and Drew Universities, and at Moravian 
College o 

Dr. Park was invited to lecture before the Annapolis Secondary 
Schools Science Seminar. 

Dr. Buck organized a 2 1/2 day symposium on Arthropod Physiology 
for the American Society of Zoologists at the recent "Washing- 
ton A.A.A.S. convention. Abstriicts of the 45 papers presented 
have been published in Anat. Rec, Vol. 132, No. 3 (Nov., 1958) . 



Part B included Yes 



ladividual Project Report 
Calendar Year 1959 

PaKt B g Koaors, Awards^ and Piablicatioas 

Publicatioas other than abstracts frosi this projects 

Leveabook, LoS Biocfe^aiBtry of Insects, (EdJ 252 pp. Pergamon 
Presa . 

FriedffiaUp Stanleys Sustained flight itx Pfeonala (by a new method) 
and its effect osa blood pH. J^ Insect Physiol. 3s 118-119 



McDeraotty Frat«k A. atjd Buck^ John B.s The laiapyrid firefliesi 
of Jamaica. Traas. Ajbs„ Eat. Sec. S5s 1-112 <1959) . 

Irrev'erre_| Filadeifo a©d Leveaboofe, Leos Effect of diet oa the 
occurrence of S-iEetb}'! cysteine aad the free amiao acid pat- 
fceni iB insect blood. Is press in Biochiia at Biophysica Acts 

Friedmais^ Stanlays Th.& purification and properties of trehalas 
isolated from Phomia regiaa Meig. la press in Arch. Bioeh, 
Biophys .. 



2o Physiology 
3 „ Batheoda 



Individisal Project RefKJsrt 

Csieadar Ysar 1959 



Part A. 



Project Titles Mechaxiisss of the eirculatory reactioa ©f 

seaaitive jspecies Co s^^&hetic joacsroaiolacules c. 

Priacipal lx^&st±gs.toTi L©«is® Ho Marshall 

Oslser JiQvestigatorss Charles Ho Raima 

Cooperating Uaitss iloae 

Mas Years (ealsadar year 1959) 
Totals 3 

Frofessiooal: 1 
Others 2 

;?roject DescripSioas 

Object ivQss To study vasod®pr«8sion and edssiaa fonaatioa ia 
rats after deKtrasa acimiaistratiou and Jis doga after poly* 
viaylpyrrolidon® (PVP) » 

Hethodes Eats have Jjeea prepared bearing percaaaeat tubes ia 
the «bd«niaai aorta feosa ■ahlch arterial i>lo.od prcassizre is re- 
corded througho3ifc iSKpertesats oia coascious laaisialSo Theste 
chroaic l>reparatioi:iB oesesioaally hav® lasted six wee&s^ but 
2°3 weelsi is more usual o Loeal effects of the plasma .expaadesx 
oa skia capillary psrmeability have beea follo^?ed by sotistg 3i.gc 
of dye (T"1824) estravasatioa (bleissg) aad passage of fluid 
iato the subcutaaffious tiasoes (edeaia fonaatioa) » Usiag e vsit&i: 
displacesieat taethod^ va caa neasure the iacrcascd voltsste of 
rats' froat pe^Jis with a precisioa of 5%o Su espsritaeats coa- 
ceraed ■with miast cells., (tJie most iaiportaat source of -hiEta" 
jaine aad S-'hydroKytryptasiine ia rats)^ we used Pugh^s 
toluidine blue staining techaique„ The Parke-Johrssoa micro- 
method for ^Xood g;iucose was worked ap partluuliurly ia r®ga-,?d 
to its use oa blood samples coateiaiag destraa or PVPo W© 
fouad that PVP does not interfere^ whereas destraa^ coatainiag 

.. reduciag and groupo^ iavalidattts the glucose detersniaatioa juet 
as it does usiag the aathroae siethod. Tha detezrainatioa of 
blood glucose with ias igaif icaat dea:trsa later fereace is poa-" 
siblo by tespiag the destraa dosage of ths ascitaal tc a sniaiffium.. 



Major Findtnigsg i. In coasclous rat© It was possible to cora- 
pletely suppress the reactloa to latraveaous dextran by the 
proper eombiaatioa aad dosage of aati-felstamine i^ith aati~ 
serotoaln drugs. Tbis coaftrms iadepeadeat result© te 
sQesthetized rats from another laboratory <, 

2<. H . pegtuss is inoculation of rats and mice increases 
their susceptibility to erogesatous h.'.stesiiae -aad serotoaiiio 
We inoculated rats with suspeasions of tfeis orgaaism and found 
their suseeptibiilty to destrau^ a histaiHiaffi«»aad serotoaitt-- 
releaser ia rafcs^ to be unchanged „ At the aame tisae^ couats 
of tnast cells decreased in th© afcia of ths paw dorsum^ wklch 
is oae of the "target areas" of. destraa scaasitivity. This 
indicates that the destran react ioa does aot depend oa the 
presence of these cells , 

3, Our major activity has feeeia iavestigatisg the relation 
of insuliu to the rst's destraa reactioiio Experimeiffits are 
described in. the literature which show both eahsacsd aad miti- 
gated reactivity to destran after iasulin., We have fouad thai; 
route of admiaictratioffi and dosage level of both destraa and 
insulie deterjaine the differeaces in effect o Hon-hypoglycejaii 
levels of imsulin. protect rats agaiust both vasodepreseion 
and edeitja characteristically seen after dextran is injected 
iiat ravenously. After larger doses of insulin^ rats are pro---" 
tected against signs of the destran reaction but are taore 
susceptible to fatal coavulsloaSc, The kno^m effect of issuXin 
oa cellular permeability to simple carbohydrates led us to 
expect an enhanced formation of desstraii-iaduced edema after 
inBulin, We have avideace iadicating edeaa formatioa may bp. 
selectively cuhaaced by insuliUj for although rats do aot 
show edeaaa of the eistremitieSj they are thristy and bluing 
is more generalised., 

Significance to MIAJID Research? The investigations descril'ed 
constitute basic researca in the physiology of cellular per- 
sneebilityo It has been our attenspt to keep the celluif.r 
reactions in perspective by studying them withia the fvsme^ 
work of the organism as a whole. 

Proposed course of the projects We shall continue to follov? 

what se®tt to be proraising leads to more complete uncVarstandiug 
of why indlvtdue.1 specien react to certain taoleculef/o It is 
iiaportant to test the effect of Insulin on the reactioii of 
dogs to PVPj ■ahich has no structural relation to the dextrose 
configuration as does dextraa. Because certain anUiestarases 
are effective in saitigating in rats the capillary hyperpermea- 
bility occurring after irradiation and thertaal bums^ 



representative drugs will be surveyed 
daxtrart reaction in this species. 

No |:ublications, honors, or awards. 



Part B, included o Mo 



PHS-NIH 

Individual Project Report 

Giilendar Year 1959 



Part Ao 



Project Tieie: Pulmonary Ventilation 

Principal Investigators Heins Specht 

Other Investigators: Howard Brubach, Roy Hiltner, Erneaf. Kaicy 

Cooperating Units: Nona 

Wan Years (caiendar year 1959): 
Total: 6 
Profeseiooal: 2 
Other : 4 

Project BescriptioR.: Studies in pulmonary ventilation and v.- :. 
physiological phenomeua subject to ataiospheric Influencs / 
abnormal pressure and gas mixture. 

Objectives: The principal objective of the overall project is ■ . 
explore new phenomena regarding breathing behavior with regn- tf ^ 
to their physiological significance. In order to accomplish'- * 
this, various otudies of physiology must be undertaken in o^ ^;«" 
to control accurately the ei:perimental situation, "^'he inft-f."' 
relation of work of breathii-.g and resistance to breathinp "l U^e 
Deen sought as limiting factors in normal as «ell as abnor-lnai 
conditions of breathing. A secondary objective has been -."he 
3tu&y of adaptation to enviroimental atresses and factors wbich 
modify such adaptive responses. 

Methods Employed: Subjects are studied for oxygen ccnsum .tion 
carbon dioxide production, breath velocity patterns ar r.< vclvr- 
measurements as necessary. New devices or adaptations haveb^'^n 
mace for new approaches to measurement of physiolo^'io.^ 1 functi"- 
The^se of Archimedes principle in body density measu/'ment 
without the use of water weighing has been initiated ais ar r- 
o.. a study of the effect of dense gases on breath velocity p 

Itojor Findings: Two studies on the effects of restraint on at - 
tolerance in the rat have shown that restraint affeicts the 
raaintenaace of body temperature and thus indirectly the tolaran-e 
of ,aypo:d.a at simulated high altitude. The observsnons are ■-. " 
further example of the subtle aann^r in which seemingly' inc©^." 
aequantal prccedures raay influence physiological measures,. 



An analysis of fche temporal lag betvj-een alveolar pressure: 
and the resultant mouth air ficw in respect to gas density, 
breathing effort and breath frequency has been published and 
has showji that assumptions disregarding this phenomenon in 
attempts to measure airway resistance lead to errors due to 
the fact that the pressure at Che alveolus measured by any ox 
several methods is out of phase with mouth air flow. Gross 
errors may result since uncorrected observed data would lead 
one to believe that flow without pressure and no flow at finitJ 
pressure exists under certain conditions. This study has not 
cnly called attention to thie phenomenon, but has also darners 
its dependence on gas density and rate of acceleration of £;hc 
breath velocity during different breathing patterns. 

Current work on breath velocity patterns indicates that 
high density gas lEisturas have a somewhat less mar!*:sd effect s: 
the pattern than expected froni extrapolations from previous 
work with low density gas mixtures but the method of asseasEen; 
raqiilres that ansch lEore data be collected and analysed bafora 
quantitative effects can be given. 

In Che course of the work mentioned above the opportunity 
was taken to utilize the various gas mixturea to initiate the 
devising of a method of body density measurement ti?hich obviats : 
tae estimation of lung voluina yet uses ilrchimedss principle of 
weighing in two different density media. The fact that both 
madia are respirable makes this method attractive and the 
avoidance of submersion makes it potentially very much sroi-!?. 
useful than previous methods. 

Proposed Course of Project: It is planned to undertake research 
on pulmonary ventilation in dogs in order to provide a backgro^ 
of information similar to that from human subjects but permit: 
active intervention in normal processes not feasible with sul;. 
ou a scale which will provide statistical analysis. 



Par4. B included. Yes 



incii.v:i.da&i Project Report 
Calendar Year 1.959 



Fart B; Honoi'S, Awards, and Publlcatioas 

Publ-.',catio5SS otfear than abstracts fretn this projects 

Bartlett, Jr», R„ G,, BsMbach^ rU F,^ Trisa&lSj, R, C, and 
Spec^tj, H.; Airway reaistaaee meaouresaeat duriag any breatlsing 
pattern ia man. J, Applied Physiol, 14s 89-96 (1959). 

Bartlett^ Jr.^ R, G., Snabach, He F., end Speeht, H,s Demon- 
stratioa of sventilatory mass flow duriag ventilation and apaaa 
in man. J, Applied Physicl. 14; 97-101 (1959) o 

Bartlettp Jr.^ R, G,^ Aitlaad, P. D.s Effect of restraint on 
altitude tolerance in the rat. J. Appliesd Physiol, 14s 395- 
396. (1959) o 

Bartlett, Jr,^ R„ G., and Young, M., M^J Free Roaming in the 
Albiao Rat and its afiiect on restraivit hypothermia, Jc Applied 
Physiol. 14; 393-394 (1959). 

Bartlett, Jr,^ R, G.: Effects of restraint on oxygen coafsiimpfclta 
of the cold exposed quines pig, J. Applied Physiol. 14: 46- 
48 (1959). 

Bartiett, Jr., R. 6., aad Alfciand, ?, D.g '^&xi±±on of body 
temporature and restraint to altitude tolerance in the rat. 
J. of Applied Physiol. 14: 785-788 (1959). 



lo PJrtysical Biology 

2o Physical BiochtsaistJry 

.?o Bethesds 



Individual Project Repoxfc 
Caieadar Year 1959 



Part Ac 



Project Title: Th© relationship betw©®n structur® sad function 

in feiology* 

Principal Xiwestigatorsj Dt, Kaloasa Laki ead Dt„ Hi &o Shroff 



Ot&ex Investigators 3 



Dro Wo Jo Bowen 

Dr, Wo Ro Carroll 

9r, Dc Re iCoiaiQS 

Dr» E. Helauder 

DTo Fo Irreverr© 

Dr, Jo Ao C4ladner 

Dr. Ro B. Sisspsott 

Dr. Y. Levin 

£»r. L. Cc Stewart 



Ho Lo ^iairfcia 
lo KQoller 
Jo Standaert 
J. Wo Ksaly 
E„ R, Mitchsll 
Eo No SiaitSi 
E, Fo Wilson 
Go Rice 



E, K&Qtoa 



Cooperatlag ?Jnifcs: 



r*r. 



Jo Oo Davis j, 
John To Xripp 



Bl (S<s3riai Noo 
LBBF^BS (Serial Ho 



23$ ) 



Prof, Lester 

Dr„ J« 

HJoS 
Br, Lo 

»o 



, Sisptc 

Cohen, 



Bro 
Dro 

Drp 
Brc 
Bro 
IhTc 
Dr, 
»ro 
Dro 
Dr. 



Reed;, Depto of Ch^tiistry, UniVo of Tesss 

U»So Piaaty Soil; and Hutritloa Laboratory j, 
of Ags'iculture 

LC-HXJS® (Serial Ho, 42 ) 

Etiy^, LC-HLSM> (Serial Ho. 
Co Gajdusek, MINDB (Serial 

Weiafeach^ LT»"JSXAID (Serial Ho, ^ 

SSarl A<, Seh®llaBbers^ LTDi-HIAXD (Serial No, "~ 34 

,Io So Folk^ OBC-MXDa (Sesrlal Ho, m 

Eo Mihalyi, LGPM^ISHI (Serial H©, .._ . l?.?" " " 
Bo HoR-vathi, MH-HIMSB (Serial No, fe ip_ 

L«o Maadelkern, Natl, Bur, ©f Standards 
Fo Co Bareter., GMST-NHI (Serial No„ ^^ ____ ^_) 
LPT-NIAM* (Sftrial Mo, ""^IQl 







Man Years (calendar year 195? 



Total; 20-1/3 
frofessioQals 
Other', 7 



J 



13»i/3 



Serial No„ HIMffi^UQa_ 
Page 2 

Projecl: Descriptions 

Qbj actiycs s 

The chief interest of this section jremained the study of tha 
correlation betwessn structure and function. 

The piroblesa of structure ±u its reiatiou to function is very 
explicit ia processes where one fo.n!i of energy is coaverted to 
another foinn. In a different f.spect., structure and function ars 
the characteristic features oi' the isode of action of anzyiaeso. 
Both of these aspects of structure and function have been under 
continued study in this section. 

Oscular contraction is &n esampl© where ch@sical energy is 
converted into mffichanicai work, k gsreat deal of study was devoted 
toward tfee understanding of the contractile misels proteins (myosin. 
actia,, tropfaayosin) and their interaction with ATP. firing this 
year studiaa have been mffide on the. ensyines<==thri3mbiii5 carboxj^p®pti« 
daee A,, B, layosia ATPase« 

The poiyiaerisation of proteins reasained undar study in ordexr 
to gain insight as to how cells build up network structure such as 
the structures involved in ssuscular contraction and blood cemgulstio;:^,. 

Methods ; 

The mathod of attsck is both direct and indirect. In the direct 
attack^ for ©a^aiBpls, the proteins of muscular contraction and blood 
coagulation are separated from their native milieu and are studied 
under arbitrary conditioas which are selected to reveal properties 
of interest. In the indirect attack studies are made on some otber 
(alrsffidy better) known proteins tc gain information before the diract 
attaixk is made. 

In these studies this procedures of biochemistry and physic&l 

chemistry are employpdo For axsaapia: paper and ioti-'->3Scfeange chrymix^- 
tography , 0ns>'jEoiogy, ultracsnfcrifugai analysis^ osmcsaetryj ligh'c- 

scattering laeasureiaents. electrophoresis^ diffusion msasureiiients,, 
X"ray diffraction, etc. 



Migcular^ contraction - X-ray diffraction studies revealed that 
durinjg the ATP induced contraction of glycerol^treated muscle fibars^ 
the ia--keratin pattern disappeared, indicating that the basic process 
in co!:ii:!L-action is the "malting" of isie ordered filamezits of the 
contractile proteins^ 



Page 3 



^ii££«IiH^iSM.* ( Coif >-fc ' d . 



Th«f-.sffi i:i^di^^g5? assign two roles to KVS, The contraction is 
initiaKgd. by M?<, In this pracass the eaergy of /iTF is net used 
up. In &. senii© it acts as a si.g^sl and tha contractile structure 
acta as an "amplifier,," ATP also acta as the ultimata energy 
source by reatoriu^j the contracted strticture to its original stafc::;. 
The ijjsaedi^afce acuroe of the contrac/cioii thua is the "Candeacy" c.c 
ths ordered regions of th^^ confcraetii© structure to go to the ratidxva 
£oxx& ("sasltlng") , 

Vm aechod of analysiE of s^&gassiuia in eerus has been sacKc-ss - 
fully CKtended to ascertaining the. raasnesiuixs content of myosin E„ 
cera^ Posner^ Biorio, acic. Laki) „ 



Polyphospbata CS?'^3P0a), » ,jr, has been found capable of causing 

rapid rela»2tioB of glyesHrol^ftreatad muscle, fibsrs •which ha^e besn 
made t®Kse by application of ATP, Several pcraiaeters of this 
phfiT^aaiimoxi -sera studied. (Bowerij, Martin). 

^^£sj£^ •-» It is now becccaing increasingly apparent that withowr. 
the isiiderstanding of tha detailad coraposition oi myosin;, its role t:.i 
muscular contraction cannot be evaluated. 

Myosin, has be©a isolated from earthwovm fsnd soKse of Its properties 
studied, (i&csainz^ Maruyaias) . 

Jl^osin preparations xroro. norsaal and frosa failing hearts of dogj 
show the eacn® scdimointation and diffusion constants and hence the 
same laoiticular weights, (torroll in cooperation with J. 0.. Davis) o 

Identification «f myosin as a sarajo:: constituent of the conduct:. =n 
bundle (■fbundie of His") of beef hesxt wes aatafclished by sedirafm':. 
and electrophoresis studias, The other protsin constituents in .: 
unfractionated e.a:tract era very aicilar to snufiCla, even thou,3a tkl-.; 
tissue ia thought to have a primarily iiervOus fraction. {Heiaader, 
mtcheli) , 

The stronijly acidic (because of the psetjenca ox cysteic acid) 
phosphase<=contaiE;inj; peptides ofots.lae.d from myosin hy oarti&l acid 
hydrclysis were further characterized. (Laki, i'lihalyi, Knoller) , 

Actin "•» The arginine^contair/.lng psptid^a obtained from acfcia 
by thff -acfcioa of tr'^-cpfsiir ;jre wnd^^t" stndy, Th*?- oatto*?"" of th,f>f?c- .-■',:■■,. 



Serial NOo NMMD -lOS 



(Coat 'do) 

The C'="te3naicuil ead group of rabbifc actin was quant ifeatively 
detesmined by using carboxypeptidas© A, (Laki^, Sfeaadaart.) 

Optical rotatory diapersioa studies on actin gave stroug in=- 
dications that on G»F transforaistion the (CK)n " "57** of G-»actin 
changes to -31**=, Since in 6 M ur@a both G° and F=-actin give (Q')j) ^ 
"look's the chasige is iatsjrpreted to indicate a gein in order for 
F^actiuo {Lakij Standaeart) . 

Heat mesLSureazents made with the Benzinger micro colorimeter 
during G- and F-actin tr©ri»f onaation iadicffite that ^ H for fch© chiange. 
of the G^protein to F-proteliii is about -i- 2000 cal, Tiiis observation 
combined with the optical rotatory moasurements Indicate that fcha 
G»F Cffansformation (at le&st in 0»1 M KCl) is driven by ths enargy 
liberated from ATPo <L«.ki9 litEinger) , 

Sti'ucfcuraljste.diias on other proteins; 

Salmdne; Th@ isolation of peptides formed during partial 
hydrolysis of salmine has been ©ecoaplishedj, and the composition of 
soiB® defcermined in an attempt to leam the sequence of amino aeida 
in the proteino (Knoller and Carroll.) 

The sfcructyral details of tha protein oolecuXe tliat can ba 
revealed by the study of tli« binding of cations and anions have been 
under continued investigation. 

The binding of Qa^', H'. snd 01'°' to serum albumin has been 

studied and th© nature of the electrostatic effect det«?.ETiiined. 
(Saroff., Lewis ») 

A study comparing the binding of anions to tha protonated 
nitrogen in model corapounds and protains is almost complet(edc Tfe.s 
bioding of chloride ions to detergents in the moacjcieric and micellfflr 
forms revealed a much lower electrostatic effect than that predicted 
indicating a high water content and consequent shielding of charges 
in the detergent micelle, (Saroff ^ Heaiy) . 

The study of the coinplejcattion of anions with salmine gave 
constants for binding -which war® unusually high (ccsnpared to d«tferg«n£ 
laicellss) indicating a clustering of charges in a manner siiich th^it 
the water molecules are not shielding the charges as effectively as 
in the soap micelle- In addition^, the quantitation of the binding 
of anions to saimixie revealed a structure for saliaine which groups 
the arginine residuas into six clusters of three residwes each. 



Serial NOo NIAblD 



Ma j oi:^^^ Find logs s ( Cent ' d „ ) 

Tfeie structure is compatible with the aggregation of saliains 
with its associated nucleic acid. (Csrroll^ Sarof f ) „ 

At present under study is the binding of anions (chloride aad 
brraiidft) to lysine and poly lysine (obtained fraoa M. Seia, Waizmana 
Institute^ Isreai) , These results will be compared to those ob- 
tained from albumin^ th@ detergents^ snd sslmine. (Sarof £) o 

Anion binding studies on salmine combined with the amino acid 
analyses of salmine and studies on its size and shape hav@ prooxptad 
us to consider the implications of the observed heterogeneity of 
saimineo An analysis of this heterogeneity has been undertaken to 
show fcbat there is a possibility that genetic infonnation is trans- 
mitted by the salmine molecule. (Sarof f, Carroll) . 

A continued study is being csrried out on the relationship of 
the SH group to specific structures in the protein molecule » At 
present under study is the relationship of the SH group to the haae 
function in h^noglobin. Th© SH content of heoiogiobin v^as found to 
vary with the pH. (This probably explains soma of the disagreetnemts 
in the literature on the SH values of hemoglobin Kinetic studies 
oa the decrease of the SH titre of hemoglobin ha^re been encouraging 
so far since the rate of decrease is about one order of laagaitud® 
fastar than that previously found in serum albumin, J-lercury in the 
hi'' and monovalent fona as well as silver are being used in, our 
analyses to remove the ambiguity resulting from the use of faivale-iit 
cations alone,, An attisapt will be va&d& to correlate the decrease 
in SH with pH with th® Bohr effect (change in pH with ©2 binding) 
and with the reversible dissociation of hemoglobin. Me are ispecu^ 
lating on the role of the thiaaolina ring in this reaction, 
(Saroff^ Simpsoflt), 

Collagen -•= In the course of studies on collagen rafetabolism in 
health and disease^, a dipe-ptide containing en equal amount of proline 
and hydro3Qrproiime has been isolated from hfaa^an urin@c. This canpound 
corraeponds by chroma togrephy and color reactions with a synthetic 
L^prolyl^L^hydrostyproline, This compound in hunaan urine appears to 
be the major form of hydrosyproline excretion, (Xrreverre) . 

FrcfiB the hydrolysate of an antibioticj, telomycin^ a naw cyclic 
iaaino acid has been isolated which by chromatographic behavior may 
possibly be a 3"hydro3£yproline, To characterize this compound 
3<»hydro3syproIine is being synthesissed, (Irreversrsj, Cohen) , 



Serial No. NIAMD-lpg 



Page 6 



Major Findings ; (Cont'dc) 

Studies on the changes in physical properties of sesrusj albmuin 
under various storage conditions for times up to 5 years have con» 
tinuedc There is good correlation among changes in ultracentrlfuge 
pattern^ viscosity,, and reaction with trichloracetate with time and 
temperature of storage ^ Th@ decreased solubility in dilute t?i° 
chloracetate is a promising Indicator of physical changes and has 
interesting aspects^ (Tripp and C&rroll) o 

Ensytaes ; 

Xhrcmbia =■= Thrombin has been purifissd via cellulose es.°- 
changers, Prelimiaary studies show that working below pH 6o5 and 
above pH l„Zf the enzyme is soluble,, This enables us to study the 
kinetic-molecular properties of thrombin, (Gladner^ Folk, Laki) » 

Preliminary studies indicate that by the action of the Lski= 
Lorand factor only one peptide is liberated fross fibrinogen by 
thrombin, (Gladner., Loewy^ Lakl) , 

A number of peptides of arginine were prepared for studying 
the specificity of thrombin, 

Gly„Arg^M. and Phe.ArgoM, are split by thrombin very slowly = 
Elongation of the chain from the C^terminal does not seesu to in<= 
fluence the rate of the reaction^ as Gly,Arg,Gly„ Et is split in 
the same magnitude of order of velocity as the amides. On the other 
hand^ change in the N-tesrminal does influence the velocity of the 
peptide splitting by thrombin. By blocking the amino group^ the 
peptide is split considerably faster^ e,g,, Bs,Gly.Arg,M,^ and 
Bz,Gly.Arg,Gly.£t, are split much faster than the corresponding 
Gly,Arg,6ly,Et, and Gly,Arg„Am, Elongation of the peptide on the 
N'-teEiainaX has the same effect, e,g,; PheoGly,Arg.Ao, and Gly,Phe. 
Arg.Ao, are split much quicker than the corresponding Gly,Arg,Am, 
and PhecArs,M, 

Thrombin was found to have wide esterase activity, B2,Lys, 
Me, is split quite quickly^ about one-fifth the rate of aplittlng 
of Bz.ArgoSt, Addition of soybean inhibitor did not change the 
velocity of the reaction which shows that the splitting of BZoLys,Me, 
is not due to contamination of thresabin by plasmin, Benz,Gly,Lys, 
Me, is split even faster, Bs,Orn£oMe, and Cbz,GlyoHis„lfe, are also 
split by thrombin. It seems that the esterase activity of thrombin 
Is toward all the basic anlno acids, (Levin), 



Serial No, NIA^ffi^ipa 
Page 7 



Major Findings g (Cont'd.) 

Carboseypeptldes <=>'- Preliminary studies on carboscypeptldase A 
have shoim that the eazyma.ttc activity of this metalio»proteia 
(zinc) can be further enhanced by the addition of cobalt ions^ in-^ 
creasing the activity as much as 100%<, The reaction is pH^ tesa^ 
perature and concentration dependent o Aether or not the @ff@ct 
is due to replacement of zinc by cobalt or cobalt snteriiig & second 
"active site" is under investigation o (Gladner^ Folkj, 



Carboxypeptidase B has nov been isolated in highly purified 
form froo pig pancreas. It appears fr<KD hydrodynasilc measuremenCs 
to have a molecular weight of 34^000. It appears to be a matallo^ 
protein containing zinc. Although its specificity differs markedly 
from the well knovm carbosypeptidase A (above), its similar molecular 
properties to this enzyme Is remarkable (Gladner^ Polk^ Carroll) , 

Trypsin =>- Under proper conditions^ trypsin can bind a sectKBd 
molecule of DFP« Using DFP^^ to bind to this second site^ we have 
been able to Isolate a peptide (19 amino acids) whose amino acid 
analysis shows it to differ from the site of the first DFP^binding, 
Since binding the first site with DFP inhibits the enzyme^ it is of 
great interest to elucidate Che complete structure of the second 
site, (Gladner^ Vlswanatha) o 

The entire sequence of peptide A liberated from fibrinogen 
during clotting has been elucidated. (Gladner^ Polk^ Levin), 

En z yme ^ compIeaKes : ^t In collaboration with Professor Lester J, 
Reed of the University of Tesas^ studies have been carried out on 
the hydrodynamlc properties and size of two large enzjrme complexes 
Isolated from bacteria; a^ketoglutarate dehydrogenase complex and 
pyruvate dehydrogenase complex. These have molecular weights of 
2,4 and 4,4 million respectively^ and behave as fairly compact 
spheres as judged by sedimentation^ diffusion^ and vlscoaity measure^ 
mentSo Each complex is capable of carry ixxg out four to six enzy^ 
matlc steps in the os^ldatlon of substrate^ and contains the 
appropriate co-enzymes in fised amount. Studies of the fragmentation 
of the compleites in ways to maintain separate activities has been 
started. (Carroll) „ 

Hew amino, acid °- Studies on the detection; isolation^ and 
characterisation of nitrogen ccmpounds (related to amino acids) in 
living systens, 

A new acidic aromatic amino acid has been isolated a(ia°^carboxy° 
phenyl) glycine from Iris bulbs (Iris tln gitana var, Wedgewood) , 
(Irreverre^ Thompson^ Asen) , 



Serial Moo KIAMD -I PS 
Page 8 



(Cont'do) 



This amino scid has also been synthesised and the N-acetyl 
and N-chloroacetyl derivatives prepared, Eaaymatic studies were 
made on these conipounds with the view of separating the stereo- 
iscaaerso 

It was found that the urines of normal infants contain an 
a-amino acid which does not correspond to any a-amino acid knowi? 
both naturally occurring and synthetic so far studied « It was 
established that this amino acid did not come from the food in- 
gestedo Aad it does not occur in the urines of older children 
and adults (over 100 exaained) . 

The examination of the urines of EBRD cases from the wilds of 
New Guinea for amino acids and total nitrogen showed very Interesting 
and unusual patterns » This work is still in progress (Irreverre, 
Ga jdusek) „ * 

Significance ; 

When part of the protoplasm or the whole cell (as in, e g 
cell division or muscular contraction) performs mechanical work* 
a network structure is built up at least temporarily, mainly through 
an orderly polyiserigation of globular proteins. This structure then 
reacts with the surrounding medium and by utilising metabolic energy 
(stored in ATP e,g,) performs work (muscular contraction, amoeboid 
movement). In order to understand this "mechaaoochemical coupling" 
(the interaction of structure with the surrounding and its disorders 
we must know how such structures are built up. In addition we also" 
have to know the detailed structure of the %uilding stones," the 
structure of the polymerizing proteins. Muscular contraction and 
blood coagulation are ezcamples of processes w&ere structures are 
built Up through protein polymerization. Such knowledge eventually 
will lead us to the understanding of certain diseases of muscle. 
Study of blood clotting, in addition to supplying clues for protein 
polymerization^ gives us better understanding of the disorders of 
blood clotting. 

When both direct and indirect approach leads to some specific 
disease (e,g, hemophilia, rheumatoid arthritis) the advantage 
offered by studying the disease is utilized to the extent profitable. 

Proposed Course of Projects 

In the next calendar year we will follow in logical sequence 
the topics outlined. 



Serial NOo NIAMD --108 
Page 9 



PHS'WIH 
Individual Project Report 
Calendar Year 1959 



Part ^^ B ; Honors^ Awards^ and Publications 

Publications other tlaan abstracts from this project; 

1» Bowen^ Wo J«^ and ^{artinj, Ho L.: Analysis of myosin B for magnesium.. 
Archo Biochemo and Biophys.^ in press » 

2o Bowen^ Wo J., and Hartin,. H. L.: Analysis of serum magnesluia in 
presence of calcium with Osroise Fast Blue BG« Soc. Exptl^ Biol<. 
and Med. 101; 7^4-736, 1959. 

3o Carroll^ W« R.^ Callanan; M, J,; and Saroff^ HoAcS Physical snd 
chanical properties of protamine from the spens of salmon 
(Oncorhynchus Tschawytscha) . IIo Anion binding characteristics o 
J. Biolo Chem„ 234s 23i4-=2316, Sept., 1959o 



4. Folk; J. E^f and Gladner^ Jules A.a Carboxypeptldase B., XII. Specific 
esterase activity^ Biochlnio Biophyso Acta 33s 570=>572^ 1959o 

5d Gladner^ Jules A., Folk^ J, E<>^ Laki^, K,^ and Carroll^ W, R,,; 

Thrombin»icduced formation of co=fibrino !<, Isolation^ purification, 
and characterisation of co^fibrino J, Biol, Gheno 234 ; 62=66^ 
Jan, 1959. 

6. Folk^ J<. Ep^ Gladner^ Jules A.^ and Z^akij, K. ; The thrombin^inducsd 
fonsatlon of co^fibrin» II. Preliminary amino acid sequence 
studies on peptides A and B. J^ Biolc diem. 234 ; 67"70; Jan« 1959 o 

7c Folk^ Jo Eo^ Gladner^ Jules A,^ Vlswanatha^ T.s A simplifisd chromato^ 
graphic purification of leucine aslnopeptldaseo Biochinio BiophySo 
Acta 3£: 256-257, Nov. 1959. 

8c Folk; Jo Ed J, Gladner^ Jules A.^ and Levin^ Y.; Thrombin^ Induced 

formation of co°flbrin« III. Acid degradation studies and suceaary 
of se<iuential evidence on peptide A. J. Biol. Ojem. 234,? 2317-2320, 
Sept. 1959. 

9. BvanS; R. L.^ and Irreverre, F.; Synthesis of Y°amino=butyryl=Y 
amlno'butyrlc acid. J. Organic Chemc 24; 863^ 1959, 

10. Irreverre, F., and Teraian^ Levon^, A„: Nitrogen partition in the 

excreta of three species of adult mosquitoes. Science ^9; 1358^^ 
1359, May 15, 1959 „ 



Serial KOo NIAMD ° 1Q8 
Page 10 



Publications s (Cont'do) 

11, Irjreverre^ F.^ and Evans^ Ro LoS Isolation of ■y°6"aaldioobutyrlc 
acid from calf brain., J, Biol- CheiBo 234; 1438=1440^ June l959o 

12o Komins^ Do Ro^ Carroll^ W, Ro, Smithy E. H^.^, and Mitchell^ Eo R<,% 
A suhunic of myosino Archo Biochffla, and BiophySo 79^; 191-199^ 
1959 o 

13c Haruyama^ K^^ and Kominz^ Do R. : Earthworm sQrosino Zeltschrlft 
fur vergleichend® Physiologie ^i l?wi9j 1959 o 

l4o Soad^ Foj> Ejcatinz^ Do Ro^ and Lski^ K,% A study of th@ tropcaayoslns 
of three cold<^blooded vertebrates of different classes » Jo Biolo 
214s 551-555. Mar, 1959. 



15o Laki^ Kij) and Standaert^ J„t Th@ miniiaal molecular weight of actin 
estimated with th@ use of carbozypeptidase Ao Archo Biochemo and 
Biophyso^ in press o 

16o Mand@lk@m^ Lo^, Po8n@r^ Ao So^ DioriOj, Ao Fo^, and Laki^ Ko ; Mgcbanism 
of contraction in the muscle fiber ATP system, Proco Natl, Acsdo 
of Sciences 45?. 814=819^ June l959o 

l?o Saroff; Ho Ao^ and Bealy^ Jo l^Jo! The binding of chloride ions to 
amineso Jo Physo Qxemo 63; ll?8=118l, 1959o 



18o Saroff^<, Ho AoS On the acyl shift in protein reactions o Ensymologia 

21_; 101, 1959 o 

and Evaas^ RoLo 
196 Saroff^ Ho &J', The conversion of the amino group of a&ino acids and 

proteins to the non^^basic nitroguanidino. group » Biochimo Biophys^ 

Acta 36: 511-518, Deco 1959 o 

20o Helander^ E.^ and EsssEsart; E, V,i The localissation of myosin in the 
conduction bundle of the beef heart o Proc, SoCo E^ptlo Biol, and 
Med. 10l_; 838=842, 1959, 

21 o Folk^ Jo Eoj, and Gladner^, Jules Ao: Cobalt activation of carbosy^^ 
peptidase Ao Jo Biolo Chemoj, in press, 

22o Asen, So. Thompson,, Jo f„g, Morris^, Co Jo^ and Irreverre^, Fo? 

Ch£mo Isolation of p<=^aminoisobutyric acid from bulbs 
^^ Iris Ti ngltana varo Wedgefwood, Jo Biolo CSiemo 234: 343-346^ 
Febo 1959 o 

23o MorriSj, C„ Jo, Thon^son^, J, F.^ Asen^ So^, and Irreverre, F,; 

Isolation of a new acidic aromatic amino acid^ aCm^carbosyphenyl) - 
glycine frtm Iris bulbs, J, M. Chtsa. SoCoSl^: 60-69^ Nov, 1959o 

24o IrreverrCj, Fo^ and Levenbook^; Lo ; The effect of diet on the free 
amino acid patterns in the blood of southern army worm ( Prodenla 
Eridania) o Biochimo BiophySo Acta^ in press. 



Serial No„ HIMD 

lo Physical Biology 

2, Physical Biochemistry 

3c Bethesda 



PHS-13IH 

Individual Psojecfc Report 

Calendar /ear i)59 



Fart A, 



Project Tide; Izmimno chemical approaches to the isolation and 
characterization, oi; proteins 

Priocipal Investigator: 13t„ Ro R, Williams 

Other Investigators s None 

Cooperatl!sg Units: 

Dr» Eo Mo Leraerv, LFH-NIiiM> (Seriial Ko„ 82 ) 
Dr. Ko J. Blochj, A5eR°51I&MD (Serip'. Mo, 123C) 



Man Years (Calendar Year 1959): Totals I 

Project Description: 

Objactivea ; 

Production of escperiraental arthritis and abnoKstal serological 
reactions in aniaialso 

Methods; 

Sensitized sheep cell agglutination^, ultracentrifugal 
analysis^, and ch^aical analysis of proteins. 

Major Findigj^a ; 

The observation that rats injected 'jith Streptobacilluig 
Moniliformis develop joint lesions and positive sensitised sheep 
cell agglutinations has been euctendedo the serological reactions 
of rats and rabbits to killed organi^ss have been studied and 
found (positive B.F.To reaction) to be largely due to ijianuniaation 
with gansna globulin in human ascitic fluio. The imnmnisation of 
rabbits with killed organisms grown in media containing huiaan 
ascitic fluid produced high DFT titres; but no elevation in SSCA 
titreo 



Serial Ko„ ^MMD-im 



Page 2 



Positive flocculacion tests in ejcperiiaental animals must I 
carefully evaluated. 

Proposed Course of Project ; 

Extension and confirmation of above. 

Part Bs Honors^ Awards, and Publications 

Publications otbsr than abstracts from this project: 

lo Lerner^, 1= M, 11^ Bloch^ K, Jo# and Williams^ Ro Ro^ Jt.% 
Rheumatoid ssrological reactions in essperimentals., II o 
Bentonite floccuXation tests in rats with €xperiin@ntal 
arthritis o Arthritis and RheumatieiQ; in press, 
(With technical assistance of Marion Robertson^ Ralph !« 
Groc«n@8,^ and Clarence C. Israel,) 



Serial Ko., HlM l:a»llCI 
lo Pliysical Biology 
2o MslecuJUii" Biophysics 
3 o Bethesda 



Individual Ps-oject Report 
Calendar lear 1959 



Project Titles It?;/ostigatioa o£ the macroiaolecular 
orgsiHlsation of li¥iirag nsattsro 

Principle lOTeEtigaters; Ro Wc Go Wyckoff (retired August 

yffld Lo Wo u 



'Other Iwestiggtoras ¥o M^ Moslojj Go CeoliE (fisitlsig 
ScisBtist from Jaauary through May 
1959) 

Cooperating Usaitss Piesre L^pineg Imstitute PasteiiTs, P®r.tsi, 

Fx'aac© (through Jfe;f) | Do Bo Scottj, N'stiosfil 
lastituta of Damtai Resssrch 



Totals 4 1//2 

Professional s 3 
Other 4 1 1/2 

Project Descriptioas 

ObjsctiY®ss To gain inforaatiori afccafe the aacromolsculeg 
that are essential coasSitueiats of liTirig ffiatter^ fca see 
hov th©y SET© arranged ira th© structures thejr form aad to sse 
hov this arraieigaffifsnt is altered fej infectioEs and d©gsH,er- 
ative diseas©o To study qertaira of these aacroaoleculeiSj. 
Each as "Viruses «md other protQiB.Sj in parifiadTorsi after 
isolation froK the livisg ffiatarialo To iaspro^s . the yesolu= 
tion of the elsotros aicroscops and to interpret th® way in 
whdch ifflagss ar® fonasd laear its liiait of' reeoli!itioE,> 

Methods S^loye<3; Th© elsctroa microscopy of fflicrporgaadams,, 
cells and tissues la saspensioa or thinly sectionedo The 
pijj sicochomic&i chsa'actsriaation of niaerosolecular cob?50e='' 
©Rts isolatad fro.® such material usipg electron edcroscopyp 
X-ray diffractioap amd similar established techniques 5 the 
de^'elopmerat of laey physio&l procedures j, iscludiiag X-r.'sy 
fiiicroscopy and lou.g waifelaagth X-^ray diffraction,, to further 
such oharacterisatioao 



Serial NooJ^©^^ 
Page 2 



Major Findings? (1) The analysis of the crystal structure 
of protein crystals by meaas of electron micrographs and. 
the use of EBodels has been continuedo The crystal struc- 
ture of the Rothemsted tobacco necrosis protein has been 
determined in this li^ay and is in close agreement with the 
crystal structure found using X-rays « 

(2) The photography of the molecular separatioi^ in 
crystals of organic compounds of molecular weights 500 to 
?00 has been continuedo It has heen determined that this 
is not a direct imaging of the crystal planes but is rather 
aa interference pattern produced by phase changes in the 
electron wa^es between those passing in between the pro= 
perly oriented crystal planes and those passing thru these 
plaaeso This interference pattern can ha'^e the saiae sp&c=" 
ing^ as the molecular plane spacing datermined hj X-ray 
diffraction j, but appears above aiid b©low foeus rather than 
in focus o The fine structure of the interference patternp 
including halving of the spacing for some positions of focus j, 
can be predicted and checks \iith the e^erimental datao 

(3) The mJ.cro~spot X-raj microscope has hQQn adapted 
to photograph diffraction patterns using long wave-length 
X-rays up to 10 !» The resulting dispersion on the record- 
ing plate has been increasedp for short plate distances j, 
beyond that possible with comsercially available X-rey 
diffraction apparatus o This permits the easy det©ri3iiaation 
of large aoleoular plana spacings in crystalSn 

Significance to the program of the Institute; There is in- 
creasing interest in imaging and interpretiiag fine structure 
using electron m3.croscopyo To interpret electron micrographs 
of some of these fine structures accurately it is necessary 
to go back to considerations of basic image formation p par° 
ticularly for structures less than 50 Angstrom units in 
sigQo It can be shown j for instance^ that periodic structures 
in this range can produce interference images which ha'^e 
periodicities other than those in the objects, depending on 
the condition of focus o Periodicities other than those of 
the object can also be present if Bragg reflections from 
the object contribute to the imagSo It is believed iss^rtent 
to try to define the conditions under which these artifacts 
take place as an aid to the interpretation of the fine 
structure appearing in electron micrographs o 

Proposed course of the Project; The investigation into the 
use of characteristic X-ray absorption and fluorescence to 



HIAS©-110 



localize elements in sections photographed, with the X.-ray 
microscope will' be contiauedn Th© use of this instrument 
adapted for diffraction together with standard X-ray dif- 
fraction appai'atus will be used to further the work out- 
lined in the prerious paragraph.. 



Part B. included, Yes 



Serial No., MIA&B-llO 
Page 4 



PHS-NIH 

IndiYidual Project Report 

Calendar Tear 1959 



Part B. 



Publications other than abstracts from this projects 

Labawj, L., Wo aiid Wyckoffj Ro W, Go? The electron micros- 
copy of tobacco necrosis '^Irus crystals o J.-. Ultrastructure 
Research 2 s 8-15 j, 1958. 

Labawp L> W,; kn electron microscopic determlEation of a 
tobacco necrosis ifirus crystal structure c. J. Dltra- 
structure Research 2: 177-184. » 1958 .. 

Wyckoffj Ro Wo Go and LabaWj L,. WoS Observations at high 
resolution on several indanthrene dyes.. Proceedirigs of 
the 4th International Conference on Electron Microscopy.; 
West Berlins Germany j, September 1958 n 

LabaWj, L., V„i An electron microscopic determination of the 
Rothanisted tobacco necrosis protein crystal structure, 
Jo Ultrastructure Research 3; 58=69 s, 1959c, 

^yXen^ M„p Scott g Do 8,5 Mosleyj, V^ M., ; Mineral! zatios. of 
turkey leg tendon (in pspess) o 



X« Physical Biology ' 
2-» Itoleculss: Biophysics 
3. B@th@9da 



ladl-yidual Frojecfc Report 
Calendar Yeas: 1959 



Project Titles The physical eha&istry o£ roess&ranes aad complex 
isesibirane syst^ns of biological interests 

FrlQcipal Zm^®@tigator: ^;rl Sollskesr. 

Other Investigators: Euth HsCliEtock Cie2£ August 31, 1959); g®rsld 
H. Sheaa; aad Stanley D. Jssses (slace Agril 14^ 1959). 

Cooperating Uaits>: Loose cooperation ia saaint&is&s^ »lth Dr. Charles 
W. Carr^ Associate Pro£®8®or_, D®|Jt» of Physiological Ghesaistryj, 
IS@«iicaX School^ UMv. o£ .9Sisin®j&ot&, smtJ with Dr. Eugeae Gzim^ Asaoc"' 
lata Professor, D«gt. of Physiology j, Uaiv, of Minaesota. 

M&n Year® Ccal<Kadar y®ar 1959) j 

total: 3-1/3 

Professional: 3-1/3 

Other: 

FrojQct Dsscriptioa: 

Objectives: A physicochoaical study of ocasbranes and fii(S^ran@ model 
$tyst@sas vith th@ purpose of providing a ratioaal physicoche^iical 
basis £or the elucidation of nuaasrous phenotaena in living organisssSp 
for instancffij ©Isctrolyte balance and electrolyte distribution, the 
accusoulatioa of electrolytes in living cells^ cell and nerv© potan- 
tials, and electrophysiology in s@sE@ral« 

Methods ©asployed: The preparation o£ porous mea&ranes of highly char" 
acteristic and specific electrochemical properties (the methods hav- 
ing been worked out by the principal investigator and hi© collabora- 
tors), and recQstly alao of oil saanbranae of eoasewhat siiaiiar 
characteristics and the inveetigation o£ these jaeafcrancSj and of 
m^abrane systesas in laiiich such ta^^ranes are functional psrtSg fey 
physlcochenical, especially eiect^oehaaiicalj aiethodSj, such a& pot4gr6- 
tial and resistance aeasurecaeitts, alao fey choalcal analytical 
pffoeedureSj, including radioactive tracer ©ethoda. 

Major findings: Theoretical considerations had Isd to ths predictiori 
that the ratios of the rates of the electrical transportation across 
persBSQlsctive jaestabraaaa of any two species of ions of the saB*® charge 
coeststing in solutionji should be pTredictable quaatitafcivelyy a) frees 
ths bi-ionlc potentials arising tJith the ®sae Ions across the saasa 
tsu^ibrane, and b) from the rs^io of the rates of the exchange of th® 
easee tiK> iotts across the Qaane {ness^ran^ against a third ion, Th@ «£- 
perijssatal rosulte vere ia fair agrecss^at with the predictions. Hov- 
averj, significant devlatlonSj, outside of the range of the e^psristontal 



Serial lHo„ KIAM)..!!.! 
Page 2 



Major findJLstge (coEt'd.); 



errors occur rsgularly «;hicla ssmst be asstmad to h® iiu& to 
alect:rophor@t£c iacssr actios be&^@@Q ions of &H® easm charge^ 
tke soIv@qC^ fiiffld fete pores o£ th@ si^abrsra® i^ea aa electric 
currsat is seat through the system. At higher curr@i&t dass" 
Itias the situation is still more cos^licated by polarisation. 
For the time bsiag It sscsas that ao rsasonabl® miount of mi- 
peritaeatal m>vk could clarify Ist detail £h6@@ highly involved 
problesnSj, ths fur£:h©r lavtastigatioa of ^ich has^ therefore^; 
b<sen diecontiaueda 

Sigaificaat progress has b®ea madcs ia tha study of "oil" 
vam&iSsateBjf particularly hy the use o§ porous '^@f loiis'' discs 
which are filled islth the oil. This asrang^isat supplants 
the traditional U^tube in «ihich tte thickaess of the 'Hsigaibraae'' 
is of the ord@r of 10 cm„ Tim lae^ technique reduc<§3 the r@o 
sistemce of the esspsrijasntal cell® by t«ec» orders of tugaltude 
and correspoadingly accelerates the retes of IokjIc ©xchang© 
across th«^i by the &sam factor. Soese ne«;ly developed coas9i@r° 
cial preparation®^ "liquid ion esehasgers", vsatss found to be 
pronising active nolecular specie to be iaeori»«rat<sd in the 
isesabranes. Thsir properties se^D to sisaftlify the system 
significastly eosepared with those of prios- authors. On th® 
basis of fisueh tedious and tMa^coasusaiag prelimltiftary w>tk it 
se^as no« justified to express the hope that «e ^lll be able 
to fmk®^ over & |»eriod of years.^ substantial progress ia a 
field «^ich @aialy for the e^periasntal difficulties involved 
has been Biore or l@ss dormant for several decades o 

The concentration jpoteatials «hich arise in cells with psrEa« 
selective iseraibranes aad electrolytic solutions agree closely 
in a mediuEi range of cos^eentratlons «ith those calculated from 
kno«n data on the basis of conventional assustptions for cells 
«lth sietQbranes of ideal ionic selectivity. The experiaieatally 
detertBlned pot^ttials at low concentrations Cssaaller than about 
0.04g> are consistently belov the theoretical values^ the dis-° 
crep4iinel@s being larg@r the lo»er the coaeentrations„ It ^as 
dessonstratsd that this imcKpeeted effect is not due to issperfec" 
tions of the meaibraaes. Theoretical cons iderat loos ruled out the 
possibility of a major importance of osmotic water siovessent but 
have led to the hypothesis that siSBibraQe hydrolysis ssight con* 
ceivably account for the observed effect. 

The fact th&t «^ater is @ia Ionising liquid and that the distribution:! 
of the H^ and OH" ions across the asMffiibrane wast under equlll.briu]i!!3 
conditions (under i^ich alone the conventions! theory applies) caw 
form to the .postulsite of the theory of the Donnan laessbrane equilibriusi^ 



V<" •«.«> »0H-»> ««-<'> 



Z^'Z^ ";;7ar'rri« 



Serial So, _Ili®sML^ 
Fsg@ 3 

hags been hardly rsgs^^ded in the zes&sit literature! on a^a^raost 
potest ialSo Cslls ii3 ^ich the Dom&aa coadition is not fulfilled 
are not equilibrium (or quasioequilibriiac) Byatsam in ^ich alone 
the conve&tios&al ^&y o£ calculating theoretical values of th®s© 
poteQtiele could be strictly valid, Fsdoi the theoretical points o£ 
view coffiveiKtlonal aqueous conceat ratios cells with ^naselseEl%".a 
meiBbrsnes have to be considered as dyaasic "two loaic" cells to 
which the dynamic theory of polyioaie potest ials, as developad re- 
cently ia thi® laboratory j, sasst be applied. It was sfeoi^ seai- 
qusBtitatively that ©sperisaesjtal cells aet up origiaally with tm> 
neutral solutions drift slowly, by the easchang® of iosss betwe^ 
the two ©olutioaaj, toward® a dlstributioo o£ the loas of the waser 
^Ich corresponds to the Domian equilibrium; the del icieaey is 
potential in such cells being due to a kind o£ internal 8hort-> 
circuiting. It also could be sho^in sssBi«quea£itetively that pairs 
of solutions adjusted beforehand to the proper ratios of hydrogea 
ion concentrations yield potentials snuch closer to the theoretical 
(reversible) values than those of cells »ith solutions of equal 
hydrogen ion concentration, fha experissental diSfieultia® in ob" 
taining quantitatively satisfactory data for publication are coa- 
siderable due to the ex:tre&ie pH sensitivity of the unbuffered 
eKperiiaental solutions near the neutral point; m>rk along th&B® 
lines is in progress. Sa principle, however, the before^esentioaed 
discrepancies bet«i^een calculated and theoretical potential values 
in Q^ibrane concentration cells B®sm to be resolved. These resulti^ 
have obvious bearings on the evaluation of essperimsntal potentislc 
in snany sy8t«s involving saaabraaes, including cells ta ®hich 
pertEsselectlve m^iibranes are used as {Qes£>rane electrodes. 



EspsrisBcatal work on aa improved aiadei for the accuaiulatioa ©f 
electrolytes^ of anions and of cations sii^ltaseousiy (Scieacej ]M^ 
939 <1956>, once iswre has eonfin&ed the correctness of the previeusiy 
developed theory. The rather involved experi^Ksnts necessary in these 
teats will require a great deal of additional ^rork before the asatesrial 
rail! be ready for publication. 

Significance to Research of the Xnstitute: In order to understai^d 
electrolyte relationghips in living cells and tissues^ it Is neces- 
sary to have accurate infonmation on astsabrsne model systeias <9}hic!«^ 
under carefully controlled kno«^ conditions^,, reproduce at lesst 
scat® of the laajor in vivo phenosBsma, The m>rk of recent ysars^ 
particularly the study of polyionie pot^itialSj, of absolut® and 
relative rates of ionic fluxes under various conditions^ and th^s 
construction of an Jn vitro model of electrolyte accmsajlattoa have 
brought u@ significantly nearer to en understanding and an in vitro 
reproduction of the type of effects «Aiich ultimately suet govern the 
in vivo ossBotlc b<shavior of cells and tissues. The work already 
carried out indicates that even fairly cos^lex sia@bran@ sys^^isS; 
sifitilar to those found in living nature, aiay prove in the foreseeable 
future stnsnable to a cra^lete and quantitative physicochemieal &asi.lysl&. 



Serial Mo» ..OTA»m»m. 
Page 4 

Proposed Course e£ Projects Furthss ©sparisneatal work oa ffi"i«c£rolytic 
accusBulstioa agsiBS& coQcoa^ratioa gradlstiits. Further ^9ork on 
iseiabranfi hydrolysis sad its Influance ca laes&ran® potisntlals, 
Reeusiptlon o£ the leork on the sbeolute rat« of exchange o£ iotm 
across P«ffla89l@ctlv® sissibraaos, froa the ea^esrleiaatal aad theoretical 
point of vle«'« Aeesleratedi cont£«3uatloa o£ the studies on oil 
!a«mhran®3 on thm basis o2 ths abov® d©@crlbad results. Ov&t tte 
long range usny oil ths effects studied with permseleetive m&ibt&nsiti 
over the last 20 years ^ such as their u©® as s!s«abraa«8 el«ctro€l®Sj or 
in the study of saaBbran® ®s|ullibria, or in the investigation of ioaic 
specificities^ etc., should also b® investigated with oil ©saibraa®©., 
as far as they lend thesm®lves satisfactorily to these purposes „ 

In additloBj, largely depending on th® availability of a suitable 
collaborator a study is planned of th& foress tikich operate in thm 
spontaneous formation of regular structures of iBlcroscopie and sc3b°° 
microscopic (but greater than csolecular) dlssienslotiSo Long range 
forces of attraction mtd repulsion bet%7een silcroscoplc and sub~ 
sQlcroscopie particles ar@ hnomi to exist <> l^ese forces are acc@@gibl@ 
to quantitative aseasureaseato by aetho^^a developed by the senior inves- 
tigator before ctsalng to Hm» These studies are dasigned to furnish 
an insight iisfco tb© physical forces ^ieh create organiged stsucturs® 
of various levels of cosplexities as tho@e existing in living synt^SiS^ 



Is B Included? Yes 



Serial NOo _HSAHL-sJJUL— .=-_ 
Paga 5 

ladlviduffil Project Reipozt 
Calendar Year 1959 

Part B; Eoaoss^ A^sasdSp mid PubXieations 

Fubllca&ions other than abstracts from this project: 

l&sia^ Marc s»d Sollaer^ Sari: Prsparatioa aad Properties o£ 
Zmproved Prot^tis^e CoIIodioa Matrls UmAtvmt»a of Sstr^s® Ionic 
Salfictlvity, J. EI©eer©ch®ao Soc, 106, 347-354 (1959) 

McCllstock; a«eh, Meihof; &«ssss aad Soll^as^ Karl: Th© E®lstiv® 
Rates o£ Elsctroaaigratioa of Dl£f@reat tows of tha Sase^ Charge 
Across P@nasel@ctlv® M^braneso Jc Slectrochsmo Soe, (in pr®s@), 



zvxal. No 
1 „ Phys 



2c ?hoto&ioic^> 
3o Bethesda 



PHS'NIR 
Individual Project Report 
Calendar year 1959 



Part Ao 



Project Title: General Project Domain of Section - 

Molecular Mechanisms of Radiant Energy 
Transformation in Biological Structure.. 

Principal Investigator: F<. S. Brackett 

Other Investigators: (by projects - a to g) o 

(a) Ro A, Olsonj C. Lo Greenblatt and E, Engel 

(b) No E. SharplesSj, 0, S, Tenmier^ and Jo Ro Mills 

(c) Eo Do Becker and R. B. Bradley 

(d) Uo Weiss and Ho Ziffer 

(e) Eo Charney and Go Ro Gauvin (Ao Shannon- summer erapJ,o3 

(f) Wo Ao Hagins 

(g) Ro Go Adams 

Supporting Activities: 

Electronic Development - Mro Lawrence Showkeir 
Instrument Development - Mr„ Charles E, Lohr^ Jr„ 
Data Processing - Mro Wmo Eo Hahn^ Jr, 
Secretary ~ Carmelia Mo Joy 

Co-operating Units: 

Department of Terrestrial Magnetism of the Carnegie 

Institution 5, 
Naval Medical Research Institute^, 
Dr. Co S. Watson;, University of Minnesota 
NCI Radiation Branchy 

Dro Lewis Jo Sargent (LC-KIAi'ID) ;, NIAMD~57 
Dro Jo Mo Bobbit & Miss Do Hanessianp Connecticut State 

and Ohio State Universities^^, 
Urner Liddel^ Bionucleonics^ NIAMDj «120 
Laboratory of Technical Devel opment j, Heart Institute 

(See Serial No. )j, 
Dr, William Carroll^ Physical Biochemistry j, NIAMD-I08 

Man Years (calendar year 1959) 
Total: 19 
Professional; 13 
Other: 6 



Project Descriptions: 

(a) The action of radiant energy on structure and 
chemosynthssis in living cells « 

(b) Investigations of the action of radiant energy 
on biologically important compounds,, 

(c) Molecular structure determined by spectroscopic 
methods . 

(d) 1. Properties and biosynthesis of photodynamic 

microbial pigments, 

2. Photochemistry of organic compounds. 

3. Chemical constitution of the so-called 
"hydroxycodeine" , 

(e) 1, Molecular structure and organization in 

biologically important systems, 

2, Investigation of the optical rotatory disper- 
sion of organic compounds. 

(f) Flash photolysis of Vitamin. D and its precursors » 

Brief Report of Research in Photobiology* 

Life processes are uniquely dependent upon light in 
several waySj, particularly: 

lo Energy storage - photosynthesis 

2. Vision 

3, Production of calciferol - (Vitamin D) 

Despite our complete dependence upon these photo processes,, 
the molecular steps by which they are accomplished are not 
well understood. Research in this field comprises not onljr 
direct studies of the/processes themseiveSj but also studies 
of simpler related systems which may throw light on the more 
complex natural mechanism. The tools for this field of 
research include both the oldest and the newest of those 
conceived for the study of radiation and electronic processef 
These include, for instance^, recording spectrometers in such 
varied domains as Infrared (IR)^, Kuclear Magnetic Resonance 
(NMR), and Electron Paramagnetic Resoance (EPR) , Visible 
and Ultraviolet, In factj. advance in this research field 
requires continuing technical investigation and the develop- 
ment of new instrumental approaches. 



See individual project reports for fuller discussion: 



Brief Report of Research in Photobiology (Cont'd) 

Progress is for tfee most part £0 be found in the 
piece by piece assembly of pertinent findings^ thus: 

In Photosynthesis; The organization of pigment- 
protein molecules into a functional network nsay 
explain the extraordinary effectiveness of this 
"machine", (a) Partially reversible changes in 
bleaching are found and a dependence on oxygen 
demonstrated. The site of this action is related 
to the lainellarchloroplast structure, (b) Digestioi-i 
of lipoid from the chloroplast allows the still inta^: 
layers to separate in fanlike fashion. Protein diges 
tioHj, on the other handj, causes the layers themselvet 
to collapse. 

Bearing on the mechanism of photosynthesiSj, it is , 
shown that anomalies in scattering by the pigment accounts 
for only a snsall fraction of the wavelength change from 
free pigment to in vivo condition and so may be indicative 
of its relationship to the organized structure. 

Alsoj related to the role of chlorophyll in photosyn- 
thesis are our findings concerning porphyrin structures; 

(a) The specific stoichiometric nature of the bind- 
ing of copper- porphyrin to bovine serum albumin 
is to be contrasted with the lack of such bind- 
ing to B -lactoglobulin. 

(b) Still more physical evidence is found from Nuclesi 
Magnetic Resonance concerning the "ring current" 
producing local magnetic fields within the mc' 
due to the induced circulation of T electrons 

the conjugated porphyrin ring. 

Many of the factSj, iideaS;;, interests^ and inquiries 
concerning one photomechanism apply to other., though 
different^, photosysfcems for energy absorption and transfer 
ThuSj, the study of the basic mechanism of vision proves 
closely allied to our interest in the molecular mechanism 
of photosynthesis. Here again the primary photo mechani-KC!^ 
is least well understood. Here again the efficiency of the 
system transcends anything in our experience. Physical 
theory is shown to predict the minimum electrical current 
(about 1000 charges /photon) that a photo receptor must 
produce in order to convey information to the brain. 
Experiments carried out in collaboration with the Naval 
Medical Research Institute on the photo receptors of the 
squidp yield values 750 electrical charges per incident 
photon in good agreemeait with the prediction. 



icepoi-E: at Research irs 'caocobioiogy luonc'd) 



The genetic changes produced by ultraviolet are 
especially interesting as this is probably the region 
of energy threshold aad may show specific selectivity 
in mechanism as contrasted with the random effects of 
high energy radiationo Looking toward such investiga- 
tion,, a co-operative study (with Dr. Elkindj NCI^ 
Radiation Branch) of the chromosomal alteration;,, as 
related to the levels of lethality in dose of ionising 
radiation is being carried out along lines closely re- 
lated to those developed by Puck, 



Instrumental Projects: 

1. DATA PROCESSING - Mr. Wnio E. Hahn^, Jr. 

Contract let to Airborne Instruments Laboratory 
for the construction of a logging system which will 
take data from laboratory recording systems (analogue) 
and convert the information to digital forci recorded 
on magnetic tape in a form suitable for direct process- 
ing on our central IBM 650 Computer. 

A machine for plotting data from paper tape on. 
a 11" X 17" graph paperj, has been assembled from 
purchased components - 

An Add Punch has been ordered for producing paper 
tape, either by manual transcription or by automatic 
punching through a solenoid deck. 

2. The double monochrometer previously reported has 
been completed to the point of preliminary runs for 
the purpose of cutting the linearizing cams which 
are now ready for refinement. Accessory equipment 
is still under construction. 

3. The grating system for quantum determination at 
two or more wavelengths has been completed and the 
thermal control system is being constructed. (This 
project was delayed by the long period of building 
construction in the areaway. 

4. Electrode technique for O2 and CO2 determination 
was again the subject of study. A new electronic 
approach was suggested by the study of Lessajous 
figures. A dual purpose membrane-protected flow 
electrode has reached a later stage of development 
(see Olson) . 



: Research in Photobtology (Cos 

Essential £o an understanding of these raechanisajs i-^ 
the role of the pigaent which absorbs the energy and initi- 
ates the train of events which result ia the essential 
chemical storage of energy on the one hand or the aerve 
stiimlus on the other. In a widely diversified group such 
as tbisj the interest in these pigmentSp ranges from that 
of the structural organic chemist to physicist's i^Jterest 
in triplet and aietastable states which nsay foe involved in 
the mechanism of energy transfer. 

Thus J, a red pigment naturally synthesized by a inn^iv-si 
of the genus Elslnoe (Ascomycetes) has been isolated by 
our organic chemists and proves especially interesting 
both because of its unusual pattern of conjugatiois. and 
also because of its photodynaEiie action. Pigments which 
promote such destructive photo oxidation are sometisnes able 
to supply energy to valuable endothermic reactions when 
pieced in a suitable setting or structural organizatioffio 
In pb-otochesElcal studies^ the frequency of the lig;ht is 
important both because it governs the energy for s. unit 
molecular action and, also^ the phenomena of resonance. 
Thus, the ultraviolet or high energy end of the solar 
spectrum produces important chemical and biologic changes 
not caused in the visible ~ such as - conversion of 
7-dehydrochole8terol into calciferol (Vitamin 1>2) j 
erythema^ bactericidal action and mutation. Of these, 
two have been studied in our group during the past year . 

Work on steroid photochemistry has been resumed with 
a thrssefold attacks 

(a) QuantiiSE requireiaents for transformation of 
ergosterol whose concentration is determined by 
digitonin precipitation is being studied at a 
variety of representative wavelengths in mono- 
chroEiatic irradiation^ This information supple- 
ments that obtained by ultraviolet spectroscopy. 

(b) Flash photolysis proves interesting as it reduces 
the opportunity for thermal change as well as 
providing information on the nature of changes of 
short duration. 

(c) A re-examination of the great amount of data fros 
an extensive study in monochromatic ultraviolet 
irradiation. It is shown that ?elluz'8 concept 
of a photo steady state involving precalciferol 
can be estanded to explain the remarkable wave- 
length dependence of yield of tachyaterol and 
other isomers. A dark reaction of precalciferol 
to tachysterol is suggested by our earlier data. 
This appears to be confirmed by the reaction 
found to follow flash photolysis. 



Instrumental 'Projects: (Coiit'd) 

5„ A spectral scanning device has been developed for 
microscopic study - yielding absorption^, emission or 
fluorescent spectra or a time sequence at several 
wavelengths simultaneously, (see Olson). 

5. An instrumental development has been completed 
for the study of chemical changes in Flash Photo- 
lysis. This has proved very fruitful in steroid 
photochemistry, (see Adams). 

Improved iastruraentation for Flash Photolysis is 
under construction^, both for studies of vision 
(Adams and Kagins)^ and for steroid photochemistry 
(Adams with Sharpless and Brackett) . 

7. Other instrumentation for research on photorecepfcc; 
mechanism has been evolved, (see Hagins) . 

8. As a result of the co-operation with the Laborauor^, 
of Technical Development, Heart Institute^ a "functl^ 
generator" designed by that Laboratory has been con- 
structed by the electronic shop and put into operati 
(This co-operation has been valuable in initiating c\ 

studies) , This type of analogue analyses of our 
spectroscopic data is proving most interesting » 

The project reported last year as: "Effect of 
Nuclear Radiation on Biological Systems" has become 
a new Section in the Laboratory of Physical Biology 
under the direction of Dr. Umer Liddel. Dr. Liddel 
continues to collaborate in our studies of molecular 
structure and infrared spectroscopy. 

Significance of the Program to the Institute: 

Exploitation of nuclear energy has faced society 
with a group of serious hazards which are commonly 
referred to as caused by 'radiation'. Actually thhre 
is included a variety of causes - not only electro 
magnetic radiation but bombardment by particles of 
differing speedy masSj, and charge. 

Empiricism has provided seme knowledge of the limits 
of "safe exposure" so far as immediate acute ef facts ar<.- 
concerned. The longer range implications of radiation 
damage^ howevers, require not only experiments of long 
duration^, but more insight into the basic nature of th;? 
action of radiation on living things. 



Significance of the Program to the Institute? 

'fhe purpose of this section is to gain an under- 
standing of these basic roaehanisais at the molecular 
and cellular level. 

Our primary concern is with electrotaagnetie radia- 
tion as contrasted with particle bombardment. 

Furthermore^ the region of more moderage energy 
(near visible) holds greater interest for us because 
of the resonance or correspondence of these frequencies 
to the Eischanisras of biological structure. An under- 
standing of these unitary processes may ultimately be 
extended to the random effects of "high energy radis 

Our researches were undertaken because of their 

fundamental importance before nuclear energy «r>f-( Rr,,^c.e 
travel focused public interest on this area 

Further planning beyond the scope of our present 
enterprises is limited by the serious lack of labora- 
tory space and uncertainty of relief. 



aettiescij 



PHS"NIH 
Individual Project Report 

Calendar Year 1959 



Part A. 



Project Title: Flash photolysis of Vitamin D ai»d its 
precursors., 

Principal Investigator: Ralph G« Adasas 

Other Investigators: 

Go-operating Units: 

Man Years (Calendar year 1959): 
Total: 1 

Professional: 1. 
Other : 

Project Description: 

Objectives: To elucidate the molecular changes accompavr 
ing the conversion of ergosterol^ or any of the so-cs'lJ. 
intermediate compounds^ to Vitamin I) by ultravi: 
radiation » 

Methods EtDployed: The use of a short intense flash of 
ultraviolet light to which the compound under investi- 
gation is exposed produces changes in that compound's 
molecular pattei-ns. These changes can be observed 
spectroscopically and, provided the flash duration is 
short enoughp the kinetics of the subsequent processes 
can be measured « For this purpose^ the flash is sjm- 
chronized with an oscilloscope and camera^ so the '::' 
course of any process occurring subseqvient to the f ■ : 
may be recorded » The samples are all maintained in ei. 
oxygen free state- 

Major Findings: Thus far it has been, dateriained that 
changes taking place as a result of exposure to the 
flash are the same as, and equivalent to in wave- 
length dependence, those resulting from classical 
steady illumination. It is apparent that there are 
changes taking place in the dark subsequent to reach- 
ing an equilibrium state by means of a series of 
flashes. These changes have been previously observ"<sd 
by Dr. Brackett but are nov? confirmed. There is 3 
strong wavelength dependence of the equllibriuaa tnlJ. . 
which needs further investigation. 



Significance of Research to the Institutes It seeras 
sufficient to state that no satisfactorily understand- 
ing exists for physical mechanisms by which ergosterol 
is converted to Vitamin D, The explanation may v/ell be 
found in the relation of steady state to dark reaction 
and photochemical efficiency „ 



Proposed Course of Project; Thus far the time resolution 
of present equipsaent does not allow sufficient investiga- 
tion of the probable excited state of ergosterol and/or 
other intermediates. This equipment will shortly be 
greatly itaproved. 

The dark reaction is at present under investigation and 
results will shortly be submitted in the form of a or-b' i ^ - 
£ion„ 



Part B included NO 



A o iriaysai-^is^ Biology 
'2 , ?ho£©bioJ.ogy 
3. E@s:hemia 



PSS-KIH 

tedividual Projoet tsport 

Cal®adap ^ear 1959 



ISLLAs 



Frojees fitlss Koi^sular structure detesrolaed by 

Friacipjsl Savesfeigaeorj Sdwia ». Becker 

Qth&T iRvestigaeosrs; Rolsert ffi. Bradley 

Qo-op^T&tiug, UiSiies: Dr, Co J, Watecm^ Uaiversity o£ msKgsofca 
CHS® Studies of Porphyrias) 

M/m Yaaris (cal<sadar year 1959): 
Totals 2-1/3 

Profesffiiesial s 2 
Ofcherj 1/3 

Project Beseriptioa: 

Objectives: Aa undsrstas^iog of the forces f»ichi» £Qd 
l>e«::«e@& molecules^ especially those of potea^lal 
biologieal Isiportesic®. (2> Dsveiopatsaf: of sp@ctro°- 
scopie stat&odo for studying titoI«eular structure and 

aaalysing for ssatsrials of ehetaieal sad biologie&I 
iafcerest. 

Methods Esiiplc^ed: fhe prioiary eeclmiques us®d ira 
work are infrarod ep@e£ros!copy (IS.); auci« 
r«^oaaQce <H^)j, aad elecKron paraiofigQetic 
CSPR). the IR snd I^IR methods supplsatent eecti oi:&er 
in. providinig detailed Imformaeiota aboue the s£rucl;ur0 
of (soleetsles o I» additiou^ bofch types of apeetra are 
highly sensitive to the effects of molecular iater- 
aetionso EPE ie used is studies of molecules taavlng 
unpaired electroas^ iacludii^ free radieals msiS. p^xra- 
:ic atoB^ atad ioas. 




Major irindisags: IHie project caa be divided roughly iato 
three parts: (a) studies of hydrogen bonding end other 
molecular It&teracticeta^ utilising both IR and WR; 
(b) S(M studies of laolecular structure in porphyrltss; 
and (e) oiore general inveetigations of laolecular 
structure by WR assd SPR; 



Serial Ho. HZAMD - 114 
Page 2 



Major fiodings: («oa8:M) 



(a) We have made ea IR iavestigation o£ hydrogen bo»d° 
lag betwesQ alcohols and various proton acceptors (e.g.^- 
acetone^ pyridia^^ dloxaae) in order to de£er»iiaa reliable 
v&lues o£ equilibriua consessts &£d energies of formation 
for such hydrogen bcadSo These thermodytusmic quan&ities 
are nov b@i»g eorrslated ^th spectral properties; such as 
frequency shift and baad widtb^, in aa attes^t to provide 
eaore definitive criteria for the esisteace of hydrogen 
bonds and possibly Improved tsgthode thereby hydrogen bond 
energies can be estisiatsd dirsctly fros spectral datSc, 

(b) We have found that the I3!R spectra of a series of 
setal-free porphyrin masters display unusual resonance 
frequencies £cr the s^tbine and B-E protons. This behavior 
has been explained i& tenae of a "ring current" ssodel^ in 
«hich local easgiiietie fields are induced is the atolecule by 
the cireuiatios of "^ electrons .'i^out the conjugated porphyrin 
ring, 

(c) A mxeber of eRperiBeraes liave been carried out in 
eoll^oratioa id.th chealsts in ovir laboratory and in other 
laboratories o HUE studies of eoveral eoe^ounds (including 
derivatives o£ pyridine^ codeia^j^ dichlorobenx^ae and indole) 
have sister ially assisted in structure! deteraiaations. In an 
EPS study of the oxidation of cblorpr<»siazine ia vitro a %?e have 
deeoastrated the presence of tt free radical interasediate sad 
hsve made e(»se aessur^KSits on its rate of disappearenes by 
further reaction. 

Slgaificssiee to HIAIS) resejsrch: TSte further dovelopsiait of 16® 

sad EPS is eKpected to be of cerasiderable assistance to saany 
RIAIS sclentlstSj, since these i^thods will add two ssore 
spectroscopic techniques that can be brought to bear on 
biological probleeas. For essaiplej, ISI® spectra of such 
coa^ieac aoleculcs as steroids sad porphyries are frequently 
helpful in unraveling their eolecular structure. SPR studies 
Biay permit the detection of free radical or parcsiagnetic intes-" 
mediates in reactions of biological eignifieanceo 

A deeper understanding of the properties of hydrogen bonds 
is clearly desirable since such bonds are of prisae IsportaEKe 
in deteraining the structure and function of proteins and 
nucleic aeidSo 

Proposed course of project: (a) Additional vork on hydrogen bond- 
ing systens vill be conducted along the lines already indicated, 
We are now planning HMR ezperia^nts to coe^lesent the IR results 
reported above; aad ere cooeideriog ths extension of these 
studies to other spectral regloas. (b)- We e.xQ now interpret in| 



Serial HOo ilIMa.p4 
Page 3 



Proposed course of projects (coat'd) 



ehe m& speefcra of porphyrias described above its order £o 
obfi&is £n£oraiB£ioa on eieeCroaic structure aad to provides 
a iBsfthod of anslyaing for certain types o£ subseitueat 

groups <Ha porphyrias c He piaa a£udies with other mtal-fres 

aad ^t«l«subeti£ut:©d porphyrisss. (c) We expect to coattawa 
our program ©f eollsboratio® with otlsar invisstigators in a© 
effort to assist theas la their asaalytieal prob1.©ais aad t© 
leara isors about the taolecuiar structure of iateresting ^jprn 
of ffioleculeso Specific probl^ts alreedy ia prograes (@,g.^ 
the frea radical iatenradlate ia chlorprcwaziae oxidstioa) 
will be cotttinuffid with a view toward early teratinatioia. 



Part B ia-sluded fee 



Page 4 """" 



PHS-SIH 
Issdividual Project l®por£ 
Calendar Year 19S9 



Fart Bs Howors,j A'^&tdB^ e&d PabiieaEioas 
Ptiblieatiosts oehsr ehaa absl:racS:» frosa this project; 



Becker >, £<.©.; Isfrarsd studies oi bydrog«a bcsadiag its raashaaaoli; 
ethaxsol^ and t-butaaol. Sjfs^. ots h^drogsn bosdiag^ Lj«bljsa©a 
fugoslavla (July 29 to Aug. 3j, 1957) pp. 155-162^ 1959 « 

Becker^ 1. D^: WM. studi®@ of l^^droges boadiag in alcohols aiid 
pheool. 3. ChesESc Physics^ 31s 269-270j 1959 « 

Seeker; S. S. arad Bradley^ R. B.: Sf£eets of "ring currents" sm 

the IS® spectra of porphyrias, J. Ch®ca. Fhyeics^ 31; I413-1414s 1959. 

Seckes-^ Eo D,: !n£rar«d studies o£ th® s®l£-as3oeiatiosi of chiorc--^ 
forffl. Spactroefeliaiea Aetag 9-. 743-746^ 1959 » 

Ssekdrj, £. D.s Ae @ff«ct o£ taol<seular Interactions em ^lE r4s£er'» 
J. Phys. Chan.g 63; 1379-1381» i959„ 



Bethesda 



PHS-KIH 

Individual Project Report 

Calendar Year 1959 



Part A. 



Project Title: Molecular Structure and Organization in 
Biologically Important Systems. 

Princtpa.'. Investigator: Elliot Charney 

Other investigators: Alice Sharmon (sunnner employee) 

Co-operating Units: None 

Man Years (Calendar year 1959): 
Total: 1-2/3 
Professional: 1 
Other: 2/3 

Project Description: 

Objectives: The general objectives of this research are 
to explore the nature of intermolecular interactions, 
the role they play in molecular organization and the 
relation of this organization to biologic activity. 

Methods Employed: Two of the methods employed are some- 
what unusual although related to techniques previously 
developed. In one o£ these the course of a protein- 
pigment complex during ultracentrifugation is followed 
by light absorption. This is done by photographing the 
precipitating protein-complex with' the normal opsics 
of the ultracentrifuge and with the normal filter 
replaced by a filter transmitting only in the spectr^i 
region absorbed by the pigment. The other method is 
concerned with testing theories of the apparent dipole 
moment of p-Quinone and involves examining the infrared 
spectr»jm of the gas of this compound under the influence 
of an electric field and the measurement of the elecerio- 
optic Kerr effect of a benzene solution of p=Quinone. ' 

Major Findings: The strong stoichiometric complex which 
a copper-porphyrin forms with bovine serum albumin has 
been found to be stable over a pH range of 2-12 making 
it unlikely that this complex results only from the 
formation of salt bridges (the porphyrin in question 
has 3 carboxyl groups per molecule) . The same porphyrin 
binds also to ovalbumin but does not form any complex at 
all with p -lacfcogiobulin. More resent results,, :iofc yet 



Major Findings: (Cont'd) 

completely analyzed indicate thatj, unlike the case 
of the eopper porphyrin^) the serum albumin complestes 
of heme and protoporphyr5.n are not stoichiometric in 
the same sense » A prelirainairy report of this work 
was given at the Biophysics Society Meetingj, Pittsburgh, 
Pa. 5 February, 1959, and a more complete report has been 
submitted for publication. In an effort to elucidate 
the role of copper in the complex studies are currently 
under way on the binding of two other porphyrins^ 
chlorin 645, chlorin e^ j, and (hopefully) the copper 
derivatives of these compounds. 

In collaboration with E, D. Beckery the infrared 
spectra of p-Quinone and completely deuterated p"-QuinoaG 
in the gas phase and in solution have been taken at higis 
dispersion and are being analyzed to assign all the 
spectral absorptions to the respective normal modes of 
these molecules. Other vjork on p-Quinonej, which is 
the basis of the original interest in this molecule, 
has involved the test of theories of the origin of the 
dipole moment of this molecule by methods briefly 
described above. On the basis of the results thus 
farj, it appears thatj; contrary to the literature^ 
p-Quinone does not have a large permanent dipolc- 
momenty nor is it likely that such a moment is i 
by the measuring field. 

In collaboration with U« WeisSj the optical rotary 
dispersion of a nutriaer of compounds have been measured 
to wavelengths shorter than previously measured and 
anomoious behavior of the rotatary dispersion associated 
with chromophoric groups other than carbonyl has bean 
observed. This work is more fully described in Dr. 
Weiss 's report. 

The data collected two years ago on the viavelength 
dependence of the scattering of light from a spherical 
algae has been completely analyzed. The analysis sho'.;;; 
that the long wavelength in vitro - in vivo shifts of 
the 680 n^ band of chlorophyll results only in very 
small part from light scattering and must, therefore, 
result primarily from the state of organization 
(crystallinity) or more likely from the in vivo 
complex of the chlorophyll to proteins. This work 
is complete and in manuscript. 



Significance o£ the Program to the Institute: 

It has long bean recognized that sub-microscopic 
(molecular) organization is the basis for much of the 
structure of living organisms. The role organized 
molecular structures play in biologic activity in 
general and in energy transfer in particular is only 
partly elucidated. Using the probe of eleetromagiaetic 
radiation with biologically active chrosophores such as 
porphyrin pigments or more simple analogues, we hope to 
make further advances in a fundamental understanding of 
these phenomena o 



Proposed Course of Projects 

The porphyrin-pigment complexes will be examined in 
an attempt to elucidate the specific nature of the bind- 
ing. If possible^ these complexes v/ill be used as an 
aid in the determination of the internal structure of 
the proteins in solution and during denaturation. 

Dr, Ellis Lippincott of Maryland University is attem-Ji: 
ing to measure the Raman Spectra of p«Quinone and deut - 
p-Quinone supplied by us and the resulting data will b.: 
used to complete the assignment of the absorption bands 
of this compound. 

Another investigation is in the early stages of planniiy; 
this involves the measurement of dichroism and/or electro- 
optic birefringence of proteins and polypeptides in solu- 
tion for the purpose of determining changes in their 
internal structure as a result of environmental changes. 



- ., t'Viysical 
2 Photobiol 
3c Bethesda 



PHS-NIH 

Individual Projacfc Report 

Calendar Year 1959 



Part A. 



Project Title: The physical and chemical basis of 
photoreception. 

Principal Investigator: W. Ao Kagitss 

Other Investigators: None 

Co-operating Units:. Naval Medical Research Institute,, 

NNMC^ Bethesda^ Maryland 

Man Years: (Calendar year 1959) 
Total : 1 
Professional: 1 
Other: 

Project Description: 

Objectives: To outline the successive events by which 
light quanta absorbed in the receptors of animal ej^es 
lead to the production of nerve impulse information to 
the brain. 

Methods Emploj'ed; Two complementary methods have been, usee 
to explore the early stages in the response of retinal 
photoreceptors to light. In the firsts the electric 
' currents produced by receptors lAen stimulated by lir^ 
have been measured v^ith conventional electrophysiolo; 
techniques- In the second^ absorption spectroscopy 
flash photolysis have been used to study the photocbv, 
reactions of the primary light- absorbing pigments in 
receptors. 

Major Findings: A theoretical analysis has been made oz 
the minitDum electrical current that a photoreceptor raiu;' 
produce in order to convey information to the brain at 
the rate which has been observed ejtperimentally by 
previous investigators. Using theorems from thermo- 
dynamic and information theory;, it has been shown that 
photoreceptors of the usual range of sizes must produce 
electric currents of the order of at least a thousand 
electronic charges for each light quantum absorbed in 
the retinal photopigment. 



Major Findings: (cont'd) 

In collaboration with Dr« H. Go i^agner ox. c-se ivava] 
Medical Research Institute, a direct experimental meaB.; 
ment of the current produced by photoreceptors of the 
squid in light of known absolute intensity has been mac)' 
It was found that the cells yielded at least 750 alecv ■< 
charges for each incident photon. This result suppori^- 
the conclusion of the theoretical analysis,. 

In order to extend these results and to investigate 
the process by which photoreceptors yield electric 
currents^ apparatus has been built to measure currents 
voltagesj. impedances^ and radioactive tracer uptake b 
the retinas of octupus and squid and to study the phor 
chemistry of their visual pigments in the living cell-. 

Significance of the program to the Institute; 

A clear understanding of the physiological mechanism or 
light sensitivity in the retina may help to explair« some 
aspects of the related problem of energy absorption and 
transfer in photosynthesis and cellular metabolism jn 

general . 



Proposed course of project: 

The immediate objectives are to find answers to the 
following questions: 

(1) Do the photochemical changes observed In visual 
pigments have anything to do with the physiological 
mechaaisra of light sensitivity? An attempt will 
be made to answer this by comparing the quantum 
efficiency of the photochemical reactions with thaf 
of physiological excitation,, 

(2) How do photoreceptors convert light into electric 
current? Using standard methods of electrochem: 
and tracer technology j, it is hoped that the ioni 
basis of receptor currents can be found o 



Part B included Yes 



Page 3 



Part_j2 Honors^ Awards, and Publications 

Publications other than abstracts from this project: 

Hagins, W. A, and Jennings, W. H.j Radiationiess mig; 
of electronic excitation in retinal rods. Faraday Sor 
Discussions 27; 180-190« 1959. 



2. Photobiolog} 
PHS-tTffl ^' Befhesda 
Individual Project Report 
Calendar Year 1959 



Part Ao 



Project Title: The action of radiant energy on structure 
and chemosynthesis in living cells. 

Principal Investigators Rodney A. Olsoa 

Other Investigators: Chas. I-. Greeabiatt and Este.la V 

Co-operating Units: Dr. Elkind, NCI, Radiation Branch f^Ci" i,fJc 
(see Addendum) 

Man Years (Calendar year 1959) : 
Total: 2-1/2 
Professional; 1-1/2 
Other: 1 

Project Description; 

Objectives: To interpret the raechanisms of energy trans- 
fer and allied metabolic steps at cytological sites ci: 
photochemical action. To determine the roisp in this 
respect, of chromophore orientation in laminar photo- 
receptors such as the chloroplast (a classical examp" 
of a heterogeneous system in trfiich energy conversicv- 
and transfer occur) . 

Methods Employed: The "ion excluding" maiobraae electrodfis 
for simultaneous measurement of CQy + Oy photorespor?9 r-^ 
znetabolic transients have been further fievaloped. 
02 + C02 freSs dilute NaOH is drawn through a aiicrr 
spiral course beneath the surface of an ultratMn hydro- 
phobic meiTibrane and passed over nearby sensing alectrod?.:; 
After equilibration via this "micro gill" CO2 is detenrctriv- 
conductimetrically as Ha2C03, and O2 is deter^mined by 
electrolytic reduction versus a silver-silver hydrojd.de 
anode. Considerable effort has been applied to reduce 
noise caused by bubble accumulation on electrodes and 
to shorten the time response for the kinetic study of 
the briefer photo metabolic transients in cell suspensio 

Changes in chlorophyll fluorescence at photochemical 
sites were evaluated by the development of a scanning 
type spectrophotomicrofluoronetero A fluorescent tnicrfi- 
scope with a superpressure convection cooled mercury arc 
was provided with means for scanning the emission spectrum 
of the cells or structures observed. Scanning at 60 rpm 
a linear spiral slit with direct angular rotation liukag;: 
to the horisontal sweep of an oscilloscope provided via 
photomultiplier a quantitative v^avelength distribution 



Methods Employed ; 

studies of the time course of changes occurring at 
relevant regions of the eraission spectra^ a disc bearing 
a series of interference filters was substituted for the 
rotating spiral slit. Each filter x«-as provided with an 
accessory filter to exclude the exciting light. This 
type of scanning when displayed with a very slow (2 tain.) 
horizontal sweep (time base) permitted as many as four 
sitaultaneous traces to ba recorded from the oscilloscope 
each showing the time course of changes in eciseion at 
wavelengths chosen. By substituting an appropriate filter 
one of the four traces could be used to record changes in 
relative transmission. Kence^ the time course of emissici- 
changes of cell structures at three spectral regions coul^ 
be simultaneously recorded with the time course of bleach; 

The treatment of cell suspension, cell-free chloroplasi:; 
etc.j, was as described in the pre\'ious report « 



ajor Findings: The performance of O2- CO2 hydrophobic 
msfflbrane electrode developed in our laboratoiry appears £0 
be adequate for application to a study of Og and CO2 trans- 
ients accompanying photochemical activity in cell suspeufji- 
Its time response (less than one second) should permit a 
kinetic analysis of COj and O5 "bursts" etc.^ and "troughs' 
Furthermorei, its immunity to "specific" enz3?ine inhibitors 
added to ceil suspensions should permit identification of 
each successive transient with an appropriate metabolic 
step. In its present form after a few minor changes to 
enhance stability and response^ it will be adopted in ths. 
near future to launch this heretofore impossible programo 



Our development of the scanning spectrophotometer micro- 
scope has made it possible to follow^ quantitatively^ -rapis 
changes in emission of fluorescent cellular structures and 
coneossaitant changes in transmission. At very highy but 
sublethal light intensities, chlorophyll fluorescence of 
670 B^ at lamellar sites disappears and is replaced by 
yellow fluorescence peaking near 540 tm. The effect is 
oxygen limited and is accompanied by a bleaching of all 
visible pigments. It can occur under ideal condition 
at room temperature during one minute or less. Time : 
studies of changes at various wavelengths of emission 
indicate the formation of a non-fluorescent intermediate . 
Attempts to identify the bright yellow fluorescent subsKapc 
are based upon comparison with the emission spectra of knci 
materials. At present photoly tic oxidation products of 
chlorophyll are indicated and/or a flavin-like compound. 



Major Findings-; 

Farther studies with cell-free chloroplasts via 
absorption microscopy provide further interpretation 
of fine structure Jjs vivo. Hypotonic swelling and 
the results of specific enzyme digestion produce 
characteristic alterations o Proteolytic enzymes 
disrupt the lamellar structure while lipolytic 
enzymes leave the organization intact. Tiie pigment 
lamellae appear to depend on a protein substratum 
and are separated by an aqueous interphase « 



Significance of Research to the Institute? The develop^ 
ment and use of specialized optical and electro- 
chemical instrumentation provides insight into pro- 
cesses of energy transfer which occur in the optimal 
biological material under study. Since the laminar 
fine structure appears to be common to other photo- 
receptor systems^ the study of its role in photo- 
chemical metabolism leads to better Interpretation 
of energy transfer, in all living cells whether light 
or chemically activated o 



Proposed Course of Project: Completion of the h^'drophobic 
meni>rane electrode development opens the way to kinetic 
analysis of light Induced 0,j and G0« transients = In 
addition^ a systematic stud5? of the effect of inhibitor 
on the numerous characteristic transients should at-} ■ 
identifying their origin in the stepwise sequence c ' 
metabolic steps immediately following light or darkua;; 

Identification of the participating metabolites in 
the sequence of the fluorescence shift will be sought 
by comparison with fluorescence of pure compounds and 
by attempts to obtain large quantities of bleached 
cells for extinction analysis and chromatographic 
yield from extracts. 



ADDENDUM: 



Dr. C„ L„ Greeablatt has been collaborating with 
Dr. M. Elkirad of the National Caacer lastitute in a 
study of ionizing radiation in maosmaliaa tissue cul- 
ture cells for the latter half of the year. This 
work is an atteznpt to evaluate the role of chromo- 
soaal damage in cell lethality. The tissue being 
used is Chinese hamsterj, chosen for its low chroaio- 
somal complement (a = 11) ^ and readily identifiable 
character of the major chromososEes , These propertiei 
facilitate the determiaatioa of visible chromosomal 
alterations o 

Chromosome complement is being studied in ceils 
just after irradiation as well as in cells which art 
long term survivors. The specific details of these 
abberations as they relate to survival is referred 
to in Dr. Elkiad's annual report (N. Col. -Radiation 
Branch) , 



Part B included YES 



Par£_B: Honors^ Awards, and Publications 

Publications other than abstracts from this project; 

Olson, R. A, and Engel, E. K,: Visible absorption micros 
copy of pigment systems in living calls using interference 
filters: Chlorella Chloroplasts. Proc. Microscope Symiv--- : 
GhicagOj, 1958^ McCrone Assoc. ^ 1959. " ' ' 

Olson, R. A. and Engel, E. K.: "Chlorophyll" absorption 
microscopy of in vivo, cell-free and fragmaated Chlorella 
chioroplasts. Brookhaven Symp. on the PhotochemicaTIpFav 
tus. Its Structure and Function, Brookhaven Symposia in 
Biology, No, 11, 1958^ 303^ 1959. 

Greenbiatt, C. L. and Schiff, j. A.: A pheophytin-Uke 
pigment in dark-adapted Euglena gracili s. J. Protczoo] 
6: 23^ 1959. " 

Greenblatt, C. L, and Sharpless, K. E.: Effects of some 
metabolic inhibitors on the pigments of Euglena gracilis 
in an acidic taedium. J. Protozool . 6: 241 19597 



Serial So. MI^ID-118 



!.« Physiology 

2, ?ho«6&ioiogy" 

3. Betfe^sda 



PHS-KIH 
Individual Project Repor£ 
Calendar teer 1959 



Par£ A. 



Project Title J lEveseigations of the Actioa of Radiane 

Energy oa Biologically Itaportsast Goapoirnds » 

Principal iBves^igator: ^onaatK Ec Sharpless 

Other iKSivestigators: Odette So Tesmev 

Co»operating Unite: Non@ 

Mara fears (Calendar y®ar 1959) 
Total; 2-1 M 

Pro£e«aio!ial ; 2 
Others 1/4 

Project Description: 

Objectives: Th«t objectives of this project are the 
establishaaaat o£ various biologically intportant inter- 
tasdiates in photobiology and the study of their kinetics 
and other physical and cheiaical properties o 

Methods Eiaployed: Ultraviolet or visible radiatlca is the 
sseans employed to effect any alterations in the saateristi 
under investigation > Spectroscopy in fhs ultraviolet^ 
visible and infrared r«igionsp as well as cheiaical pro*" 
ceduresp are the lisajor atsthode used to evaluate any 
changes %}hich occur. 

Major Findings: 

lo (With Mrso Odett« So Teaeaer), The photoeheraistry of 
srgosterol and related steroids to fora the various VitS" 
tains D^ is one o£ the seajor photochessical reactions of 
biological isaportanceo 

Detenaination of the quantum requiretaents for the dis^ 
appearance of ergosterol has been candied out as a func° 
tion of both concentration and wavelength . Evaluation 
of ergosterol requireiaents in quanta per ssolecuie has 
been carried out by determining residual ergosterol after 
irradiation by the digitonide procedure. Sxtrapolatioes 
to io«; doses of irradiation gives the quamtiue requirenstent 



corrected for the competition of the photoactiva isos'; 
io-cmed. This quantum requirement ( ) has been furtl- 
evaluated as a function of initial concentration of ■: 
terolo This value is in general a linear function oJ 
initial ergosterol concentration. The final extrapoLr . :.^ 

value J, the qi.!eo.ti 
extraneous light 
) , A isummary of 



to infinite 


dilutions gives 


tfc 


te desired 


requirement 


of ergosterol 


corrected 


for 


absorption i 


and intermolecular 


factors ( 


the data so 


far obtained : 

Wavelengths 


is: 
A 










2650 






1, 


,6 




2800 






2, 


J 




2967 






3, 


,1 




3021 






I, 


.0 




2537 






2, 


,9 (3 



-4 

2„6 X ID molar 

Confirming values will be required for some of these figur- 

2. (With Ulrich Weiss) . The alkaloid thebaine has an 
absorption band at 285 vap which has contributions from 
an aromatic ring and a conjugated diene system in the 
molecule. Irradiation of this compound in the ultra- 
violet region under anaerobic conditions causes this 
peak to drop to 507. of its valuCj, presumably due to 
alterations in the diene system^ Irradiation in the 
presence of air results in the same initial drop of 
intensity to the approximately 507o vilue^ followed by 
a gradual disappearance of the band presumably caused 
by a photochemical ly induced oxidation of the aromatic 
system. The irradiated product is now under investiga- 
tion to determine its structure. 

Significance to research of the Institutes I'he proper under- 
standing of the behavior of biologically important mole- 
cules on a molecular level is absolutely necessary to the 
extrapolation of their effects to a cellular level for the 
evaluation of their effects in health and disease. 

Proposed course of project: Some more data is 3ti?i.l required 
in the quantum yield of ergosterol disappearance as func- 
tions of wavelength and concentration. The reversibility 
of the ergosterol transformation will be investigated by 
irradiation of intermediates such as lumisterol and 
calciferol and analyzing for ergosterol. 

The work on thebaine will continue^, both to determine 
the nature of the product and the quantum efficiency of 
the process. 



;rt B included 



PHS-miH 
Individual Project Report 
Calendar Year 1959 



Part B. RonorSs, Awards j, and Publications. 

Publications other than abstracts from this project: 



Greenblattj, Co L. and N. E. Sharpless: Effects of Some 
Metabolic Inhibitors of Eugle na gracilis in an Acidic 
Medium. J. Protozool. 6: 241,, 1959. 



i."iij.v-ii<. rroject tieport 
Calendar Yea.r 1959 



Part A. 



Projec'c Title: Chemistry and biosynthesis of pigriieRts 
and other natural compounds o 

Principal Investigator: Ulrich Weiss 

Othar Investigators; Herman Ziffer 

Co- operating Units s None 

Man Years (Calendar year 1959) 
Total: 1-3/4 
Px-o£ess tonal; 1-3/4 
Othar : 

Subproject Title: (1) Properties and biosynthesis of 

pbotodjrnaiaic microbial pigments . 

Project Description: 

Objectives: Isolations, in chemically pure forra, and 
study of chemical and physical properties of indix-- 
ual members of a group of closely related red pig- 
ments produced by fungi of the genus Elsinoe 
(Asconsycetes) ; investigation of their photodjrnamlc 
action: stvidy of their biosynthesis. 

Methods Employed: For isolation of chemically pure,. 
homogeneous pigments^ extraction of the fungus 
mycelium with organic solvents is usedj, followed 
bjf countercurrent distribution. Elucidation of the. 
chemical structure of the individual compounds so 
obtained will be attempted by the usual methods of 
chemical degradation and interpretation of physical. 
especially spectroscopic^ data. 

Major Findings: A preliminary investigation of an 
Elsinoe species (carried out elsewhere in I9565 and 
published) showed that the pigments can be extracted 
from the bright red mycelium by acetone j, that the 
deep red crystals obtained on gentle v-farming of the 
crude pigment with ethanol consist of several chemical 
species having identical visible spectraj, and that the 
pigment complex exhibits typical photodynamic action 
against bacteria. The characteristic absorption 
spectrum in the visible region is very simila:: "o 



Major Findings 2 

that of 3j, l-O-dihydroxypsrj/leae-A^ 9-quinone^ showing 
that the chromophoric part of the pigment molecule must 
be closely related to this quinone. The IR spectrum is 
in agreement with this conclusion. The pigments frora 
Elsinoe are the first derivatives of perylene which have 
been found to occur in nature; the osuly derivatives of 
this ring system isolated previously from a natur.?:' 
source^ the two erythroaphiris from aphids^. do not 
as such in the insect^ but ere formed by enzyme accj . .■ 
during isolation. Other representatives of this group 
have been found quite recently. 

Work at NIH was first directed towards improved 
culture methods for Elsinoej, since growth and pigment 
production in the 2% malt extract solution previously 
used is slow and aeration by mechanical sha)<ing is 
required; hardly any piginent fortas in still culture?;. 
However „ all attempts to modify the insdiisis gave results 
inferior to those obtained with 2% malt extract. 

A satisfactory method for paper chromatography ci i. 
pigment coicplex was worked out and proved valuable in ci . 
subsequent investigations. Through its use it was fouu. 
that the crystals obtained earlier by warming with etha"- 
are chemically different from the native pigments; how 
the visible spectra of both types of compound are altno 
identical . 

For separation of the complex into chemical individu: 
columa chroaatograjahy using a variety of adsorbents wa 
not satisfactory. Countercurrenfc distribution gave vs:: 
promising rasultSj, fractions being obtained which appaa. 
homogeneous on paper chromatograms . This approach is s-. 
present being investigated further with larger amounts ;> 
crude pigment, hoping to obtain sufficient material for 
elementary analysis and study of chemical structure ani 
physical properties. 

Significance of Research to the Institute: As th.;. ^ ...^ 
representatives of a new group of natural compounds^, ths 
pigments from Elsinoe have general biochemical interest 
as has their photosensitizing (photodjmamic ) action v 
Such action has been established so far only for a sme ' 
nuniber of natural pigments. The various species of El 
might also offer good possibilities to investigate exp-i^ ; 
mentally the biosynthetic pathvjays by which their p:lgffisni. 
are formed, and to test plausible but hypothetical scheav:' 
which have been proposed for the biosynthesis of certai'/. 
related groups of natural plg-?'^--'^-? 



tails to produce pigment under these conditions^ 
probable that a precursor is formed „ which is co; 
to the pigment by a reaction presiimably involvia;^ 

pheric oxyg»n„ Isolation of this pi-ecursor from :, 

cultureSj, and conversion to £he pigment in viti Ot mig!! 
be an improved way. to obtain the latter^ and would gl.^ 
inf ormat ion on a late stage, in the biosynthesis of th 
pigment. The fact that some available species of ?; 
produce yellow color under conditions where mosv 
other synthesize red pigments suggest the interss _ .. 
possibility that the yellow pigments are biosynthetic 
precursors of the red oneSj, the yellow strains being 
genetically unable to carry out the remain5.ag steps. 
It is suggestive that the erzytaatic formation, of the 
erythrosphins from their naturally-occurring pale 
yellow precursors proceeds through bright yelloj? avd 
orange intermediates of unknown structure » Ken^e, 
study of the yellow species might give" information or, 
the bios5mthesis of this group of natural substancee. 
In view of the recent great interest In biosynthetic 
pathways, this information might be of general value, 

Subproject Title: (2) Chemical constitution of the so-callsci 
"hydro3cycodelne" . 
(With Dr. Lewis Jo Sargent (LC-NIAMVv 

Project Description: 

Objectives: To establish the correct chemical const: i- 
fcion of a derivative of codeine known to have f.^^'' 
interpreted by its discoverers- 

Methods ijn^loyed; These were the ones of chemical ti 
formatilon and spectroscopic investigation gener?'= ■ 
for study of chemical constitutions c 

Major Findings: A compound first prepared in 1916 by 
reduction of 14-hydroxycodeinone with zinc and acid 
was interpreted and named at that time as the normal 
reduction product^ hydroxycodeittec It was shown later 
by Dr„ Lo Fo Small and co-workers that this interpreta- 
tion cannot be correct;, but the true structure of the 
compound remained unknown,, The present study has 
established that it results from reductive coviplirig o 
two molecules of the hydroxycodeinone in a nor-sym^ 
fashion; a structure for the product has been prop..^ — 
which is in agreement with its properties and permit;.' 
formulation of a reasonable mechanism of its forma:'- 
This type of reaction seems to be novel,, since nc ■ 
example could be found in the chemical literature 
asanuscript of a paper by Sargent and Weiss, de 



Rhis subjectp sny contribution to a better uaderstaad- 
ing of their chemical behavior is worthwhile. Ic t'b.e 
preseat csse^ an old error recorded in the chemical 
literature has beais corrected; iti additionj, lin 
apparently aew type of chesuical reaction has bean 
observed, which may be of interest beyond the case 
at haado Compounds of this new type may perhaps be 
found in other instax-^ees of reduction by zinc and 
acidj, and may so far have been overlooked in the 
complex mixture of products which is often obtained. 

Proposed Course of Project; This investigation is corcp^'; 
and no further work on this topic is conteaipJ.ated, 



Subproject Title: (3) '.'hotccheHjistry of aikaloidu end 
phenanthrane derivatives » 

Objectives: Investigation of the transformations of 
organic molecules by ultraviolet of visible l:lght. 

Methods Employed: The organic compounds were irradiated 
in solution^ using natural sunlight or laboratory 
sources of ultraviolet radiation. The escperiiients 
with the latter sources were performed by Dr^ Sharpie; 
iaethods and findings are described in niore detail ivt 
his Mnual Report. Isolation and characterization of 
the resulting compounds were attempted by the usual 
chemical methods. 

Major Findings: (a) Irradiation of thebaineo The or 
alkaloid thebaine contains a homocyclic conjugated di,:; , 
system somewhat analogous to the one responsible for the 
photocheniicai reactivity of ergosterol. The photoser^-- 
tivity of thebainey anticipated for this reason^ wsi 
actually found on irradiation with U7 light. Xhe iviL,:.. , 
absorption band at 2840 K^ known to result from approKi- 
©ately equal contributions of the diene syatera and thfc 
aromatic ring of thebaine^ decreases to about 507» of 
its in5.tial intensity on irradiation under anaerobic 
conditions j, indicating disappearance of the diene 
chromophoreo Isolation of the reaction product (s) in 
pure fona is under way. (b) Irradiation of 9-brojEO- 
phenanthreneo The photochemical dimerization of 
9°substituted anthracene derivatives has been studied 
repeatedly J, but the behavior of the analogous 9-substi- 
tuted phenanthrenes on irradiation has been given little 
attention. Dr. Ziffer has found that exposure to sun- 
light of a benzene solution of 9-bromophenanthrene 
results in its conversion Into a crystalline coajpound., 
which differs from the parent substance by its high 
melting point and low solubility in organic «< T v-pn- r, 
Its chemical nature is u|ader investigation.. 



mental iiapor'cance for an unclerafcanding of ligl" 
biological processes. Although photochemical reac: r 
have long been known £0 oecur^ interest in tiiem was 
sporadic^ and systetnatic investigation of the uausual 
structures often formed by such reactions has started 
only ia recent yeers^ so that knowledge on tiiis area 
is far from sufficient. 

Proposed Course of Project; Biucidation oj: the chemcal 
structures of the phototraasforjnation products of 
thebaine and 9-broniophenanthrene is planned. The 
investigation will be ©Kfrended to other compounds 
which are sensitive to ultraviolet or visibla light. 



Subproject Title: (4) Optical Rotatory Dispersion vjith 
)3r. Hlliot Charney. 

Objectives : The objective of this research is tha ?- 
of the optical rotatory dispersion of organic moic: 
having chromophoric groups other than carbonyl^ tc 
find out whether such an investigation is experimtv 
feasible^ and, if so^ whether its results are of vtl . i. 
in organic-chemical and biological research. 

Methods Employed: For the study of the wavelength 

dependence of optical activity throughout th« visihl.: 
and ultraviolet regions of the spectriim^^ the Rudolph 
Spectropolarimeter is generally used. This instrursenL 
requires manual setting of the wavelengths at which 
measurements are to be made. Recently,, an accessory 
equipment has been Introduced by Perkin-Elme]: Company 
which malces it possible to route rotation of polarJ.Et;^ 
tion through standard UV recording spectrophotometers. 
This equipment has been loaned to the Photobr.ology 
Laborator3'^ for one week and has been tested ifor its 
ability to give inforsiatiDn not readily obtained with 
other devices. 

Major Findings: Study of the changes in optical rotation 
with wavelength has recently yielded results of great: 
value in organic and biological chemistry. In partic;' 
the anomalies of the rotatory dispersion occurring 
the neighborhood of absorption bands in the ultrsv 
have given much valuable information on chemical c 
tutloHp configuration and conformation of organic 
pounds. However J, these studies have been mostly res:... 
to aldehydes and ketones. In those compounds,; the 
absorption band causing the anomalous rotation if of 
low intensity, so that sufficient light is transmitted 
for polar imetric measurements at wavelengths fair] 
close to the absorption maximum. It would be desv. 



Serial No. ISflAMD-^Ii; 
Page 6 



Subproject Title; (4) Optical Rotatory Dispersion with 
Dr, Elliot Charney 



to extend such studies to compounds with more intense 
bands,, particularly to phenolic and polyenic substances » 
Both groups include many products of great interest to 

chemistry J, biochemistry, and therapeutics. 

Work on this problem was proposed In 1957 to Br= ,) , 
M. Bobbitts University of Connecticut. He utilized 
the Rudolph spectropolarimefcer of the Ohio State 
University for a preliminary study of the optical 
rotatory dispersion of morphine^ codeine^ and thebainc: 
down to about 2950 S, finding pronounced anomalies in 
the curves of these non-ketonic aromatic compounds. 
The results were subsequently confitTsied by Miss Haness;l;. ■ 
Ohio State University. However^ these measurements 
could be made only by exploiting the possibilities of 
the instrument to the utmost^ the difficulties being 
due to the fairly intense light absorption by the 
corapoiindSy and to the unsatisfactory stability og the 
light source provided for work below about 3100 A. 

With the recording instruraentj, conditions for satis- 
factory Investigation of such compounds were worked out" 
by Dr. Charney. With their help^ it was possible to 
establish the occurrence of anomalous optical rotatory 
dispersion in a variety of non-ketonic compounds which 
had apparently not been studied before. They are; the 
alkaloids thebaine^ neopine, quinidine and^, probably, 
nicotine^ and the dienic steroids ergosterol and lumls- 
terol . The curves of two compounds (codeine and andro- 
standione) that had also been studied with the Rudolph 
instrument gave results in good agreement with those 
nreviousiy obtained. Several compounds included as 
negative controls showed the expected absence of 
anomalies. 

Significance of Research to the Institute; These preliminary 
findings suggest that it is not necessary to restrict 
the field of investigation to ketonic compoundSv ^^'^ 
that the scope of the method can be widened to include 
substances whose light absorption is due to phenolic 
(opium alkaloids) ^, heterocyclic (quinidinCj, nicotine), 
and dienie (ergosterol„ lumisterol) chromophores , 
Similar findings on some benzenoid compounds have 
been made at NHI. If confirmed by more detailed 
itivestigation., this may mean that a large number of 
compounds of biochemical or medicinal Interest can be 
investigated by a method which has been extremely 
'^ruitful In tha study of cnrbc^y^ ccmpcimda . 



Proposed Course of Project: On the basis of our preliminary 
results^ a further exploration of the optical rotatory 
dispersion of non-ketonic compounds seeras worthwhile » 
The study might also include investigations of proteins j, 
which have very high optical rotations caused by their 
helical conforiaations. 



ACTIVITIES OTHER TMN RESEARCH 



la January J, 1959^ I joined the Panel on Biocheaistry 
and Nutrition of the Research Fellowships Revievj Branch, 
Division of Research Grants^ MIH» In September^ I v;as 
transferred to the newly founded Panel On Biophysical 
and Organic Chemistry, 

At the request of the B and N Panel ^ I prepared a 
detailed memorandum on the question of NIH s?jpport for 
research and training in pure organic chemistry^ coining 
to the conclusion that there was both need and justifies 
tion for such support from MIH funds « 



Part B included YES 



J) -119 

Part B. Honors^ Awards j, and Publications 

Publications other than abstracts from this project: 



Bobbittj J. M.j WeisSy U.<, and Hanessian^ D.,: Anomalous 
Optical Rotatory Dispersion in the Morphine Series. 
Note in the J. Organic. Chem. 24; 1582^ 1959. 



PHS-WIH 
Individual Project Report 
Calendar Year 1959 



IS-!: A. 



Project Title; Effect of Kucleer Radiation on 
Biological Systems 

Principal Invesfcigator: Urner Liddel 

Other Im'estigators: Ellis S. Kespner, Charles W= 
Malichj, Frank M. DeFilippes^ 
(transferred tc NIAIB ap;-^"-"- 
imately 1 Kovember 1959; 
Lewis^, R. J. Holthaus 

Cooperating lfcis.,i. JDepartment of Terrestrial 

iJlagnetism^ Carnegie Institution. 



Man Years M^uiCiiX'-di. year 1959) : 
Total: 4-1/2 
Professional: 3-1/2 
Other: I 

Project Description: 

Objectives: (1) An understanding of the effects of 
neutrons and X-radia£ion on biological systems 
(a) by sCudy of the molecular interactions which 
occur as a result of these radiations (b) by 
study of cytologicai changes which occur under 
radiation. 

Ifethods Employed: Radiation of cultures of micro- 
organisms and subsequent eKamination by microscope 
and other physical methods - e.g.^ induced radio- 
activity, spectroscopic ai\alysis where feasible, 
incuding absorption and electron paramagnetic 
resonance. 



Serial Ko. mmo~l7_Q 



^iajor Activities: A portion of the laboratory space 
assigned was made available for use in the late 
winter. A 50 curie Po-Be neutron source v/as in- 
stalled in a graphite nsodarator in March and 
preliminary raaasurements made on the nature of 
the neutron fluK. Biological studies included 
radiation of pnetsmococci cultures to see whether 
radiatiors had an effect on growth rate. The 
results were inconclusive because the neutron 
fluK was too small 

The second phase of laboratory rehabili- 
tation scheduled for con^letion in March, was 
finally completed in December. This will enable 
the installation of a Van de Graaff generator 
which, with tritium targets^ should provide a 
neutron flua: at least 100 timas greater than 
the Po source. This installation v/ill provide 
the maKimum source feasible in the space now 
available under the conditions of the space 
loan. 

By use of a radioactive analog of an 
asaino acid, phenylalanine, it has been shown 
that the final selection process in the syn- 
thesis of proteins of the yeast, Capdi da utilis, 
takes place in one of the two metabolic "pools" 
of the cell. This could be interpreted as an 
operational identification of a template 
mechanism of protein synthesis. 



Fss ' ms 

Individual Project Reporfe 
Calendar Year 1959 

SeriaJ. No„ WIAMD-121 

omR 

MatheEatical Research 
Eethesda, Ifesylsmd 



Vsxt A 



Project Title: J-iathsimtics of kiaetics and reaotion-traaspost 
eystema^ 

Principal Investi^tor: John Z^ E©axon 

Man Years: 

Total: 1 1/2 
Prof sssioaal : 1 
Other: l/2 

Ob jeetiv@ ; The chief ohjectivis is to conduct a systeaaatic 
study of a^thamtical problems of cciBgplex reactiBn systais 
and systems in which chfisaical con-v«rsion and traxislocation 
siiEultaneously occur,, The study is thus concerned with probleBs 
of rate behavior, traaasport, the relation between energetics and 
pfeencsEsaological rat® equations in general and irreversible 
thermodynasaics ajsd chisaical Isinetics in particular „ This project 
is strongly o3?ient©d towards actual probleias of current interest 
in bioloffiTo However, there often arise probleina of inherent 
Bsatheimtical interest and ess^aples are cit«d, in -Mbst follows, 
^daich are of relevaaca to current laathezoatical research^ 

^jor Results and Siffltifi-canee : . 

Matrix Theorejas and Linear Analysis: It vas reported 
3.ast calendar year Idiat it had 'b&m. proved that the Jaeobian 
Matrix for an arbitrary non-linear ehesiical system is necessarily 
siaailar to a syissaetric usatrix^ Some of the consequences for rate 
behavior of metabolic systems and relExetioa-tiEe analysis vere 
sketched „ I'his theorem has been broadened so that it applies to 
certain diagonal stochastic i^,trices of cuirent interest in 
zwQdom-ijalk problems ^ It has also been shown that the signs of 
the Ejatriz el^uents eaa be uniquely assigaed^ This assigoiBBnt 
is essential for the discassion of stability of equilibx!lxB& 
states in ccaaples systesas and the possible esi-steace of maltiple 
stationary states „ These ssatters are of obvious importance in 
•triggering B^chatdsuis'" in physiological systems and in the 
dependence of stationary states upon initial states „ Further 
progress of a practical nature has been laade in terms of applica- 
tion to rela3®.tion~tiBse analyses „ In particular it has been 



Seriaa. Io„ MMm- 1,21. 
i^gQ 2 

shcn'm that each conservation eonditioa (os* smtsilal balance) 
implies the existence of aa eigen-vector corresponding to a 
zero root aad that tlae 'botality of such vectors are linearly 
independent o Thus the gsneraJ. results ars carried oirer to the 
reduced Bystom of rate ewuations with vMch the sspsrimenter 
usually deals ^, In addition to aniswering some general questions 
about s«i,-fce behavior tbeae results &£& of considerable interest 
in terms of the relation of thermodynaialcs to MiaeticSo For 
exaarple, a largo clasB of rate functions for autooatalysis are 
consistent irith thjeraaodynasaic eojiilibriim results „ But th© sym- 
Bsetiy reqairsEsent on the Jacobie^ matrix sslscta uniqujely oxi 
a<3xaiesable rate funetioa„ Scaas of thase results wes?e presented 
in an invited lecture before the Aiaerican MatheHSttical Association^ 

Area Theorsn : Also reported last year mi&s a BS>thod for 
caaputing the ares under a curve in tes^ss of the coefficients of 
the differential equation of t^iich that curve is a solution <> 
Extensions of this thsoraa (prcsapted in pert by collabox^tiv«! 
work on clotting iKechaixiam vith Dr„ R„ H^ ShtOaas, EIAMO) have 
been obtained: Special deductions frtm tho general theorem 
enable certsln results to t® -wxitten down from the inspection of 
th® reaction Beh&sm^ In particular by inspection it can be stated 
leader -what conditions •fcSss final yield of products -will be inde- 
pendent of certain reaction steps in -the scheso® and independent 
of the initial conditions, Sob^ aspects of the general theorea 
have been ext^ended to non-linear sys-bess and .applied to the 
kinetics of prothrombin to thr<sabin coziversion„ toe consegvienee 
of general checdcal kinetic interest is that rate constants can 
be estiimted frcsa final yields in non-linear systeias for which 
no anal^ical solutions for the oaate sgjmtions o,re Imo^ao 

Superposition Properties: In the clotting study referred 
to above it -was found that inhibited and uainhibi'^ed curves of 
thrombin yield can be 8uperimj)osed by ssaltipli cation by a scale 
f&ctor as can uninhibited curves tinder different iidtial conditions „ 
A Eathejsatical stuQy of these resx:LLts showed the following: the 
superposition property for solutions of linear differential 
systems is -well known „ She problem here is the eoavwrse^ It h&B 
been shown that superposition implies azid is implied by hosaogeniety 
degree one of the differential, syst^a and the linear uase is a 
special instance of this class „ In tesras of the cloti-dng problem 
it resiilts that the throsbin yield as a function of tio®, t, 
prothrazibin, P, and inhibitor, I, is factorable into tl^e producrc 
of three functions of these variable©^ e^g,, F(t}H(P)G(l)„ Shis 
result, independent of any specific kinetic schCTie, used in 
conjunction tfith certain rsthei' non-rei3trictive kinetic assump- 
tioae, affords independent kinetic evidence for the existence of 
ea inteiMjediate between prothrombin and thrombin, allows the 
irreversibility of certain steps to be established^ and predicts 



cerfcaia €s.periiDea"tel.ly vsx'ifi'ad z^latdon iDetwaen uninMlJitred 
atid inhlbilted jd.ald-'airp.aB,, Ther® xm.n jjoaswl "by tMs auslysis 
tixe foilcrvriag proM.<aa of scjb^ interact in asatilx algebra: 
ftlTBU a EJ^tris vith elesKSnts a fuaction of a va?5.sble r^.^ tixxdey 
what ecmditioa&i wi3JL the spestruja of the siatslx consist of 
t%K> subsets, one set iadepend^nt of :<t A set of sufficient 
coaditions ha''/e iDeen found,. In appiieatioa these conditions 
pliace iEBSsdlats restrictions -upon the kis^tic Echsssae- proposed, 

ApprojdJKate Diffusion Sq^ation: ¥ork haa contiimed on 
this as inaJ.ca'fced In ttes 1958 report „ 'Xb® rsv^tion bstiiv'eea the 
aaalyfcical and coiimutational probleais in tMa work -was presented 
before an ISM Syjaposiiaa on Cfcartputers in Biolo^' aad I^Jedieine 



Ifert B 3.15} ;lnclu(;t-jd 






Bart B: 

Pa'bllffiar b lOKi B 

Easts J^ B, Slid Bsarca, J^ Z, Thermodyeasjics of HoaasostasSs ^ 

Gaapter in MlnessI MetatsolJLaaj ©(aited by Es^xmer snO. Ccsaaj? ^ 
Kew Yorkj, Acadsaic FseaSf 1939 „ 

Stettea^ DsWltt^ Jr„ and Hsaroa^ J^ Z^ Intellectuial Levsi 

Ji^sasured by ACB and Serum Uric Acid CoaesatxatioB^ Seienes 
3^ 1737 (1959), 

Aawnoff, S^ emd Hsaroa, J^ £» Kinetic Models of Ac.c»itaB8 
ActicxEs.,, la pi?ess„ Ax'chi'^es of Bioelicsis„ aad Biopbys. 

Haaron^ J„ Z« Considssatioa of Apprpslsate Solutions of tSse 
l^imtion of Ccfutiriuit;^^ Acesspted for poblieatioa in 
special JM SyjapositaB ca Caaputsrs in Biology and JJediciue^ 



PSS-NIH 

Indiiddual Project Report- 

CalenSsr Yeex 1959 

Serial So„ rJi\l>1D-122 

Mathessatlcal Research 
Bsthssda, Maryland 



Pro,3eet Titles: l&thssaatieal tasma^Mtlfm and analysis of prdbleaas 
relsrvaat to esperissisntal neurophysiology 

Principal lainestisator : Wilfrid Bali 

Ksa years: 

TffSal: 1 1/2 

i^ofessiozml: 1 

Ofelser: l/2 



Pm^eeij B®9j»iptioa: 



(1) To develop explicit aiatlsssaatiesal foxiajilatioss of 
various neurcpbysiological hypotliesee „ 

(2) To elaborate tiieoi^tic&l pr^jdictioaie tbsfe are well 
Emited for esperiffiental testing ,, 

(5) To aaalys® and reassess certain cui-reat saeuro- 
pliysiolog:j.cal concepts involT^in^ sijEultaaeoue 

eoasiderstion of tta"®® differeat kinds of iafojnaatloa 
(a) neui'oaaatomical cljata, (t) electrophysiological 
d&t&f as v^U. as (c) a ms^hsm-ticsl. fonmlation of 

tbsir intSE-dspendjeriC® „ 

(k) To coatri'iyate to tbe intei^retation aasd tlite desi©! 
of some of the experiESruts conducted by nef»i3?o- 
pl^siolog-ical colleagues at 1JIH„ 

^^tJhodg eiajployad; 'ite ae:uropliysi ©logical pro'oleai ssmet be 
redlsicea to its essentials and then be foMsulated nstteaiaticglly., 
S^picallyj this formulation is a partial differential equation 
that ffiust be sol<?ed for a imrlety of bouEd^zy and initial "coriiiitions. 
The uB<s of laplace treasforssis has been very fsrultful in solving 
several cwnt?eat problffiatSo 

ani3. SigpAfieance ; 

(l) Tha Bprsad, of electxle current from a neiiron scsssi 
into ■braneblng djsaaritio t3?ees has be^Ka fosmalatsd nsatlasffiaticsllyo 



Seria-l Ko, SIIAM0 -122 

m^ 2 

Because of its importfiaace to the interpret.atioa of recent 
expsrlEJsnte perfosraed upon motoaearons with intra.- ceU-iilai* vEic^^D" 
eleetrodes, considei'u'ble car© has b«en devoted to th® pz^paration 
of a paper (Srpe-slESSa'Sjal Keuxtilo^, 1959) wMch inclviaes a 
careful assessussait of as0UBiptioai5, derii^tion of -bteoryj* ps^ctical 
foxmvilae, aad detaalad applic0,tion to the best easperimeatal data 
cura'ently aTrallable fross anatoasies,! and elestaTopbysiologicai 
sources o Caa the basis of tla® cuafrent teta^, the s^stilts indicate 
that lao'feoneuroa dendrites play s dominant role (vather than the 
mibsidiary role aseuESd by others) ia dsterffiining Importgait 
HOtoaeuroa properties, 2his has iuxportant inrplications for 
ciax'rent concepts of synaptic ©xeitatioa and reflex intsgratioao 

(2) Sigaifleaat gains M-vq "be^sn iaa!3s oa the tmr® general 
problem of xcsabrane potential spread over- aeiiron aasB. and 
dendritic surfaces in ssspoase to synaptic current gansration 
awi' thes® 0ux^fac®s,. In addition to nxsasrou® -useftO. special 
cases j» the solution to a -visry gensral pixjbleaa bas bsea obt&insd: 
tbg Bja^-gtle gen^ra'fcor curreat can havs sa arbitrary tisse coarse, 
its intensity can have any oiae of a larg® varj.ety of d-istrLbutiona 
over the stxca asd dendrites, mid. the initial condition of the 
laerabrajis s^arfaee need not be the sweating conditioa^ Several dif- 
ficulti<S3 axjd sfflibigidtiffis in tha theory of synaptic excitation and 
in the intex"pr^tatioa of i^eeat easpsrintenta sro no%r being analysed 
in tenass of these theoretical results,, This tteory predicts^ 
for example J, the differences to be e3cp©ct®d betweien e. synaptic 
potential generated predomiBaatly in the dendrites and one 
generated predoslxiantly near the Bcsm^ k publication is in 
preparatipnj fnxth&r sigaificaat applications are ajitieipa-fced„ 

(5) Mr„ Kara Shahn and I have developed a procedure that will, 
enable ua to uss th© IB& 65O ccssiputsr in the study of several 
questlone of relevsmcs to the e3ctracell^lla^ electric potentials 
recorded by aeurophyaiologists „ IMs procedure provi.des us with 
the electric potential field to be erL>ect^d for >;arious distribu- 
tions of point current gexisrators on the mjxfece of a sphere „ 
Most of the difficulties h9.w; aov been ovQ2?ccsm^ Application of 
the resrults to amizxjphysiologieal problems remains to be doae^ 

(k) Col3^boration is in progress vd.th Drs„ K,, Frank and 
F, G„ Kelson,, HIIDB, in the design and interpretation of 
exjjeriassints witti uXngXe aotoneu3?onB of cat sisinal cord„ 

Broposed^ Courge ; Current results have x-aised furfcher 
gxiestione „ Work x^rill continue along ths sasiG general lines „ 



Page 3 
SforfcB 

Eall;, W^, : BsuncMns desidarf.tie trass and motoneurca aesfera'ae 
i-esistlvity, Erperisaen-S^l Keusx>los7 1; k91-3^» 1959 

Invited I®elim'ss: 



Deceaiber 29., 1959 j"-n a syBj^JOsii^ eatitled; 'te.tlsess.t.ical 
Moi^ls in tbs ld.fe Seiencss" spoaso5:*ed "by Sse Aisst^rican 
S-^atistical. ABEOciationj, Ths BicsKStric Society, aad Tbs Xns'&itute 
of Ifea3£3!50,tie0l StatistiGB„ 

Sbveaibsr 25;, 1959 for '32^ Bfeeuity Sajairsar of Soutl2H®st©3ia 
Msdical School, Sallys ^ ^scas^ 

laarch Ij-,, 3.959 for the Neuropbyslologj'' and Keuroanat^iSy S^aismr 
of the Wa3,t«r Reed Aissy l^dical Centor 

l^Tch 5, 1-959 ^oy the Riysiology end Hmrisacoiogy S^sinar of 
the George VTasMKgt*:^ Uaiversity^ 



IncU-irldual Project Report 
Calendar Year 1959 



Ifethesaatical Resees'cla 



Part A, 



Project Title: Stu<a3r of iodine kinetics in the tiiyroid system 

mid radioiodine tre&tsient of tJiyrold abnoraaa-lities 

Principal Invest! ©itor : Moa@s Berssan 

Other Investigators: Charles Lexmllen 

David Eeeker 
Biefcard Eemia 
Martia Soneaberg 

Cooperating Units: Clinical ISadocriaology Brmieh^ 

KIAMD, Serial Ko„ 

Nev York Hospital, New YosJs 

Sloan Kett«ricg Institute^ Hev York 

Man Years: 

Total: 1 1/2 
Professioiaal: 1 
Otlxer : 1/2 

Project Ceserj-ption : 

Objective ; Tbs objective of this project is to develop a 
general model for th® kinetics of iodine in tbe thyroid systasa 
that vill ejjplain the various thyroid disorders fotind in jatients 
and that •will agree -with the various experimental data collected 
on these patients. It is aiso the purpose of this project to 
study the sites of action of radioiodine when used for therapy 
and tbe effect on iodine tum-ovsr rates produG©4 by radiation, 
drugs and hypoph^ectcsay. 

Methods employed : Bs.tients having various thyroid abnor- 
malities have been studied. Kinetic studies using radioiodine 
have been made on these patients over periods up to about 2 
years „ "Riese include studies before and after treatisent of the 
patients. The id.netic studies involve nieasui'ements of radio- 
activity in blood. PBI, thyroid, urine at 'i?5iricus tiiiasB after a 
tracer amoimt is administered to the patient, Fran the collected 
data caleulations are made of the turn-over rate of iodine from 
iodide to organically boimd iodine in the thyx-cid, the secretion 
and degradation rates of thyroid hooiiaone amd other variables. 



Serial Bo, lOlAJ® - 123^ 



Appliestion of s^tis£S!a.ti£^i EiodslB and ccas^mt^rs to th® 
analysis of tbjs data is End© in an effort to deiaoas-ferate if tbs© 
lEodels used ar@ eoas.1 stent witli ths data aad to deriv© sessiires 
of uncsrtaiaty fos- tSs® constants of tbs mod^sISo Th© msthodologsr 
for this typ® of aaalysi® is stiii 'bslxig dsvelopsd and tfes final 
analysis of 'Uas tots, is still psadingo 

TJa® a?ijnoK^ili ties studied are due laostly to byperfelm'odism 
bM. oaacsr^ Effscts ps^uced Ijy 3?sdiQtlca?. twiatc^at '(l^^^ and 1^33) 
hOTOphsfssebc^y, thyroid ^id pituitajey hcsssonss My© "beea s'teadisd^ 

Majos' FindiBgs; Tcis fiodiugs i-sportisd on iaat yxsar hsvs 'bs«n 
found to apply to «^ditioiml ];atiiss3,tii ^ho wjdisrwest bypoptiyseGtosay^ 
It vas foimd -Ssat ia scsi© pati^its trfe?o populations of protein 
iKsmid iodJ-ae sasst aKist ia order to explain the kiEetie dats.„ Tb.® 
populations Mve Jiot "bsesi idaatifisd^ 

Sigaif icmee of g^rogy^ to the Institv.te ; Sins© the 'fchyroid 
is a Sost 3j£po3ctant or^nln tls® r©®alatioa of physiological 
pafocesses, its detailed sodss of aetioa ar© of iatersst for untisr- 
stasadiag tte tls^'oid as ■sfell as othar metabolle systeas in tfee 
body^ Farth®Sfflo?@, tJae develoxsaent of s^ aoalytical procedure to 
treat the hosjsostatic u^elisfflisra of t^ tbjwoid say also ba spplie- 
aMe to oiihar hcss©ostatic Hssctaaisrss in th© bodje 

Pgopoaed covtbo of pz-o^eet; 'Bj© eomplet© asxalysis of tie 
data collected over tlm i^et few years is stiDJ, pending tb® eoajple- 
tion of the dsr-relopasat of tSse ise-fenodology^ 'When this is doiae, the 
aaalyeis outliEed ©arlier Tfill be doae,, 

Mxf satperi^sats ars bsiag plEuned t-o iavestisa'te two abjj.or- 
smlitise fouad ia tbs collected data^ litis vlll "be done in 
coila'boratioa 'rdL-Sdi Dr«j„ C„ G„ I^millen sad J^ E^ Rail of the 
Clirdeal Eagocriaolosr Branch of the HIAMD^ 



Bart B not included 



HiS-JIIH 

Individual Project Ke}x»rfc 

CaleiidaY Year 1959 

Serial No, NIAMD-123 

OADR 

Mathesaatical Reeearch 



Project Title: Analysis of Kadioisotopa Tsucer Data 
Principal Investigator: l-ibnes BesraMi 
Otlier inveoti^tors : Esra Shalm. 



Cooperating Units : Clinical Sadocrinology Branch, 

Serial Ho„ 14?C 

Man Years: 

Total: 2 
J^ofessionaO. : 1/2 
Other: 1 1/2 

Project Description: 

Objective ; The objactive of this project is to develop a 
nath®3satical and coaputational methodology for the systaaatic 
and routine analysis and intsirpretatS on of tracer data on nteaSj 
state biological systems „ The nethods tmder developisont are 
intendsd to provide a i-ationaie for ho5? -bo choose a physiological 
model for s set of data and ho^ to trsst the data for the model 
chosen,, It ia also intended to program the procedures for routine 
use on analog and/or digital computers, and isake them svadlahle to 
other investigators, 

Methoda employed ; 

1) fethematical Theory: The developtaent of a 
theoretical hasis for the procsdures of ansilysis to be used„ This 
includes theory for model construction and data fitting, 

2) Digital ccsirputer - programs have been written for 
fitting eatperimental data to models using high speed ccmputers. 
The programs are being developed . to be applicable to a variety 
of models and sufficiently flexible to take the eajperiioental 
data directly, 

3) Analog computer - The application of the lanalog 
computer has not been as extensiv® as anticipated in view of the 
success in using the digital computer „ It \fs.s still used, 
however p for special pi-oblems,. 



Serial Jfo, lUMm -123 



Ma.jor Findings; 

1) A caoprehensive program to do a least squares fit of 
model constants to various foiins of experimental data and general 
enough for a vide rangs of models has "been vritten, and was applied 
to special problems. Further develojsaent and testing of the pro- 
gram is still in progress, 

2) A iBsthod for obtaining the xmcertainties of the model 
parameters that will take into account non-linearities of the 
system 'betea-vicr is being tested, 

5) Applications - The methods developed have been applied 
to probleias of several investigators. These include 

a) Glucose .^tabolisB. - Analyses of C^*^ labeled glucose 
kinetics data obta,ined by Br, S, Se^l of the CliiiiGal 
Endocrinology Division have been made„ These saaalysea brought 
out inconsistencies in proposed models, inadeqsiacies in the 
collected dat^ and suggested additiomil ejcpsriseatation to 
Justify new model proposed, 

b) Initial application of the methods to iodine kinetics 
data on a series of pe.tientB collected by this investigator 
are in progress, 

c) Assistance vas given to & number of investigators in 
foxffiuiating their probleaos mathssnaticallyj and in the analysis 
of their data. 

Significan ce to the F ro graia of the Institute; A great deal of 
work is carried on using isotope tracer techniques. Interpretation 
of the collected data is usually made by postulating same compart- 
mental model and solving for turn-over rates and caarpartaoent sizes 
of the model o It ie assumed that the values obtained may either 
reflect mechanisms of action or indicate the sites of nozaal and 
abnormal processes. Such an analysis of data is limited et present 
to very simple systems because of the caaplexity in the analysis of 
multiccmperfemeatal systems. It is hoped that the methocLs developed 
here will enable investigators to s-fcudy more ccsiplex systems, 
erts^ct more information from their data and have a measure of con- 
fidence for the models they propose. 

Proposed course of Pr oject- The development of saathematics for 
rigorous procedures in foimilating biological models and in analysing 
data vi3jL continue. General compater progrsans applicable to a 
variety of problems vilJ. be de^'eloped. Application of developed 
methods •will be pursued, 

Bart 3 included. 



&sria,l Ho„ KIM® » 123 
5 



Vast B 

Publicstioas : 

BS2B!S52j, M^j SeliO©nf®ld^ B„: lafoxsaation ecat^jat of tracer data 
^jitJi raspect to steady state systsss^ S^nss^ainxn. on 
Infos^astioa "Bieory in Bl.o3,ogyj H^ P„ loekey, editor. 



J C, G^, Bermaa, M„ sad Sall^ u„ £„: A a^thaisatieal , 
apprcaoh to t^ kinetics „ J, Clin,, Invest „ ^: SS-'S?^ 
Jsa, 1959o 

I^wsllea;, G„ G,,j Baii^ Jo S„ and B€-nasa^ K„: Studies of loclo- 
allKUiaia Ms-fcabolissa^ II, Tbs effects of thyroid hj5Tmo-n&^ 
J„ Clin„ Invest. ^ 88--101, Jaa, 1959 „ 

Beffissaaj M,,^ &a& SsliG©af©idj R, : A note oa -unique sjodsls in tracer 
kinetics „ J^ EKpsr„ CC'11„ Hessexch^ In press „ 



S. 



Wm$&&t Title: lt»ial ®f Hsssr teei^^aesamtii© te^gs 

®€ter 2®!?ast:iga£®»ai: to©. tog®s: L. Biask^ fest J. BI©Bfe# 



©seals i-5/6 
■ Ft-^t&mlm&lz I- 5/$ 
©e^es-: 

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paeess^ la ssm. 

aids e££e£&@j, i.€ ^^. 



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Fa^t B isclBded Y®s £T7 H© /^^ 



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the sase aa ^^en flrat i»bserw4 durirag cbe 

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thyvoU hmctimt nm &lm st^istd. Uthm^ mvitml 

tM ^<&&sm&thaMmi'^ d^@e as&d the dastresstigia e£ ths 

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at»li«at&«idett|r<^ sulfuric aeld, wcus £o%md «X3 te<ir€ mtl«' 
iuiXssm&taTCj ^x&pax:ti<&& in snisml studies. This c:«ts3^u!nd 

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of £!3l@ 3£u4^. 



6a Klssssro tTitmsla&lenOf syathasise^ fey Dsr. J. ftt&d 
©£ Sqw»b swsd Cosspaiayj has fesaa a^fettaisfcered «© 5 
r&mjffistold ajj^&rifiis patients , fo?3sr to ® share tturm . 
stvstlxmtlim ^s£ I oa « ^9i;atolie steady. TJbe e&ti- 

«Md iQmtd to fee ©a©«foureb to ®G««tbiir^ S^aK »£ 
dsMsimaifc&aisiSffls ©ad too to fcteea KIj^s Ktot ©f SffiaBSciaoloaa . 
iaaeiEg tSsie 6 t© SO day fxariod ®f tkm® ©feofft casra ti:l*ls, 
i^ serlows site e££e«s«:s ti^aire «n<sot£ait:ffisra«!. ISose» of 
3 t® 10 ujg. we^Q ©apl»ye4. Oaa ©£&er ^tieat ^auticlpeeed 
In a Ketefeollc atatl^, sseeiviisg 20 it^. 4isily. IS® 

v&t® db®&t^m&. fto ealclwis b&isaca ^s^dt&d m h^ ponilti^^, 
liae iSat«t ijrffis Qot: aafmlmt-99, AMltim&l situdlea with 
tMs oMpotimd am 3nitlei|»a^(l, 






aK-glt?i&ia. Sn £mS:i«£a£lL43a^«;o]i^ offset 
it t^as fotfflBfil to te s&s) «t^l<^ftlea& «£ oa(s»S:taiM ta m.&-h«tU 

d:is«i£»QtiQ«eeii la m® ts&m <m tag. <£jiiiiy> tfitest tl».p«&lffia«: 

<2©imios«B<l e assfcric uloar £owr Bwatfec aftssr itib® etas:*: of 

Sas!ii«sga4«i imlm ©Ida k^sRsy :fc'@£ 4Qjjs»si£3 ija t&a fsce, oeek 



5 



g*£i<2as;is. is feos sSa® pEewlded Seed!© Sor f«re&®r . 



3) Ssayfcli, C. J-, Itsalbs, J. J.;, Clark, S. S., teaia, B, C, 

SJists^aeisss imd Arsbritia » S^vie^ of Mesricaa es.d 

Review) J Part 2, Mis.Ssfeera.llad., 50:366»494g S95®. 
Part llg i^jm.lntaiV^^^d. f 5£:S34«S01j iSSf =, 

eaf large d®ises ®f pretSaisoae on aeu£e sfeauaafcic fever: 
Obsarvaeiosas cm. the £s:es£is&ae ©f 1? ps£i©Q£s ©itls: 
cerdisis wish a l^f&ar follow-up. ii.M^A. J.Sis.CMld.^ 
97:56l"570j, I.9S9. 

5> Sogers^ B. IsS., ®ad Blacky 1. Ki- : aislleal Isservlai?j 
Rhs^3a®J;©id aySSsrlfcis. Saa.Firacait, 19?IO2.*10S^ 1959. 

6) U£s, J. ?.;, Vas Scoefc, S. J.,, BsratoBj, H- W., Edgsesafe, J. H, 
&mius, J. 3.f ©Ua;> J. a.. Bell; M. H.^ aad Saufessa^ H. g.2 

the liJla£lor.al SasUtut©® ®f Health. Aaa.Satara.lSsd., iS59p 



Serial K®. 
Page 7 

1} BtsalS; J. 3, % S9r«:ic©s£as-©tds: Clse^stjeyp g>hyEl©iogy.e 

8) Wmistf J, 3,: €»^tieosit&mlAB: Ciistieal usee aisd 

9} Bmto;, J. J.s Se3K-c®l«tesi© md aarss®i<i asrSteitls. llsid. 

II) SiEaisfty J. J.; Rse@giait:4ea aad treatssfst ©f areferlfcia la 

14) Blacky R. L.j Sias-^?er anf r<iK2iaaS: sesossre^ &&ti.vlti®& in 

the rfeeiaaatle «IS.s«fflses. &3rch.©£ Fl^s»Ms<3.& lelseb,^ IB^ESSIS- 



:^. Baibasda 



ladivldissl Project R&pott 
Csl.«ndaff Year 1959 



SBSLA- 



FPOjecfc Title: "fise Beafeoalfee Floeculstiaa Tast ia 
Ebfi?mafcol.d Arthritis 

Friacipa'l. Investigator: fJr. E«s£ J. Sloch 

OKfeej? laves tiga tors; Bsr. Joseph 1. B«aia 

Coaperatittg Waits: Bts. E«h?isi Lsraer II CW?S St J^ 

smd Wc. lerold lieu 

Msm Years (caXfittdas: year 1959): 
Total: 2/3 

Frofessloaal: 1/3 
Oa&esr; 1/3 

Project »©8criptioH8s 

©bjectiims: 

f© coBtiaw® the js^alwsta^i of pj»® ls®a£©alfc® 
flocculstion eest: <1CT> as a diagnostic aid ia iHteuasstoid 
srthritls . 

floecMiat ioa Yesfc. 

Svfflluatism ©f tli« soMltlvifcy aad specificiey 
of the BFT as a sarologle i!»soi la rheumatoid eirthritis has 
been coneiau®d ia the ^s^ year. A fiofcal of 1500 tests 

deftttite yhewateid a^tfeffltls Imva h^a tosted; ©f these 
175 were positive and 25 «cre ass«£ive CfiFFffJmiBsately 
903^ psitiv®). 1te»ty«fflaw«® patients witfe Jwaaaile onest 
of rh««saatoid e^t8»ritie lk»v« i««a t®sted; of these 3 iUV 

Part B included Yas /TJ Ho iZJ 






lisd a positl'^ ?:©s£. Mli^agfe jtwenile rfeesffiaatoid 
srefcurisi® is sssaaidias's^ Sa Ise the Ciai?!.^li©od catmt&T^&xt 
of She adalt disease , gerologlssjl tes£© get^trslly teaa 
to be positive la ealy IS^ of tbese pafeteiats. 'jSfee 
eollsgea diss^es osmtlu^&d to e&atributa l&e a^s^t higher & 
incideaee o£ |«jsis:ive esss:,, Xk sysfiesie iufsua 
erythesiaecsas 10 ©f 18 patieaea feadi p®si£i^e fcesSsj i« 
sclerodsraa 6 of 13 ga5:i®aS:a had pesi&l^® £®Bt®. itessag 
patients ^th ©osj^slasssaatoid arfchrieides £&e follo^issg 
results siere obtaiae^: t&t^teea pstlcuts wltla 
©Btedarfchrttis fesd "aegati^rc tests j eight |>aties5fc3 ?fitfe 
anlq?lo8ing speadvlitis has negative testa. 4 t©tal of 
67 patients ^itSa geut aad/©r hyperurie^ila ««&e tested; 
of these 4 had & f^sitim test. €h3s o£ these patients 
&elosgs to « fimllj' ia'^iefe taaay EsssSsra Stanre a ftasitiv© 
BFT ia the abseaas of j©i®t losses® j t?s3 eth.©? patieats 
prsbabiy liava.,sfes«^at&ld artlii-ifci® and gout; la tb® feursh 
patieat the pssitiv® BfT asy ba due fc© abaaEESl psnst®lai® 
p7ed»ced b]r a.dis^^aed liver, thisrtem p&timits ^th 
fs<i£^'ia;sls aod ^r^ritis ^^^^s tasted; «»£ ^®ess <mlr «>®i3 ^<^ 
a ^0@iti^G tost)' a&^lmis^ s&^'&zaI p&tl9mt& MS & &»tm 

arthritis. ®a& #£ 15 patiestte ^tfe Ml^^^s 
|jr>a a IK^sltl'^^ test, - 



C-gRsrse ®f 



i£&^ea^B«iU 



i;^gi;^^w#^^:ftM4f)i^;jMggaE 



tlie £F? Sasua tesa fterfossBed, at x^s^ly int®r9al&) 
-®a aii fffeemsatelS fm&i«^f» adsalttiad t® ©ur m.tt. Tks 
eliiaical ei3u;se asid eeEelsgieai €tcey©f 2© patimts have 
bctoa eorrelated. I^r® did siat &^p&m c® il»s £2^^ 
si^ifieoat dlisage in 1^? titer d^ing r^s8i<ms or 
e(3Qse7bGi:ie»ns o£ t&a dieees®., Se^^eral ^e:i^t8 &ad 
>iy eoBSZa&t titers ovei: 6«v«rsl ]?«d7s. ffii«ra 



l£«.s£^&.^IJi..M!g^gMiSsiLl^i^^> 



Alaska. It ma £suad t2iat t&e l&ei<3@ee:e of pesltiw 
reaet&eaa tn appas&&tly &eaithy natives ^»as 6iM.lm: S& 
£Q4md 1b tbe Ualt«d States populetisas setsdisd: 

2»3%. 0<3ra f«6m 2^ ialMt»&t<mta of the 
ti XsUnds «e?<B also tasted md df 
5 C2.i%) ^re pooistiva. 



Serial Fro. „mjmjsUS&. 

Paga 3 ■ 



ware tesesil. "Shese sesra Isava elso li®<m SesSei^ ^ 

8s. Ball, In Esjglaadp Msisg t&e ■ seRaitlsad steap oeil test. 

Svssa£«atly fche sresalts ®f Shis atady wilE !>e '©©rreiatei 

The BfT was also tssed I® &sst sara @&t&is^ed 
Is^ a Iteieed Sea&es FaMle Saaitih Sezvics Health Bmvisf iJaia 
la its 7@ri0u@ studies. 



The Csi^i&iegy fabe.,La6aa gjaafci^ fest CS.L»g.T.> aad 



■* Several iaipssfcigesors have rs^rt®^ s 

sigaificansly greafcer iEcideace ®f positl^j^e eerol©gic«I 
teses for r'^vsea&sM ard^itis la f&^ly s^s^sre &f 
siaetsiatoid pffi£i£»es tStao ^a^g matched c«aesel ^smsps. 
Th^ c^lllasy f^j^ ?.at€m £isa&iea test es^lo^ittg host: 

We, ]^7old mn, a C^S^? e&ud^e duriag eh@ 
susoer of 1959» esperiamtcd with tt^is pffoc®d«r© tiader 
©«ar sizpewisioa. le «as foeasd tisat a IjS dlitt£i®a ©f 

At 56® G for 39 !sisu£ee, |»i^9vided e^ti^m m^ltt^ns for 
tsstisg asrt^. ^ tbis ^Sffis added a latoK susp«sis&<»a 
(diluted so tbaK 0.% tal. of LaCem ia iO mi. ®f 1g«ff®sf gsva 
2fl% light £7@stsmi3sleQ is& a Golssssi Jtmtoff Spscexophot&saetor) 
sii3c«d vlth aa &^ml ^^l^mt of Iseaeed g^s^a sl.g»bullra 8oluti<!ta, 
"Hae eixtaro of diluted e&smi aad gass^ globisii^ es»at@d 
iQtss wae drowa iat® a caplilas^ tube aad allef^d to stmd 
et Tooa temperature for osse hour. ' At t&is tiise' it ims 
observed for SigslutlostieiB. S^^eaty ef 7@ s»fttlsii££ ^Ith 
elas8lc«l or defiiatto ri«eus^toi<l arthritis Md & fmeitiv® 
C.L.F.7.: stR of 18 patlaats witla prol^sble or sKsssitils 
che»aatoid srtlsritis 'ms:& slso positl^. Tkare «9ere 
3 poelti^ tests in 72 ^tieats with aoa^rfsMssaBteid 
arthritis. Fiwa ©£ 191 &spl&ye»a, 2 of IQ& hlxs&d doaors 
sjsd S3^3s of 22 Bor^al wlm&teers were positive. 



Page 4 



Cesspat'lsoa ©f cbe results obtatecd la £fee 
e.L.F.T, and BFT s-swalM agree^teat Ik ^6 of IB p^aelesafca 
^tli classical ©s- defteifie rfeetssgatold asshffi&is sad la 
14 ©£ 18 eases ©f psobaMe or ftossifole raeissstoi^J ssrfcSssfitis. 
rkB CL.F.T. asspeasad fc© lie 8ligli£ly ss>s« eaasltlw ttea 

lose ®£ espeeifileie^. 

Prewi©«s esiseslssssses us las '^'^ BFf iisd 
isdiaaaed dsst raEetiws of rhet^aSoid arS&riels ^£ieat» 
did Bo£ isave m mmaml liaeideaca »f peslfei'spffl tmta. hy 
this p^ocsto^a. IrawEWff, ^i£h C.L.F.'T., ? ®f ©4 rslafcive© 
®f fffeeum toid p®ai^£s (m) isad 4 of 33 R-oXacives 
©£ pattmtB wi£!i jwt'SSBlI© sfeesmatotd «s:fcteitis 1121} 

gsots^ Issd a ^sitlv^ t-ass; {!%}, Tfesse rssulte steggcsse 
.tliafc all® proasaes ®f irfee-^'.a£oid factor ia ©esijja tmy b& 

It muR pr&vixsssaly ?ep®r£@d £has: ?at@ injected 
! bf ^ssitlv® serological tsis 



tmst^ for s^e^ts^told 
sxi&tiZls. I^ess ©earsloglcal restjslfcs tj®se sis© prddiEced 
ia rats as & rmpsm^ tus killstd asxtlgesiis o^lch 41d not 
p£@due@ jolat letsitoas. I^asHmisatioa of 2-@bbl&9 wltb 
killed fiEstigeae !»s©4ae©^ Mgls BFT fcieatcs. "asa factor 
ia zabMfc sesua raspoasi^ia for fclie KFT reaceioa ^ss 

fey iai»aa»lpstcal m& phjrslcal 8aQ£&9<rJ8 a® I® distiact 
itold fas£@r mud s^ppesred S:s l)s a^ 
aatibody to Inmaa profielo©. Sate iajecssd wifife 

h^mm proteins d©^l©p«Ka Jolac leslozts feat tao elwatad 
BFT titera. ma psesassce «»f buram pnotoia in the eoi&ur® 
irodluBS used f©r etais raieisoorsaBiiaia appsarsd es^mftial 
for £&e pcodac^isa of eiei^atad B?¥ tl&«srs ia «&s sera 
of iajse&ed or istsasaissed r^&Ke. 



S|IS£Lot^^Slll^SS.JS^Ii!«^MJ^ML^ 



^s^g^oia^ 




toid factor i» a, aaterogl^tollQ ^lc& 
1st ®f eaailer proteios linluad Ij^ disulfide 
t of i^eumtold aftr« wltiiL stili^dryl 
eosi^lets lee» of serologic astlviti'. 



i?age S 



of Wal^nstJToit's sgsssogl®fe?s'ilaemis, sad tbsfe tiais sfs© 

is 2i]S* Espsrisenss fey Ste-. S.. S.<saiisaQ fiffilSi^'"m«I 

Mrs. teisiss Yisldiag ia^icseed K&aS: SreaSsjsafe ©f rlj^^afcsld 

lea«3 Ko ioE® of seg^iogie ©cti^iey ia 'dm WE. 

ar£liritl3 aad ©as t^tfe sclerosersas feoSla ®f ^sasae feed feigh 
BFT Sitees. A oa© ^e&k erisl of 730 ®f,. ^^gaicillssise 
dail^ failed £o fiff«e£ tte BFT eltQs ia eiefeeif |i!®£i®aS,. 
A fercisa? Goiarge of 1.5 g®. of ^^sicllla^ia© dallj tsas 

SKJ cfe^ag© ia BIT titer; slowly ^sreasissg 6;@fcal leukesyEe 

Poaieillssaiu© did a®£ ®f fsct the Sitar af ss.-®tl3aar serKsa 
ffiacsegiofesslisty i.e., is«S)Sggi«seis8iia, Et®ff ^id It 



, The BFf is aa IsffsorSaaJ: tsol f©r 9:!i® eliaical sad 



s<s--^^^ "^- ..^«M^:iiE^. 



Xsdivldss^l ?s©jec£ Eepart 
Calsadar Year 1959 



1) BS4JC&, E. J., ^d 3«ala* J. J.: Sisple^ srspld 

disgaostic test foP rlis«?sat©id ssrtbsrieis-— b®e£salf:a 



2) Sloeh; ^. J.: Rsesmt ^sSiflcaeioos In sest^l^jglceil 
eeste for E&essaaSaid ©reSffltis. B^lX.Sli®«^.M9. 

3} l^rKsr, E, M., IX, Block, E. J., sesJ WiUi^^, 1. E.: 

?iaaeti©a and boatoaiee fle«eula£:i®a test; £b sat® is^ltfe 



I. CllaieaJ, Javestigfitj.«>as 

Cslea4ar Year 1559 

FsgS &♦ 

fiif^Jaet: title: SJsgsraa's Sjfndro^ 
PrtKci^l laves £lga«»r: Sr. Sure J. BI©c«a 

gobstrS SSe^ma (NXBR), li©lsffi?sJ Oglesby CKXKSS)^ 
Joseph J. ksaim, IJiili^ R. Sarsoll, 
Richard A. Msisgrea (IKJI), Sheldoa Salm 



llaa tesrs (eslsadsir ^es? 1959): 

T®tal: J.-2/3 



To study tlie cllQieel^ 86?ologlc&i^ IfflEsastslogical end 
psthaXoglcsl 8p@a£rt^ of Sjog^^s's sytidr^ais ssti its 

tiesu@ diseases. 

SSethoda Bgjpioygd; Major Ficdiaga ; 

Cliaical. Ag^^«c£B . 

«f Sjogrea's ayads^sw «?ars 0tudied aafcii eh© psaseat £is®. 

related £0 dsyatsss of ssi^c^viQ imsi^z&&<m acid sj^a^toas of th@ 
?ela£ed cotmectl-ire Sriesua dlsesSQ. CsKsfiinsaCoxy eztfinlstatiess 
sod iafeoratosy studies ^srs pe^rf^ra^d by et^pesaeiog tsalts 
ia the 0^tlialsB8»i9gy Sorciee (^. Richard Qglssby) &b^ KlSSl. 



Part B included Yes / / K© / K^ / 






The diagtsosis of l^sraeocoajuaecivielo 8i«(S£ ^eaa 
Slhmesnt&vf keratitis deatoastsTAted b]^ bloaiesosespy, 
docfffifisad tea? flev ds^ostrated bf Sehtsim^t Test #1^ 

a&d aboormtl sSiaiKlag o£ Che bulbar canaj^mctlvs «ad ostraea 
by SeagaioiRsse eesin. Beatal sisaaiinatiett dlssldssd defuses 
of the oral micow ss^s&^anos, muia»sal ^e&es^i o£ deatal 
. eaxrlee Isa scsae pati@£ilu3 sad eaisrg^ssat' ®f taajor @£sllv®s:y 
gleadA. Fairotid ihsm %a&B sssasusred ln^ use e£ (±e Laslaley e«s^ 
and iadleated gsas:&e«3 rsdustioa 07 cew^X&te abseacs of 
ealivar^ flow la several patlea&s ia t3sA» gsaap 
(Dr. Sobsrt: Seepfsaa). te amit(&t&tf sialography of Kbe 
paro«:ld glaad^ sialatssls ^a@ dmsasesra&Sid ia «U of the 
gtatieats «xsasaia«d |!kr. Ir^ia Shi?), ^^sosisataly 40 oth@? 
patltaaCs hsv@ b«ea e^ssisiod ^ these teshaiqisea aad viil 
8«rvfi as a eoatzol gr@«p. Aecordlsig to C&d Asstezieaa 
Sheusatl0a Ascoslatloa criteria fosr tha dlag^ls of 
sheus^toid aiffths'ltiii ghe iuitial gm^sp of 21 patieats ^ss 
di^ded into f«mr gr@i«p3. Ia tlie first gc@»p «ffe?e S patl«at@ 
wdth dsfialte or eisesical t-heisraatold arthritis; la ths 
&©c®ad gro^ ^®r@ 3 psi£il@ats «?ith possible ;heu£93t<9id 
srtkritlsi f:«^ ^4 s^les^dsmgi md S,^atl@atfl had oaly 
&vsil s&d ooisia? Ei^alfestatioas e£ Sjogrea's 

Latoratosy BaaataaticMBs. 



M uaastsal fiadlag la this gsm^. of petlsnts ^as 
a oaaihlaatioB ®f tmi fixed urtoairy sp«sei£le gravity aad lo« 
blood area aitrogea. This say be relatad to tha ehreaic 
iatsks of lars® ^sscuats of ^ater. (^Is pSiase of our study 
is belag candnct^d b^ Sr. Sheldoa Kaha » KM). SIh o£ 
these patieats h£td 1&& «^itG blood call, oouats aad wmy 
had eeslaophllia. 'Em patieats had thnnsboirytopsaia 
^thotst imrpura. The erythro&ytci sadissmtatioa rate vms 
QlevBt&d in 20 of 21 patieats. 

"Sotsil e&wm protsias tj'ers le@s thsa 
the first 3 gr®i^«j ®f patieats mtd ?jaK© iacs 
patieats ia grswp 4. Coaceatrstlea of 

below aoraal ia eh© mtira group. Sorua gldbulia eooceatratle© 
^ae lacressed ia alS. 4 groups; a aarked 
noted ia the gmm gSobulia 4soa«eat?a&ioa, mpo&ially ia 
the last 




Serial M©. ,MS^MzM^^ 
3 



tfee tsoaeoBife® fl©e«a3.ail:i©a i;«s£ (SfT) ®®s 
fosifcive ie 20 ©f 2i f^tieafc® .sad the 8®oslSls®d s&eep 
ceil isggiut:i3S£ti0a £«wt mm p®sl.«:lve ia 13 of 20 tested. 

^ al.S:s®csa£Elfegati®a Skarougfe a sse'Ksg© ^^ssiss,' gradieat. 
S©r^ fi?6® She 4 groups bs&sved steiia^ly. 'Has sesol^gs-ealiy 
acti^^ f irffiefc.i«»s «s®a£:ais@d Icsss tfesa 10^ ®f Sise a^feal 
profcef-sa mt®& ia escfe eisp^rteeraS aad caateiaed all ©fete 

iij®isa©l®gicaliy hy g®l dif fwst©^® using jsst ^mfcises^sj a© 
I9S gemm g,Uhi&lin, 



artteltls. Cfltsse 22S assipless®® feav® feasa described 1b 
®f|s.EWsi®!Bt©ly 3^ ®f fftefffisatoid artlariais patisae®, 
©socially Etos® «?ieis ^«Ef Mgk tilies's ia s®x®l®sical £®sts 
fer srbieisfisafceid &ttMltis}, WuKtSim^msK^p ell®©® islssra* 
e<mtrif?sga£i®ia ©Sadies ia<lles£a4 th&t £lse iEcssas® ia 

ia t&e 7S class ©f p-mtmiMs. 

gasSEa sS®^5tia 0t. RUhsizd A. tlategaraa), Slgaificaat 
£it:©r» «j®rs ols£aisi©«l 'E?i£& 13 of 2 S. sara fswa paeleats la 

witteBt Sjogrea'® syadssai® had slgaiglefflafc £ltesrs to Shis 
feeessaiqua. K3«s<w«!?j> ©Kly 2 pafeieuaes hfsd ^sifciv^s X..S. cialS. 
pr«f5>««rs£t0«i©j ia ©a© ©£ £&ese pastierats fifjis ^ssas 
asso'siistssl with elteieal er^id®fflc« of sys£@ssie hs^^m 

Fiva ©f tb.0 suseiasats Isi £&ls gm?3^ h&4 ps<©ieiv© 
direct Cesar's asses, SteaSies ®rs pE!©ce<a4ias eai isSeatif^ 
l:he flisaititei^' csatteg r®4 &l®©d cells ia K&®e® |j©£tmts. 

tm evidaae© ®£ ellalcal &%r®id diB®ejy& iu £&es® |?ati®acs» 



Serial K©. .Mim^^^c^ 
fage 4 



feSEl^^a|.J^di^. 



QSteaees of fei^sa salivary glaod Isava tet® lassaeSusiv©. 

efest sera of ^£i.@aes ^i& Sj©gE^«s syads^ raas£®d ^idj 
ealiiss ejserasts sad hissed oi^>l®seafe. ^qs© studies are 

iajs«£ed ^iKh fas^gssates ©f lacrimal glsads ©^l®ifi®d 

ia Fr®wsd*s adjevaafe. ^ftsr fiv® ®@aks alias® aateais shmsd 

Siee©l®gie <EKajsiEsKi©a ©f eissis® f^rsj^ tim&m sskim&ls Is 

Sj©gsrea'8 s^drssse is a little kemm v®ri®ia£ of 
sfeetaaatoid axre&j?it:is . Fs^iimlQasry sto^ia© sisgsese t&at i£ 
ffisy fee dtsa £q aa aatoiLasasss pr©c®ss ^icfe iaasrfgires ^tls 
tfe© fsiacHioffi ®f glsads srespsissible gos: ffisisfissiag vasiom 
saseoais ®s^>sraa©B. A f^atesr tiad^rstsadiag ®f £!iiB s^droaa 

related dissasee. 

aatib©diss £® saliwifj aad laeriaal gl«ad c®as£lt«@a£s 
®ggl«tiEa£i®a £ec&ai«j«e3. 

2) Ciiaical atssdlm uil2 eoa?:teise ia ©^dcs? £o 
def la« ^s f«U fip««t^^ ©f i:^ie sjradr*^, iaeimiiEis 
et^ssfy of pael<mes vi& ©®iy ©sal ©r eeaiar <5iseas©. 

4) Studies wtm eoasints© e® <2eliBeffie® shs pastesjj @f 
de^,t:al caries aa«S ®&ra«t^ s«€» ia s»aeieaU '^leS 



samltegiea?^ E^saifastatiuas aassig t&e f«®iiy 
— - a^feSi Shis spadeoss®. 



Serial lo. .m:sm&_'_UJic_ 

3. BQ£^es<Jffi 



Salute- ¥ear 195? 



FOTt^A. 



©tlser Xisiwsi:ig®£©rs; Sirs, Jamfe i^Mjias sad J. I^wd lali; 
sad Isr. Sssgis F^sdeaba^g;? loskefeiies- 

ia l&® Psiblie Healtia Sesviec 

mas ^ejsrs <eal«4ar ysair ItSf )i 
Total: 2-1/4 

|a3V^5is>epSi'tesau tst tm& sad ©tlsei- aaisisi®, £o invsaitigsaa 

<fefc«ii:®isa Steig- se«la8:lsas^lp to tSie as-^rifetdes and 
©^^g diseases- 

gj^gggl •-' ^se? bit^shsfialeal trsit:® i& hwsa&s sad 



Paes B Is^sl^etS ¥a© ^J 1?® /"^ 



Fas© 2 



Ehs bl@s^d gEossj^j sad ©^sr Bfst^ss t© b@ descsifesd 
hQlaWi £a.II. ^^itlsisa tMs eisssificaties. fsrtsia studies 

t^iiit&iQ Vh&a® tz&t^ is the laeldseeee fotmd aad Isk sosts 
eas@s the sel®€a:i\^s f@ress ssay te related £0 disease. 
l!: iis of ia£ar®@t !:<» ^e&@s;i^&@ i£ pepulatissLe llvieg 



g'^£<ss. Sid &4dtti^f G)£^ies of t^ 4ii&ttthistiJ^ of t&iess 



& £leld ti'ifi t® £1^8 Csa&ral Pacific ^as ^&S& Iss t&s ^ssS 
e£ early 



Tliese are <s f^sily «»£ s@?t^ ps@teia® <c^is& 

&USSQ8 mad Kfease ars Bgjdejf gaastie cssfcs^i. Esssa raea 
tj^^jas bffi^TS Issea diaafisveraa by as MMSi> seiemeiat; and 

Iss £&a Marshall. Is leads, 'fha prevalesea of Sffpa l-l %?es 
fo^t^d £© &® Sjigla is £^ loffig®ij3!j> ps3S®ie, alfcho^s^ aot 
i^ite so el«s^ae©d is ^® smaller jsssE&er ®.f sssra studied 
fessa the ©ah©!.* satoll,©. Several iadlvidtasLe ^£ii ea 
&sptogi®^ia ^ere fensadj £his ^.^uld p2:seiS2!3&l.y fee ©f 
^b5?si@l€3gicsl iispoiraaase uadsr sosae e®adiS:ioa@ of red 
l>lmj«S c©S,l. &r®sMo53--£j, 'ia t<K© aasas 1£ ^ae fousd eSsafi 

ph^isiist'fpi& ehsm^ dia^iag ihe os/asssi of tli@ te^ y&Bss. 

Q^sdl^i :lQ ce^sj^msticm '«rl£h Sir. K. E. Slnsls^, £t was 



^ape&globin re€«ti^d &s» ssisrl^ sios^^l s^stsats af ti&r 



Serial 5^3.,. „^mtMi^ 
3 



t» hme onlj ©a© ©f £te tofitogl©&i» £y^®s. sscl^, 

&a"ffa also fessB st«^ie^ £a ® varisfty ®S ffisljsails asd 

flse a^l®ti®se4M test: fsr •%%©^aa&9i(l 
rlie^ssesateid asttolfcl®. It has saesaeiir fe®« s&@a^ feg- 
E®as£i©5aj, se4 £^6 the istslMfciiag saaee^ial t^m^U 

say fflot bs a£ e&a sa^© i^esss, l^is sis© te^ &te«£©^- 
lay S£eiai«^g. Ia a prfflltolaasy ffiE:t&% ©f Mi'lfasaj, 
Sskii^j. 4lask»a Sa-dis^f &n& Mie£@@ai!i%i i^pil/Sti'&sm^ 
it feas feee© sfes®a £to£ Site e# gsse sg|!^siffs m be 

Mzicmm liava esfcal gaaasa gl®fe?5ltas tMmi atesis a&as; af 
ssoveal ^it& MffiS'ie^s.s. lis© slgaiflcfiaesg ©f £lai© 



E£ tes besis ©tew by €arfel@r sad ©e&sssr® &mt 

S«ms persosis ^e& l^tsteMa ased ®£&@7 es^gosrs si?@ also 
high esesr@££;3?@; h&t ^<g geae^ie £9ia is^ thm® eases is 

ima immd fh^t mimslf f 0% oi eliess «©k« feigls <m&^®ti&T& aa 



Sesrlsl S3>. sl^sl^^p _ 

swl>Ject®^ to £fflll©?s£ i»a 1954 follc??isas Kfe® d©fe«!aat?©ia 
Of a Euelmsr dsvice oa aearlrsf Bikiai afcal^l, it hm 

MI3 mapfjt. Ssw«?®ff, it is wnWrnlf ts&ss e&ie i® £!i«* 

differeace betmasi fetjs ®K^s®d ©ad un®3g|s©g@«! girsaps, 

ia a ©Mil E!i«ssrm4esim g®§>aia£4©a^ fjss® & ©aafffef afc®ll 
^£|5 EMsrly nmnml Iwsls ©f r^eSl®£i<sa. i^ alsejeaate • 

is 0is«s«at iss ^emis or Seasiiteasg Asis. Stts^ies to 
deas^aia© if g^iis is && &m its progvas®. 

-ms sMllay 8^ gaate pljesij7l£&i®aaj..bis»4dc (fit?) 

d^mlc&Uy ffltoilasr £© sms i^ttemgsaie taesrials md is: 
hm hmn SMgg®8taa £&&£ ttasre is m mm&^MtiQn hstt^&m 
^ tmt^n&a^tsmt® ^lywaTs^lsm smd ^hymU disease, 

SS ^f>0arffl to fee ^ffssma ira Kaceea aailatfc©, £li(e steg^j© 

r T.^ ^ to majwetles t^dj tec. Jac©S. tofe&ia® aad 
i^^f^/'^iil ®''*^ ^* ^«^«®°« ^®^»^* «^ »wa5 proteins 

hm.6s hm& h&m e©srTOtea@«$ ^itlb etes® s®aa «s© |Kip«>s 

aad pp«8r-gel elecS^jptoresi® studies o fs^iatlms ^^ 

di£fer«€eo i^ bMisa® |»a&£ams tew sls<» J>«aa fo^alT 
«*««,«»<, ^. 5 Tf^'^^^f*^ ^ ^® P^sitfjm &£ fete teU'Sfi 

M m4^z to &'««s®iMs if eiiig is S0, s&sdie® cm mmlm 
fmiUmammntB^UtBi. ^m mU^imi of ^kmm ^irLtiojis 



Page 3 



& ffiS^aw of sfiasdies ok geaeelca sad 
t»isS a^efeslUs has twaett eoss^Jtetsd,, 



^S©i«S asrtihritis ^fas fmasi to &s psrsseas 
iia fc&€s Maskm E©ki» emsssmltf ef WaisOTig^t. A 
essTisrl^isag fiafiiag «fE8, the l®w iaeidiaaas ef &stm^ 
Sii'thzttia o£ bm4s md wristo ia she SskliBO. thin ims 
£®?sRd to lift sigaificasely l&m^ than la as iissstem ^ita 
Fopulaeieta eoCTecfeed fesr age md ©ass. . & &i# prevalesaca 

eests ms f®«Bfi is elKS wlllag® of ^alawslg&S. 

pse^rties «f £lie feapfcogloblas tssisg sgwelflc emssFstsa for 

2, A sfeia^-, &S £ls® ffiotigeale relations ©f eke hmslusgiffiMap 

3, F-urt&esr ©fc^iea m. tiia dlstsri&afeism ©f 

4, A at&tistt^l &m&y of sreg^rodsietiTO cs§«ci«:y 
1e psSiejat© ^icfe Efes^saatoid ssoads^Iifcis. 



Serial fc. S.^^rli;!£ 

C&lmSMiT fmt 1959 

Fssblie&tt&«0 <ith&^ thim &hBt^t^tst tt@m %hi® pirstj «>££:: 

l^aluresai-G said ferssed fe^ sfcsefi'feoeaecjss tei^si'-ftteus . 

e^e Alaskaa fur sjssI^ gsa^sad ©cjuirsrsl, imd imssmt. 
fe«^£0glol>4a!S aasd l5<Mogl®feiias ©f Alaska l8ki»® aad 
tost© pljessgrlthloc^rbtelda maa® AUsiism Sskim© sad 



aad ladisa iS'©|3Mia«:.teai3 ®f Alaska. iSata^sr© M|ai®"W9^ I9S9. 



Sestet SI®. BlMm-li-SC 

1 



©f Mgh iev«3. MSB cacrstors ta BttcffsaesiasMt, 

L©B®sy;» A., Ce^, S., I^^ie^ H. 1.,. Jx.^ 

®£ Harsfeall I®S,©ffid®s:s la 1959 » five yess-s sftesr iuspsajay® 



Ss-, Bl^lsars ^as e^assd MBimt^t Editor &i ah.^ jsis^alj, 



2. ^-fiferieis sad EfeMs^tls® 

3. Bdtlieeda 



Xadividssal ?s®Ject ^p®Tt 
Cffilaaclfir Yesr 1959 

S»r»j®c£ litis: fe 2||^ Sffeets of S£®E-©i<3i^ 

©the? Xavestigatosrsj ©s-s. ^icS&n M. ^3s^&iE3 aa^ 



ISm Yasrs Cealea^e yeesr 1959>: 

i^re SiKtsdled, waiBig 4irec-£ eixKyese ass&ys,^ i®*togxt'i eracer 
todtsslquies^ jsa^ op@ci£ie ch^aica.1 ss!t&lf&&&* ia^.T^i3 ^eri? 

■ VfSm €fe£f^tiea. • 

Ics? eesiceasxacieaas of varioiss Isffi^sgo^iiall,:^ eci;i"s«> 



Pars: B tecl^wkid Yes i27 »» £Z7 



Page 2 



& laOO^I:©!*! ^srteaisa isa ^€®ga ®f ^tasmy msm^ else ■ 

ia fes-aijB^ splsaaj i^sselSj, tess'Sj, lives j, SSs^^ss^ aad 
ki&a®^ ®f g^ me. Bs®f pl^lt&^ is sis® usjder ®£«dy. 

Sm& 1, s®ii, m. SKs%£ilis« bM f&mt (S. f^sgilis) smiled 
S»S7 ^i«&s@ siseittig^ 



£^i©2f lixsk ia S^ sfeffii® ®£ fe^slrogea trsssffi? i® fe@ call's 
easi|!®£ifeiT^l^ sm?ei?se4 ^ e®s©glie3."al. This su^esfcsd e©«q 

^ife^a S defieiosi: rats, feo^ver, did ssos rewsal d4ff®re®e®s 
is: Sl^S cytscte^sxK C 7@«i»etsse or ^e &sgr@e of stexisid 



tisd^ (Of 1&G effect ^seevdsle^ li&te?@s&JLEis 
SlUet&&cm ia rasptmss l» a^ded «ytmSit^m& G. Its iiv^r 
and kidaey, t&e dagi?®® ®f steB»id ItshiMSioa ^as a&sspiy 

AMiti&tml data ma^ hB9» es^fimed eite presos&ee of es 

slte^aate s©u!ts ©f elestsess fcgaasp®rt M^s^ms fl.sv"0|»r©£@is« 
ead cF£®c!feJ?e®ia S ^Msh is a®t affeeeed lag- stemid®. ae 
imi<'cyt®chs«^e e irad«£c£fis« of ^-shle? (^ie& trsasfers 
diseetly fees fls^jfrafeaia t® ejtQchr©^ 0) fmo pt&^vmdf 
sewdied md©^ ^ried ©©adieisms, sad fesasi eo fes %mmspm^tv^ 
e® stemids. Mffase ^ssy ©f s3£er®siMal eysoehs^ 
re&scease, eF8®efe:KaE® t^idasa md B?SH ead ssacaimce diapfesnrass 
revealed a& seesoJLd is&^lbitis^. md&s mm {ss^^bst 
diff«r€sit) e0Hs4iEiims; suee^inage oxidaele^ e®ul4 W i^ibiesd. 



^•Jsale Bis^Uod by Ws. hmi^mc® Cs^-wia^ MI^. X^, 



Serial 
Page 3 



d©5?eXo|!iSssat; of seszoid tolersace, gratresmeat ©f 
r&te ^itk ia?ge ^q®s o£ stezoids did sot slt@r tk® 
aetiviey ©f Sf^ cyfiodarosa C reduct&s® ®e £lie irespeasas 
e© steroid ®r eygs^lw®^ 6. 

To ss:|»l@re fus-efesr Che ?3iaei@nshif) @f this 
eff®et: £© t'sssosr suppressii®a ^s ©re stiad^iag assveral 
Ss&ape ef scssold sesi@i&iire a&d steroid resisCsrat @®us@ 

£i^sr@»^. Frelieii^ry results euigaee t^t st@sr@ld 
)leS:fissea md e@miti.^ltf cm be e®?s@ia&ed vith t&s 
£@tidase activity @£ £^e tts^r prspaxaticsia. C^HSSS 
RESULTS MS C^J^IDMSD FSSLXMSMMY M9 G^FI^SfUL). 



Mditisml phj9lj»l&^U sad patlis>l®sic 
correlates are beiag iss^^stigatsd. 

Is £r«isla tisstae bsiasgeaatss sa 
sueelaate ©sidatias; &m ^ iss^ibited &y steselds, &ut 
@»e% higit@r (50 « 75 1> eosaceatrati^is of ste?®id fir@ 
req«alred. Here tb® ef£®st hm tmt txasa esi^letely 
localised; h&t dexas astt ae^^ar to h& b&ts&&& Sl&mpsotmlti 
mtd eyteehro^s h, fhie effeet is very labile Ise eosstr&st 
f9ith the etfible SF^^'Stereid rslati©s@Mp. 



Lagtaee oad ^^tmat^ Ostldatim. 

St«3die8 ^sere eoratiat^d ^Itb etesold effaets @a 
b&tl^ pyrtsvate aad lactate osidatioa. 

aad particular sfstsmTthA piE®dustl^ ©f C^^2 ^^«» 
C| labeled pyr^s^ate C^^ is <^asia£®aely i^lMted 
by tast©et®raas, m^, aadr®se©o@^3»|?«di©ae, ■ 
Al sadr®stadiea,S«l?«dl©iM!p aad to .s©ssidee&fely iessar 
eateat Ss^ a acssbsr ®f ©they stessid®. mie ©ffact 
i« obeersrsd under a variety of ^aditieas, feat Is 
best seea «itli 1&& eeaeentratiea ©f tissue pregsratioa, 
mm^lijaltiag caaceatratims of siabstrate. la the 
s»£eseBea e£ frtm lfi»3 e^ ia^£ »? i«ar.f. _ 



©f feeai 10*3 gis> 10"2 jj i^i j^j^ ^t 
^H 7.5»S.G. Fraetieoatioa studies r^ea&ed the effect 
to be asset otrifeis&g £a tba sell f^astlea fsedlaseatiog 
betifeeta 0«800 X Q i&mf^v&hl& fcs^aetivity ®f croda 
>. 



* Br. Alesaadar Sesstsda is eag^aged ia these iewestigatioas. 
Dr. Michael ?©teer ®f ml is eeaperstlag ia tifes aaiKsal 
ejsparSjniaaeatiea. 



4 



pysofihaB^^tQf &v m^ did ao£ alee? £fes. effect. 
Safelfeitioa was deeseassd &y lip®le acid, 

Esssslfcs with e, Ifflteled laeeae® failed 
t0 ?@9@al GSQSis&eat iahthttifsn. 



M®lthmT && site sosr ^ekeai^ ®f 

B, ' l^sfcaSe ^ ®a£<lai£i@3B; Bsl^ la^ge e©Bc^itffa'ci<^s 
©£ liver fess^gsaate ifc ws |5@ssifele ia afe©«£ 70% s£ 
esperiffissats e© sfes» 2»f©i4 sU^sla£i@a ©f Ci^„ 
psrod?se£i®a £sm g2 labeled laet^Ss^e^* hat si©t'^C2 
_^^^vgt® fe^ ■vs^i®Bs a£@rsids. SafortaaiiilfT" """ 

alse sffeel: could fee ceasisseatl]^ @1>sar!m4 mss-e 
n©£ e©Bpi®tsiy sacsessfal. 



Stis^Ietisa was dlsiaislEsdi ©sr a&esllebsd 

It: was gssies-ally usjaffeefcad by 'S?iH geaer^aSiag 

of WE ©ST SfHH ejslmiefid £&® effect. St was 
partlculas'ly iaeerestisg ehst she sdditloa ®f 

soatsoi mte of CO^ pzodusiii^a asd this m^v&mio^ 
was reli®i?sd % sSsroids. 

l-he pls^iologie is^iicstiaas ©f ehis 
fiekl® esensid effect tee^ a®t 



&dsres»siQetealged md g^ja&es^issd rats 
did o»t ^p^ss to msp©ad dtffsreatly fgea etss 



C2 i®tel@d pE©pi©na£s «msl Cj sad 
labeled asetate gave results sisilar t© c« 
labeled laees&e. 2 



Serial K©. WHm-lSac 
CBle&Aav Year 1959 

Esm&^Bg M&sissdBg sad Fublicstisots 

1) YlsMlsg, K. L., aad SasfelaSj 6. M.: M effect of 

2) Yleldiag, E. I.., 3Bd f^ias^ 6. M.: SffiMM£i«m ®f the 

3> ¥ieMisss5 H. L., sad Te^ias^, G. M.: SteTOid seaslElw 
Ssstd stsEoid iassasieive else£r©a tsaaspoee paelsa'ays 
CiSmuseript e«mgiete «=• £© b« ©asse t® J.BI^I.Cfe^si. as 



Serial Jio. ,^^^^^2^2.^ 
3. ^Chesda. 

Prajsst Title: Jisveaiie El8®a^£©id AstbriSis 
f^iaeipai Iavss£lga£oir: Bsr. E. L^ea® ¥ieid4ag 

istm Years (eals^dar y®&T 1959): 
T®e«X; ^ 1/3 

Pr©fegBl®Ml: ^1/3 
defect: © 

A. Secs-eh fos* iuf€c£i@«s agent In ji3.waii@ rh@$s^totd 



Study eilaleal saslfes&atloae fisS emirse ef i:h€!%ssat@ld 



©Kcssticms, and £:i8s«es, ^asa possible^ ^sre eul&usred 
£&© ssrict ceiessris ®f seuSe febrile aeelviey @&4 wera 



yart 8 iaeluded Yes /""^ Mo j^27 



Serial &. ^H»ti^^,.»»^ 
3. 






1® S&s Fte&lie feslsfe Ssst^ee 

Mas Yeag-a Ceialt^dar y©sff If59)? 
T®£al; 1/3 



©isTQGt easj?®® «sd eh«aslcai assays tsa esosela ose^sets, 

Baiag miseis aKSs-asts iv&sa ©ae aersaiai sad ©a© 
®etl^iKi®s ware ^sfcesMaasd as ladiceis ©f ATP te®gfe«!0&3a 
pm£ei53. AB?ssa ®sitl>i»isie9 ^se.v® ideaeiaal toe 
(In sSi© fece ©f S22SGI® ^s^resoftfciy ie is mae ki^wa ^ich 



Fa!c£ B teciadsri ¥es £37 K© £X„/ 



Serial, m. Mi^sUOlL 
Pag® 2 



PggiK?sed Sougae of Projoct:; 



£a ©rder t® pass®® £lj©a© lsa£@ffes£lag fiadisags, 

es3ssi£islly idea£ie©i «:© hs^sa e^^tosls TOageaite 

develops ia ^m® m&t&g ie is ®sps3£®^ eo pursia® Sfee 
ato^e £i&dmgs. 






S^g&sssidSj Maryiaad 



C&lemdss fear 1939 






i:iAES <Gai©E«tsr fear 1959): 



The isatsbolic ps-eessass that SS'sas^ir® i® ehss cello of ela® 
sBucesal lifsissg of £53® stossch,, iatsstiiaee SiS^ gall glad^r asi® fer 

wi£lj tfe® physiologic aetivifciss ©f %h&^® areas. ®r© asr, fully %%&Sisv<^ 
stood o Al'chsagh the biochesaical reacfeiOKS gfest ©cots^ i^i£bia Eha 
lives jsei«l paacr^sis ha¥© tesa studied ia grsfflter dagail,^ £hsre srsiaajlji 
jaaiay gaps ia oaxi: &i£S5^le%® o£ these osg.sffiS5 S®®* ^^ ^-^ ^Ssi© leag- 
£sr© paroass ®f tMs unie to I'o-v-eseigffiee pasteays of aeeaboiissa of 
ttes® tisssseOj iasiag ©£ first ©ates-ial fjwja aaimal ©cs-iircss .'.•md 
even^uslly biopsy spsGisasBS frosa hijsMin SBbjfsets with. sxi& y^lttaut 
MsQS.&es of dasse £;issuss= It is then lateaded f,o apply £M is'" 
forajsatiOB gsiiaad ia ttese st-a^4i@s t® s;te furfilisss- in^^stigstioa of 
tli® faac£i©£5S of tfees® orgaias;, ia iaai?aaljs mr^d teaaiasj aad £o in® 
v®seiga£ism of Isosngs^aal srsgulstiea &i thsis: fssacsSoaso 

Tte iaifeiai pbasa ©f sSiis prsjfflct aatails site satfclRg up ©£ 
assay j!«i;'ho4s for sSiidies of £5.e$.us setaboliasa^ aad of aaiEsal and 
feaasati physiology. 



Pass 2 



The ensyises th&t segulaee the reaetic^a sequsacs iavslved 
in She e€»s.ve7Sioa of hesaa to bile pigssaats^ &n6. thets to £ecal 
ezcraeory pigssagS; have not beea studied estei£sively° Uuder^ 
stsuSlag o£ tbesa ensyissss ssay ^i^ll bear @a sxseli siibjects &s 
the ^efesais^ uadaK-lyiag the produetioo ©f jasjndics aaa she 
«a£es'^epe£ic cir<Ksls£l©a of bile pigseafe ^a£aboli£sso M® 
have iaitlagad Oisr study with t&s raacsicjss ia wMch biiivasdia 
is redaced £o bili^ebia. Le^>©:^g sad Wysdhaaa aSijdied tfeis re» 
actios iB 1.93® sad dmaOiiStrased rsdaelEig activity ia aaey 
feissaes ©f tiis gai»@@ pig asjd in the livers ©f th® E^ty aaissis 
tfesy sttEdieda Ifeelr assay ©£ activity wsj® aaly qualit&eivej> 
hsmev&s^ We havs ^svaiojsad a qtsfiEtlsative sssay for this 
activity sad havs sfeos?a the reaction to be d©pandsaS ob ©2'^, 
TPMH ia sooa to b© tSiitsdo Cfe? fceatstivis fosiasulation oS r.!js 
react ioa is: 

3llivsrdic ■4- B^m -i- H*' Bilirubin -y Hf^'^ 

Additional evideoee ettggests that ths ■ze&ct±o& is essysss 
catalysed aad we ar© nsfs engaged ia ©a ae;ee^t to ptsrify %h& 
easyas® in &x4&x to study its profsertiaso If purified easyms 
catt bs peepsaredy a best o2 related iavcstigatiesa csa be 
tsnde?tskes. fhese iaclud© tha develofsjseat of a ©pacific en« 
sysjatie assay for biliverdia ia bl©od_, assd the usa of tbis 
rea&tioa to geaerats bilirubia in vitgo la arder to study ita 
setabolissa ia greater dsfisil than is feasible at pr&ssatc 

Bo d^gYloge__^.tgbg|_iag__ 

A cliaicai test for iatastiaal. sbsosptive capactiy^ now 
in geaer-sl u.se, involves tba feeding of d-xylosa aad the de^ 
tersiinfition of tke quantity of sylose excxrsted is tbs uriaa 
during the essuiag five hours » If is assuiaad that ay lose 
matfibolisisi is aot a signifisaat vasiabl® ia tbis "syloss 
tolersace t«st»" Although the pathway ©f Esstabolissa of 
d'»:Kylose in msssaals is not ala-cidated at gss-sseat^ Segal et al«5 
have suggested tbat ia csan tlss liver ssetaboliaes as asach as 
40% of ii^travesiiously adsaiaiste^sd d*ayloss<, ,Ia ordosr to 
e^losrs this farther., we ar® iStudyiag the EStabolism of syloss" 
l«cl4 jjy tissue extracts aad ia tis® liver of rats duriag per« 
fusioa atadieSo Initial results suggest tfest in tba rat ths 
liver is not & sita of oajor tsstabolic activity. 



Serial HOc^l^^j,!^ 
Page 3 



¥s ss-e sxploriQg the possibility of «£Sieg iQ^raluminal 
biopsy tubes to obfeaia sasples of iRtestinal ssaacosa oa wMefe 
studies of easy!^ ectivl£y and patlxe^ajs o£ i^tsbolis^ csn be 
perfoirsedo We axe csrreistly engaged in perfecting our skill 
in the use o£ thB Btf.bia biopsy instrus^n&o 

^^^° Isi vitgo Pby® i®j^a jxal^ Sjgadiss 

Ao lran®p®y£__f]|mc|ioa8 

lo IsoXated^^ ,9ggg^5_^-^o|„ .^^^^j^^li^.tMSi '"' ^® have 
siodifled an apparatus tssed by others for t;bs study of £ra»S'^ 
port &cro»s isolated ssgisancs of gssioea pig saiail iafeesttaso 
We are still eagaged ia psrfectitsg the eecfeniqu©^ If: Is ouje 
inteation eo tsss it tia<sre&f£er to study the effects of ansys^ 
in^iibitorSj, herssoaefSj aad othssr factors oa tise traasport of 
vsrimss coi^masds across the eisall bm^l Bmc@s3a 

Bto NiE«nberg=; we have isolated a s©il bacfceriuis thst caa 
utilise octanoic acid as a sols eotirc® of rmtzimstnto Wa piss 
next £o radiate ebis orgastisaa in ara attes^t to prodtsce smd 
isolate & sssita^S: '£hs£ no longer !^tab@lig@s occaaoic acid^ 
even tko«gh it yreKalns Khs capacity to trscsport Shis fasty 
acid froffi sbs esitejraal sse^i^iH itito £he cfill., Sucb. a wsazasit 
would permit us to iavesfcigate fafcfcy scid fesansport iu bsc* 
, eeria in sojbs dsgsilo It iss hoped thafe uaderstaadiag sosiise 
aspects of i"3se traasposrt. rsaclssaisaa la bacteria will provide 
lasight iai:o sl£d.lar f^mctlong ia aissssaliSQ tissues » 

In collffiborstimi wifcb. Dffo l&jfffcinsjre^ who has ^veloped s 
teehaiquie fer perfiusion studies of tfea isolafeed rai: liverj, we 
ls©p« Ko sfciady no£ only d-»sylo©s a^tabolis® (li-B) but also bile 
pigmsQt sasfcabolisaaj, ffiadoeriu® influeaees oa vairious liver 
itxa&ti&ns^ aad details of £lie (Ba£©rohepatic circtilatioa of 
various coa^oandSo Pffeli®inary sKwdies are ao^ in pt'ogreeSo 

IV o Clljalcsl Studies 

^° B^;Sg;ao8tic^_aad^^iave6t£gafc 



Tlse followi©g deeesrsaiuiations esais »ow b« p®rf»3fss©d in oti?" 
laboratory: 

<l)o Feeal fat excretion; (2),, Ser^m vitaisia A ffiad csroteae 
levels; (3), Blood sad uriaary xylose Isvels for fcte syloss 
tolercace tsst; {4)„ Scrim biiimilsiti I.e's/elSj aad (5) Uriaary 
S-hydroKy-S-iodolsaeetic acid (mm beiEg set '4p)o 



Serial MOo^^lM^_J.3IG_^ 
?sg® 4 '" " ——».■ 

We hev© pesformd two iaeeisfeiaal biopsies^ bath successfally. 
We-plsB £0 Bet up seehods for the datersaiaasion of fecal es* 
cre|ion of lUl-tTioUin md l^^^-^oe&ic aeld^ &s ^511 ss 

are stadyiag patisaes with £Ms syi^dr^e \©"ll®'£.gmia.e t&e 
fraquaacy ©f iateseifial eaalabsorpsi®© is tis® psressEse ©f ©!« 
eval:®^ le-rols ©f blood saroecmis, Bsa paEiaeKs wieh fuacfeisrniKg 
c.ai-eisoid3 aad ®ae wi£fe a metastatic tessor e&at may Iss & -aoa» 
f«aac£ioaiag csreitssid asre bos? uadsr si;?i(gy osj the wardo 

This is beisig stJ^died in aa a££ss^£ fco ^g@ce, Mldsss-eo isiiSBS" 
fseeted bioeheiaieal lesions related to feh® dtgestivs system, 
Ws fesvB isexsdied ok-s sasszbe? of s Smslly a'ith polycyseie diaeasa 
of tfes liver assd/or ld.&sys» 



Fare B iaclaaded: 



Serial ^J<s •'...*[X**^.;;13S£» 
Clinical Invest! gatiojas 
Arehjfitis & RS8ffi«!Mfei®Bi M. 



FES - HIE 
Caleadar Yaar 1959 



IS^l^ 



PSOJSCT TXfIS: Seudies on Aleaptoasjsri© &&& Oclaronofeic Artteitls 
ia Jgau aad Aainaals s»d ssa PfefflnylksKonuria 

mimtmh imEStrnk^m-, mvt is. la bu 

OfUR IWESf IGAfOlS : J» Eo, Seegsailler^ ¥» ZsaasMai aad W. ©'Bxiaa 

CO0FSMTBS6 TOI!£S: D^o Eiclisird Aisld^ Pediatries Bepto^ 

Georgetown Ualvessity ISsspita'lg Wash«., B, Co 

mH ^S&RS (Caleodai: fear 1959): 
Total: 3-1/3 
Pirofeaaiemalj 2-1/3 
©Khar: 1- 

FBOjaCf MSCEIFJPION: 

A- M£^.£gBI£^..^i^^SHS2l|g.A0fagi£is 

Objectives iu studying pasleats with alc^Sionuria hava been 
@@v&?&i: (l) to dati&xmln® &he enact as&iare o£ ths tsietabolic ds£ee£; 
ia this amditi&ui <2) e© study slie hereditary jpateera of this 4iS" 
aassj aadj^ if fwssmible;, £© efevelop a test wfeich ??ill d<a£ec€ tlse 
heearosygous seas© ia relatives of alcspeoiMsricss csffryiag the £rait; 
(3) to study the f®^iss£i«m aad depositioa of tk& plg^aafi dsrived fsros 
ho-sogeatisic acid aad to dBsfcesmiae hm? it process sAe pa&h.olosi<«='Sii 
chaagas ia the coaaecfcive sissues^ par£icularly £h© joints | (4) to 
study th& cause of tks arthritis nearly always associated wiS:li Chi® 
coaditioa^ aad (3) to attes^t: various i^aas o£ treamane of this 
!tsat.abolic diseasa. 

n&'aiTe of ^fect in elta^pfcoaayia ■» quantitative analysis of 
She ^nsyjmB itsvolved ia tyrosine jsesaboiism have beea made in liver 
mxd kidney hasfjgsQates fxroa aatopsy specisaas of anoehax' patient witli 
alcap^oasariSo Again it: has b«en possible to shos? £hst alcapeoauric 



serial Ko.,js^l3^„ 
Page 2 

tissues differ frcses the aoirmal only in having ao detectable 
hotnogentisic acid oxld&se activicyo ttms,^ it has beeo cleairly 
denioaet:raeed in ewo faaiilies that £he de£ece In this toetaboiic 

disease consists of & deficiency of hotssogeatiaic acid oscidase 
aecivity in the liver and kidseyo 



?;^S'S»E®.£|,aleaptoauric patieata » 
hOQK>geatisic acid k@A been ^sasured i& lm« 



'^le ceaeeatratioa o£ 
lme@ joiQt synovial fluid 
of an aleaptORuirie patient and of the rib cars:ilage o£ anocheiir 
patient with this disease o the levels in each of these tissues ^ere 
lower than ia the blood, indicating that hoiso^atlsic acid is not 
maiBtaifssd &t a high coaceatration within these tissues in alcap<» 
toa»riao 

Infaeritaitce of aleaytoa«gia «■ fhs recent sisggestion o£ 
Milch and Milch that this disease is inherited m a do^nant trait 
with inectsplete penetrsace^ rather than aa a si^ls autosomal re« 
cessive inheritaneej, is sot supported by ea ess^ination of the 
pedigree of one of our patients » li^on careful qaeacionitsg it ^&b 
disclosed that ghere had been a coasanguiaeoi^s tsar ri age which was 
not inentioned in earlier interviews o With this complete inforssation^ 
a aisipXe recessive inh@ricance adequately e^lains the expression of 
the disease in- this feailyo 

1' S^agJEiBntel oeteoRosis ^aad arthritis in guinea pi^s ■» 
As part of the st«dy on the tssehanisa by vhich the sccutaislatioa of 
hosaogentifiiie acid leads to the developi^nt of ochronotic arthritis v 
In slcaptonuriaj, - the distribution of hcKsogentisjie acid in the tissues 
of guinea pig^' has been ststasured at different tisses by a specific 
ensyisatic laethdd after chs intraperitoneal injection of this acid. 
Very lov concentrations v&t&-tcnmd in the csasclej, liver and the 
other orgj&nffi; but values ahmat as high &s in plasma %%re present 
in the cartilage ®ad skino fhi® anus«al predilection of hosogeo" 
tisic acid for the connective tissues ia in agreeaient vith the 
deposition o£ the ochronotic pigssent in these &asm areaso Further 
studies are b^ing smde of the nature and syntheses of the pigi^at 
and its relationship to the asaociated arthritiSo 

2i«MS«£iEJSSSi^»^Hk£.Ui^ " Setffliled st^ies of the 
syse eysteia of liver which catalyses the ioxmntion of hoisasgss 



eysteia of liver which catalyses the iet:mi.ti&& of hoisasssstisie 
acid froja p««hydro3£yphenylpyruvie acid (the keto acid ©f tyrosine) 
©re beisag conticaedo VitsMn C is involved ia this ensysEetic re- 
action aad scorbutic guiaea pigs have a defect in tyrosine saatsbolisffi 
and eKcrete p«»hydrosypbeaylpyruvic acid when fed this aaiiao ecido 
■01® Esetabolic defect is corrected by vitamin C^ bwt wntil recently 



Serial Ho. niiSm-lSlC 
Pfige 3 



it was not kemm how chls vitasain maistaics nocmsl ty?o&ise 
raeeaboliSiBo Bmm insight into Che laeehanism amsR in @t;u4ie© wltk 
purified liver enzym&u^ It «&s fotmd that ascorbic aeid mtd 
2^6->dichlorophe!!iolindQp!£eQol bad tha properi;y of protecting on@ of 
themy p«hydro35y|sb.eByipysuvic acid oxidee® (the eissysse which catalyses. 
the osidasiou of p"laydroxyph©i:syi.pyruvic acid to fecsaogentisic acid) 
fr<^ beiag inhibited by its s>ub3trstao In the pr«SGuce of ascorbic 
scid^ csid&tioQ contiousd; in its absence, the oidLdatioa sioued Sams. 
and stopped. Kee^atly we have been able to det^ostrate the way the 
vit^iia acts ia viWo Scorbi2£ic gssiass pigs war© found to have as 
Bsuch p^hydroxyphsaylpynsvis acid ©Kidsse as aorsaal aaiamls^ bat wteni 
the scorbutic groiip wsis injected witi& p»hyds©sypfeeaylpyruvic acid^ 
over half of their iiver p-hydrosypheayipyx^svic acid oxidase was 
inactive oae hour later a la eootrast^ iajeetiou of ths substrate 
did not isihibit the osid@se in sorssal guias® pigs» It app&&s& 
that ascorbic acid sets in yivq to profeect th® oxidase fro® in» 
hibition as was found tti the enayma studies la vitroo 

It is of iaterest fehat scorb^itic guiiafiia pigs givea 2j6» 
dichlorophsttoliadophsnoi several hours bafore &n injection of 
P"hydroxyphenylpyruvie acid were also protected; thtas^ the Ajm has 
B<m& eati-scorbistic ©etivity* Further studies on tfea ability of 
the dye to correct other aspect© of acnsvf are ta progress ead 
hii&tologlcal ssasainatloa of the tissues o£ dya treated animals is 
being m&d&o 

s^^vial fluid ,aad other £ias^esj» fha specific eosyaatlc mthod 

to ms&mxse Esmtl sssaaskts of koffiogentisie acid in plastaa has be@n 
oodified to jBSka it suitable lor the analysis of hcmogaatisic acid 
ia tissues o The method has been utilised in sidles of th£t dis«- 
tribution of hsmag^ati^ic acid in ths tissusa of patients with 
alcaptomsria aad in studiais of tba distribution of this acid la 
guinse pigs. 

hoaoge Btisic acid " tha assthod for hojsogentisie acid has also 
been taodlfied to leaasura tte derivation of ho^ogentisic acid., i,@„^ 
the lactone and sssthyl aad ethyl asters. The fsetabollc fate aad 
distribution of tlssse dsrivatives- has besn studied in guitwa pigs la 
tha hopes that cm.e of thas« coa^ounds i^ould ba ststabolised slowly 
to hxxmgeati&i.^ acid and thereby lEaiataia a higher plassaa level 
ov@r a longer period of tise than ^hsn h^sdgantiaic acid itself is 
given. Such a scHspound is needed to Induce ei^arii^ntal oehronoais 
in ani^stals. 



Serial 'SOo__jm.^m^l3li. 



B* Phgny Ike totm ria 

Clinical Sgudias; 

lo Besfc aeshod of datec£l<m of hagerog^gogs tgait - Several 
aaalytical atsthods have bsea propos&d t& degeet tbd carrier of the- 
pheaylke&oaoria er&it; ebess iQclsds £h@ fisistlQg blood lev^l o£ 
pheayialsaifis,, tha plasssa level of pheaylalaaxES afesr an oral 
phsajrifil^ai:^ Solerasce ssstj, or t!»s xa£lo of tha pheffiyi&IaQltsfi to 
tyrosine after aa oral load of phsaylalaniss ,i In collsboratioa viith 
Sro Mc&ard Auld of she Gsorgsgo;^ Uaiverslty E@t:s^ded CMldraai's 
CllQiC;, ^E^ Ere cs(^a?lQg' all chess sase&ods ^slng o%sr aof? saalygiieal 
lae-ehod eo ess %^icfe is th& ssoat reliable.. Ifha experii^ntal group is 
coa^sed of parea^ of pUeaylkseo&urlc childraa baiog follo^^d by 
eSie Clisieu 

2= gffseei yeasss of dlas log ia pfaeaylalaalae In prevasSiag 

sBBBtal retardatio n l a ptsgRylkfttonarle c bildrea « Studies on thd 
effectiveness of tha lo«^ phanylalanina diat la eresttinag pbeaylketosurlc 

childr<sa require repsa£ad uesasureesac of t!b& level of blood pheayi<° 
alanine sa& tyrosiae» Tha variable rsspossa £o £isa diee^ parSicularXy 
in older infants ^ isay be largely due £6 t&e greater difficulty is 
stain tainlQg a lo^ l@vei of blood pheaylalaaine i£t this group o We 
have bees aaalysls^ the blood «sach isonch of @aver&l children beiiag 
followed at the Issearded Chil'^ren's Cliale^ Gaorg«£o?m University 
3ospi&alv Co evaluaee ete effectiveness of the die&o 

3° Sarl^^_diej^o3ia_of_ ph ea^'lfeeeo a arla la a ewbora^^^iafaat ■» 
A sibling bora in a fs^ly fd.eh Iqo'ctl phesyllcesoiasris has a 1 in 4 
chance of being a£fecead». The nev ensysusitic sethod for blood 
phenylalanine ^vsli^ed in this laborat:0ry„ particularly ths saicro 
modlfieationj, makesi It; reasonable to ssaks the diagnosis within & 
day or t%so after blrth^ snd such analysis should be done in newborn 
infants i» fessilie© ^fith knosm ph«aylka£oa«ria« We ase prsseatly 
doing serial analyses on blood sables oa an iafaat with this back- 
ground in order to aak& the diagnosis and st&rt the special diet as 
soon as possible if this child should have phanylketoauria.. 

jG hggi gal Stadjgs; 

tk^ principle of the aethod to s^tasare phsmyl^laaiae in blood has 
been adapted to ssa^sre ssall queatltied of several othsr aroisatic 
asino acidSj eusfe aa p-fluorophenylalanina and g^fehietiyXaianiney sad 
the ffistaboliss of ehe latter is being etadled ia guiasa pigSo "She 
effect of B^thieaylalaQiaee, isia aatl!2eCaboli&,e or phsaylalanias.^ oa 
the coRf.«;atratioia of pheaylalaaina asid tyrosine ssjtaboiites in blood 
find tissues ii} al&o bei&g investigated, 

Part B indued: YEB 



Serial No„,js*^^-^ 



PHS - MIH 

Individual Project lepore 

G&Und&T Ye«kr 1959 



Farfc^; loaors^ Awards sad Publicaeions 



l^ Zaaneai^ ?» G.j, aad La Su^ S. H*, llis tyrosine oxidatioa 

systsffi of lives o IV ^ Seodlas on th® iahlbitlon of p-hydrosy- 
pfeEoyipyruvie acid oasidasa by excess sabserse®.. Jo Biolo Ctem»,, 
234: 2925«293i, 1959 » 

2.. La Dss^ Bo Ko,, sod Miehaal^ P. J^^ Aa enzymatic spsctrophoto- 
sssstrlc BssKhod for the detsrsiaation of phsaylalsaiK® in biaodo 
Jo of LEb» sad Clin* Ksd« , lu prass, (Febo i960), 

3« La Dta^, Ba No^ fhs itJ^reaiKus of early <3i®g©osis and tjreafeaant 
of pfeeaylketomsffia, Aaaals of lato l&4o 

4a Zaanonip Vo G»3 and La m^ B, ^.., Smdies on the defect in 
tyroi&lsie &@t&biolissa in scorbutic gasiet^a pig5.> <l> 3iol<, Chesao 
In press (Jtea IMO). 

5.. Ilia DUj, B. He, *fyff®siaosi»., chapter in Biochemistry o£ Molecutlag 
M^a^£p edited by J, Steabury,, Jo B. Wyog&fflr<3»aa mid 
Do Fssdricksoia, ^w fork^ McGifsw-Hill, la s>resSo 



Clinieai Investigafeioa® 



PIS •« HIS 
Individaal Pffoject Sapors 
Cai<a«dsr Year 1959 



PHLA 



PB0JSCT fllMi MeeaSjolic and Tlasrapeu£ic Sgt«ll«as of Qamty ktthritiB 

PRIiCIPAL IWESTIGATOS: J. I. S«5egisiller 

OfME IMVlSflGATOSS: Arfefear I. arayg®!^ lofea J„ Buxaa (MM.) aad 

CODPESATIBE UNlfS: Lab^rfiSory o£ C&esai&aS. P&astaacologyi, MI 

msi YSmSi iC&UttdBX ¥®as: 1959) 
lot&l: 1-1/2 
Professional; I 
Other: 1/2 

mOJBCT DESCEIFTIOM: 

Sfcudiea cm dstegs tot ^Im tmn&s,®vmnt oi problem cases o£ 
gout hav© been contimaado Freviosss work in tla® Mafetoaal Heart 
lastitute on a groap of coiEpecssiids chsjaicalty rolat:®^ to pheayl-^ 
btstasoQs had shsmn &hat aatirksus^tie activity could b@ correlated 
with ohemiaal straccare and that ufficosusric activity eoald be 
related Co ihe acid aS'Socistioa coesteot (pKffi) of ths dr^o A 
nvinsxy jEetabolits o£ pbeaylbataaocie, oxyphisnbueffiaooe,, h.&4 h&en 
®howa to pos®s8S poteat aatl^rhe^aeatic «ictivi&y;, but very lifctl© 
uricosorie actlirityo Upoa tSwB ijatroduction of a h&to group iufco 
fche sida chaiu of ©KyphaabutaKOna. tbe sresuliiag coa^ound (G='2970l) 
was fouad so |>ossess poKeat uricosuric activity,; but lifetl® aati- 
x'heuBistic activiay^ Agaixj fcbe itricoausric activisy was correlated 
with an increffisad acidity of the coaagjomid i6?ith efes pla iSroppiag 
from 4c 6 for osyf>heiibaeasoae £o 2o3 for kfSto»03syphe!abu£asoaao T&ere 
was a cor responding decline in the biological imlf-life frtsa 72 howr© 
£o 8 hours ^ieb, prevented the high se^isai levels aesded for an 
aBftirfeeumattlc sffcc*: wish tte parent eosa^foundo Shis dru.g bas bsea 
given to 9 patleafes for Bhoxt perioele of tiass snd has been very 
well tolorstadl^ wieh no fcosic s±S& effects So date;> Siuce th® oahstr 
pofsent uricosuric agents now available harss. a cosssi&rably shoE-Cer 
biological half-lif®^ on tha ordar of 3 hours,, tba S krnir half-life 



Serial Ho., BJIAJtB" 1133c 

Pago 2 



of keto-osyphaabutssoaa gives proisds® of providing a saora 
sustained uricos^jric »c£loe ^Ith less frequent a^ministraeion of 

ffea eat&gonistic action of salicylates ©a thst ttricosuric 
effeee ©f so3sasoia®i^ss sad seifiapyrasoas has bcsea further storied 
aad fouad eo exist a£ even Im? &js©s of salicylates « 37 eoa* 
trass;, sea£ssaiiaopfee% %jlsich is th,e activo e^feabolic pro^taet of 
aeetanilid aad acsfeopfeeaeaidiRj gives oo such eai:.ag0Bistic fflc£;i®n;> 
'Hhile at tbe @aa@ tiess providing a» aaalgssic action, 

A aew sjsshod has been devised for datenaiEatloa ef 
saaasoaia in biological fluids &t physiological pl^, afciliaing 
glistajaic &hy<irogaaas® Evidence has b©©a obtaissd fos tli© preseaca 
of free aasasmia la noxetal lussma plasssa^ 



Pars; B iacluded: YES 



Serial ^Oo^^^J^^W^ 



FES » MIH 
Individual. Frojecc aeposrfe 
G&Umd&T Ysar 1959 



Fa^^o Honors p Awards sad F^jbliesitioixs 
FTOLICAflOMS: 



Lid41.e3 Lo;, Sse;^iil©r, J<, I.^ sad Laatsr^ i,*^ the eazj^satlc 
specferophosoaMitric sssfchod for deter^iisatiOBi of wsic acid^ 
Jc of Labo aad Clino 5l84oj 54: 903»S135 1959» 



Rheiaasttc ©isaassS;, 3. Ao M. A.,, 171: m, 9^ 1205«1220,5 1959; 
m.: too 10^ 1345-1.356., 1959 j 171-. Soo 12^ 1680-16915 1959 » 



Serial Ko-JS^^lS^S^ 
Clittlcal Investigatiosis 
Arthritis & Btemasism Bstc 



PHS « HIE 

Caleada? Year 1959 



P§OJEGT Tills : Afea®?s3Sli£iss of Fsrisje IfeMbolisis Associaf.e4 witfe 
6©uc 

ERISCIPAL immtmhW^i J. S. Seegiallles 

orasi lERTESIIGAlORS; Arfehar !» araysel sad Lois UddU 

eOOPSMTXSS UMIfS: Hos^ 

mm YSMB CCalsadair Isas 1959): 
Total : 2-1/2 
Professicmait l«l/2 ' 

PaajSCT DSSCRIPflOH: 

TSs© ffisfcabolic ©origin and dispositioa of uric acid ia gmifcy 
ps^tlen&s h&^s bee» studied further by adsidaistsriag isoeopically 
labeled precursor© af tsric acid along wifib iab©lap<a uric acid ifeself 
aj&d folla^ng eha iBcorpora2;i©a o£ label iato arinsary uric aci^ ia 
aorcsal aad gowfcy sufej^actSo A subseaatlal porfcioa of tlas pstiaafes 
^th gout show an sscessive syagthesis 4a euovo of uric acid as 
messtsrsd by Kfe© ©x£®at of iECorposafcioa ©1 glycin©*l-ci'^ iato ai?ia° 
ary tsric acldo Additioasl petieists can be sho^ni to be producing 
etxc&m&iv^ ajsouats ©f uric acid If £he glyciaa iaeorporatioa d&ta 
is corseceed for ehe dya^sdcs of tha us-at:e pool, This iacresssd 
production does not show wp in fefes tsrinary -aric acid excretion 
values becaugj® of its esstra^reaai dispositioa.. Shere still reiasias 
a portion of the gouty patients wfeo show bo drnmoBtzeble differ" 
eace in the extent ©£ giyciaa-i"Ci^ incospoTstioa into urinary wric 
acid from that o£ norsssl iadlvidu&ls. 

A phasmacological agsBt wkich suppresses tli« excessive u?ic 
acid syathe&iiS found in son;^ gouty subjects has been studied further > 

6""Diaso»5-oso"l.*ffiorla«tciae (B0II) x^ich has beea shows ia this lab* 
oratory to suppress uric mrJA ayatheesis ia few© gouty subjects., has 
been odjainistersd to a tot^l af ssvan gougy patients « Two pstieats 



Serial MOo^HI^^O^sC. 
2 



vho shoasd n& drop is sena uric acid oi^ in uriasry uric i^cid ex* 
eretioa never £hsl«ss afeossed a s-<ib£»£antial redectidn ia Ch® lEeosporaeioa 
of glyeioe-l-ci^ ittt& eriaary ssric aeido. Thi9' stsppsession of paffiaa 
biosyatheeis was evidently Esasksd by th& large urece pcsol in ebss® 
stsbj«ctSo Uadesis'able effects of BOH csasistad ©f daodeaal ulcers 
ia two pati€ai£s aad ulceraeioos of tfea oral ssaecsa is £iva of the 
savaa psfeieats ®ft?sdied, Isegiae ^s« ©f fcfeis drag foi* suppressiag 
tbe <2ric acid psodsic^iiosi ia goaty patients appears £s 1^ isE^sssdisafco 
It is conceivable,, boweves^ fchst maz& specific IssljibitMJrs of pisriae 
biosyn£b^3is miglst cesrry with efeem a raore favorable sherspeutic iodeso 

Aq ®3^eri@S!B£:aI gooi for studyiog the hissi&ostai^ic emstrol o£ 
pt^rioe sy&thes^iQ Ib the tuisisa has b«ea £ou8d is t!se actios of & drtag, 
2°echylsiaiao-i^3j4«»ehiadiasol©g a tiicotiaasiide aa£agoais£ ^ick feas 
been iss^d expesisstntally in the treats®Qt of q«q.g&z<, Other workers 
notad that its nsis. in the ht«aaa resulted ia ^t lacrosse in both ssrura 
urato values Kid in daily urinary uric acid, ?lie origin o£ this 
increased uric acidy whatfesr from cellular brsakdswa or de novo 
synthesiSp vas not clear <> W€ hsve b^@ei able to coafira this fitadlag 
aad furtheraiora to show that tfee iacresised arie «cid prodisctioa is 
tks con£jaqueaoe of aa incraesed purine biosyathesis induced hy . , 

2-e£hyla!aiaothiadiasole» -th^ extent of incorporatioa of glyGine»l><J 
into wriaary uric scid ia a noa~gou'cy individual was brcugbt up to 
the range observed isa goaty isubjeets by sdiai-Ristration of 2-ethylaiBino«» 
thiadiasolso Furtlserts^rey its effect was cos^letely preveated by 
adtalnistration of large dos@,s of nicotinessideo iMs drug w&s fouad 
to feave a cosspsurable effect ob urinary allaatoiis and uric acid pro- 
ductioQ in the gtsisMsa pig aad ia vitro studies of its acti<Hi ore 
sow underway o 



Part B Included: tBS 



^£'iel Woo ntfiim^iMc 



FH8 « mm 

CalsBdajy Year 1959 



?a&'t B: E©E©rs. A^ssfl!B^ a!a4 5tiljlic©sifiHB® 



uric ecld prodsaetlea ia tbs k^^s) lo^Cia^ by I'^th.jlsm.^ao'" 



455-464 J 1959, 

urie acid ajfatinagls is tk& ^utj feiaasa by &e ass of 6««liaso»5* 
oso*L"ao2riffiMsiae <SOS)« Jc of Glia, Iav®a£o^ la press <Maro^ i960), 



Ssria! No. HlAl®^I35a 



lo Clinical Iflvestigaticns 
2o Met&bolic Diseases Branch 
3o Bethesds 

PHS=NIH 

Individual Project Report 

Calewdar Year 1^9 

Project Title; Studies in Boss© MetsboHss 

Principal la^estisstors Dsto 6c, Dossald Whedoa 

OthQff Iftfestigators; Dro Lee Lut9?ak and Dto A^aseis E^ Tsshjiaa„ Jro 

Cooperatisig Units; Non® 

Men Years (calendar year 1959): 
Totals & 

Professional; I 
Otheri 5 

Project Descriptioss 

Obleetiyes; io To i^irestigste the factors affectissg aineral 
storage and loss in deiain^alizing bone disesseSg with particular 
attention to the relative isjfltsences of gdrejsal cortical steroids^ 
gonadal steroids and dietary levels of ^inerslSo 

2o To iff!'? estimate the rates of aiReral deposition and aaouBsts 
of bosie usdaroiRg sctive exchange with body fluidSo I.r various bose 
disorders o 

Methods Employed; 1„ Metabolic bsisaee studies sinder rigid 
dietary eositrol in patients with ffarioMs^eJs:isi'SS'sili»legboBe digeases., 
s}Otin<; the effects on nitrcgeRo ealciuH and phosphorus balances of 
gdrestal cortical st^'oids, of @on@dal steroids and of various 
dietary levels of calcium and phosphorteSo 

2o Deteraination of pool sise« turnover rate and dspositioii 
rate of calcium in patients with various bone disorders^ using tracsr 
doses of rsdioaetive eslciuso 



Part F inclMded Yes 



Pssge 2 

Mai Qg Findings; The sajor eurrecst regearch interest of this 
project is in deterKiifliag the tole of th© le^el of dietary calcius 
intake in the pathogenesis and treatssent of post-siteRopaussi and 
senile esteoporosiso Bata fro® cJietary SMr?eys by othei'So fsros our 
owB ajetabolic balance studies QXtendinq over ise^eraX laoaths iii ©ash 
of several patients and fro© our own radioisotopic detersisietions 
of "tsone fori^tioa rate" has led to the fotisulation of afi expanded 
concept of the alteration in bone sfetabolisis in osteoporosis „ a 
coecept ??hich accords nutritional factors (particularly availability 
of ©ineral supplies to the skeleton) at least eqsjal. ifsportanc® ??tth 
previously recognized horaonsl faetorsc This expanded concept (described 
sfl the Sffleaaary section of th® Annual Reports) and the data surreetly 
available to stspport it are being published in this esonth's Federation 
Proceedingg (ti-asscript of SysaposiM® on Effects of High Calcius 
Intakes,, Aaeriean Institiste of Kutyition„ held in Aprils igs^^o 

One facet of the results of these studies thus far hints at 
a probable significant part of the sineraHzation defect in eettaia 
osteoporosis patientSo The generally higher calcium intake requiressent 

in these patients to ashieii?© calciasa storage (much higher than for 
^oung norstal individuals) and the considerable variability in this 
requirasent 'su'ggest that an intestinal absorptive defect aay be 
present in certain patientSo Studies of fat snd sinarsl absorption 
are being collaboratively initiated in these patients to assess this 
possibility, ■ 

Radioisotopic studies for the aeasuresent of bone foraetiori rate 
are continuing, in patients with a variety of bone diseases to deterEine 
the influence of various hormonal and nutritional factors on bone 
iaetsbolisuBo' Currently special interest is being devoted to the 
effects of vitsmin D in osteoporosis and to the ealeiuss intake level 
in Patjet's diseaseo 

Significance to NIAMD Research; Senile and post<=@enopa«8al 03teo= 
porosis are t;«in forss of skeletal demineralizstion i«hieh are asstming 
isicreasing isspcrtance as the proportion of older people groRS in the 
pepalatSoR of the UoSo end of the worido Surveys for incidence nof? 
in course sees to indicate that approKinately 30% of wossn ove? the 
age of 50 years have roestgenographically visible osteoporosis of the 
spinso Csreful review of results to date froaa E^nagauent of these 
patients by hormonal therapy onlygtogether t«ith assessiaent of the 
investigations in this project to date, sake it seee evident that lon^^ 
accepted concepts are inadei^uate and a new approach is neededo Evalua^ 
tion by these studies of the significance of nutritional factors in the 
pathogenesis and BKinagesjent of osteoporosis is indicating their 
importance with progressive weighto 



Psge 3 

Radioisotopic studies of botss forsastSoa rate are yleldissQ data 
bearing q& the ftsndaiaentsi differences lei hmie @&t8boIisa in ^srious 
bone diseases and oe the mode of action of ssuaserous Nofkotos snd otheic 
agents on setsboiie processes in bonSo 

Pfopoged CoMgse of PgoHeet; Metabolis balssBce, isstopie and 
gastrointestinal absorptive Cthe latter collaboratitrely) stadiss ?i?in 
be continued in an effort to detersiae th© effects and BBode of action 
in eaielisa ssetabolissa of adrenal cortical asd gonadal steroids @»d 
of the mineral Imel of the diet, and also to obtain understanding 
of the processes of bone fortaatios and resorption in rorioMS bone 
disorder So 



Serial Noc _mmi*U5G 



Issdividjsai Ps-ojeet Report 
Calemdar Year 1959 

Pgr| ___Bc, Honors J, AwordSo and Publicatioas 

Pablicatioiss other than abgtcEcts frcei this jMrojeets 

1) Ls5t«!ako Lo: The ©stiaaties of 7adio@etl?@ csleiM®"=45 by liqtsM 
sciiitillatioffl eo»Kti®@o kmlo Ch®ao 3i:340„ March l9S9o 

2) 8h©don„ Go Oo: l*y@s®at coneepts of the physisslooy of toosis® iss the 
sgiesg hussan: iiiflu®ae@ &f hormonal aad @th@7 factors in cst@ap@Te»8is< 
Procseedissgs of the Fourth Isjtas: national Coagesss of Seifontologyo 
ns6lS-627„ Tipografia Tito Mattioli, Fideazao Xtaly^ Fe&o l959o 

3) Whedoa„ Go Do: Effects of hl^h calcium intakes oa feOBgs^ bleed 
and soft tissaeSo Assa^ieais Xeistittite of Ni3t?itioB S^posium os 
Th« Effects of High Calciea latakeso Fedo Pp©eo<, Ooco: i9S9o 

4) Wh«do£3o Go 53o5 Ost@op©ro8is: atrophy of disas@o Special book 
pu&liestion of res^rch eoafereaee "Boae @g a Tissae" held at th© 
LaekenaH Hospital^ PhiladelphiSo Peuasylvaniao October 30=3io l^SBo 
(in pi-@ss)«, ' 



Serial Noc 



Esi Invest ioj 



2o Metabolic Diseases Byasich 
3o Bethesda 

PHS=WIH 

Iiidi^idraal Projest Report 
Caiefidar Ymr 1959 

Project Title; Total Energy Metabolisa; Studies in Health, a^d Oiseas© 

Psrisjfiipal Investigator; Opo Go Doaald Whedon 

Othar Xm-yestigatOTs: Oirso Eo Ro Bugkirk, Ro H„ Tho®psofi„ Ro Moore/ 

Lo Lutmak and Ao Tashjiafl« Jro 

liai! Years Ccslendgy year 1959) 
Total: 5=2/3 

Pyofossioaai. 3-2/3 
Other; 2 

Project Degcription; 

9h\mtives % lo To establish a techaique of total energy bslarace 
Mhich HBB h@ sppUed to serious clinical probl^ss and to fuitdsseatal 
physiolG^icai pscobli^ss of eaergy sjetaboiiss not nam tsBdersteodo 

2o To study the irsfiusace on tots! eas^gy consuaption and balance 
of c-arious factor So including cliaaate snd the endocrine horaoneSo 

3? To iRvestigate th© characteristics of energy balaae« ssd 
their influence on the nutritioissl state of patients in pertisient 
disease conditions, such as obesity asid cancer o 

Methods Eaplpyed: Indirect hasan ealorisetry by sseans of complete 
contitiisoiis expired air analysis in the Metabolic Chafer « metabolic 
balance detersBinationSc caloric analysis of dietary intake and exeretCc 



Part B inclwded Yes 



Serial No, . SIAH^'IS&C 



Ms.lor FJ Bdtnjgg s lo Change ia coaeept ef s b asis ss^^gjo.!!?. 
pheaoaenoa s Last jBwe*s MetaboHe Chtsailier study of the iisf lueae® 
of cold en^ironeent on the eetebolie effect of food Csp@eifie 
dynacsie affect or S[^) produced such an Ki]@sp«cted flndif^ that 

the @xp3r iiasents were repeated in additional jouag male sttbjeetSo 

using instr^sentatiofi t^ith issp^otred s@nsiti?itya The added studies 
haw® confisrmed the finding first reported by these NIAMD iwestlgstes-s 
that hufflsn beings differ greatly ft^m dogs in the aiiiizatioa of 
thermoge^esis associated s?Sth eatifsg for body heat bslancso The studies 
on dogSt, performed by SebKer diurisg tS5s classic period ef ealorisetry 
in^estigatioWc. had shown that food-irsdHsed thersjogeuesis iy«3ld 
replace cold-induced therisogeaesis sfid snisals fed in the cold 
«90uld be kept froa shiver ingo The cJirrsnt hu^H studies „ oa the 
; other hando hs^e shown sisa^atiorj itf tha ti^o types of thgrEogenesis; 
that froas food did sot replace that frea coldo It will be aecessax-y 
to scdify the stateaents iR sost Physiology textbooks oa cold=S>S 
therso^enesis ^terrelationshipg,, ishich ssiggest that Rubber's work 
is applicsibie to all hosieothersic speciesg sssd isdicat® iiiter»species 
differ enceso 

2 o peli a eatioirs is^ith fidel ity of moBgent°tC'=^goasHt aetabelie 
changes reveal characteristic featsa res of phSROiaeiia ob scured hf 
older methods ; The unique cspecity of the Mstsbolle Chamber's 
iestrusentatio!! for tracing the patterns of ftrndsisentsl physiological 
pheiioseas hae been dessonstrated in this stsdy of the iefluessee of cold 
on SOEo Not aereiy the degree of energy expaidlture but the kalcidoseopl'^. 
fores can be eutliited by the Chamber's systees of coiitiieuous expired 
gas sasapling for @iriissai changes in oxygesi end carbon dioxide eoraeentrstiotfi 
if} eoRjuaetiois f«ith cositirauosis recording of other physiological data sueh 
es body tesperattsre at ^ario'^s sites, heart rate,, etCo Varictas s^thode 
applied in the past to human energy studies have all bee» bssed on 
internal sasaplig^ of expired air ishich totally obscures aomer.t to 
snoaent ssetabolic «%iafi(3®So Althotsgh cyclic vsristion in oxygens cogss»ptio:< 
associated ^ith shivering has been suggested in pre?ioi3s inter^^l 
sai^ling studieSg delineation of setabolie changes with fidelity 
which is provided by the Chassber continuous sasspling proeedtsre hag &ad& 
possible the following observations in studies thtss far: 1) determinstlof! 
of the exact daration of and interval between ^eriows bursts of energy 
expenditi;re associated with shi^eringo 2) recognition of a sustained 
under lyis^g increase in sjetaboiisaln the cold distinct fro® the psr iodic 
peaks associated with gross body shivering„ 3> the 'finding of marked 
inter^individsjal differences in the setabolic response to coldo bolls 
in lag before itsitiation snd in ^agiiltude attained^ 4) definition of 
the duration and totul asoisnt of metabolic chaage associated with 
ingestion of food CSL®)^ and 5) accurate separation of the taetabolie 
responses due to SDE aj5d to cold, and reecgititaon of an altered SE® 
Bietabolic pattern in the cold as coEapared with its form in a eoisfortabxe 
esivirofissnto Coispssrison of eontinssous body tesperatwrs sea snr events 



Serial Nqo ^_,JXI»-lJi5= 
Pa^e 3 

with o:Kygen coRsuaaptioR ©apha sized the large capacity of the foody 

fer thesTKal dsfflpissgo Cyclic cham^es in ojsygesi eofissiEptiosi wesre ml 
reflected by changes in assy of th© asasurcd body tessparatisTsSo 



3o MiSfeHSMlS_i:es]aixaL©osit«a|>!y^^ 
llJ»£i®a.,.oCgSgSMBg.£M,ob^s|Jls It is anticipstsd -= with support 
for this sRticipstion f?o® prelteinasry obsa'TOtiosis ~=° that dsliueiatsoH 
of pattersss of ©etaboHe respons© t® ©K®rcis©o tR sssociatioR with 
biocfseaicai ®©asMT®ssntSe will piroi/ido iBsight ssto ait€arati©ag is* 
asetabolic processes ira states ©f iapaired physicalo eardio^aseBlay 
or metabolic functiGJU Ssueh corabiaed jrespiratory-mistaboiic SRd 
foiochessieal studies in eollaboratioj! with three other Bjstabolic 
reseasrch groispSo to^e been iftitiat©^! in normal subjects &nd in pstlests 
with aoderste to ssarked obesity in at? effort to sppraise the metabolic 
shifts or aitsratloasoccwryis^ in respons© ta ©xerciss aad to caioriis 
restrictiowo As asi exaisipl© of the nm aress bsing investigated in 
Hhat is now an early phase of these dynamic studies issy bs cited ih@ 
elevations noted iis sensa ketone body and isBesterified fatty acid 
levels during the weeks of walking cs:ereis@o These bioehessical ehas^gtBs 
hint that during long periods of coRtisstting aoderate ©xereise there 
aay be set in eotion increases in the proeesses of fat ^obilisatios? 
8nd_ possibly fat aetsboiisKo Studios reported last year by this 
gr'owp sho^wjd the proBounced effects on carbohydrate setsboiSsi® fnm 
prolonged iaactidity„rei!rersible by extended e^erciseo 

^o !fedMicatlgn..andxefiKSa®|^^ 
body water Q One of the eseasm-emejits ®ade during the course of the 
various energy balance studies is total body ^sater,, an iaportasst 
eompOKent of body coapositiojio Although several, ssethoda sre avsilabl® 
for the det0rM£ii3tion of total body watsr, only the dilution procedwrs 
using tritiass^labelsd water appeared sttraeti?^® ftoa the points of 
vie^ of vsiidityo reprodsjeibility snd awilable instrusisntation Cliqaid 
scintillation cetanter)o In order to msq the tritium ©athod it was 
necessary to modify existing proeedsures to separate wsr^esiiiently the 
dilisted tracer from the body fluid aaapleo A ssethod isB^olTing Sow 
teiKperatiiire,, loii? presstyre sublisetion has been adopted and has proven 
quite satisfaetoryo Shell-f roses gsraples of tritiusj=l8lj©led plassia 
or urine are frosen st 26°C and a pressure of 1000 microns of sareMryo 
The subliasts is condensed in a trap at =70°C(, thawedo assd radioasssyed 
in the liquid scintiiistion countsro Care siust be tafeen to ©otsplete" 
the stabliaatiofl process becsias® a serial sassplisg experisent iadicatCfd 
an enrictaeat of the tracer as sublimation progressed to eoapletiorso 
Errors of =iC^ body water eould result if this preesation is not obserwdc 



1 



Page 4 

5' Iffstr HBewtation res,eggch ; Initiated for the pu^posa of a 1 tet I. $»'•;■ 
iadustria! coRtisiawJS flow gas ajsslysers specif ically fsv ietaboHc 
Chamber research (50=fold isicrcssse in seas'iti^itf desired) o efforts 
to iaprove the stability and sensitivity of the ojtygej? sasiys^ ces? 
mofw be coRsidered an iisstrussetitstiois reseavch accomplighsfjssto This 
is aanifested by tfse desirs of two other resesrch groaaps Cttot at MIK) 
to adopt th© circsiit ssodificatioJi now in use isi Metsbolic ChaBbey 
stwdieso Aided by advice fsroE the WAI Laborstory of Techsicai B«?elop- 
faesit„ the oji:yg©n instrument has b©ej! refissed to- the poiist ®t.er® 
0,02% chs.nges is? ojjygsn cojseent't-atiois can be aecjaratsly detected 
in air streams of 100 liters pes* siiR?it4So The carbon dioxide saaSyssr 
«as raodified sosee tm<s ago sfsd has performed gatisfsetotily ov^: the 
past t??o year So b«t steps are mnder Ksy to iesprov© this ceH e?en 
fwrtheTo The staff of t?3© Cha5ab«?r h^ss silso '"dm.'8loped a data hajidlisi^' 
system tc dfflai with the ^'oiusij^iouEg dai.u .siaJ8@?st®d or th© strip^ehart • 
rscordars sjid to facilitate caleMlatiofss? paper tape fvms the system 
mil be fed to the NIH^IBM eoaputsr faciiityo This origlHaUy designed 
system has jwst beess installed and is currer.tly m%A®-c testo 

Sag.?? ififf a JJ!LS, jcg.. ^'^»'^^'^P_?.gg.^£.Sll° Stitdios of hussan ©netgy setsbolissa 
in the Metsbolie Chambsy ars concefsjed wit h a variety of basic prohlesis 
of physiology stid s^etabolisas §sid are thus' directly related to th© 
principal interests of NIa^MO researcho 

The study on specific dynemlc effect and the inflssefice th'Sreaj? 
of cold en^iroRfflent represents im^estigation of a fuisdasssstsl physiologies 
phersoBienoa of ewergy setabolissao A distissct species difference ass 
sho^in on the part of hMsan subjects froa dogs on which the ciassSca:^ 
studies of Rubner had been perfortsed; the differojce revealed in 
this study will require re«risiojs of the cpinicij of physiologists held 
since Rabifjer thst therKogenesis from food iisgestion can readily 
substitute for that fro® cold (shivering) in wssintenance of body heat 
balane@o Respiratory-cjstabolic studies of sistabolic response to eserei''-^* 
it! normal and obese subjects^ with associetad biochemical aeasarsssrits,, 
sjill provide insight istto rsetsbolic processes in various states of 
iaspaired physiceij cardio-wsscwlar or ^stabclie farsctiosio iRstrumentsitiois 
research is proyidiag sodificatioas of respiratory gas analytical 
apparatus Khich ^ill be wsefwl to other inyestSgatm-s in this amd in 
related fieldso 

Propo8ed^,,Co8igs e^of Pr^oieet; Studies will he contisjuad slong 
the principal lines described in this rsportc 



PHS«NIH 
Ijidsvidusl Px-oject Repoirt 
Calendar Year 1959 

£S£L-§,^ Howoirs, Awards^ and Pifblicatious 

Publications othejr than abstracts fro« this projects 

1) J^hedoM, Gc D„ New hn^m\ es^&vqy t&el&holism researcho Jo km-, Oietetic 

2) GraRde, F», S8oaagle« JoEo, Bsiskirk,, E., Ro and TaylOT, H, L„:, Body 
t^jperatwye responses to exefciss in ssh an restricted food and 
water intsk&<, Jo Appu Physiol o 11''^'^', l^S^o 

3) Kreidero M„Bo„ Isepieteo, P, Fo„ BMskiyk„ E.c Ro sRd BssSo f>cEo? 
Effect of continu&es cold exposiss-e on rsoeturaal body t^aperstures 
of K8.«io 3.0 Appio Physaolo M^^^* i'^SQo 

4) IaiJpietro„ PoFo, Goldsian^ R„ ?.., Bu&kiTk, S,e„„ ss?d Bass, DoE„: 
Response of He^o a«2d gjhite wii«s to coldo Js Applo PliysSoic 
145 798. l95«>o 

5) Bass, DoEoo lasapietK-o. PoFo„ aed Buski?k„ EoBc,: Cosp-ar .Isojs of 
basal plasma snd blood volismiss of ^'egto and ^hite esaleso 

Jo Applo Physiol o Hs^OU J^S^' 

6) Goldiaaa„ Ro and Bwskirk, EoR^t A method for underwater i«eig55i«Q 
and the detersinatiott of body densityo Hwiwiis Biology t.Cin press) o 

7) Buskirk, Eo R<. and CoynsilisaK^ JoEo Speciel essercis© problesss 
ira oiddl© age. ^UBt«Jii.Jci©|!C£^B£J^|c|je^0^i££lse^ 
Sjggjrto Jfeyper srtd Broso Cisi press)o 

8) Mooye„ Ro sad Buskir!{„ Eo Ho: Exercise and body flaidso Chajtter^jja, 
§^Pi§B5S.3 A 3 M MM&JSL33^^Sl M M.MAMMl- f^arper and BroT^ cIk pyfe; 

*)) Buskirk, Eo Ro: Underwater weighing^ Uumn Biology „(in press>o 

10) Buskirko Eo Ro: A discjjssior? of problems relatsd to the caloric 

cost of li^iR^o Bulletin of the Ness Yvtk Academy of Medicine a C in pre,:. 



Serial N©., HSAIg)!-l3?C 



lo cubical Ss!"7es«,ig®tio3s • 
2c. MetsbolSe Oiseases Bi.*8!ici 
3o 



PHS=NIH 
Indi^idusi Project. Report 
Calenda? Year 1959 



gagt Ao 



Project Title: Metabolic Effects of Adrenal Cortical Steroids 
Pyiiscipal Xn«f®stigator ; Bto Go Donald Whedon 

tors; Djo Leo Latisak and Dto Arasn Ho Tashji8i5„ 3i\ 



rfift^-T,«vatiB« n«4t« . "^^^^ project coaipleaeists (snd is coopsrstife 

Coopeiratifig Ojiita , with) "THai of New Aati-Rheisaatic Or«f|So" 

MaR Yssrs Ccaleadar year 1959); 
Total: I'-l/a 

Professioaal: 1/3 
i 



Project Dsscriptioji: 



To evaluate the setabolie effects of trarioiis new 

synthetic adr^al'-ccrtical steroids with respect to sodiu®,, potsssiusa 
aad nitrogeci ©xcretioit airjd iss selected instances with respect to 
salciuia and phosphorus balanceo Effective ai!ti=inflaH5^tory action 
does not qualify a new steroid for wide clinical trial in rheusmtoid 
arthritis unless certain ssetsbolic side-effects cars be shmm to fee 
ainor or absento The particularly uKdesirsble effects eest often 
encountered are sodium and t^ater retesfstiong and potassium and raitroges; 

lOSSo 

MetSio dg EiEfilo ved; Under rigid dietary cojitrol short=terw aetaboSic 
studies (sisi weeks) are laade of the effect ef neis syssthetic adrenal 
steroids oh the uriaary excretion of 5Bitrogen„ sodiua awd potassiuie 
and on the blood levels of the latter two eleaentSo When short^tetr® 
studies suggest scceptsMiity of the compourtd with respect to ttse 
setabolissa of these elegaertts,, raore lengthy studies are carried cut i.n 
selected patieats for the long-tera effects of the steroids ob the 
complete setabolic balance of these eleaents and of calciuss and phosphorus; 



Part B included 



No 



Serial Noo ._iMM:?"i3?C 
Page 2 

Rfe^r Findiia^s; Durljig the past year a single 90 day balance 
study oa a patient ^ith active rheijisatoid ax-thritis has hBen 
conducted of the aaetabolic effects of a new synthetic steroid„ 
compound 128, a 6=i-fIt3oro inated coE-tisone cosipotisido This 

effective anti°rfsei!Biatic cospoand cawssd mly a teaiporary diuresis 
of sodius and a modest incrsas® in petassiM® excretion so that, 
if additional studies were to show siisilar ?©stiits, the siuiissal 
degree of electrolytic side ®f facts ?»©ul«3 tend to encourage furthsr 
therapeutic trialso The atrikiag finding with this st®roid„ 
however 4 was redaction in isrinary calciuss^ a ehaisq® KOt previously 
noted with aay adreaal steroid yet subjected to isetabolic balance 
sssayo Cospletios? of balasce analyses are eagsrly anticipated snd 
further studies planned because of the great value which ssoafid 
result if s caIciuffi-=gtoring adrenal steroid should be coBclusi^ely 
identifiedo 

SigKJfJgjt ace to NI AMD „Res,<^rch; This study is cooperative ?«iih 
"l^ial of New ABti=Rh©a®atic DrussT" NIAMD„ and is iaportaat 
prisiarily in isdicating whether effective anti-rheu^tic steroids 
Bay be safely given to patients over coasidersbie periods ©f tiee 
with respect t® metabolic effectSo Of additiosiai isspoirtance is 
the fact that deteraination of the se'tabolic actios ef stsroids 
under study ©ay yield information sshich will give Mseful leads 
to cheaists engaged in the synthesis of variouzs cortisoKss^iike 
steroidso 

PropQ!sed„CeMr,s^e of „Pra{eet,; This project will be continaed 
iijterssittently slostg the present lines as facilities perEit„ !«ith 
particular stress on electrolyte effects of the new steroids assd 
atteRtioR to the long^tera asisieral effects^ particularly of thos© 
cofflpouRds which appear destined for broad cHsjical nsso 



Serial No. ....MMtzlMS^ 

lo Clinical, in^estigatiojis 
2o Metabolic Oiseases Branch 
3o Betbssda 



Individual Project Report 
Calender Year 1950 



Pyyt Ac 



Project Titis: Study of the Ko?Eal and AbBorssal Physiology of the 
Forsed Elasents of the Blood 

Principe 1 Isites tigs tor; Oro FVederiek StohlataiSo Jsfo 

Other Inwestigstors: Noae 

Cooperating Units: Nciis 

Man Ysas'S CCalei3dar year 1959): 
Tctsi; 1 
Professioaal; 2/3 

Othe?: 1/3 

Project Description: 

pbieetlveg; Study of factors contributiRQ to the prm3«ctioR 
aed d@str««Jtian of formed clsjaents of the blood la iiersal arid 
disease ststeso 

- Methods Employed : Aside from routine deterdBinatioR of forasd 
elefflSBts in the peripheral bloody these coasist of ^asiaressesst of 
red cell sisrtrirEl t?ith Cr"'^ sad differentiai agglutinstioESo red eell 
production with Fe59 yptaJcOo 

In additiofflo the ability of the sarreea of patiests mth refractory 
type anemias to respond storEalSy to standard stianlio phl8footoiBy„ 
hypertraiigfusion and steroids is being studiedo 

Assays for ^ythropoietine fvom suitable patients before and 

after ^aricas forcss of treatiseat are being conducted is coajaHCtlor! 
with our basic research projecto 



Part B included 



Serial NOo JSb®jiM£=.-^....==^=«»=.. 
Pags 2 

Major FiKdingg; lo CorsdatiOR of p'lasB?a amd uirinary erythro- 
poietine levels with bose caarrcw erythroid celluiarityo It has been 
established that this relatiotsship holds e^en in thos«s instanees 
in which ther© is a hypercelluiat saarroK which fails to e^Qntaate 

in the delivery of red cellSo 

2o In a study of patients with refractory anemia airad abnormal 
erythropoiesis it has been established that (1) the erythroid elesersts 
are tareiiiiig o'lrer at a noraal to accelerated rate with death of cells 
'isj assrroa, (2) the Rsrre>ti responds ROTaally to physiologic stismlio 

SignificaKce to NIAM D Res earch; Aneffiia is a cogaaon coKplieation 
of arthritis asid certain metabolic diseases and say b® refractory to 
treatsento A better understanding of the regulEtion of erythropoiesis 

is of basic interest and shotsld eventually result in improved therapyc 

Proposed, CoMirse of Project; Investigation of patients with 
polycythesBis vers, anemia associated with rheyesatoid arthritis or 
thalassecsia with respect to the relationship of hypoxia and for a 
postulated red cell feedback in controlling red cell productioHo 

Further study of refractory aneasia and the effectiveness of 
steroids in therapy^ 



Serial No. ,.JS^^d^m^__ 

Part B: Hor9ors„ Awsrds, assd Publications 

Publications other than sbstracts fross this project: 

lo Stohlaian. Fo, Jr^: Erythyopoietineo Pediatrics 23: n35--<?3&, 195^. 

2o Stohlssan, Fo.Jro: Observ^atioess on the physiology of erythropoieti 
and its role in the regulation of red cell prodiactiofio AmrbIs 
of the Ne^ York Acadsasy of Sciesjceo 77; 730=>724, i%*^o 



Serial K©o ..jglffgl-l'^^- 

2o l^etsbelie Diseases Branch 
So Bethj 



PIiS43IH 

X6di?idusl P!fOj«ct R®p©rt 

CaieHdar Year 1959 



Part Ao 



Projeet Titisn: Stady ®f Blood CeagulzstioH end Diseases ©f Hosorrhag© 
stid ThroebosiSo 

Priacipal Investigator: feo N„ Ro Shulsaa 

Other Investigators: IfeSo To Co Bith®ll„ Ao Leitnes- and Rd Astero 

Cooperating Usitg: Dtc John Zo HearoB, Office of B^theratical 

Research; DrSo Ro Ko Shaw „ Jo Do Davidsofte 

Wo Rail, md Esil FtBi^ Cancm- Cheaotherapy 
SQCtion^NCIo 

Man Years Ccffllcndar year I9r)9); 
Tots! : 3»l/4 

-Professional: 1-3/4 
Other : 1-1/2 

Project DescrSptioN: 

Objjc^ivgg,: Studies of the reactions and intoractions of 
coagiElstion factors Jj. j[|tro aad ^ ji|© to dofin© fMrthssr the nature 
of the blood coaf^latioa @@eh@nis!a and tho factors of sigiaificane® 
ia the pathogenesis ®f obseusre coagulation disorders in ordsr to develop 
better a«tbods of elinieal thsrapyo 

Me^hed,g, ,Eaff?.oye^|: Techniques of protein purification and chsraetsa- ia 
tios including plassa fractionation for specific coagulation factors 
and elect? ophoretic and ultraeeittrifugal aaalysiso ERzjmtloqy of 
proteolytic enayjses and their inhibitors Sneluding techniques required 
for refiised kinetic analyses using protsin 'end synthetic s^ibstanceso 
All research techniques for quantitative g^sur^nt of the varices 
coagulation factorso Variofis techniqiass or inorganic and organic 
che@istryo PharBsacolcgic sssd physiologic tochstiques applied ia san and 
aniESslSo 



Part B Jficluded Yds 



Serial Ho. mAm^l^c 
Pag® 2 

S^^MMM." '^^^ coscbiaed ^ork of «»a^ i«v@stig@tors iedieates 
that at Icsst fi«fe aad posgibiy eight or ^or@ diffsrGnt cosgulation 
faetors int^act dyeing the itiitial stages ef bios'd coagislatiosi to 
products throegiioplsstic acti?ity„ the activity ®hich converts prothrosabin 
to tbrosibiao Diseases easised pri^rily by abnori^lities in throfi3l)o» 
plastie activity are the differeut types of hesasphilia aed the 
so--c3lled he^ophilisid stetsso It has been possible to idesstify . 
the £tuaerou$ factors iiavolved is fort^tion of thro^oplastic activity; 
five diff«^efit cosgesiital hecsorrhagic diseases with clotting defects 
related to an abnoreslity in thteaboplastie activity have beee 
attributable to a diff<3s*eflt specific deficiency in each csseo In 
spite of the fact that sany factors hsvs been iaplicated is the 
foTEiation of throaboptlastic activity, there is rmarkably little 
iiifoTisation cotscerrtiisg the bioehesiistry of throssboplastisi foroaticii: 
©y its activitya For isistaRceo it is aot kns«»!t «fhet!jer the various 
factors act in sequential ensyeiatic steps or cotabine stoichiosaetricslly 
to for® throsaboplastlHo whether throabeplastin acts only esizyssaticaliy 
on prothr^^ia or eossbistes «ith it stoiehiosaetrically as ^@11„ or 
whether the 8o=saned "deficiescies" of throabsplsstic swbgtaases 
represent a trtse lack or the preseace of abnorsial antagonistSo Res^rch 
directed at these problems has led to the follosfismg fimdiags dsaring t^s 
past year: 

So Following our finding (see 1958 report) that two of the factors 
involved in thro^oplastin forsatiois, anti^heiaGphilic globulin iM^) 
and Factor V, coald fe® irreversibly inactivated in vitro by agents 

which stroBQly &ind caleius (bo^o «thyleMedia®iB©=^tr8<=>acetic acid 
<E0rA))„ i^e have sho^n that it is possible to sake anifsals artificaily 
deficient in these factors by exchange transfMsioB with blood treated 
with EDIA,> This has persitted for the first tisi© an e^aiaation of 
the turnover rst@^s of these factoirs in "noresl" snimlSo the only 
previmss infortsstioa on the half-life of AHG and Factor V has hmn 
obtained by tseaswritsg the sur viral of these sateriels in eossgenitally 
d«fiei®Bt petieats; bat the raises obtained have been eacesftaiBo for 
ex«Bple» bec8us« it has h®m impossible to assess ths effects ef 
dlfftisioi) of these factors stto e^travaseslar si^ceso By comparing 
resslts- we obtained in ani^ls having acutely-^indueed deficlesacy ststes 
with results w® obtained in patients having eonganits! deficiency 
statsso it was feaisd that the half»life of these factors (approxisKitely 
8 hoars) was sieailar to the rate at which they coald be returned to 
the cireulatioa in noreal eniealso indicating so far that the turnover 
rate of these siabstances caa be extreaBsJy rapid even in aorBal snisffllso 
StQdies of the sinrvival of two d^.er factors involved in thronboplastin 
foreEation<, plasjaa throsijoplastin eoaponent (PTC) and Factor VIX^ in 



Page 3 

coageeitanf«<Sefi©i®at pati^stSc shewed that the half°lif© ©f these 
gabsteraces tos also isf th@ otdte of- 8 HokySo The issplieations are 
that all of the materials iti^roi^sd in thro^oplsstin fortsation are 
rapidly utilized Jjl llM ^egatdlsss of their ia jf||£o stability; 
for A!S sKd Fsetor ¥ ar® ©xtr«^iy labile Jn XlJS£ s««5 cosisia®© 
rapidly whm blood clots„ sshss-esa PTC ®nd Factor VII are ©:Ktr©e®ly 
stabi® iai^SE ^^"^ pr©«©at ia as high corac^tration ia serea as plasess. 
It is iiit«r®stisg that all fosr of these throsboplastic factors ha^e 
®«^ JjL ligo 'tarnover rat® approxi^'tely B to 12 ti^^s faster than the 
traraovsr ret® ©f clotting faetoits ROt in'^olVsd in thresbopiastiM 
activity <8ogoo protteoa^iss and fili'riffiogeffl)^ Further- studies of 
this typ® -proaig® to pro^id© the type af ifsforfsation which is 
nscesssry ia order to d®^olsp b©tt®r methods of tr©stiigg hesaophilis 
and allied eonditionso and to pro^ldo oluies @@ to tha biochegaicsl 
Qsture of tho initial stages of blood eoagulatios^o 

bo In addition to the theoratScal implications this work has 
provided soss ^©ry practical iaforaiat'ioH eoneeraisg th® use of the 
'aBti=co®§«slsj!t„ S?rA„ in obtsiflipg bloed for trsnsfuaioas irato Humtu 
beingso The findietj that mimls tm be ®ad® aytifieiall'y d@fici©at 
in AIS and Factor ¥ with ESTA blood indicates thst EISTA should ml hs 
used to obtain blood for patlewts ^ith AW or Factor V def Jciescy 
or for j^tSessts who will r©e@i9® aaassire transfssiata (swh as heart 
punp cases) b@eaiss« in the forsss- instances th® blood collected in 
EDTA would fiot correct th® deficiency and ia the latter isstsnce the 
transfused blood oosald ii^duce s s^io^s hea^orrha^iic £t@t@o 

Co St®!3ffli;9s frois our observations ism 1958 report) that caleitas: 
is mm ic»t.®grsil part of the A!3S nsd Factor V imlBCUles„ vie ha?® been 
i^ssisri»g the ealcius eoistosBt of differ dat pitsssa fractions in order 
to det«9r@in® whether it is foasibie to detect the specific clotting 
factors osi ths basis of their ealeiusi' cos9t@nto Ths aethodology of 
plasm f^setioisatiois sssd esiero ddtarsination of esleiue has be@n 
worlred m% and prsliQinary results indicat® that, it ssay h@ f®Qslbl@ 
to detect specific deficiencies of clotting factors by calcia® deterMtsjtioa 
slen© and possibly folios ehaages in thedistribwtioa ©f pUnm caleisa 
in different protein fraetions dtsring blood ct^swlstioEo These studies 
my prove to be helpful in relating ifaolecular strQCtnre to function 
of clotti.^ factors and in detereaining the nattsre and sequence of 
bioch^aical reactions which take place during the forsstion of throsab©° 
plastiQo 



Page 4 

2o Cliaieal g^dies of itauyttal eoafl^Iatios diacardgrgo 

So AlthoM0h the hslf^lif© of Factor Vlt !n ss eosgeaitally 
deficient patient sas found to be only 8 hoars (see section loSo 
above) it has be«g found that b(»)efits froa sdainisteriag plas^ to 
sach a pstieat last tov 3 to 4 day$ after all traces of the adaiaistered 
factor have disappeared fre« the eirettiatioe by in ^,i%vp, testSo 
Aithoagh ere do tsot uradar stand the aeanlng of this^ «?e have bees able 
to Baietaia prophylactic therapy by weekly iafMsions of plas^ in a 
pstiesit «ho otboTKise ia^ariably bleedSo sad th^efore have iRdicatiosss 
that conceatrates .©£ Fscter ¥11 my provide practical affiinteasssee 
therapy for gussh patasMtSo Because Factor VII asd prothroabiss are 
both reduced by dieuaarol tfeerspyo th^r'e have been suggestions that 
these ti«9 factors Bay be dsrivatif'es of the game preeursoro It kbs 
iateresting that ssssive doses of vitaslQ Kj did Rot prcdace impro^e^ 
aeot ia our Factor VII dsficiesst pati«xt in contrast to its therapeatic 
effectiveness in coHgenStsl prothrostiin deficiencyo 

ho The ©eds of inheritasce of Factor VII deficiency was studied 
by snrveying the imily of a Ha^ajo Xiidian patient with this disease 
nhish necessitated a field trip to collect blood sssples and obtain 
a carefal faaily protocol on the Arizona Reservstiono (Hsr finding 
that this disease is fion^eex linked and dosinant isith variable penetrsnc3 
is ia agreesent «ith the one other genetic study of congenital Factor 
VII deficiencyo 

Co Study of sev^al patients nho de^elpped unususl h@s»rrhagie 
eanifestations ^hile en dicKearol drugs, although adequately controlled 
as deterained by prothroEsbin tiae valtsesg shosed that they had« in 
addition to the u^al Factor VII and prothrombin defici«icye s defieiestey 
of PTC and as abnoreality in the thromboplastin geoeration test »hich 
suggested the lack of snaSditioaal factor as wello There are 
conflicting reports iss th@ lit^ratnro ceacerning the factors ^liieh asf 
oecasioeally beeoae deficient durissg dicussarol th^apy<, O&s sttsdies 
indicate that PTC as well as Factor X are affected and that these 
factors reisaia depressed long after proth70si)iD and Factor VII returss to 
noraal aft^ discontinuing dieus£srolo 

do We have been intensiirely investigating an unusual coagKlatioi* 
abneraality which has proven to be an acquired complete ABG deficiency 
without an anti-^AIS antibody in an elderly f&isale patient ^ho has nc 
detectsbl® underlying diseaseo Since all other cases of AIS? deficiency 
occur eithss- congeiiitslly ia sales or in feasles who have developed 
an antibody against the factor following {nregasncy or in association 
with lupus erytheetstosuso the clinical and laboratory inforsation 
obtained oa this patieat asy lead to a further uaderstandi^ of the 70l@ 
of AK5 in blood eos^Klatioa and possibly provide inforsation concerning 
the eentrel of plasaa levels of this factoro 



Ps!ie 5 

@o Is: th@ coMrss of o^aluatiffig ams chasotherapeutic sigents,, 
the Casjeea- Ch^jotherapi- group of the NCI found that th® drug 4-iasino- 
pyraaolo-pysriaidiae (4APP) produced aarked psrolongstlon of the pr©- 
thr©ia>iB tim in tr©st®d patisntSo Our Sttvostleations of this 
abtiorsHslitjr shewed that 4APP prodjscsd an scute transient drop In 
pTothrosi)iR„ Faet€ir V,aeid Factor V!l coisc@ntrstions »hich conM not 
be pr©tf©!it®d bf raassi^e d©s©« of vitasis! Ki aad which comld h® 
attrlbutsd to hepotoeeilQlsr d^sa.g®o Apa?t ftes establishing th@ 
precise sjatare of the toxic ©ffsct of this drMg^ these obserratioRs 
are of research interest because 4APP saj prove to be aa excel l©nt 
agent for prodaeiag csBtrolied specific deficiencies in laboratotry 
anliealSo 

fo The high incidesice ®f throiaboessbolic cc^pllations Ib 
patients reeei^iag stm-oid therapy hs9e beea attributed by gosse' 
investigators to an elevated level of Factor VII<, Osissg a n^u aote 
scRsiti?© teshjsiqtie which we demised for seasjsrissg Factor ¥11 w© 
foMssd that Factor VII reasained perfectly Horsal ss did all other 
ki^o^S! ccagisl^tion factors e@ ^fl! as platelets io patients asid eositrol 
subjects ql^&R hi^h doses of steroid horeoaeo Although i«@ ha^eist 
yet foMHd the reasos for the apparent hyper cosguable state, we know 
that previous explsMtions offered are anteflsbleo 

3o. KiagMs„.E3l«^|®s..oLJK^jEMI^ ^>«ea prothrosbist 

is traissforsesd int© throsibiffl by biological activators there is little 
if aisy change lis its physical propertiesg for so-ealled biothrosbis? 
.has practically the sssse molecular tjeight and electrophoretic sobiiity 
as prothro^Snc. Tha:"©fors„ it hadJSJt beejs possible to eoB^lade fro® 
physicoffsh^ical stadias that traasforssatiosi of prothrossfoin into 
biothrosabin issTOlves forratiors of other prothrombin deri^ati^eso, The 
kinetic stadies done in ossociatioi^ with Dto J, Zo Hearon, tshich 
d^oisstrated the forssatiosi of s^eral prothrofisbin t^efi^atives during the 
conversion of prothrosBhin to throesbin (s®s 1<?50 r sport) « hate beew 
extesded aad refined darisg the past yasrp Tb® cwabined esperisssstal 
and Eiathessatieal analysis of the parothro^iti conv-«9-sio^ systess sM 
ite iahibition hf proteolytic e^ayae inhibitors has resulted i» the 
folle^is^ basic ssod@l for the r©eetio»s; 

ProtkroBsbiffl --^ Darirotiw©^-^ DeriTOtis-©^—-:*'^ Thr<3®bie? 
. Inert SosspoMsd k4 | + Inhibitor 

Disri^tlv^Iahibitor CoMplex 

Oth«ir dwi^'atlires cmU possibly ferei feefer® D®ri(insitiv©^ foraso, 
Sat© ceastaats kto 5u„ and k- mxf sifith the coacsatratioes of eoB° 
versioQ factors wheressii k^ Ss the Keashaagiag rat® coaataat of cosplm 
fo^esstiosso ' Th« kis!@ti© details of thSa sMiqa® fora of ©o®p«tStiv® 
isjhibitica will b« deserib®^ in SSto Hesrora's reporto The fact that skcJ-; 



Page 6 

derifrs%i9e« fora accounts i>r a number of the puzsling attributes of 
!(iroth70ie£>iQ con^srsioB in biological sysXims »hich hitherto ftare 

djcplicable osly by esss^ing that certain stoiehioiisetTic steps t$€r® 
involved rrath©? thea that they wrare parely eaayTsatie reactiosiSo The 
iiapliestio»s are that the s«?@?sl prottecsbiis derivatives mif hs^e 
sepstMt® bioehi^ical aitd physioli^ie fraactioiss',, 

BsalllMSCSllSLaiaiOM-^M'. ^o '2'h© types of M M&£L a^sd 
is. y,!,?^ eosg^ilstioEi stadies b@isg don43 rap^esofit a fandssesttal 
approach to the nsid^srststidlng of '^@ Esatacs of diseaises of h@@os:rha^@ 
and thrcaabosiso These diseases comprise a ^jor segneat of hsssitologie 
disoyderso which hay© hm& a categorical i^tsrest of NIAMDo Farther 
ptrogr^ss in diagssosis asd treataisnt of a euabar of hssorrhisgie diseases 
dopetsds on undarstsnding the fistur® of th@ iietab@iisi@ and isiteractioti 
of varioas coagulation faetorsa Such studies st@ appropriate to 
this XastitutQ aad hatr® direct bsarisg oe ge»@ral problems coacerniRg 
the c8otabolis@ of physiologically active p70t@ii»o 

2o Studies ©f th© ©ff®cts of proteolytic esssyses a»d their 
inhibitors ^n prothro^i^ to throsi>i£! con^srsioi: ha^e coatinued to 
deesuiastrate ths great ^alue of eollaberstiv® ^thearaticsl analysiso 
The eosielusioQg reached hme resialted from s eocBbised experiss^atal 
Slid aathegsatical ssalysis in ^hich s^the^sstice has not only provefs 
the validity of the (jorkisn^ hypothesis dt^wn ftt^ laboratory invrnti^^ties^^ 
but has provided additioeisi co»elt!sicf3s nhich have been eostfiri&ed 
exp^iffi@»tailyo The sath^tieai -analysis has also suggi^stsd ^mi leads 
fosc fisrther r ©search o 

3o Isi the studies of calciu@°4:i£!di!ig iagents in relation to 
©osgulatioR factorso the deaoMstraticis thsst at least two of the ®ost 
labile eoagtalation factors eontsis! caleitia as sr integral coapenesat 
suggests that farther studies directed at relatisg sioleeular structure 
to f»iictio^ 0ay prove valisable in uesraveliiig soto of the ccapleKitiss 
of blood ess^JslatioiJi The prodactioa of specific deficiencies of blood 
coagulation factors la lIlS. i^ laboratory anisials asiag Na2S0TA as 
an inactivating agent ??ill fscilitat® e2q}cariie«!3tel analysis of the 
pathogenesis aad therapy of ■ th© siatMrally-^ccHrring disordsarso The 
ttsrnovm- rates which have hmn established for the sevarial clotting 
faetors studied not eaiy provide a useful practical guide i» clinical 
therspyo but also have provided leads for further investigation of th© 
bioehceaical pvoeessee lisvolved i» th® initial stages of blood eostgulatio^o 

4o Consjsitation m coagalstion problems throKghout the CiiUlesi 
Center has provided ap'scialissd clinical and laboratory ssssrvic© is 
an isportant area of medicine and has furnished s nuaber of interesting 
cases for hessatolcgie research stisdy as neUo 

f&mMml^mMMIm^^^ St^m of the &ioche®ical m& bio«^- 
pisysiesl ehsraet^istics of the resetioas involved in thre^^eplastic 
foreatioffi and the eonversiosj' ef psrothrooi^ia to tiwt^s^is «viUb® e&atint!^ 
slong the lines indicated ia the p-eseat reperto 



Serial noo .^JS^fel^ 



PHS=i?JIH 
Individual Project Report 

Cal@s3dar Year 1959 

PUXSo Hoaorso AssardSo «>e<? PHfel. Saaions 

Pwbiieatioas ©th®' t^5al8 abstracts f^c^ this project; 

lo Shaw, Ro SCo, ShalasBo ^o R^o Dsvidsoa^ Jo Doa RsUo ''^o a«^ fT@i^ E^ 
StMdies with th© esqjsriiMiBtel anti»t«a®er agesit 4=^Ki!Wijyrazol@» 
pSrisaMitiSo Cbrcsv <isj pr@ss)o 



lo Clinical Investi^stioit ' 
2p Metabolic Diseases Sra^eh 
3o B@th9Sda 

PHS-NIH 

iQdi^idtial Pt eject Report 

Caleadsr Year 1959 

Project Title: Study of the Icgi^BOlogy of Blood Cell Deficiencies o 

PriHcipal Investigator: D?oNo Ro ShuicaB 

Other iB^estigffitorg: Drso Ro AsteiTo Ao Leitiser and To Bithell 

Coope7atin«| Oaits ; Bto John fiarr is^ Cleveland Metropolitass Genera! 

Hospital 

^is Y^rs Ccsieadsr ymr 1*?S9): 

Total s 3=1/^ 

Professioaal: 3=3/4 
Other ; 1-1/2 

Project Dsseriptioa; 



sees tfhSeh at 



To study the pathogeaggis asd feioehesiistry of iosBsiaolofie 
diseases nhieh are estased by sitstibodies forised against aotologotig bleed 
eell3„ assd to determine the sigtsfieance of this type of isaBinity in 
idiopathic blood c®ll deficieaey stateso Of. spscial iaterest are th« 
bioeheaiical rsactioas which mmit in foraaatioB of coespleses betRIC?^ 
cells, aistibodies and drug hapt@a@8o asid the physiolot^ie processes 
which result ia seqsseetrstiosa of cells ^ith attached antibodies » 



M^t^hodiB .Eaaloyed; T©chisiqK®s of qtiantitativ© iomBOchesistry 
inelading preparation and phygieo°eh€^ieal eharseterisation of purified 
asstibodieso sicro««t:alyses f&t Ritrc^eao histasiHeo ^^^ alkaloid dra^So 
precise aessure^ests of eoaple@05t fixatiosio and qsaatitative ae8stire=- 
aents of celltslar agglstiaatioa and lysis o Methods of p?evo!cing antibody 
responses in aan and anie^ilSo ssethods of separating specific cell typss 
froa nhole bloodo electrophoresis, isotope taggiisg techniqceso ssd 
Coon's flt]oresce«t tsg^ii^ tschaiq^es are usedo 



Part B included 



Yes 



Serial No, J|^14^___„ 
Page 2 

Mai Of Findings; "Ayto-iiassiiaity" Cantibodise forced in an 
individual which react ^ith the individual's mn tissues) has bee» 

iraplicate<! milk increasing frequencj^ as the basis of diseases 
involving cellular d@striJcti&no So»e of the most incisive examples 
are hegistologic diseases in which a single iyp@ cf cireulating blood 
cell is destroyed by a specific antibody which appears to react with 
one particular cellular antigeno Althra^h these hematologic itt^ine 
diseases are relatively well defined, th^© are a nu^er of sajor 
questions which have not yet been answered o For instance, do antibodies 
really dsvelop againist substances which have always been present 
in the individual; do so@e antibodies forced against trtsly foreign 
antigens attach to eellSo not by forsing a specific antiigen^'aetib^sJy 
coaplexg hnt by a aore fortitions process of non-specific adsorption 
on a receptive cell surface; or indeed are certain soeatic antigens 
essentially foreign to antibody^foraing tissues? 0«r studies of 
heaatclogic auto->ie^ne diseases have been directed at anst^ering 
these and siesilsr qufsticns. 



1» CoBpqr i ?ori..j£antibo^i.Te§etio||^^ 
ajslJratq thr^o^peytojenJcj^grHa'' Folio^sing our finding (see l%n 
report) that the coaplesc reactions ^hich take place bet^se^i q^inidine. 



antibody^ platelets, and eospleaent in qisinidin^ thro^oeytopenic 
purpura are the saete as the reactions ^hieh taks pises betttseen stiboph©£i„ 
antibody^ red cells, end coapleaient in stibophen h@@olytie aneeiia^ 
we have continued work with the rare antibody induced by stibophen 
in an atteapt to resolve the question of antigen specificityo Beeauge 
we have had a very litaited supply of seruffi containing antibody at 
lOT^ H concentration or less, these studies have required de^'elopissent 
of methods for jgieas^ring extr^aely Ion concentrations of estechoi 
Cstibbphen and sodiaa catechol distslfonate) by sp^etrophotdaaetric, 
spectrophotofluoro^etric, and isotopic labeling techniques eonbined 
^ith iffi@uno»electrophoresis for application in detersinlng the kinetics 
of antibody complex forsations Results so far indicate that the first 
8t©p of the ©ver-^ll reaction "Khich resMlts in sn antib®dy"»drag-een<^ 
cosfiple®a8t co®plex is the attschKeat of drBg to ssttibodyb- This is 
an important finding, for if the first step of cosepl®:^' fer^tion 
is coAination of antibody with drag rastber than cell with drrag„ 
the iisplications are that the cell°Mirug complex is not the antigen 
but that the' antibody»drug co!^1®k msj h® non«>sp©cifie&lly adsorbed 
on cell (Bissiibrasies just as other tson«antibody plasma proteiE^s are 
adsorbedo 

Another very interesting finding pursuant to cur observatioK 
that hunan but not guinea pig eoisploisent is fixed by stibophe-n^ 
antibody^cell coepSexes «»as that only the second coaponent of ce«pl©«@nt 
is lixi^ in the reactiono Cospleasent components 1, 3 and 4 ares^t 
fiKsd and not involved in the hsaolyigis prodwced by this particalsr 



Pag© o 

» 

aiitibody^ The fi:Katio0 of a single cossplsasat cospoHssit (C g) by 
a heisolytic antibody is s wniqTO reaction in iRBsmnology^ and contifstscci 
study of this reaction promises to provide further iaforraation concsrh ■ 
isig the chealsty and sigtsificssnce of the diffwest cospleasent 

cosspojjentSo 

Other inforisstion obtained is» this study was that 1b the order 
of iOO ®ol©cales of stib©ph©i? ©sstibedy par cell are necesssary for 
cellalar agQl«ti!3ation<» that i„000 or l©ss antib«>dy solecwlss 
attached with cospi^ent per e©ll are necessary for heasolysis; 
and that @ttachs!@nt of smll sesoeBts of co@pI@^fnt (too littla 
to causa hemolysis) ps-evants the c«lls fro® sgglatSRating 
ev@n iti the presence of large amounts of antibodyo, Farthar studies 
of this type will pre^rido issf oration cosic®rniBQ the physiological 
significance of agglatiaating versus coaplesent'-fixifi^ antibody 
cosspl@KeSo 

ll^gMmgPrio" ,of the pja^<Bi^^;jfeiH,jo^@|T^MIL£M.c^lJl° !» ««? 
1950 report we prssented details of studies of an nssusraal fora of 
autosensitivity ia ^hieh a ninute asouiit of the patient's own red 
cells produced large painful ecchyaoses ^hes^ extrairasated into the 
skiflo We fotiitd that these lesions, which wsr© produced by as little 
as 6 aicrofrafias of red cell stroBsa, coMld be precisely dKplieated 
by intradsrffisl issjectioas of as little ss 1 fisicrograea of histatsine 
er by iajectioji of aisy agent which released skin histaeine (such 
as basic ssaiaes or trypsin) » The eonclHsioas were that scchyasoseg 
were aediated hj histamine released as the ressslt of aw antigen^ 
antibody reaction oeci;srrijng intrsderiaally^j Since then w© hatr® had 
opportufflity to study another patient with a sissilat dinorder in whofa 
large painful ecchyaoses were produced by intraderssl injections of 
as little ss 2 sjicrograss of red cell strooao BoMevere this patient 
did not develop ecchyaoses when histsisaine or histassine»rel@asin@ 
agents were substitKted for red cellso Various atteaspts to actually 
aeasure antig@n°antibody cosibinatioa in both cases by the seost 
seniBitive biological assay techEaiqnes available have be@f! i»nsuceessf»lc 
Becanse fissed tissue antibodies do not lend theaselves readily to 
|n vitrt j t analysis, further studies of epectrua of SBaaifoststions 
which are present or can be provoked in patients with autoerythrocyt® 
sensitisatioR siay shed some li^jht on the nature of these obscure 
antibodies asd their effects on vascular persseabilityo 

3- Mog§lhi<| ^hroabo^YlojegsJi? .purpsrjsJIll, Although soase 
investigators have reported and continue te report that the usual 
eases of ITP have in their s<srm a platelet agglutinin and that this 
agglutinin i» of diagnostic and profnostic value, our studies on 
30 ITP patiaits so far indicate that the incidence of circulating 
platelet agglistiain in ITP is not ssors frequent than it is in any 
group of pati€mt8 who have received transfusi©E«„ that the presese© 
•f platelet ogglwtiains pea- s'e has no bearing on the level of the piEtelet 



Page 4 

count or resposse to therap^r in XTP, and that e?©n aa occasional 
norasl individual say ha7@ 8 platelet sgglutinitio. We ha«1 slreadi' 
shown in drug pntpurs that tkroehocytopenia esc occur »hen the 
antibody concentration t«as too low to cause platelet agQltstination 
or coaaplesent fixation, the cospleaent fixation test for that 
particular antibody being ten tisses sore sensitive than platelet 
agglutination test (see 1*>5B report) o f?e ha^e continued our 
attempts to deatonstrate an antibody in the usual cases of XTP 
by coapleaent fixation techniques, so far without successo 

4o Egtablifhaent of a .nea sYndroa^^ Our studies of 2 pati «sit3 
with OR unasaal fors of IIP ha^e periaitted us to differentiate their 
disease free all other types of IIP and to define a new syndroaeo 
Both patiessts were asiddle-aged females who had sssdden onset of 
fulsinsting purpura associated with a coaplete absertce of platelets 
approximately 6 days after being transfused during an opsrstive 
procedure (gastrectoay and lysis of stenosed aitral val^e respectively) o 
Both «ere found to ha?e a plassa antibody shieh fixed cosplesent 
mth, agglutinated^ and lysed all norsal platelets and inhibited 
clot retraction of aerssal blood. Both patients i^nifested severe 
hypotensive reactions to oors^I blood adainistsred during the height 
of the diseaseo 

Because he>sorrhagic ^nifestations in one patient '^ere life 
tteeateaing and steroid therapy was ineffectiyej, splenectoay (effsctire 
treatBient in usual cases of IW) had to be coiasideredo However „ 
with evidence that platelets were being destroyed in the circulation 
by a cosp lesent^fixing antibody^ it mm decided that splenectoay would 
not be beneficisl, but that retsoval of antibody by exchange transfusion 
Bight effect sore rapid recovery providing the unknown stisaulus 
for antibody ms a transient oneo Therapeutic results of a 90^ exchange 
transfusion were better than anticipatedo Hemorrhage stopped coopleteiy 
before the- exchange was over, platelets rose rapidly after the exchange 
to norsal levels within 2 days and the patient ressained wello During 
the exchange acre antibody was reaoved than could be accounted for 
by dilution alone^and this along with a fall in plassa eoKplee^nt 
and a rebound of antibody titer during the first poat°treat«ent 
day suggested that treataent v^s unusually effective because antibcdy 
had also been sequestered in vivo after ettachisent to transfused 
plateletso 

The second patient hsd less severe purpura and am initial 
antibody level approxisaately l/lO that of the first patiento She 
was sjsnaged cons^vatively without splene,etosy„ and over a period of 
three weelcs her antibody disappeared and platelets returned to norisealo 



Pag® 5 



Thess patients sere aot only Einique is hmin^ a eoKplesBesst- 
fisisg^ »os«>drug«{!ep«n4a»t 8Bti°platlet antibody (which has 5i«?er 
bden described before) and ths saae urus^s! clinical ^nif@st8tieas„ 
but also ii9 shewing a teisarksble peculiarity after vecov@tfo TH@ 
platelets which returned in both patiesits »;oiild not react isith the 
antibody of either case in spit9 of the fact that all norsal hvimn 
platelets (30 different indi^idsials so far) and the platelets 
froa 10 different anis^ls react with the antibodiesio 

Further studies ha^e bean sised at trying to differentiate 
the two Bsajor possibilities tSiat the recovery" platelets are coated 
with so©e substance (blocking antibody or otherwise) which prevents 



Continued work on this unusual tjR^oeie proaises to add to our 
knovsl^dge of auto-ieesunityo 

heaoalobjit^wria o A child who had the presenting sysptoas of dsvk urine 
after eiqposure to cold was foussd to ha?« s Donath liindsteiner Hessolysin 
(KLH) with BO other associated disease proeesso This hemolysin dis=-- 
spp<3ared gradually o^sr a one-^onth parriod after which a high=titer 
cold agglutinin developed and subsequently declin@d° These observations 
are of interest because thar$ is only one other reported case of 
idiopathic DLH in a child and no previous doeussntaticn of the decay 
rate of DLll or its being followed by developaent of a high tit^ cold 
agglutinino Information of this type say help clarify the nature 
of the K.H and cold agglutiniR„ two exaasples of clear -cut auto=antibodies 
which are coapletoly obscure as to etiology and physiologic significancG 

S|fiaiil£iJ!£aJi2JiMOsa^£S!Lo "T^e studies of drug^epsndent 
and idiopathic coeplesent-fixing antibodies have led to a clearer 
understanding of the basic ifisaunoreactions which result in cellular 
destvuctlon |^ yiyo and have provided explicit inforeatien cojicffirning 
the significant factors t$hich cause cellular datsage ia diseases of 
sensitivltyo These studies have nusterous iaspliestions in the gsn^al 
field of .ijsfiunelcgy and hare bearing on a large group of diseaiseg 
of suspected sensitivity <0ogo, rh^u^toid arthritis, collage;^ 
diseases, nephritis, etCo) in general B@ilicineo 

The finding for the first tisse that IIP can be caused by a ccssple- 
aent°fi}ting antibody is of special significance bec^iuse up to now 
there has been no proof that ITP iz an is^usiologic dieeaseo The 
establishaont of s am throsiboeytopenie syndrooe will help to clarify 
the pathogenesis of an obscure group of diseases and provid® a rationale 
for further esperiaental approaches to effective therspyo 



Pag© 6 "■ - — — 

fsmH^^SmSM-MMMi^^i ^ntthm studies of tfs© hioohmistxy 
i^SMSioch«®istr3?„ kitsetics, aud plsysiological sisiuificaBs® of iwMM©- 
ffeaetloRs «shich are cUwieally sigraificsiit ^ill b® corstiM©d along 
lines indicated in th® pireseist repert. 



serial N3o ^iM®il^^.^-«^ 

iKdiirldMal Project Report 
CsSeadsT Ymv 1950 

PgrJLBo Ho«oyg„ Awards, and Publications 

Publications other than abstracts fr©® this project: 

io ShulEsn, No Ro, Clinical iaplication^ of e quaistitati?© study of the 
M yi^g.o ss3d |£ yitfo reactions of sfi antibody responsible for 
throsabocytopenic ptirpura^ II Pensiero Scisistifico, in pyesso 

2o Carpanter, R. Mo, Jeaden, D, Jo. Shalaaa, No Ro, Tar^ffla, JoSo, 

Toxicology of a Triaryl Phosphate Oilo 1= Essperiaental To5:icology„ 
AoM Ao Archo Indust, Health,, 20:234 (l9S9)o 



i. Ciiaical Xnves&igaeioss 
i'HS-MIH 

^aa Yews (calssaiste' yew 1959) s Bstiese I^s Cealftadaa? y«a3f 1959) s 

Totals 3 1/2 
Professiomds 3 
:s 1/2 



FEoJact Cescs'ipeiOEsis 

K^^k has cantlvsmd &m the idestif ic®ti^ .^ad ehssaetasizsitlom oi a 
p@sticalM.)& ioOaparotela, This jK^otQiQ «[^ch esa bs Sotsnd isi irelMively 
small s»BuQt in mmal th^xoid iciseua ia tsiaistsallj^ atoadsae in c«^£:ain 
tses^pi^n&alsl,® irafe thjsoid etsso^s. It; lias mn? ^eea shotm th&t this 
pasrticuUse ioda^^oi^eln c^ to Isoiaeed fey a tsm eeeboique of difSesmtiai 
c^68-i£uaMlO!S3 iss etose fiirsseiffiffis. In Shis Ceehnique th« suviaisisg ImeSuKi: 
s^otor is t!3®d €iid @ ^i£dl $sm)mt &t tteeag^exiat® £a 0.88M antexosa is lAjssr&d 
oato Im^ vohssm of 0,92^ suoroaeo ^ItipSe spins are laada dssd the 
aadioexafis eollseted ®td aussAjmd. USilii^ticm ®£ ^is feecSmique fer 
isolation of uricoso ccntsiai&e particles of a 7at liv«p hossog«a&t«a ios 
gttva a calcsslatod paorticlo siss of £vaa ,063-0 o^ siienms. IStis 
woll vith vaitaes ^tainfid firom eleetsrora saicsroscopy by l^£ e£ ^? 
og 0o0S-0c2S ©icsroES. 

Fuarth^ studies ifi th® isoi®t«d pesticsslM.» iodopsvteia vera done 

cIutffiMieogsr«i^y on diethylcssiaoethyi coiSuioseo Hie psotQin isolated was 
hydvolysBd end amino acid mmXfB®B pesfoseondo An excess o£ glutataic seed 
asp^tic acids «Kas found and histidiaa cstd asgiaina could not bo 



identi£i«&do Sedintentation in tlio prep^atioa nlt?aceatsrifuge «m& tsm 
meat o£ I*^^ sho^ffid die 8o£«|killsed paiTticulsta iodopsxitoin to to of 
yelativoly sasall sisse CS«,> < 4S) o 



Part B incXssdsd Yes C I m { ii } 



So 



PliS-iHH 
Individual, Psojeet Mp^si^e 
1959 



Pi^ject Titles Sesr^sn ^ysosiae Sisdixag ?70i:@lQa 

Psiracip^ iMV'Ssti^t&Tt Jacob BiDbbiBs» M»Do 

Otlie^ lesvestigaeossi J« S» Mil, M<.D.» Wc ^»-i&t a&d 3, 

Cfejopesafcing Uaifees l^a B, So BlaB^^» MJ^^t^m (Se^i^ ^. UlC ) 
S33!d l^. Wo Bei«s^jates, S^dVo of Mtchigea^o 

^aa 7@^s Ccal43£^^ ysm: 1959} i f&&i.m& Ds^ (eeloadaor j&m 1339) s 

Toiual; 3 

Prof esslossal s 2 

Fzojfice Sascriptioas 

Ih^e tsas h&m tms^i^ isvestisatlosi of gb® eecteiques uClUfisd for 
idastificAfeioei ef tkf^mija» bissdiag psoe«isso I& hao baea sIuRm shsit 

buSweaa pH'e of a»9a elaessvpbosresis Brvv^as ist addieion to m intor-aliO^ 
thysoxioe bl&dissg psrotols^ sxui ^Imaiua, a psealSitMaia psot:«ia vhieh bltasio 
substaneial quais£itl«s o£ tb^^sozlms, Zn ffixaoaius eaietoaate a pseHssixasj 
•etiisata shows ehst eha eh^sosioa &ii»!l»S capsei&j o£ p3«el!MDia i& ^«»t 
l.SO 1^ of tfayeo3dS8 p«ff stl of sosroa. la coopesatloa with D?. B« S, 
Bluabsts* stu4ie8 cf ^Tsexiss bindlas proteins ia storda gel alectaero^ 
phoresis has &®«8 p^arfosnado Sea his repeat foar dataile. A eolSsiborativ® 
project iaveati^ted by ttroodiaBexuica olactioi^iosrasis ea papa« oad stearch 
gel the coTsespos^daaee batwosa the vs^iotss protaiaa. 



Fuarthatr t^osh has baen <ioae ^tilisiss dialysis systasis fo7 aotic^atisxi 
of thyaroxiaa biadiasc If proteia is preseat oa both sides of a dialysis 
Si8sabira«a» thysoxiaa will dialyas rel&tivoly r^dily« Utilisiag this 
tachniqua, tba affect of varioua b^fas: ieas baa ba®a studied. It has 
been sh£»m that ia the p^aaeaca of serea, betsrbital earkodly iacreaseo th@ 
rate of dialysis of thysoKisBo TarihydsoKy flsBiiao nethaae, borate <B2d 
phosi^iatft do Qot show this effect at a siailair pHo Tbie stroosly e^gfiasts 
that barbital ictoTfosEfs with the associ^ioa of thysoxiae aod at leisat otse 
psrotein is sertoio 

F«?t B included Yes [ J Ko ( x } 



M&itloml studies hev-e hmxi dosta In coili^ir®timj 'U'ith B?. 
Bss±®rBaXt83 of S^i© uaiv€®sitj c£ Michigan.. A imilw ims sfetadiad isfeo 

This ch^actee^lstle «ms fooad la the propealtus eiul cme <»£ tisre® ehlldsrss^o 
X& vms afisocia&Qd vieh <e£stl?@S.y aosaal thy^eid Stmntton mA ^ss^ 

^p?o;£i{a3tely twic® th« 8WB®al vsl^, Sswdies ©f tba kitaafticB og thygtwsins 

ssid mtd o£ dfi$s^lati«3i of sJi^^vosiss a?e govemed % the lavel of irm 
thysoxiae is e«s%^o 



lo Cliaicai Investigations 

2o Clinical Sadocrlnology Branch 

3 o Betheeda 

FES-lim 
Individual Project Report 
Caieodair Year 1959 

f&ict A : 

Project Title; Studies in Carbohydrate Metaboiissa 

Principal lavesfcigator; Stac&oa Segal ^ M. I>, 

Other Investigators: ^IberSa Blair 

Cooperating Unless 

Man Years CCaleadar year 1959); 
Tofcalg 2 
Frofessiocal: I 
OtheK^ 1 

Project Descrlpfcions 

Various Aepeets of Carbohydrat* Metaboii^a Have Been Sewdied 
in ehis laboratory ia tits past yearo 

lo Salicy lat.@_ jffeet s _oa_^«eos® , metabol 1 mi c Uslas the 
rat disphragns technique 8iil|.c]faees bav® beea ^ho«m to tuarkedly 
iacrease the oKidatioa of C*-^ glucose » At the same tiae the 
dru^ stieailatee glycogen breakdkwn m&lnlj in the asrly period 
of incubationo Active sauscle phosphotylase was reduced 85 perceato 
No effect was seen on glucose uptake « 

2 o Factors ■afffecfcia^ galactose^, taataboli^ ia ^^ne In 
collaboration with USo Vale Topper of the Laboratory of Biocheaietry 
substances affecting galactose imetsboliOTi J^ M^S ?rere studies 
ia QOrmal sac and galactoseaic children o I'he hormoae psrogesterone 
has been £"ouad to stiaalats galactose setabolisa ia gaiactosasie 
childreao Ethyl alcohol has an inhibitory effect ©a galactose 
jaetsbolisia in the nossai subject o 

3| , ?atht-?ays of g l'Beose igetabolisa ,1b maao Studying 
6|,ngly C*^ labeled glucose and their rates of conversion t© 
C''"'*02 has eauabled us to construct & biological ajodel of glucose 
raetabolisiH from v^hich the assoimt of glucose being metabolized 



Part B isacluded Yas £TJ Ho /"~37 



Serial Mo<, .^J.;^S^?t^-^ 
Fage - 2 



by fko -cariows pattefays aay be essfcaatedo Cur calcalatioBS 
shew that about 10% of overal3. glucose setaboiiea is carried 
out via the peaSoss piaospfeate pacbsray of glucose Eaesaboiissao 



IML^' Hoaorss Awards^ aad Publications 
Fufelleatioas other tbaia abstracts froa thie projects 



Segal, So, & Albeffta Blair o Effect of salieyaSe oa musele 
pisosphoxylaseo Eatwre mi» ^609^ l®5^o 

Pescfej L, Aoj, Segalg So., & Yo Jo Toppes:, fr®gesearoae 
effects ois galactose aataboli^ in pre-pabe^feal paeiejats 
with congeiaifcal galsc£os«!^is aad ia rasa aiaiaeeined om a 
high galactoss diet: „ J„- GliiSo Inveaeo la press Jaao 1960 » 

Blslr, Ao, & So Segalo TIj® isolatioa of blood glucose as 
poaaseim glucoaaeeo Jo Lab and Clioo Me4o la press June 1%©= 



2o Cliffileal 3iadocrisM>3logy Breach 
3, 



PHS-'HIH 
ladivi^aal Project g/»i^rt 
19S9 



Firoject Titia: ISQl&t«d "OayvoU Calls 

FriEseipfsl X:sE?e&t:ige£@^; Ira 2sstesaa M. 3 c 



a teers CCaleadaar jem I9S9) : 
Totals 1/2 



A GfMj &t e&@ ftt&ctioa of calf fia4 @be®p tO&Tzoid giaads 

asd i>y esrypsli^ s£> dsat dieporsios e£ ehs tissue iai£o 
sisss^e cells t@ke@ placiSo l&es® cells possess th» ability to 
'fossa iodopffoteiias. ISse cliareiiseear of t&sse iod^protelBa is wsv 

iav@e£lgatiG£„ 



la collaboratioa with He, Jasss B. flsM a study of the 
tono^aosphato pathway ia the thycoid s^^sad is ia pxogsresfic 
Ibie pftthway i@ tmder control of thyroid stisBtlatiag 



2mt B iaelodsd Yes fTl Ko £117 



Serial HOo Bliaig)-144C 
Page - 2 



PHS-Hia 
Individual Project Report 
Calendar Year 1959 



ParfcJB; HoaorSa A^jas-d®, and Publicafcioae 

PublJLca&ioQ& other fchan abstracts froa this projects 



Field, Jo Bo 9 Johasoiis, ?,.„ Herriagj Bo» sad Pastaa»- 1= M„ In 
?ltro S£ljBulat£o!% of £he Hejcose Monophosphate Pathway ia Thyroid 
by Thyroid Stimulating Homone„ {la press) „ 



Serial MOu _..y,X.«ff?-l4vC 

ic Clinical Inveseigations 

2 c Clinical Endocrinology Branch 

3 o Bethesda 

Individual Project Report 
Calendar Year 1959 

Part A ; 

Project Title; HisBoral i^ntagonisCs £o Insulin 

Principal Investigators Jamee Be Fieldg Mo Do 

Other Investigators^ Phyllis Johnson «ad 3e££y Rerring 

Cooperating Units: 

Man tears {Calendar year 1959) ; 
Total; 2 1/3 
Professional; 1 
Others 11/3 

Project Descriptions 

The technique utilizing the glueoee uptake by the isolated 
rat hemidiaphrsgBs as & sseasure of insulin activity has been further 
modified so that a significant effect can be obtained with as little 
as 2 K 10'°^ units of insulieo Usisag this procedure e^tenBive studies 
vere done on en insulia-resistant patient who received M,000 units 
o£ insulin/day. In this patient it was possible to desrionstrate a 
high concentration of insulin in her pls3^ three laonthe after her 
last kno^m ingulin injectlono The insulii^ w«s Identified on the 
basis of its in vitro stimulation of glucose uptake and glTSogen 
deposition by the rat hesaldiaphragm and the abolition of this 
effect in the presence of insulin antibodyo Frosi a measure of the 
dieappaarance rates of insulia-I^^^ it was coiiclwded that the insulin 
vms endogenous in origin o VHaen plassaa was fsractlonated by starch 
block electroi^oreelSi, insulin %Fas found in alpha globulin and 
between the p and a globulins o Adipose tieaue fr^a this patient 
appeared to be le@s responsive to insulin than adipose tissue 
obtained from a normal parson and several other diabetic patientSo 

Several more patients with chronic iiisulin resistance were 
studiedo Xnsulln antagonist ^as dsaonst rated in all of thesi and two 
were subsequadtly treated ^ith steroids « Xn osie there wae no change 
in the insulin requir@iient while in the other there was a dramatic 
decrease,. Using the most recant fficdlflcations of the rat haiidiao 
phragm technique it was possible to dtssonstrate a circulating insulin 

Part B included Yes .£^7 ^° £ZJ 



Serial Ho. „JI#^:i45G, 
Fag® - 2 



antagonist in stsam patients with cferouic insulin rssisKance wbea 
previous, less senaieive methods failed to defceet an aafcagoaieto 
Several more patisnts with acromegaly aiid diabeSee were studied, 
but evidesace for am insulin antagoalst was found la oely oneo 

Vreliminarf sttadies wlfch tha rafc diaphsagss technique suggest 
that £ks fasting pla^a Ineultn like activity in ths normal Is 

approsiaately I x 10"^ unitSj, a value essiewhat lower than previously 
reported by others „ 

Studies have also been initiated oa the pathways of 
carbohydrate metaholtmi in endocrine tlssu<s„ In t»o pancreatie 
islet cell adenosaas ££ was possible to doaoastifate the existence 
of the hexose ffionophospisate pathway o This pathway could also be 
desnonstrated in thyroids adrenal j testis j ovary and parathyroid 
glandSo In the thyrdid it was possible to dejaonstrate a stimulatory 
effect of TSH os glucose setabolisa^ especially the hexose saoaophosphste 



Sexiial HOr. ^va»s1£5£: 

Page " 3 

ladividusl Project aeport 
Galeudar tear 1959 

Partes Honors^ Awair<i»s, and Publlcatilons 

Publicatloiss other than abstracts ft<m this projects 

Studies on the circuiaciag inssAllB itthibltor fcmnd in ssme diabeelc 
patients S2£|:tibi£iisg ehxoniG isssulia resistance „ Fleld^ Jo B^. mid 
Woodsoa^ Mo Lc. Jo Cll.a„ Invest „ 3Ss 551 » 1.959, 

On fche Nacisre of the Metabolic DefectCs) ia MabeteSc Fields Jo B. 
Arso Jo Medo 26s 659. 1959„ 

i^ction of predisisoae in iasalin-ireslstaat dtabeteso Oskleyy Wc Q.^ 
field, Jc 3c» Scwton, Go Eo, Rigby, Jo Bo, mad Cualiff®, Ao Co 
Brito Medo Jo Is 1601^ l@59c 

C5bserv.«£ioas CoEceraiisg the Diabetes Kellitws Ass«cia£e«i wifeb 
Werner*® S|mdroseo Field, Jo Bo MetoboU^j, ia press 

In vitro seiawalaKion of else hexose taouopbospbate patbway in 
thyroid by thjxotd ©timMlatiag borfsosiSo Fields Jo Bo^ Johnson^, Po^ 
Herrings B° and Pastang lo Bioch^o aad Biophysical. Resaareb 
CcsmiunicatlonSo la press » 



Serial No.. bt a^, jA&r; 

1^. Clinical Investigations 

2„ Ciiclcal Endocrinology Branch 

3 » Beehesda 

FHS-NIH 
Iisdividual Project, Report 
Calendar Year 1959 

Parfc As 

Project Titles Hvaaan Leucocyte Carbohydrate Me&abollssa 

Principal lavestigator; Arnold So Welaberg^ M„ Do 

Other Inveatigatorsj Batty RerriEg 

Cooperating Uaitss 

Maa Years (Csieadsr year 1959) t 
Total; 11/3 
Professioisal: 1 
Others 1/3 

Project Qescriptloas 

Sisaan leucocjtesg obtained by ve&ipuncture and sedij>aeatatio£3 
of fcbe red blood cells with f ibrioogCBj were incubated ia vifcro 
wieh vajfious plain C** labeled sugars aad feorsaOESSo MeasuraaeaS; 
of glucose uptake, galactose uptake,, prodiictio« of lactic acidg 
aod of C O2 w®5^« dose in norsRal ceatrols, diabetics, gslactos^aicSa 
pareots and siblings of galacfeos^lcS;, and patients being treated 
with corticosteroidso Also, iasullra responaiveaess of leucocytes 
was wessured by detesnaiaing the sfiiaiulation of glucose uptake in 
aoraalSp diabetics sad patients on steroidSo 

Results of these studies to date indicates 

1) Hisnaa leucocytes frcss Qorsal and diabetic subjects 
actively assitsilate glucoseg and tbis process is laarkedly 
stimulated by insulis la conceatratlotis as low as 0,1 ii/aai of 

be so increased productioss of 
Ltb. the effect on glucose uptake .- 



ipcubatioa saedixfiSo Tbere appears to b^ 
C'* O9 or of lactic acid coincident witJ 



2) In coageoital galacteseraia tliere is virtually no oxidation 
of gaIactose-1-C-'^ to C*^02s ccaapared with a very active metabolisa 
of galactose by leucocytes frese KorfisalSo Thus the ensyiaatic defect 
previously docuaeated ia liverg kidney, ssiall intestiaeg lens tissue 
aad red blood cellsj also cata be demonstrated tislng husaan leucocytes o 



Fart B included tea JZZT 



Fagdi 



A tagt has be-;an devised which can be sasi'iy doae ufcllialag either 
whole blood or le»jcocytes iacubated with galactose- I-G^^o To date 
we have used this teat on 5 galactosssnic 'pasienea •ulth strikingly 
positive find.lngSg -syhercas 8 no-jsoala and 2 infaiifcs with milk 
allergy were entirely noraalu 

3) Attsm-^tB are in progsass to a£t®aipfc to Identify fcfee 
carrier state (lieesrosygate) In galacfcos^aia, by utilising the 
above test with whole blood and leucocjtes fr^a the "aoisffial" 
parents and sibliaage of g-alactosssaic ©bildrea,, fo date^ in 2 of 

3 ismtlies stwdied ^e have found a significant decrease la galacfcosise 
oxidation ia bofeh pareafcSg, aad in a sibiiasg in oae faraiiy, which a 
sibling in the 3yd f-amirj? 'a-ae found £© be pexfectly aowaslo Ttiese 
studies are being exteaided at present <> 

4) Finally J w® have beea interested ita Che icflueiaee of 

«orfcicos£eroidffi and groKfeb hoxssonej isj vivo and ig vifcroj oa the 
iH vitro insulin stiraulafcioa of glucose uptake in rshite cells « 
Rssulfcs to date suggest assae effect og fcbis stmialationa bus 
further work needs to be dosae to be sare of the significance of 
the changes o 



Serial JKo- __,_ 

lo Glteicdi Investigations 

2 c CilniCiii Endocrinology Branch 

3 o Bethesda 



PHS-Hll 
Individual ProjscK Report 
Calendar Year 1959 



Part As 



Parojecfc Titles Studies of Labeled PsroSeia MfBtabolifiai aad of Tfeyroid 
Fh.ysiology 

Princi|>al Imrisseigiseor; Charles 0= LewallsSs M„ So 

Ofcher lavestlgafcorss Louis Buisce 

Cooperating Uai£ss 

Maa Years {Caiesadar ysar 1959) s 
Totals 2 
Professional; I 
Others 1 

Frojeet Descsipfcion: 

Methodss Sterile^ pyrogen fraej, eleefcropboreeicaliy 
homogeneous seriasa alb«®iiQ is ©rep-ared by preparative eIec£r©pfeosresiSo 
The psroeeia is labeled with I'^^i ^^^ biological tracer experisaeaes 
performed in pafeiessts as© io expsrjfjaen£dl aQl-mal® uadez- coaditi©as 
of varied eadocrlne afcafcuSo 

131 

Kal is injsKted isitra^aaously iri subjects of varied 

thyroid status aad serial deterEsiiaatioas of Shjroidg plaCTtag 
and ascreted radioactivity are gierfeteed„ 

Coaditi^i^s affeetiiag the iodinatioB yield and th® distribution 
of label in I '"" lodoalbj^ia have been inxrestigated— the latter by 
paper chroaatographic aad high voltage elecferopheretic anal2?3is 
of eisz^ic digits of the iodoalbmsioo 

Resultss By a modificstlois of MacFarlaae's jet iodiaatio© 
method (I) it feas been possible to prepare ©a lodoalbisain in good • 
yield la t^hich more than 95% of the orgaaicaliy boiis^d iodise 
occurs as ©onoiodotyrosiaao Biologically tkis preparation show© 
a metabolic half life of 18-21 days and less than 2% of ra| 

Ci) MacFaslanSo A* So - Labelling of plasma proteins with 
radioactive iodine « Biochesso Jo 62s 135s, 1936 o 



Fart B included Yes iTT ISo J^U 



Serial. HOo ____m&iim^i^7C 
Page - 2 

degraded ccaaponeatSo la the course of investigation of the products 
of easpaic dlgesfcion ©f this preparation by paper ehrtaaatographyj 
an inconstant iateresting clircssafcogEaphic "artifact" was observed 
in a bufeaQol ainaonia sysfcesa in which aonoiodofeyrosiae raigrated 
as 2 distinct senes, oee with an Rf of about ,'i.O and one with an 
Rf ef abosfc 0.05, These soaes when eluSed showed identical 
behavior in oshsr chrc^stograpble Bjstmas and when subjected to 
high voltage elactrophoresiso Slsee this pixesioiaeaoa has 
considerable bearing on the problea of Identification of the 
^aiT4o acid products of digested iodoalbtimin it kas been 
investigated in soae detaii aapioyiag I^^^ labeled iaonoio<iotyrosiae<, 
The double zoning could not be regularly reproduced by sny of the 
followirtg procedures: 

{1} Variation of tesspsrature disring ehrt^atography of a 
degree ccsapatible with ^abient teaaperafciire fluctuation;, 

(2) Variation ia pH of the startins sone from 2-126 

3) Salt loads ia the starting sonee as high as 100 micrograsas. 

(4) Inclusion in the starting zone of anionic dm'^a»f'®s£s„ 
cationtc detergentSg or phenols 

{5} ?ariafcioa of distance of the starting sone fro® the 
end of the paper 6 

(6) Variation of conditioning time, 

(?) Variation of anuaonia concentration in the developer o. 

t@) Xsepregsation of the paper with hea'vy metal cations » 

C9) Chrersat«ratioa of the developing solvent with aqueous 
astmonia to the extent of separating phases in the developing solvent o 

It has been possible to reproduce the phenomenon with 
regularity at constant t@sperature regulation incorporating 
certain saetallie cations in the starting sone provided the laolar 
cation monoiodotyrosine ratio, the concentration of ^isaonia in 
the developers and the conditioning time are properly adjustedo 
If the raechanissa of the phenossenon can be clarified^ it msy shed 
some light on the not infrequent occurrence of "unknowns" in the 
chromatographic analysis of biological taaterials containing 
iodlnated compounds o 



Page - ■;. 



PHS-NIH 
iadivldual Project; Report 
Calendar Year 1959 



Part^B; Honors, A'^ardSg, btiA Publiestiona 

Publicatioas other than abstracts fr<^ this projscfci 

Lewallen, Co G„3 Bermaa, Mo^ and Rallg Jo £»; Sfcudies of 
iodoalbxsaitt metabolismo !<. A isathssaatical approaefe to the 
kiaefcieso Joumo ©f Clin« Invest = 3Sj 66^ 1959o 

Lewallea, Co Gog Rails ■So So;, and Benaanj Ms Studies of 
iodoalbjsain IIo The effects of thyroid hosmoneo Jo CilQo 
Invests 38; 08, 1959o 



Serial. lOc „.,BI,S1^,t.l^ffJ..„-..-...„,..^.,., 
I^. Clinical In'westlgations 
2c Ciiaical Endocrinology Branch 
3o Befchesda 

PHS-NIH 

I»divid«al Project Report 

Calendar Year 1959 

, Project Tieles The E^ysical e%eiais£ry of Profceiias 

Priocipal InvesEigator; Harold Edelhochg Fh.Do 

Other Investigators? Henry Metzger^ K. B,;, Roland Lippolde 

Cooperaeiag Units: 

Maa Years (Galendar year 1959); 
Total; 2 1/2 
Professloaalt 11/2 
Others 1 

Project DescripfcioKS 

A procedure based on differenfclaJ, centrifijgatioa (iii the 
Spioco Model L) has perisitfied tlie purificafcloa of calf fefeyroglobulisa 
aucfe fcfeafc oaly a single sjmm&XTlcal boundary is obaeris'ed on 
sediffiaafcatioa o* a I<,-5X solut:loB.o The llgSae scaeterissg aiolecular 
weight; of this preparation was ia cioss agreesseat with tfee 
sedinie!n£a£l.oB«-'dif£«slojii value^ which provides another criterion 
of Its Ejolecular uaiformisyo 

The denaturaSion of thyroglobulia has beea studied by 
both kinetic and molecular methods as a fuactlou o£ pB snd 

tensperaturso 

Afc ail pH values observed the dejaaturatioa of fchyroglobuliiij 
a@ follcwed by it© i;%@olubilit.y near its isoelectric p®int« obeys 
firs£ order kinetics » In the neutral pH 2©Re (7 to 9) the rate 

varies slowly with pH CCh]"®"^^) whereas it cfeaages rapidly with 
tesaperatttrCg i„eoe th® activation energy is 1603 000 k©<al©/BSolec 
However about pH ll^ the rat<3 increases ■v&ty fast with pH--about 
10-foId greater than at neutral pH 'ifalues-»-^hile the temperature 
coefficient is only fflbowt one hslf that observed in the neutral 
range o 



Part B included Yes /°T7 Ko £ZI7 



Serial Moe. SliMJsl&SiL 
Page - 2 



In order to facilitate interpretation of the kinetic 
data ie ie useful to know the molecular coHfigus-aticsal changes 
that occur on denBtuT&tton^ We hmire therefore perfoscsed 
sedimentatioia, viscosifeyj turbidity and optical rotatory 
Qea£iur€aaen££i on solutions of tbyroglobulin which have been 
heated to various teBapes:afcures--at several pH -values.. 

In an earlier stady en the eplittlssg of ebyrogiobttlin 
iafco subimits (by alkali) it was poseiilated that an activation 
energy barrier governed the rate of dissociationo This 
hypothesis has n&w bees cos£iEt»ed by shoviag that an increase 
in taaperature acceleratee the rate (aad affects the equilibrltsa) 
of diseociatioso The dlesociatios reaction however appears to 
precede the deoaturatiotto Surprisingly the deaaturation pbsse 
of the reaction 3e©ng go produce very little additional 
configurational changes above that encountered by heating to 
a tfflraperature just short of altering the solubility properties 
of thyroglobulin solutions <> 

la the ne&tral pH sone the ^n^lleet tsolecular unit 
formed fr<aa native thyroglobulin 'il9S} by denaturation has 
a ssdijeeatation constant of 12Sc In alkali CpH >llo5) the 
denatured molecule has an S value of about So It is interesting 
that both of these taolecules appear to behave &s globular 
proteins when esasined by sedimentation and viecosityo Optical 
rotatory data tends to confizsa this picture in that the S"-12 
molecule is foriBed with practically no change and the S-8 with 
only a esiall increase in levorotatioso 

It would appear therefore that denatured thyroglobuliri 
does not show the molecular imfolding nosssally observed f?ith 
protein denaturationo ©sly chyajotrypsinogea sho&rs behavior 
Bimilar to fchyroglobuiin In that essentially no chaage in Its 
macremolecwlar properties was observed whea it ^as denatured 
by heat at pH 3o 



Serial ^o -, ww ji?4v\ . -s ao*-' 
Page " 3 



IndtvidiiSil Frojece Eepore 
Calaadar Year 1959 



Fart^Ss Honors ^ Awards ^ aad Fubliaafcions 
FabiicafcloRS other than abstracts from this projects 



Edelhoehg Hs The Benaturation of ^epslBo I¥o The Effects of 
TesBp&ra£t«e o Siophyso BiocbiaSo ^cfc^o Ea presSci 

Sdeib.0c.h5, E'i llie Froperfcies of TkyraglobwliBo lo I1ae Effects 
of Alkali o J» Molo Gfeesao la FressSo 

Edslhochg, Hj, aad Lippoldfeg R„ E„j The Froper&ie® of Thyroglobuliac 
lio Usa Sffecfcs of Sodi^ iOodecyl SalfatSo J. Biolo Cfeaao In Pressc 

118.11,5 -Jo So^ Robblass, Joj, asid Edelliocfei, H^,, Anssals Sew .York i.ca<£So 
Scio la 



2o Clinical Eisdocrinology Braises 
3 o Bethesda 



Individual Frojecfc Repore 
Calendar Year 1959 



l&xt As 



Project Titles Syathesis of Aaslogs of Thjrosiaeo Syatbe£ic 
and Mechsalffitic Studies 

Principal Invest igatos-t Haas Jo CahrsaaEm» PhoBo 

Other Investigators! Aimaiaarie HofePj, PhoDo 

Cooperating SxilSss 

Man Years (Calendar year 1959) s 
Totals 2 
Professioioals 2 
Other; 

Project DascripCioa: 

S^fchesis of partially and ccppletel^^ bindgggd_aB3l|5'^s 

of thygoalae TOsh as /ill),, tXI).., v(III>/„ ir(f)j 



CH„CH,COGH 




HO^ \~0-f 



The two quinoaes CV) aad (VI) required as iatermediafces for the 
prepasra$:]Lon o£ ccaapouHda (I) through ({IV) have beea synthesizedo 





Parfc B included 



ir~~~-y 



Ho il_7 



Serial Ho<. gr^Kni^ i /.or 
Page ■» 2 "" 



Cosapouad {?)„ not pre^iot^sly deecsrlbed la fche Ufcerature, hs 
been syaShaalsed as foliowss 

— B~f /-SO., 
HGi HO-f \bIH-C1 



Cv) 




«2% 



,Ka 



CcBBpouiid i'FI) couid not be sjtithesised ia an analogous manner 
due to she presence of a stroagly hindered phsnol groupo Se 
was prepared as foiiowss 






M'2 



^ 



'="====«> 




Cvi) 



Although this synthesis led t© a sabsSaace i?ieh different; 
physical constants thaa fchosa described in the lifierafcure for 
iyt}t, eiesaental aiialysia as xfell as ehe Infrared spectrum Indicate 
that It is the desired quiaone, (This laay be a case of polyaorphisuJc 

The quiaoass (V) aad CVI) will be reduced to £he 
correspondiog hydroquinones » It is hoped that these can ba 
converged to the analogs {I) through ,(IV) in a series of 
reactions analogous to ehose comsEonly used ia the synthesis 
of thyroxine and its analogs ^ 



Page 



f-f;R?lSn!-1^«r' 



Influauce of the strwcfcure of 



aide chain en f.he 



nonengfflaic^ coaveygioa of_^ degsatlao^ aa-alog^s of _ .^iiodotyroslae 
£0. enalo^s.pf £b,;y^ros:ine; It b.as been fotTOd previously th&t 
in the aoaensymic iric«ba£loa of the prepioiaic acid aaalog of 
diiodo tyrosine tk& correspoiidiK^g aaalog ©f efeyroxlae is foziaed 
in pissrtteulariy good yield, la con«;r©s£e felt© 2-Esethyl and 
2-phenylpropioaic acid snslisgs yield oaly ■^'ery little or no 
analog of ehyroKinso Ilais raises the ques£los wlsetber thtB 
iohibitioa of the cosdensiag reaetion was due «;© sterie or 
el@c£roQic icflaenceSo (Methyl and pfeeajl are bulkier aad 
more ■ electron releasing tbaa hydrogen) » 



An analog of ditodotjToaine with a propionic acid side 
chain ia whicb an elactroa afctSSSMSS S^roupj, •viZo-CSOEi, is 
attached to the carbon afcoaa 2 of the propicaic acid side chaia 
was therefore incubated o Galj a very small ^aoimt of the 
cosrespoadlng .aaalog of thyroxine was fersed '^■hiefe indicates 
that the inhibitioKi of the condenssiag veacfl&n by the aathyl 
and phenvl groups is soft due to the elaeSron releasisg propertlas 
of these substituents^ 



itesis of ^ the startisg aategial ;^yil),, for the forej^pisafi; 

eaperjEient and o£^ sevcraJ. neif^__anaIogg of thg;gggASS-,.l?Jj-^X-»_ ''^^^ ■' ^^'^ ' 




COOH 



VII 



-O 




CH^CHCOCH 



VIII 




CH^GKCmSI 



COQH 




Substance (¥11) a aot deserilsed 
follows: 



in the literatwrej was prepared as 



iO-»/ \- 






■^ H< 




SOOC-Mc 



CI-|«C='COCJC„,Hj 




COOH 



CHg»GHC(MSi 



®^ CVII) 



?sge ~ 4 



Subsfconce \'¥IV'(> was svafchesised as follows; 



c ce^»H> 

I 2 5 








"■CHC^JCgHg 



COOCgHj 






2 2. 3 3reaati«% 



<VIII) 



Substances (IX) and (X) were ob^.aiQed by parsial er ccsapleta 
iedination of !yiII)o Preliiainary bioassays skived that both 
'Cl'i) and (X) induce raet^orphosls in the eadpolec 



Mecbaniem of the elvaaination of the aliphafclc side 
chai n in the aoaeasiwaic Incubati on of diiodophlpre fclc add s 
Further study of this mechanism led to the hypothesis that the 
propionic acid side chain is ellsiQated as a cyclic compound 
(propiolactose) which then reacts further, either vfleh a proton 
to foxn hydraerylic acid CXI)j or with the quinol ether intermediate 
CXII) to form dihydracrvjy.e acid <XXII>5 





CsO 




'h-^h 



,€0OH 



XSI 



Serial Ko« .^^^t^MisMML^^ 
Page - 5 



Miseellaaeoas iavesti^atioass It. has bean fosmd that a 
sodixsa borohydride reduction of ^g-hydrosj'pheiiylpgi'n.tvj.c acid to 
the eorreispondlng lacCic acid is possible only under certain 
experimental conditions {due to keto-enoi fcautcsasriss aad fco 
fche forsoation of as eaol borate complex) & Favorable condiKioas 
£&z the reduction ha^e been detenainedo 

Ifc has also been fouad that e-hydsosypheayipjrnsvic acid 
is rapidly degraded by alkali uadey mild conditions (Ool S HaCSSj 
room taapo) to ioxm aliaosfc q«an£itisti'??ely ^-hydros:ybenzaldehyde<> 
Syntheses of this and related keto acidsj, involving She use of 
aiUali (such as -eporeed in the literature) must therefore be 
rejectedo 

Tetraiodothyropjrrtj^ic acid also shows keto~enol tautraEerisan 
as evidenced by a typical ketone specCrt^ and the fosatation of 
& bors.te coaplexo 

The photochemical reaction of iodQphesole «7hich leads to 
the elisinatioa and reincorporation of an unknown iodide-" like 
substance C^atag Bioch^ Jo, 72, 214 (1959)] has been repeated 
with thjnroxineo The phenanenon described by Tata could not be 
reproduced o 



Parens Honors, AwsrdSj sad ?ublics£ioas 
??ublica£l3QS other efeaa sbsfcracts frsaa this project: 



Mafcsuursj, Tog & Ho. J« SahamaaBo Model Eeacl:i®as for th® 
Biasymtfeesis of Thyroxiaeo lo Structural Ijisflwesiea of £hs 
Side CliaiE ia j?.aalos@ ©£ Bilodoeyroslne ©a their- Convessloa 
X© Analogs of T&yrosineo Jo im. CkesBSo So«c8 SI, 8?1 C^S59) = 

CaiiaBSQsa, He J;> & To ^tsuura. Mod&l Re&ctt&ns for the 
Bios^mfthssis o£ Thy7o:!sine. lie The Fate of £he Aliphatic 
Side C^ain on the Ck>ave?sloa of 3„5'DiiodophiGretie i^cid 
to 3j5s,3's,5'"Xetraiod®efeyropropionic Aeido Jo ISo €h©jSo 
Soco. 82, (1959). 

IteCeuura, To;, & Ho Jo Cahrmanno Model Eeae£i@as £or the 
Biosynthesie of ThyroKioao Ulo The S^ehesis o£ Hiiade?ed 
Quiuol Efihere mnd their Goznrer&ion to Hl&dered Aaalogs of 

ThyroxinSo Jo Aso Oisso SoCo, 82 j (1959) o 



Serial Hoo EM^zMSS^ 

io Clialcal Xnvea£igatioas 

2o Clinical SadoeriQology Branch 

3 c B&the&da 



PHS-HIH 
Individual Project Report 

Calendar Year 195S 



Part A; 



Project Titles S&udies on the HechaBisn of the Hepatic Capture of 
Iiasulln 

Principal lavestlgators Glemi Eo MortMor^p Mo Dc 

Other Investigators; Fraak TiefcaQs PhoDoj Hancita L^ssax 

Cooperating Ifeitss latexaediary l^etaholiaa Sectioa« Laboratory 
of Biochestlstry aad Metabolism, HMMD^SS 

Haa Years (Caleadar year 1959) s 
Totals 3 
Professloaals 2 
Others I 

Project Rescript ioa; 

Obieetives s Iw studies previously reported by the above 
invest Iga tors. It vas gho«m that lightly lodiosted Insulis is 
rapidly rei&oved frosa circulation by the iatacte cyclically 
perfused liver o Siace iodoinsulin appeared to be coacentsrated 
by the liver prior to its degradation, a separate afcep, occuring 
before insulin proteolysiat, was postulatedo The aisi of this 
study has bees to elucidate the raechanleai of this step or steps o 

Methods; 1) Cyclic perfusion of rat liver with oxygenated 
blood or plaaaa,, 2) Partition and identification of degradation 
products by trichloracetic acid precipitation sad paper chroHiatographyo 
3) Radioactivity assay. 

Major Finding s; In an att^apt to inhibit selectively the 
process of ineullu degradation^, and thuB "isolate" an initial 
concentrating laechaniaaj, a series ©f liver perfusions were carried 
out at about 3®Co Such studies revealed thafc.^ whereas iodoinsulin 
was resioved fress eircislation and concentrated by liver tissue,, 
its subseqvtent 9 degradation was ecsapletely abolisbedo Virtually 
all of the iodoinsulin radioactivity removed froai the perfusate 
could be sedimenSed after centrifugiog the hosaogenised liver,, 



Part B included Yes /" / Ho 



Serial HOo HJJ Um-ISOC 
Page - 2 



thuQ dasonstraeing its biadisg to a particulate fraction of the 
liver cello CirystialliQe iasuliCs but: not ACTH, prelac&ivis or 
gro»£b hoxssoBe» Ctaspeted «rith lodoiQsulin for £he si£e of bindlssg 
lodoinsuliSs rendered biologically inaetivs by alkali tTeatstetits 
wae not bcHsndo 

Siace eon^rol e^cperisaeots iadicated that ths liver retailed 
appreciable proteolytic activity at O^p our failure to observe 
degrsdatioa by the perfused intact liver s^sggested the preseaee 
of a blocks, £usc£i»3sally ister^sed between the process of biadiiag 
and degradation. Farther evidasce eupportiag the existence of sn 
isate£^e4iat® @tep @Berge^ irtm & series- of esporiments witis EBT^ 
C?erssae}o This c^aptmad was slio^Q to cause a substautial reductioa 
ia tke rate of iodoinsullB degradation by the perfused;, intact 
liver o However mo effect was observed on the biadiog of iodo* 
iasulin or its degradation is liver bQaogeoateSo It is tsBptiog 
to speculate oa t^e nature of this iatexssediate process. Certainly 
this data are ia aceord with sos&e published reports coaceraicg 
the deleterious effects of cooling and Verseue oa ms&braae function . 

A coBparison of the i&taet: liver aad the h^sogeaate with 
respect to the ability of &CIH and glueagos to inhibit iodoiasulin 
degradstioa revealed significant differeseeso Heitber peptides 
iahibited in&ulia degradation ia the iatect liver^ yet both cauae 
iehibitiost iii h^sogeoate e^per^e^&ts. {^ea the degradation of 
heavily iodiztated imsulia and alkali-treated iodoinsulis vere 
each ecmpared ia the two tsy&t&nSt, the ezteat of proteolysis by 
the intact liver i^as far less. 

Si^if icaa ce to KIAMB Researsh s The eventual fate of 
ias^iia iQ its reaction ^ith the intact liver cell is its 
destruction by proteolysis. The results of the above studies 
iieidicat@E, hotreverg that its access to the proteolytic ensg^es 
involved is not one of passive diffusion through a semipermeable 
meaabrfineg but raay entail a series of isore complicated steps. 
The finding that insulin is bound initially and selectively, 
svtggests a useehanisffi 'thereby certain cells may sequester insulin 
rapidly. PShether raechanisms elucidated here will eventually find 
coussterparts in insulin^ responsive cells, s»ch as tsuscle and 
adipose tissue^, is a question of considerable importance. 

grossed , ^Coarses See C^'tologieal l>ocalisation of Insulin. 



Serial HOc ___. 

io Clinical XEveasigafciong 

2 o Clinical- Endocrisiologf Branch 

3o 



HiS»HIH 

Indivi&jtal ?r©Jecfc Report 

Caleudair Year 1959 

FKOjece Titles Cytological I^calisatioa of Xasulia 

Principal XavestigsSojcs Glena So HortJaore, Mo ©o 

Other lavestigatorss Frasik Tieta®^ P&^Sce Seorge Slensierg M^ Bo 
So Wo asaaeres, PkoDo, Maacita 



Cooperating IMitss 1} Ittt&mtedl&xy Matabolism Sectiosg Laboratory 
of Bioch^ietxry 4 Mstsbolieaa Z) Secfcioa os 
Hlstoeheraietry, LaboratoT^ of Patliolog^ sad 

Hietochmistry 3) Section o£ Biod^^aistsry of 
AiBlno AcidSs Laboratory of fiiastsaeology aad 
Toxicology, nmm^Mj^^iQZ 

Ksu Years ^Calendar yeas- 1959)1 
Total: 3 3/4 
ProfeesiOQal: 3 
Others 3/4 

Project Descrlptloa; 

Oblecfclves s To identify the cellular site or sites of 
lasulin blading o Should technics prove satiefaetotyg aa att^apt 
will be made to correlate the isetabollc respe&se@ to Insulin with 
its cytelogical locatlouo 

Methods; i) Preparation of Isolated cell suffipensioaso 
2) ©rgaa perfusiono 3) Paper electrophoresis o 4) Frosen fcisssue 
sectionlngo 5} Fluorescence saleroscop^o 6) Radlosutographyo 

MJor FiaaiSig;ss Preliminary I radioautographs were 
iBade on sactions of li'^er perfused t^ith iodolnsulin et 3^Co 
Although the distribution of radioactivity waa fairly uaifowsj, 
there appeared to bs. somo. localisation along the einusoidal 
borderso There was no heavy coiicsatratloa of radioactivity 
adjacent to ths feipffer cells and it fflsy be concluded that the 



Part B included Yes £x7 ^ L.^ 



Page - ?. 

bulk of iodoi'asuiia was botmd -la. tfee viciaity of fcbe paffeac&jssal 
cell cords o 

To Improve cytologieal resolution a. B&vt&s of espevlsseiaas, 
@aployin(g a f lisoreBcias dsrivacive o£ lasislira, -vigtsiallsable under 
fluorescence aicroseopy, has been startedo Srysealliae liasuliQ 
was ■ reacted wieb flaorescein isothtocyavs&t&^ yielding an is2B«iin- 
f lUorescein dezlv&'cX've ^ieh is strongly f iuoraoc&as: and reesias 
biypoglyceaic asSi^ifcyo Thus far wq h&vsa he&s. suece©sf«l la 
«isi2allsi£ig fluorescence bound Co isolated liver cells in suspension p 
Since adeqya&a e@n&role have not yet: been co^pletetS;, no. conclusions 
can be drenm wieh reference to the sites of specific binding o 

Mggtiilcaace__fco_HI^g&..aesearch; A current feheory of insalin 

action is that it in s«»Qe way accelerates the masbrane transfer 
of glucose c Since the primary alia of this study is to localise 
the sites of Ins^ulin binding and perhaps to -visually £ollmi its 
fate within the celij infonsation gained in this %?ay sight prove 
useful in strengthening or isodifying tha aWve hypothesis. & 
technic of this kind laight be applie4 to other protein or peptides 
a@ well. 

^groqosed . Gaaraeg To continue the i?ork as outlined sbo^ej 
eventually extending it if possible, to Insulin responsive cellSo 



Pas-: 



FHS-HIH 
ladividual Project Eepore 

Galendar Year 1959 



gar|S:.,,B; HcaorSs Awards 9 and Publications 

Publications ofcher Shsa tsbstraess fr©ai this projeaes 



llor£ii&@ire„ Go Eo and Frank Tie£Seo Studies on the Fate »£ 
Insulin" X*-^^ in the Perfased Eat U'pesr.o Metabolissa 8s 4^9 {1^59) < 

Hortimore, €0 Eo sad Fo Tletsso Studi|{s on the Mecbanim of 
Capture sad Uessradation of laoulin-I*''*' by t&e Cyclically 
Perfused Eat Ll^&Xo Amto Mo Yo Acado Scio 82s 329 (1959) c 



io CirlalcaA laveseigafcioiis 
2o Clliiieal Endocrinology Bra. 

Individual Froject asport 
Calendar Year 1959 

Project Titles Bladiog of Cortisol by Sertaa froteia 

Fsriacijaal laveseigatosfs Saul Wo RosetJs ghcSoe, Mo 9o 

Otilser Invesf.'igatosj SuSfe JolmsoES 

CooperatiKj, Units; 

Mats Years (Caiandas- year 1959) s 
To&als 1 1/2 
- FrofGssiosaais I 
Ottei:; 1/2 

SsEperissents are to progress &n She atteasptecJ Isalation 
of the a&rtleostevold hinging globuli&Cs) of hisa^a aettm., Usisig 
txltiwi labelled &9ir£l&®l aad tk@ tedmique of protein cbr^aetograpby 
on aoioa eKchaoge cellulose C^o^@^ smA f eter&on) , ^^ ^^^ beea fcmad 
tSiat the cor&ieosteroid blading gloJsaliaCs) can be eluted over a 
relatively nBXxaw raoge of pH and ionic et^'enigeho Most of the 
corticoBteroid blading activity has been located in Cohn fraction 
IV-4o whose tttes- of the specific globulisCs) is 5-l©s that of 
seswao Further fractionatioa of coltgm eluates eud e£ IV-^ is 
in progress aad attempts are in progress to ii5olate sm4 cbsracterise 
a discrete binding species by preparative starch gel elestrophoreeiSo 

It has be@» shsmi (Daughaday, Sandberg and Slauss^ite) that 
protein-bound Cortisol nigrates asiodsilly at a ]^ %iaiere alburain ie 
isoelectric o Tliey have suggested the likelihood, sa a consequence 
that the binding globulin is a glycoproteiBo Experiments in our 
lab using neurerainidaee suggest that neur^ainie acid is not involved 
in Cortisol binding <> SIsillar behavior in electrophoresis and towards 
neurasinidase has been aho^m by Ingbar for thyrostin® binding protein 
CT3P)p ^e presence of TBP activity in prealbumin es^ the absence 
of neurjsminic acid in & pure prepar&tion of a prealb'^min (Schultse) 
iBsy be pertinent and \i±ll be exploredo 



Part B included Yes iZU ^^ .iEU 



PHS^HIH 
Qileatkur ^t^x 1959 

S«rial No. JL5|^ 

Off ic« of thtt Director 

faxt A 

Projoet ?itl*: Adaiaisevaclfltt 

Principal Invest ifiators: Oro Fle^rd S. Da£t» Direeeosf 

DVo Go Doneid Vh«doa, Assistane Direc^r 
Mr. Ho G. Bay lis, 3a8«evtive Officar 

Project Dascriptien: 

Adaittiaferafciog j The prograss of the Hatioaal Institute of Arthritis 
and Hetaholie Diaeasaa eneoD|»asa three sajor ereasi (1) Basic Laboratory 
Kasaareh; C2> Clinical Invest igat ions g and (3) Bxtrsatsral Prograns (Besearch 
Grants, Trainins Grants, Keaearch Fellowships, and Graduate Medical training 
Grants). In addition, the Xnstituta is responsible for providing business 
aanagcaent services to the InterdepartBental Gesnittae on nutrition for national 
Defense, a wsrld^ffida survey activity « the Office of the Director is responsible 
for planning and directing the overall adainistration of the Institute, in 
conducting, fostering and coordinating investigation of the cause, prevention, 
diagnosis and treataant of arthritis, rhetaaatisa and aetabolic diseases; for 
maintaining effective operating relationships «ith other Institutes, astd with 
other units of the Public Health Service, vith the Departnent of Health, 
Edueatieo end Welfare, othes GoveroBental Agencies, and public and private 
organisations carrying on related functions. The Office of the Director also 
participates in detemining policies governing the national Institutes of 
Bealth. 

The Director, with the cooperation and advice of his staff, sponsored 
several cooperative confereneea in collaboration vith the Aaerieen Sheuoaitini 
Association and the Arthritis snd Rheunatisfs Foundation, Chief aaeng these 
were the Conference on the Cooparative Pathology of Arthritis and Bheunatieai 
which was held in Hashington, D.Co, and the Congress on the Host Besponse 
MeehMiiaai in Sheuauktoid Arthritis, which was held in Atlantic City, New Jersey o 
Also, the Office of the Director participated very ectively in planning and 
coordinating the Second Pan Anerican Congress on Bhawutie Diseases, held in 
Kashington, DoC. and Bethesda, Maryland, June 2 through June 6o 



Serial Moo 154 
Extramural Prograas 



NATIONAL INSTITUTE OF ARTHRITIS AND T4ETAB0LIC DISEASES 
Annual Report - Extramural Programs 
January Ip 1959 - December 31, 1959 



Daring the past year this Institute has eohtinueds at an 
accelerating rate, to foster research, and training for researcb.s 
in those areas of medical science for which it assumes a prime 
responsibility. An increased number of research and training grants 
from the National Institute of Arthritis and Metabolic Diseases were 
awarded to esqserienced research teams to support a broad and x^iell 
coordinated attack on the problems associated with arthritiSj 
diabetes, gastroenterologys physical biology, ^stic fibrosis, and 
various metabolic diseases. As in the past, the diabetes and 
arthritis programs constituted the major field of enqshasis, but in- 
creased activity in the other programs has been encouraged as a 
logical step in rounding out ■ihe over-all program of Vcie Institutes 
As had been anticipated the expanding physical biology program is 
demonstrating its values, not only in terms of direct contributions 9 
but also in developixig an awareness, in "Uie various disciplines, of 
the desirability of using the techniques of physics and physical 
chemistry to complement o-Uier less precise methods. It is felt that 
this program is serving to help achieve a more desirable balance 
between purely clinical and basic research. Closer cooperation 
between the clinically oriented investigator and his counterpart in 
the basic disciplines, based on mutual respect and dependence, is 
thus being promoted by the current program. 

Research grant awards made this year supported research in 
both clinical and basic areas which, as in the past, ranged from 
investigations of incidence and etiolosr to treatment and rdiabili- 
tation. The various disciplines involved includes physiological 
and biological chemistry, general metabolism, nutrition, endocrin- 
ology, jjathology, hematology, pharmacology, embryology, bacteriology, 
physiology, biophysics, biophysical chemistry, surgery, general 
medicine, and others to a lesser extent. Budget increases in both 
training and research have permitted a controlled e^ansion in alQ. 
facets of the program and it is anticipated that further expansion 
id.ll continue as a logical and desirable consequence of the over-all 
objectives of the Institute program. 

It is gratifying to note that as the training program approaches 
some degree of maturity, a considerable number of former trainees 
(about 50f) are establishing independent research programs. There ap- 
pears to be no doubt that the existence of training programs in key 
institutions throughout the countiy has had, and will continue to have, 
a healthy effect in focusing attention on the major research programs 
of this Institute and in creating an atmosphere conducive to academic 
medicine. 



Page 2 



Research Grants Activities 



As of Iteceraber 19599 this Institute was supporting approxiiiiately 
16^*0 research projects by means of grants having a gross annual total 
of about $25,5689000. The average grant was thus $15,5000 These 
grants are distributed among a total of 205 institutions located in 
^■2 states, the District of Columbia, Puerto Ricoj, and six foreign 
countries. They support both clinical and basic research related to 
arthritis, various metabolic diseases including diabetes and thyroid . 
disease, liver diseases, nutritional diseases, cystic fibrosis, kidney 
diseases, gastroenterology, and also research in physical biolo^o 

The National Advisory Arthritis and Metabolic Diseases council, 
at its three meetings in the calendar year 1959 reviewed I368 research 
grant applications having a total requested amount of $23,783,2030 Of 
these, 8O9 were reconmended for approval in the amount of $12, 257, 657 <, 
The approved applications consisted of I38 con^etitive continuations 
in the amount of ^ZsZSZgQ^Ol - 576 neir applications in the amount of 
$8,989* 361? - and 95 supplemental requests to-Uling $976,253. Total 
council actions relative to review and approval for the year are sum- 
marized in tatailar form belowo 



Research Grants 





Noo 


Requests 

Amount 


A 
No^ 


pprovals 


Council Meeting 


Amount 


March 1959 


li.y^ 


$ 6,969,63^ 


296 


$ ^9291,338 


June 1959 


932 

562 


13,310,786 
10,279,, 679 


932 


13,310,786 
6,950,622 


Nov, 1959 


372 


6,533,890 
$23,783,203-' 

Total Approvals 


257 

1.925;^ 

- 18^4-^' 


3,786,088 


Total 


1,368^^ 


$28,338,83^-^ 
-2,770,392^' 




1.7^1 


$25,568,^^2 



1/ Excludes 932 recjuests for reaffirmation of previously recommended 
support in the amount of $13,310,786. 

2/ Includes 932 requests for reaffirmation of previously recommended 
support in the amount of $13, 310, 786. 

2/ Applications viiich were approved in fferch 1959 or November 1958, 
but idiich were not paid because of a lack of fuxKls, were recon- 
sidered at the June 1959 meeting. These figures are subtracted 
to obtain a true total because they appear twice in the boc^ of 
the table. 



Serial Ko. ^IMp •> I'M 
Page 



A number of exannplesj selected at random from approximately 
16^ active research projects supported during the past year^ serve 
to illustrate the nature and variety of this complex prograwa All 
are related directly or indirectly to problems which lie within the 
categorical interests of this Institute. 

In the field of diabetes, the biochemistry of insulin in vivo 
continues to te the subject of intensive research. Dto Robert H« 
V/illiams and co-workers confirmed the fact that insxilin is rapidly 
degraded by virtually a3J. body tissues. The mechanism of the degra- 
dation is not fully understood as yet but the studies indicate that 
it occurs in at least three iifaysj i,e.j> enzymatic and non-enisymatic 
reduction and hy proteolysis. Glucagons, ty way of contrasts appears 
to be degraded only by proteolysis. A better understanding of these 
processes might serve to explain the wide variance of insulin re- 
quirements among diabetics. The search for effective oral anti- 
diabetic drugs continues along with research as to the mode of action 
of those that have already enjqyed some success. It has been suggested 
that carbutamide and tcCbutamide stimulate release of insulin from the 
pancreas^ thus promoting hypoglycemia. HotjeveTj other evidence shows 
that this is not liieir only mode of action although it is generally 
agreed that small quantities of insulin (erKiogenous or exogenous) are 
required to make these drugs effective. Dr. Piero P. Foa and co- 
workers reported -tiiat experiments on a similar drug, chlorpropamidej 
indicated that its hypoglycemic effect is dues at least in part, to a 
decreased liver glucose production and offered no evidence of a pan- 
creatropic action. They did not ascertain whetoer insulin was neces- 
sary to the hypoglycemic effect of chlorpropamide. One of the newer 
oral drugs under investigation is phenethyldiguanide, called TBI, PEDG, 
or PEBG. This drug is independent of insiilin. Williamsp Foaa and 
others have variously reported that it inhibits succinic del^?drogenase 
and cytochrome oxidase, thus leading to tissue anoxia and inhibition 
of oxidative phosphoiylation. As a consequence, products of the Krebs 
tricarboxylic QTcle accumulate. DEC is b^ieved to inhibit gLuconeo- 
genesis and to stimulate anaerobic glycolysis. In spite of its rather 
severe toxicity, this drug msy be of considerable value in the treat- 
ment of diabetes T^mi used in small doses in conjunction with insulin. 
Dr. Foa, in reviewing experimental evidence relative to the sulfonyl- 
ureas, states that these drugs apparently act by suppressing liver 
glucose production, but only when insulin is injected or released in 
"peiTTiissive" amounts. He cau-fions that long therapy with such drugs 
should be attempted with caution, since the suppression of hepatic 
glucose may be a sign of liver injury. 

Dp. Stefan S. Fajans has presented evidence indicating that 
some of the oral antidiabetic drugs may have an important use in tiie 
prevention of severe diabetes in persons having a^rmptoraatic diabetes. 
Prolonged administration of tolbutamide in such cases brou^t about an 
improvement in the glucose tolerance test, even though the drug was 
withheld for two days before the test. 



Jariai Mo. 2KAMD »__i54 



Dpo Arnold Lazarow reported that eleetronmicroscopy studies on 
renal biopsies from human diabetic subjects early in the course of 
the disease reveal a significant thickening of the glomerular base- 
ment membrane, Ihis is observable prior to the appearance of clinical 
symptoms of the complications of diabetes. The "same investigator has 
f . ."I^^* sub-diabetes in the pregnant rat produces a statistically 
significant increase in the virth wei^t of the fetus and a three-fold 
increase in fetal mortality. Biese abnormalities in the experimental 
S^"S:^T«^^'^^^ !° ■*^°^® reported for the prediabetic state in man,. 
Dr. David Barker and co-woz4cers have dex'ised a technique for introduc- 
ing heavy elements into protein radeculesj tcLthout seriously damaging 
them. This permits Hie use of X^-raj^ diffraction techniques to st^dy 
the atomic arrangement xri.tiiin the molecule. An unders-tanding of thB 
structure of the molecule leads to a better und^standing of -tiie 
nature of , and reasons for, the highly specific reactions that are 
ch^cteristic of protein molecules in general. Drs, Truman S. Licht, 
Milton Stemj Hariy Shwachraan, and Andre J. deBethune report -Khe 
perfection of techniques for use in the diagnosis of cystic fibrosis. 

Inili2tl,°i^^ ^^ *?f "^^ ^'°^^ °^ ^^^« ^^^°^^s P^ti««^^ has 
an elevated sodium chloride content, these investigators have reported 
two rapidj sensitives and accurate methods for determining liie salt 
content of sweat. In one, the pd is measured potientioraetrically, 
while in the other the electrical conductivity of diluted sweatis 
determined. On the average there is from two to three times more 
sodium chloride in sweat from cystic fibrosis patients than frcan 
nomals. Dr. Zacharias .Qlsche reported the development of a method 
for fractionating the fuco-mucoids of the urine of children with 
cystic fibrosis. The method makes use of organic solvents and contin- 
uous flow electrophoresis. The above exaii^xLes iUustrate the use of 
^^J?£,^^^ physico-chemical methods in medical research problems. 
The mr© co-sponsored (with NIAID) an international congress on <?ystic 
fibrosis in January 1959. ^e conference was attended by seventy lead- 
ing investigators and was designed to suggest and stimulate new research 
concerning ihe basic nature of cystic fibrosis. It included discussions 
of possible new avenues of approach to prevention and treatment. 

* y^°!f P5*^°^ogical conditions, including cirrtiosis of the liver, 
gastrointestinal hemorziiage, acute hepatic failure due to viral hepatitis, 
and the ingestion of liver toxins, lead to dangerously high blood ammonia 
iTtl ^°S%°^ ^^ inability of the liver to deto:iy Snonia produS 
h«v^?J^n/Sf^*^^?•/• ^' Najarian, H. A. Harper, and H. J, McCorkle 
have found that the intravenous injection of arginine reduces blood 
^r^i?'^ f'"''®^^^'? ?® production of urea, an ammonia-containing com™. 
pound ^ich is excreted m the urine. Dr. J, K. Isl^ and co-workers 
have developed a technique for measuring absorption from the colon usin^ 
radi^ctive sodium iodide. In a group of 5 patients with ulcerative 
colitis, 1.6/« of tiie sodium iodide was absorbed in I5 minutes, ^ile 6,2^ 
was absorbed from the colon of 1^ normal individuals. The technique 
promises to be useful in evaluating the condition of the colonic mucosa. 



Non- tropical spmae is a disease associated i^dth long-lasting 
diarrhea, weaknessj, and weight loss resulting from failure to absorb 
certain proteins properly« Drs. Mo H. Sleisenger, T, Po Alngrs and 
others have shown that a diet conpletely free of gluten (cereal 
protein) provides a ready means of controlling the disease and recom- 
mend such treatment without reservationo Further research is being 
carried on to stu<^ the fundamental pathogenetic mechanism tdiich 
leads to non-tropical sprue. DrSo D« W, Elliott, R. Co WiUiamSj and 
Ro Mo Zollinger have been able to show that moderate to fatally- 
severe pancreatitis may be caused ty a back flow into the pancreas of 
a mixture of bile and pancreatic secretiono /l^ihen tlie common duct is 
blocked, as by gallstones, pancreatic secretions may enter the gall 
bladder ^ere the pancreatic ens^yme trypsinogen is converted to 
trypsin, a powerful proteolytic enzyme. The mixture of bile and 
pancreatic secretions may then reenter the pancreas where the tx^sin 
can cause severe tissue damage. This is probably the first direct 
evidence to support a theory i,1h±dla. has been held by some investigatorso 
Further work is contemplated in an effort to develop surgical tech- 
niques to correct conditions 'hiiich permit the above sequence of actions 
to occur. In other metabolic studies Dr. C» W. Vermeu3.em has shown 
that in animal eij^jeriraents a hi^ calcium intake reciprocally reduced 
urinaiy phosphorus concentration and actually decreased the incidence 
of urinary tract stone development. Dr. W, H. Boyce implicates certain 
unusual muco-proteins in the virine as being inipor-tant factors in stone 
formation. Both of ihese findings tend to allay fears that a high milk 
intake in adult man might encourage urinary calculi f orniationo 

Research in the field of arthritis continues to center around the 
rheumatoid factor and the search for more efficacious methods of treat- 
ment. A conference on the pathology of arthidtis and rheumatism which 
was co-sponsored by KIAI® was attended by k5 investigators, many of 
whom were grantees of this Institute. TJie conference was successful in 
fostering an exchange of information between veterinarians, paliiologistsa 
and clinicians, and in more clearly defining the similaii-ties and dis- 
similarities between human and animal arthritis. Several new types of 
the disease were reported for the first time. In studies on ihe 
rheumatoid factor, Drs. Robert C. Mellors, Ralph Heimer, Josue Corcos, 
and Leonhard Ko2°ngold demonstrated for the first time ihad. the factor 
is present in human tissue and, in fact, found evidence as to the site 
of its formation. It is hoped that furtlier study will make possible a 
more specific test for preclinical arthritis than is now possible. The 
rheumatoid factor, according to these investigators, is produced in 
certain plasma cells and in germinal-center cells. 

Drs. W. Rcy Slauiw^ite, Jr., and Avexy A. Sandburg have reported 
the isolation of a new corticosteroid-binding protein in human plasma 
which they named transcortin. the protein is an alpha-globulin and like 
some other plasma proteins is believed to be part of a medianism for 
transporting certain hormones in the body. Althou^ it has been known 
for some time that plasma proteins bind steroids, not raary of these 



Serial NOo 



proteins have been isolated and characterized.. A nei-i method for 
determining the reserve capacity of the pituitary to secrete AGTK 
has been developed hj Dr. Ge Wo Liddle and co~wdjkerso Essentially 
the test consists of using the agent SU-4885 to inhibit the produc- 
tion of corticol ty the adrenal ^and and this brings about a com- 
pensatozy increase in ACTH secretion in patients vjith a norrrjal 
pituitary gland. Tests on n^ny patients indicate the reliability 
of the new technique. Dr. Tsatsiparlis has studied the effect of 
Zoxaaolamine in 43 gouty patients. In either single doses cac after 
prolonged administration, the drug proved effective in reducing 
blood uric acid levels and increasing xirinaiy exretion of viric acid. 
In I6f} of i:he patients undesirable side effects such as nauseaj 
diarrheaj or headache forced discontinuance of the therapy. The same 
investigator, with Dr. C. McEwen, reports that -Uie use of colchicine 
as one of the most useful aids in diagnosing goat has lead to an 
interesting and perhaps important finding -Uiat intravenous adfflinistra-= 
tion of colchicir^ apparently benefited several patients with acute 
non-gouty arthritis. Ihe intravenous mode of administration ^i^s 
found to be far superior to oral administration both as to sijeed of 
action and lack of severe side effects. Dr. Herfort and co-workers 
have reported apparently successful treatu^nt of six arthritics by 
means of surgery ^ich is referred to as "extended sympathectongr". 
The operation relieves arthritic pain and facilitates rehabilitation 
in patients ^o are willing to cooperate in the prescribed exercise 
therapy. Dr. C. McEwen and sevei^ other investigatosrs have continued 
studies to establish the nature of the relationship between rheumatoid 
arthritis and systemic lupus eryUiematosus. In one group studied^ ten 
relatives of lupus patients had positive tests for ifeeumatoid factor 
while three had clinical rheumatoid arthritis. Five relatives^ two in 
the same family^ showed hypergammaglobulinemia. 

IXiring 1959» more than 1,000 scientific papers were published 
by investigators ^^o were supported vtn^lys or in partg by research 
grant funds from this Institute. 

Training Grant Activities 

The contiraa^ searciiy of qualified young scientists tdio are 
interested in careers in academic medicine emphasizes the current 
value and future potential of the training program. Increases in 
the budget have permitted a reasonable expansion in this area, but 
the saturation point is not yet in sight. During the past year a 
conscious effort has been made to encourage vtore training for research 
in the basic sciences in order to better balance this phase of the 
program with the more clinically oriented training. Thus, several of 
the older projects have been either modified or eliminated to bring 
them in line ^?i-Kh current policy. Four committeess composed of men 
who are outstanding teachers and investigators in the traditional 
areas of responsibility of this Institute, continue to provide guid- 
ance in the training program, not only in the review and approval of 
applications, but also with respect to the over-all aims and policies 



Serial Hlo. NIAim -» 154 
Page 7 



of the prograiTio Two meetings of KIAI'® t3*aining grant program 
directors were held during the yearo About 50 directors from 
the arthritis and 25 from the gastroenterdogy programs met to 
discuss means of in5>rovJ.ng methods used to attract competent 
investig-ators to these fields. Discussions were held to establish 
uniform thinking regarding hoi^ the time of a trainee should be 
divided between laboratory and clinical training. The necessity 
of both types of ta^aining was agreed upon. 

Ehring 1959 there were 168 active training grants t^ich were 
dis-tributed by category as follows s ArthritiSg ^3» Diabetes, 53» 
Gastroenterologya 2?; Hematology j, 17| Metabolism and Endoc2^nolos''9 
10| Physical Biology j, 10 1 and o-&er» 8. Comparison of these fig- 
ures with those of 1958 reveals the relatively greater growth of 
the newer programs in Gastroenterology and Physical Biologr. 

1959 



153 



Requests 




Approvals 


Amount 


Ko. 


Amount 


$ 3,588,370 


l^^' 


$ 2a^l9 686 


Previously reeom= 
mended 


1^2 


3b366«7J^6 


Total 


276 


$ 5s508s432 



Ihese figures represent continuation of esdsting grantSj 
supplemental request s, and new applications. Of those reconsoended 
for apprcval, 276 have been paid or designated for payment ty the 
National Ir^stitute of Arthritis and Metabolic Diseases^ in the 
amount of $5»508,4'32. Ihese training grants are distributed among 
8^ institutions in 36 states, the District of Columbiaj and Puerto 
Ricoj and support apifl:'oxiinat^ly 329 indirect trainees. 

Direct Traineeships 

Those awards for support during advanced trainings are made 
by the Institute directly to j^-sicians of demonstrated potential 
and a eon^jetence in an academic careers %^o are further qualified 
by at least three years of postgraduate training. They effectively 
coiTiplement the training grants program through provision of support 
in research method, and related clinical and teaching skills. They 
are available in rheumatology, diabetes and me-tabolisras gastroen- 
terologys hematologj'^j, j±iysical biologyj and related areas of researcho 







Serial No. NIAIfD < 
Page 8 


Requests 




Approvals 


Amofunt 


Noo 


Amcrunt 


$ 567,291 


87 


$ 494,808 



154 



Koo 

105 

Of the direct traineeship applications recotnraended for 
approval in 1959 s. 11 have been paid or designated for payment to 
date, Igr the National Institute of Arthritis and Metabolic Dis- 
eases in the amount of $4489 552c. Biese direct traineeshipss 
although made to individuals^ geographically represent 40 . differ- 
ent institutions in 17 statesj the District of Columbiaj Puerto 
Rico, and England. 



Ihe research fellowship program is an iniportant component of 
the total training programo Postdoctoral and Special Fellowships 
provide individual support for research training in the basic and 
clinical sciences to persons upon whom degrees of Doctor of 
Philosophy and/ or Doctor of M^Q.cine have been conferredo It is 
complementary to the traineeship program, providing additional 
research training to meet the needs of individuals ^ose research 
interests are basic science oriented in the several specialized 
areas as above listed under Direct Traineeshipso It is a mechan- 
ism "hereby, the biologist, chemist, or physicist is attracted to 
research endeavors essential to medical science.. 

In addition to the above mentioned fellowship programs, a 
veiy modest allocation of funds has supported fellowships of the 
Predoctorate type as a feeder to those categorical in nature. 
Emphasis has been placed upon the Post and Special Research Fellow- 
ship Programs as a means of most effectively carrying out the 
categorical aims of this Institute, utilizing the limited funds 
available to it in the fellowship program. 

Recrusists 

Type Ho. Amount ^ Nog, 

Predoctoral.^^/ 67 $ 201,670 11 $ 339115 

Postdoctoral-' I30 792,090 33 201,063 

Specials/ 42 352,548 25 209,8ii9 

Averages 

Predoctoral $3,010 
Postdoctoral 6,093 
Special 8,394 

a/ These are estimates based upon the ava:*age atfjard for each type 
of fellowshipo ~ 




NATIONAL INSTITUTE OF ARTHRITIS AND METABOLIC DISEASES 

ANNUAL PROJECT REPORT 

CALENDAR YEAR 1959 

Sunmary Sheet 



INTERDEPARTMENTAL COMMITTEE ON 
NUTRITION FOR NATIONAL DEFENSE 



INTERDEPARTMENTAL CC»5MITTEE ON NUTRITION FOR NATIONAL DEFENSE 
Annual Project Report 
Calendar Year 1959 



This Committee was formed in 1954 as a result of presentation of 
a plan to establish the CoJHaittee by the Assistant Secretary of Defense 
(Health and Medical) and correspondence from the Operations Coordinating 
Board (OCB) staff which affirmed the desirability of forming such a 
committee and the usefulness of inter-agency coordination of various 
projects and studies on nutrition to avoid duplication of efforts among 
U.S. agencies. A Memorandum of Agreement was signed in 1955 by the 
Secretaries of the Departments of Defense, State, Agriculture, and Health, 
Education and VJelfare, and the Director of the International Cooperation 
Administration. Subsequently, the Atomic Energy Commission became associ- 
ated with the Committee. The program of the Connaittee was reviev/ed and 
again approved by the OCB in 1959. 

The nutrition program directed by the Interdepartmental Conaaittee 
on Nutrition for National Defense (ICNND) , initiated in FY 1955 as a part 
of the U.S. Mutual Assistance Program, has contributed to our mutual 
security by the following means: (1) It has provided technical assistance 
in improving nutrition, food and health in the Airmed Forces which has had 
a beneficial carry-over to the civilian populations. (2) It has increased 
efficiency of mobility of these Armed Forces due to improved utilization 
of their own food resources and development of emergency- type rations. 
(3) It has assisted in defining the major nutrition and feeding problems 
in the various countries. (4) It has assisted the countries concerned in 
establishing nutrition services by training local personnel and supplying 
the nucleus for a nutrition laboratory. (5) It has bettered U.S. friend- 
ship through medical, scientific and technical channels. All of these 
have supplemented the over-all U.S. foreign assistance program. 

The proposed projects for FY 1961, estimated cost $350,000 include;; 
(1) Completion of nutrition survey in Colombia, initiated May 15, 1960 
($30,500). (2) Nutrition surveys in Thailand arid Lebanon. Official in- 
vitations requesting assistance in conducting nutrition surveys have been 
received through the State Department. Resurvey of the Armed Forces of 
the Republic of China (Taiwan), requested by the Chinese Government and 
U.S. MAAG, to evaluate the effectiveness of the rice enrichment program. 
This program was initiated as a result of the high prevalence of 
malnutrition noted in the Armed Forces during the sur^/ey conducted by the 
U.S. Army in 1954. (Surveys, Thailand, Lebanon and Taiwan: $202,500). 
(3) Since field activities of the surveys in Chile and Vietnam will not 
be completed until the latter part of FY 1960, $17,000 will be required 
in FY 1961 to complete the processing of data, analyses of food samples 
and preparation and presentation of reports. (4) Follow-up assistance to 



Korea, Ethiopia, Iran, Pakistan and Turkey, in response to requests re- 
ceived £roB! these coiintries ($24,000) o (5) Follow»up work in the Kear 
East-Africa regioxzal station, which will cover special laboratory and 
clinician consultant advice in Libya, Ethiopia, Iran, Turkey, Pakistan 
and Spain ($46,000) » (6) Meeting of the Fourth Armed Forces Internation 
Nutrition Conference in the United States ($20^000) . (Previous ones 
held in Iran, 1956; Turkey, 1958; Pakistan, 1959), (7) South American 
Anaed Forces Nutrition Conference, inc lading, Colosbia, Ecuador, Peru and 
Chile ($10,000). 

To implement the nutrition program, upon receiving a formal request 
through State Department channels froa an eligible country for assistance 
in conducting a nutrition survey, the request is coordinated with the 
Departments of State and Defense and the Inteimational Cooperation Adialn'' 
istration before it is submitted to the Coasoittee for approval. When 
approved the ICMD organizes a nutrition survey team, consisting of 
outstanding specialists in the fields of medicine, nutrition, biochemistry 
food technology and agriculture to conduct the survey. The duration of 
each survey is approximately 90 days. Data are collected, brought back to 
the United States, analyzed and discussed by the Committee and a final 
report with practical recosiBtendations for improvement is sent to the 
country. 

The follow»up program provides technical consultation to assist the 
participating country in the implementation of the reccKsmendations and 
related probleESS. 

This is a cooperative, reciprocal program. The participating 
country furnishes personnel equal to or twice the number of the U.S. team 
for training in survey techniques. It also furnishes logistical support 
such as laboratory housing and transportation. To date, 25 United States 
universities and colleges, and the U.S. Araiy, Navy and Public Health 
Service have furnished over 100 doctors and specialists for the survey 
team. The program affords an e^scellent opportunity to learn much from 
these countries regarding nutritional disease. Indigenous foods, food habits, 
customs and practices. Such infonaation is useful to our Armed Forces, 
U.S. Operations Missions and the Foreign Agricultural Service for planning 
current programs and In the event of an esiergency. 

Since January 1956 nutrition surveys have been completed in Iran, 
Pakistan, Korea, The Philippines, Turkey, Libya, Spain, Ethiopia, Peru, 
Ecuador, Vietnam, and Alaska. 

Institutes of Nutrition have been established In the Armed Forces 
of Iran, Pakistan, Turkey, Spain and Peru; and governmental Institutes of 
nutrition in Ecuador, The Philippines and Ethiopia are actively cooperatin 
with the Armed Forces. At the Third Armed Forces Internation Nutrition 
Conference held in Pakistan, representatives from Iran, Pakistan and Turke; 
reviewed the great progress these countries have made in ration improvezaef. 
and development of rations for use in mobile situations, ^iuch of this 
progress can be viewed as spectacular. A few illustrative examples point 
out some of the accoanplishaents : (1) Iran ; They have conducted additionr/ 
nutrition surveys to evaluate the effectiveness of their ration improvemer- 
It has been possible to lengthen the training day for troops from the 
four^hour limit imposed by inadequate nutrition to a full eight-hour day. 



A canning plant which has been reopened and is supervised by the Veterir 
Corps of the Iranian Anay has given an economic boost to the farmers in 
the area; it has resulted in the development of the first field ration, 
plus supplemental canned foods which have vastly improved troop Eobility, 
A poultry industry has been initiated by the Director of the Nutrition 
Laboratory, with production of 20,000 poults per year. This has stimulate; 
similar investments and has been used to supplement the feeding of the 
troops, (2) Turkey ; New laws have been passed to provide for improvement 
In nutritional allowances and composition of the rations for the Turkish 
Armed Forces « Schools for training cooks and bakers and sanitarians have 
been established for their Arsaed Forces. Further surveys have been con- 
ducted to evaluate the effectiveness of ration changes. (3) Ethiopia ; 
The personnel trained during the initial purvey have been requested to 
study more estenslvely the nutritional status of the Araed Forces, and 
the CoBiBittee has been requested to assist in planning a ration for the 
Ethiopian Armed Forces. (4) Korea and Taiwan ; Tremendous progress has 
been i&ade in reducing the incidence of nutritional deficiencies by bette?: 
utilization of their aam. resources; in addition, excellent progress has 
been aade in developing field rations. (5) The Phil ippines; The Philippi 
Armed Forces for the first time have established a food and nutrition 
council to assist the Armed Forces in better ration planning and feeding 
of their troops. (6) General: The standard "Manual for Nutrition Surveys' 
published by the Conaalttee in 1957, has been translated into 
Spanish (by the Spanish Armed Forces) and into French. This has been 
exceptionally well received by the Arssed Forces and also civilian groups 
in this and in other countries as a standard reference book. The surveys 
have indicated the absence of any significant asounts of radioactive 
substances in urine samples of people or of food easaples in the countries 
visited. The ICMHD participated, upon request, in the United Arab Republi; 
Food Conference in Cairo^ Egypt, in Woveseber 1958 and in the Mid-East 
Annual Medical Sy«po8luiB in Beiimt, Lebanon, in April 1959. The CoBBBaittec 
was instruaental in bringing together interested groups of American and 
Turkish students at the University of Illinois and American and Ethiopian 
students at the University of Wisconsin. (Uesbezs of the faculties of 
the Universities of Illinois and Wisconsin were on the Turkey and Ethiopia 
survey teaas) . The Secretariat has on record innusaberabla letters of 
appreciation from representatives of the host countries. 



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