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<isif 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´ 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).
(4) Black, So Ceanmrsleo @£ sspastle acid t& h^a^seriiie, lle«h@d in
Velo 6, Astlele lit, Cln press) o
C5) Black, So eiys&s&txt klnasa, mtSxeds in Snsysselegy^ ?@1<, 6, ^eicl«
46, Cin parens)-
2. Bl&chssslstTy o£ Asiss Acids
3o Sa&hesaa
1959
0.4
S»0JGC& !te£«?ipgi«m;
lAtj tmts!b»llaa of & e^lie hexapas&i^Sa^ as-esleidia J. «hieh ie produced '@?
^^ BccAUa® Wgvia is &eli^ £;&«^d4 with fibe al«3 of pa|K6? chsoaMt«snsphy» i«%:
e&'t$ct!S«S;> gives sles e® aa UE&ss^«m C •
» And c©-fact:9»89 gives slea fc® aa UE&s^wa C**.iai«sled
^ifllds C -pl»ayalastiae upoa aeid kfSralyslSo ^is eas^pstsad appears t@ hs
t&tMv clesely ?ela&<5d £e G^fisiieidia Jo ^& a@e ta» Ite identieel ^^th ie« Sm
Ma el(»e3ly ffela^ed is lad&eated by ehe f«et tUst s&e&t^ acid hTds^lyeis «£
eiiOisr esie gives riee «;o phesr^lelsoiae, proliacp valisse (f>^ighias aad leiseis^o
the iKffiu&aeian sis£ure £e? 6he psr®d»eeiea of Cbie
aiapU£ie^6 A clear solugicm &i soluble easj^ass plus ^79 end Sfli Sssrii^s
4UBi&$ aeids, L-C**»ie©l©«K:lo83 I.-C -alsainss l.-C*^««;y3?®sia©B L-C^^-lcBciaSe
I.*C^^'>thige^tiiu2, «hiek @]fe i^t fehe e®aisw9adat£ sasiacp eei^ isf 6?saiei<Sin J„ wr@
late til®
Serial Mo. KMMJ..^_mL
Pag® 2
Sy the use ©f eh»3 eachniqtae ©f P oinerganie pyrephr-sphsS:® ®k-
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
I IMM ilT*^^^^^l °
uri^i ttanai&s&e&
^s:t«d ts Jo M&lo
«.*x «.«*„.« *,«_s^,teisM. ^ ~ C«-«.i.. «.'-
Cia press}..
Serial ^o HjLMgD-lQ2
2. Blocheedstry «»£ Asaine .^ids
3c SeShaskIa, Hetf lsi»3
Ptojeet Ke: 66301-32
?BS»H2R
1959
Ml^°
Scejese Hele: the cy6<»l^ie&l l@«filiaaUoa of
ensysB8fe7ti»
Pirineipal Ss^siigatei?: Si^o So «»
II WilUeaa^.
Mm Yaaras > 50% fttee ea hj&lasml&is& eStadiea; 45% oa
inrejeeta^ anS S% o« tiaae apsat with 8&a74 e£ Civil Scsrvie®
L>«So
g|p®1eat If, Peacrlociaas 1^ s^isdies &a ^ha cy^iagieal locaUsati«ii e£
^ the Ceeas f luoreseaat aatibo^ tseh-
ee iaeii^de 61m lAMUsatim «£ ehft eusyae £ellsw>»
Xa tiuMe 8tttdt«» the iaradia
%?ith Group C aatiesra ceuplad to
«bicb 4t elabesatefi in altu
gletelia ea^lo4 to fluoraaeeiao
entitled '^eudiea aa Stn^troeeeal ag^alunmldssa and Jtotihy*'
alusooidaaao til, Use j^rsdoetleB and aellulas- locaUsatioa eS hf&lvsGoi-'
fdlloving atr^teseeeeal iafasUco, by So W. Eonasrt and Wo A,
£0? !!»uS»Hcatiaa asid substltted to Jiwnal of
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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
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paeess^ la ssm.
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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
e^ eSKm0<i @mm ^teercias® to joint s:«i>»ili0s w&A tsadercass
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tM pi&Ti&& «f g:raKl:«@t xfeeimaUc &ctivl^ ist «11 4 a&s.m
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-
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i^ serlows site e££e«s«:s ti^aire «n<sot£ait:ffisra«!. ISose» of
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tMs oMpotimd am 3nitlei|»a^(l,
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5
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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:
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cerdisis wish a l^f&ar follow-up. ii.M^A. J.Sis.CMld.^
97:56l"570j, I.9S9.
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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⁢ 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 oaso
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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