Document 2J74Xomjd5gg8ydNMKM6Gqqq6

: 3. ` The resting potential of the Jo- doris giant neuron can be separated into at least two components: one which can be predicted by the ionic hypothesis and another which depends upon the electrogenicity of active Na transport. This separation has special interest in light of reports that the Na pump not only makes a general con tribution to potential, but may have more specific functions such as the mediation of synaptic effects (15). The ratio PsiSf*K appears to vary with tem perature and provides another mecha nism by which the potential is regu lated. A similar division of the resting potential into components may apply to excitable cells other than the Ani ' sodoris giant neuron. Our data support the hypothesis (6) that the special characteristics of the resting potential in different nerve and muscle cells re sult from mechanisms, such as the Na pump, that modify a predictable ionic potential common to all excitable cells. M. F. Marmor A. L. F. Gorman Laboratory of Neuropharmacology, Division of Special Menial Health Research, St. Elizabeths Hospital, Washington, D.C. 20032 References and Notes 1. A. L. Hodgkin, Biot. Rev. 26, 339 (1961); Proc. Roy. Soc. Ser. B Biol. Sci. 148, 1 (1958). 2. --------- and B. Katz, 1. Physiol. (London) 108, 37 (1949). . 3. A. L. Hodgkin and P. Horowicz, ibid. 148, 127 (1959). 4. P. Belton and H. Grundfcst, Amer. J. Physiol. 203, 588 (1962); J. C. Blackman, B. L. Ginsborg, C. Ray, J. Phvsiol. (London) 167, 374 (1963); J. Del CastiUo, W. C. de Mello, T. Morales, J. Cen. Physiol. 48. 129 (1964); V. D. Gerasimov, P. G. Kostyuk, V. A. Maiskii, Biophysics 10, 300 (1965); H. Grundfest, Fed. Proc. 26, 1613 (1967); G. A. Kcrkut and R. W. Mccch, Comp. Biochem. Physiol. 20, 411 (1967); H. Kuriyama. J. Physiol. (London) 166, IS (1963); E. M. Vaughan Williams, ibid. 146, 411 (1959). 5. D. O. Carpenter and B. O. Alvins, J. Gen. Physiol. 52, 1 (1968). . 6. H. Grundfcst, in Electrochemistry in Biolopy and Medicine, T. Shedlovsky, Ed. (Wiley, New York, 1955), p. 141: H. Grundfcst, Advan. Comp. Physiol. Biochem. 2. 1 (1966). 7. A. L. F. Gorman and M. Mirolli. J. Exp. Biol., in press; M. Mirolli and A. L. F. Gorman, Comp. Biochem. Physiol. 25, 743 (1968). 8. R. Fay, personal communication. 9. J. D. Robertson, 1. Exp. Biol. 30, 277 (1953). 10. D. E. Goldman, J. Gen. Physiol. 27, 37 (1943). 11. R. B. Moreton, I. Exp. Biol. 48, 611 (1968). 12. S. R. Cross, R. C. Keynes, R. Rybova, J. Physiol. 181, 865 (1965); H. Grundfcst, C. J. Kao, M. Allamirano, J. Gen. Physiol. 38, ,, 245 (1954); G. A. Kcrkut and R. P. Thomas, Comp. Biochem. Physiol. 14, 167 (1965): R. P. Kcrnan, Nature 193, 986 (1962); L. J. Mullins and M. Z. Awad, J. Gen. Physiol. 48, 761 (1965); E. Page and S. R. Strom, ibid., p. 957. 13. P. F. Baker, M. P. Blaustcin, R. C. Keynes, J. Manil. T. I. Shaw, R. Steinhardt, J. Physiol. 200, 459 (1969); I. M. Glynn, Pharmacol. Rev. 16, 381 (1964); J. C. Skou, Biochim. Biophys. Acta 23, 394 (1957); H.-J. 2 JANUARY 1970 Schatzmann, Helv. Physiol. Pharmacol. Ac, 11, 346 (1953). 14. D. O. Carpenter, in preparation. A similar phenomenon has been noted in Aplysla giant neurons. 15. G. A. Kerkut, L. C. Brown, R. J. Walker, Life Sci. 8, 297 (1969); S. Nishi and K. Koketsu, J. Neurophysiol. 31, 717 (1968); H. Pinskcr and E. R. Kandcl, Science 163, 931 (1969): 26 August 1969 ~ Photooxidation of DDT and DDE Abstract. The pesticide J3DT [1,1,1trichloro-2,2-bis(p-chlorophenyl)ethane] and its metabolite DDE [1,1-dichloro2.2-bis(p-chlorophenyl)ethylene] can be photooxidized in methanol. Photolytic generation of free radicals that may ab stract hydrogen from solvent, react with oxygen, or abstract hydrogen from un reacted substrate occurs. Further de composition of short-lived intermediates yields many compounds. Oxidation products include benzoic acids, aromatic ketones, and chlorinated phenols. The DDE also undergoes photocyclization to give dichlorofluorene derivatives. The persistent insecticide DDT may be slowly degraded by the action of light and air. Photodecomposition of DDT has been discussed widely (1). We now report .investigations of the photoxidation of DDT [1,1,1-trichloro - 2 - 2 - bis (p - chlorophenyl)ethane] and DDE [l,l-dichloro-2,2-bis(p-ch!orophenyl)ethylene]. If nitrogen is bubbled through a methanol solution of DDT during ir radiation, the radicals generated by photolysis abstract hydrogen from the solvent, and chlorine is lost. The rate of loss of chlorine from the trichloromethyl group of DDT at 2600 A is shown in Table 1. Both DDD [1,1dich!oro-2,2-bis (p-chlorophenyl) ethane] and l-chloro-2,2-bis(p-chlorophcnyl), ethane are obtained (Eqs. 1-4). The wavelength of the irradiation deter mines the nature of the products; at shorter wavelengths chlorine is- dis placed from the aromatic ring. Prod ucts of photodecomposition were iden tified by combined gas chromatography and mass spectrometry, as well as by other techniques (Table 2). The struc tures of the products support the se quence of reactions of free radicals postulated by Mosier et al. (1). The products formed in the presence of oxygen are complex and result pri marily from the reaction of oxygen with radical intermediates. A reaction se quence (Eqs. 5-8) is postulated for the formation of mcthyl-2,2-bis(p-chlo rophcnyl) acetate, a photooxidation product of DDT in methanol with ox ygen (2800 A). The reaction sequence (Eqs. 5-7) follows that suggested for pentachloroethane photooxidation (2). A similar sequence could give rise to p,p'-dichlorobenzophenone (Eqs. 9-12). Solvent molecules may be involved in the re actions and add to the reactive inter- DSW 201622 STLCOPCB4059689 <* Tablc 1. Percentage yields of irradiation products of p,/?'-DDT in methanol (1 g/litcr); R is p-C.iH.Cl. Irradiation (hour) Yield (%) R.. R* R. CHCCI, CHCHC1, CHCl'PCl a mcthanolic solution of DDE (wave length > 2600 A) (3). The fluorcne structure is also postulated for other irradiation products, for example, (4) and (6). The planarity of DDE prob ably facilitates cyclization to a five- givawsim n a decarbonylation product also detected among DDT and DDE photolysis products. Further reaction of primary photo lysis products complicates the overall 1 76 8 4 membered carbocyclic ring. The photo- picture but affords information on pos 2 61 11 5 cyclization of 1,1-diphenylethylenes has sible degradation pathways. Oxidation 4 44 13 6 .not previously been recorded. products of 3,6-dichlorofluorenone (7) 8 24 15 6 kshlocobeszophw' and (8) were detected. Polychlorinated jfCR?f^swSfi5^mp6rtah'f*iphotcxidation.f phenols, dichlorobcnzoic acid, and a <^roducfc^fKDD'K*s^ritfv-11M3f?7probably trichlorobcnzophenone indicate that mediates to form compounds such as ^6?W?8^^^^^^1iSm<tmiit:s7mTlar'to these compounds react with liberated (1) (Fig. 1). chlorine radicals.. When irradiated in methanol under JiwTne^fF^^^ffiay'alterhatively 'de*^ We used a 450-watt mercury lamp in nitrogen, DDE undergoes reductive de- ^gompose-.-orTeabF^vith'^olvent. : Such a water-cooled quartz housing to irradi- chlorination by a free-radical mecha- ^eactions~probabty''S'ccStfht^f6r'tfie for- ate methanol solutions of DDE or DDT nism. In oxygen, DDE also undergoes >mation*-of chlorobenzOte'-aHcir^methyr (about 1 g/liter). A suitable filter aphotocyclizationreaction. Wehave <M8W>bSni*ate,*'*'* chloropfienbls?^and'''' eliminated light of short wavelengths obtained a 10percent yield of3,6-di- otfaer--fragmenTatfbh* products.-^Photo-* (Corex > 2600 A or Pyrex > 2800 chlorofluorenone (3) by irradiation of ^CRidatiomcrf^vp^diclfrdriaf-iehzophehone" A). Oxygen or nitrogen was bubbled through the solution in separate experi- . ments. Table 2. Photolysis products of DDT and DDE in methanol; R is ClCcH,. The compounds listed represent the volatile components of the photolyzate separated by gas chromatography; m, present in moderate amount; +, detected; --, not delectable; Ph, phenyl. Products were obtained by chroma tography of the methanolic solution after it was concentrated in a rotary m/e Structure DDT O- N, DDE O- N- evaporator. 3,6-Dichlorofiuorenone, which crystallized from the reaction mixture, was removed by filtration. The Neutral fraction remaining syrup was dissolved in ether. 136 PhCO.CH, 140 RCHO 170 RCCLCH, 180 Ph.C = CH, 184 RCITC02CH, 204 (2) Acids and phenols were removed by + + + + + m extraction with sodium hydroxide. The m sodium hydroxide extract was acidified, and the free acids and phenols were + - extracted into ether. Acids were then 214 PhRC = CH, + + extracted from the ether w'ith sodium solution. Portions of the ^ Ph,CHCO=CH, -- + -- -- free acids and phenols thus obtained 232 PhRCHOCH, + + . -- - were methylated with diazomethane be 236 R.CH, 248 R,C = CH, 248 (3) 250 R:CO 260 PhRCHCCLCH, 266 R.CHOCH, 280 (4) . ' . + --. -- + m + -- '+ -- m + + -- + --. ' m' + + + + fore being subjected to gas chroma ' + tography. m The neutral extract was fractionated by chromatography on a silica column. + Infrared spectra of individual fractions were determined. Each fraction was' 282 R;C = CHd + + T m examined by combined gas chroma 284 PhRCHCHCl, m + -- -- tography and mass spectrometry (4). 284 (5) . -- + We identified a number of products by 294 R.CHCOXH, 314 (6) " 316 R,C = CC1. (DDE) 318 R..CHCHC1, (DDD) 324 R,C(OCH,)CO;CH, (1) 338 Unknown m. +. '-- -- ' -- m m +' --. -- '-- -- + + + -- zn m '+ + -- cochromatography and by comparison with authentic specimens of known physical properties. In many cases, structures could be postulated from mass spectral fragmentation patterns. 352 R,CHCC1, (DDT) ++ The compounds obtained by reductive Acidic and phenolic compounds (methylated) loss of chlorine have fragmentation 142 ROCH, -- * + patterns related to their precursors, 170 RCO,CH, . + + + DDT and DDE. The fragmentation of 176 Cl,.CeH,OCHt + + DDT and DDE has been discussed 204 CLC,,HaCO:CH, + + (5). The number of chlorine atoms 210 Cl,C0HjOCHt 228 (7) 280 (8) 294 R,CHCO,CH, + -- + + + + + -+ + present in the molecular ion of an un known could be deduced from the rel ative peak heights of the . molecular weight (M), the molecular weight plus 68 SCIENCE, VOL. 167 DSW 201623 STLCOPCB4059690 Hydrogen abstraction light . . RaCHCCIa------ RaCHCCIa + Cl - (1) Duplicate Plating of Immune Cell Products: Analysis of Globulin Class Secretion by Single Cells RaCHCCIa + CHaOH------- RaCHCHCla + CH.OH light . , RaCHCHCla------ * RsCHCHCl + Cl (2) Abstract. Antibodies secreted by individual immune cells were collected (3) focally in very thin "original" and "im RjCHCHCl + CHaOH------ RaCHCHaCl + CHaOH (4) print" layers of agar containing the In the presence of oxygen target antigen, sheep erythrocytes. RaCHCCIa + O------- > RaCHCCUOa) (5) Identical treatment of both layers led 2R2CHCCk(6=)------ 2R-CHCCI.-6 + Os R-CHCClaO------ RaCHCOCl + Cl (6) to mirror image patterns of hemolytic plaques. Development of one layer for (7) immunoglobulin M hemolysins and' the R-CHCOC1 + CHaOH------ * RaCHCOOCHa + HCI (8) other for immunoglobulin G hemoly RaCHCCla + Cl------ RaCCCI. + HCI RaCCCla+Oa------ RaCOaCCla 2R=COaCCIa------ 2RaC6CC!a + Oa (9) sins produced unrelated plaque patterns (10) indicating that few, if any, cells simul taneously release substantial amounts of (ID both yM and yG antibodies. RaCOCCla------ > RaCO + CCla (12) (R = p-Cl-C.H.) A comprehensive understanding of the synthetic capacities of individual immune cells depends upon the facility with which the products of single cells 2 (M + 2), and so forth, as 35C1 and Most of the photolysis products may be collected and analyzed. Single 37C1 occur in the ratio of 3:1. Many could be examined with these tech- cell isolation procedures permit such of the oxidation products are known; niques. Compounds of higher molecu sampling, but the numbers of cells that therefore, mass spectral and chroma lar weight, such as those described by . can be examined are relatively few (7). tographic comparisons could be made Fleck (7), are probably present in Fluorescent antibody techniques also with authentic samples. Mass spectral small quantity. The seqence of reac permit reasonable inferences about studies were extended to model com tions of free radicals postulated by immunoglobulin synthesis since they pounds, and the photochemistry of Mosier et al. (1) explains the photo detect residual globulins in or on the p,p'-dichlorobenzophenone was also in lytic pathway. There arc many in cells of fixed tissue specimens (2). vestigated to obtain information on the stances of photolytic fission of a C-Cl Studies of single cells by the isolation further breakdown of photolytic prod bond to form a radical which can sub procedure showed that up to 19 per ' ucts. Substitution of an oxygen atom at sequently react with solvent by hydro cent of immune cells may produce two C-l of the 1,1-diphenylethane system gen abstraction (6). We postulate that, classes of immunoglobulins simultane affords aboundant fragments at m/e in the presence of excess oxygen, pho- ously (7), while with fluorescent anti 139 (3'C1CcH4CO) and m/e 111 tochcmically. generated free radicals body techniques instances of possible (33C1CcH4). from DDE and DDT add oxygen. Re double producer cells have ordinarily Molecular ions were obtained in the arrangement and reaction of unstable averaged around I percent (2). We majority of cases, but difficulty was en intermediates account for the forma now report a new replica-plating proce countered with (1). An intense base tion of the photooxidation products dure which allows duplicate sampling peak at m/e 265 suggested the oxonium which we have identified. of the products of single viable immune, ion structure (1A) presumably formed Jack R. Plimmer cells without necessitating isolation of by loss of a methoxycarbonyl residue Ute I. Kungebiel single cells. from (1). The*presence of this residue Burton E. Hummer Adult female New Zealand White in the isolated material was confirmed Crops Research Division, rabbits were immunized intravenously by the infrared and nuclear magnetic Agricultural Research Service, with 10-ml portions of thrice-washed resonance spectra of the isolated frac Beltsville, Maryland 20705 10 percent sheep erythrocytes on days tion. The compound with m/e 338, which References and Notes 0, 9, 18, and 24. Suspensions of im mune cells (2) were obtained by proc was present in significant quantity in the photooxidation products of DDE, could not be assigned a complete struc ture on the available evidence. How ever, nuclear magnetic resonance, in frared, and mass spectral data suggest that it contains a methyl dichlorofluorenyl carboxylate moiety. Mass spectral data alone were used for assignment of structure where trace quantities were obtained and authentic material was unavailable. 1. F. J. Gunther, Chem. Educ. 22. 238 (1945); E. E. Fleck, J. Anier. Chem. Soc. 71, 1034 (1949); J. Roburn, Chem. Ind. (London) 1555 (1963); A. R. Mosier, \V. D. Gucnzi, U L. Miller, Science 164. 1083 (1969). 2. C. Walling, Free Radicals in Solution (Wiley, New York, 1957) p. 448. 3. J. R. Plimmer and U. I. KJiugcbiel, Chem. Contmun. 648 (1969). 4. Perkin--Elmer GC270 instrument equipped with a 30-m open tubular capillary column (inside diameter ,051 cm, coated with SE 30 on Chromosorb W(scot)]. 5. J. Jdrg, R. Houriet, G. Spiteller, Monatsh. Chem. 97, 1064 (1966). 6. J. R. Plimmer and B. E. Hummer, / Agr. Food Chem. 17, 831 (1969). 29 August 196* DSW 201624 essing the whole spleens of these ani mals on days 5, 6, 7, 8, and 27. The cells were washed by centrifugation, and those from two rabbit spleens were pooled and suspended in 20 ml of cold Hanks solution. Very thin layers of sheep erythrocytes in agar were pre pared as follows. A mixture (at 45C) composed of 18 ml of 1.1 percent liqui fied Bactoagar (Difco) in isotonic 0.05A7 iris-buffered (pH 7.4) Eagle's minimum essential culture medium 2 JANUARY 1970 69 STLCOPCB4059691