Studies on the Melanocyte-Stimulating Hormones of the Neurointermediate Lobe of the American Bullfrog, Rana catesbeiana I Electrophoretic and Chromatographic Separation and Identification of the Intraglandular, Acid Activatable, and Secreted Forms of Melanotropic Peptides WALTON W. DICKHOFF' AND CHARLESS.NICOLL Department
Accepted April 2, 1979 Peptides of the neurointermediate (NI) lobe of the American bullfrog (Rana catesbeiana) were separated by polyacrylamide disc gel electrophoresis (PDGE) and by gel filtration. Those which had melanotropic activity (MSH) were identified using the Anolis skin bioassay. Five forms of MSH were detected on acrylamide gels. One region exhibited characteristics of an acid-activatable aggregate. Two regions had electrophoretic mobilities similar to mammalian a- and P-melanocyte-stimulating hormone. A high molecular weight form, which was also acid activatable, was separated by gel filtration on Sephadex G-50. A secreted form which had electrophoretic properties similar to mammalian /3-MSH, was detected by PDGE of medium from a culture of NI lobes for 1 day. Tritiated amino acids were actively incorporated into the secreted form during incubation of NI lobes. These results indicate that the pars intermedia of the bullfrog contains multiple forms of MSH but it secretes predominantly only one form which resembles mammalian ,&MSH. The gland contains a high molecular weight form which is converted to smaller forms by acid treatment.
Amino acid sequence determinations reveal that pituitary melanocyte-stimulating hormone (MSH) occurs in two fundamental forms: an a-form composed of 13 amino acid residues, and a p-form with 17 to 22 residues. The structures of 15 MSHs from six mammalian species and two species of dogfish indicate a substantial degree of sequence homology among the hormones from different species and among CY-and p-forms (Scott el al., 1973; Lowry and Scott, 1975). A peptide core consisting of the residues His-Phe-Arg-Trp-Gly appears essential for MSH activity (Harris, l%O); most peptides with activity contain this sequence, and this pentapeptide alone has intrinsic MSH activity. Adrenocorticotropin (ACTH) and P-lipotropin (P-LPH) ’ Trainee on USPHS Grant NIH-TOl-GMO-1021 during the course of this work. Present address: Department of Zoology NJ-15. University of Washington, Seattle, Wash. 98195.
contain the active sequence of MSHs within their respective structures and have a small, but significant degree of MSH activity. Adenohypophysial hormones exist in several forms in the pituitary gland and in plasma (see Yalow, 1974), and numerous investigators have detected multiple forms of MSH activity from neurointermediate (NI) lobes of vertebrates (Burgers, 1961, 1963; Thody, 1969; Preslock and Brinkley, 1970a,b; Hoekstra and van der Wal, 1971; Shapiro et al., 1972; Baker, 1973; Loh and Gainer, 1977a). Heterogeneity of MSH peptides may be related to molecular evolution of the family of pituitary peptides consisting of (Y- and P-MSHs, ACTH, and P-LPH. Combinations of these peptides, which are not all functional homologs of MSH, in NI lobe tissue complicate the study of MSH physiology by bioassay alone. The aims of the present study were
313 0016~6480/79/110313-09$01.00/O Copyright 0 1979 by Academic Press, Inc. All rights of reproduction in any form reserved.
to investigate the possible heterogeneity of MSH peptides (identified by bioassay) in the NI lobes of the American bullfrog, Rana catesbeiana, using electrophoretic and chromatographic techniques. In addition, the major form of MSH that is synthesized and secreted in vitro was identified.
to detect neurosecretion in the posterior pituitary. After electrophoresis, gels were removed from the tubes and oxidized for 1 min in 2.5% KMnO,:5% H,SO,:and H,O (]:I:7 by volume). They were then stained with 1% Alcian blue in Walpoles acetate-acetic acid (Lillie, 1965) at pH 5.5 at room temperature for 30 min. GelfiltraCon. To obtain an estimate of the molecular size of the peptides with MSH activity, NI lobe homogenates were subjected to gel filtration. Fifty miiliMATERIALS AND METHODS grams of NI lobe tissue (34 NI lobes) was homogenized Animals. Adult Ranu catesbrianu of both sexes were in 1 ml buffer and centrifuged at 3OOOgfor 15 min at obtained commercially (Western Scientific, Sac- 10”. In order to remove components that might preramento, Calif.; West Coast Aquatic Enterprises, Fol- cipitate in the chromatography column, the supemasom, Calif.; Southwest Scientific, Tucson, Ariz.) and tant was then filtered by suction through a 2-mm-thick were maintained unfed at room temperature in plastic bed of Sephadex G-10 and washed three times with 1 cages (35 x 46 x 20 cm), tilted to contain water at a ml of buffer. Sephadex G-50 fine (Pharmacia) was hydepth of ca. 2 cm at one end. Animals housed for more drated and packed with 0.05 M phosphate buffered than a few days were treated with Terramycin (Pfizer) Anolis physiological salt solution (PSS; Dickhoff, to inhibit bacterial infection. 1977) at pH 7.5. The PSS was used as the elution buffer The American chameleons (Anotis carolinensis). and the diluent for all steps in this procedure. To which were used in the bioassay for MSH activity (see minimize protein absorption onto the gel, 3 ml of 1% below), were obtained from the Snake Farm, La Place, bovine serum albumin was applied to the column and Louisiana. They were kept at room temperature in a two void volumes were collected prior to application of the tittered homogenate. The column was run at 4’ large aquarium and fed Tenebrio larvae and supplied with water ad libirum. The frogs and lizards were in a gravity flow, descending manner. Fractions of 2 ml each were collected and stored at 4” for PDGE, bioaskilled by decapitation. Eleclrophoresis. Proteins of bullfrog NI lobes were say, or protein determination (Lowry et al.. 1951). separated by polyacrylamide disc gel electrophoresis For PDGE analysis of gel eluates, fractions were by ultrafiltration (Amicon, (PDGE; Ornstein, 1964: Davis, 1964). Running gels, 50 pooled. concentrated mm long, were prepared in glass columns of either 5 or UM-05 filter), lyophylized, and redissolved in 3 mm diameter with a large-pore stacking gel of 5 mm Tris-glycine buffer (pH 8.3). In t’itro srudirs. For the organ culture experiments, length. For PDGE at basic pH, a 7.5% running gel with a Tris-glycine buffer (pH 8.3) system was used; col- NI lobes were collected and cultured in dilute umns were stacked at pH 8.9 and run at pH 9.5. For Waymouth’s medium (Grand Island Biological Co.) containing antibiotics and insulin (Waymouth’s, 7 ml: PDGE at acid pH, a 10% running gel with a p-alanine buffer (pH 5.0) system was used (Gabriel, 1971); colsterile distilled water, 3 ml: penicillin, 50 U/ml; strepumns were stacked at pH 5.0 and run at pH 4.3. tomycin, 50 &ml; Polymixin, 1 /*g/ml; Kanamycin, 10 Pooled or individual glands (weighing l to 2 mg CLg/mI:Gantrisin. l &ml: insulin, 1 &ml). Three NI lobes were placed on lens paper rafts on stainless-steel each) were homogenized in large-pore gel and were applied to the top of columns. For 3-mm-diameter colgrids in contact with 1 ml of medium in a 3.5-cmumns, 1 mg of tissue in 0.3 ml was used: for those of 5 diameter plastic petri dish. The small petri dishes were mm diameter, 3 mg in 0.5 ml was used. Samples of placed on damp titter paper in a 9-cm-diameter petri culture medium were prepared for electrophoresis by dish. The dishes were placed in a water-jacketed incubator at 30°C and gassed with a humidified mixture of adding a few crystals of sucrose to produce sufficient 95% 0,:5?? CO,. At termination of the culture. tissue density for overlayering with large-pore gel. Columns and medium were stored separately on dry ice until they were run at room temperature with a current of 2-3 mA each. After electrophoresis. gels were stained in were processed by PDGE. 0.1% aniline blue black (w/v) in 7.5% acetic acid for 2 In the short-term incubation study, six NI lobes hr or more. Columns were then destained in a Shandon were collected, weighed, and individually placed in 0.3 transverse destainer with 7.5% acetic acid. Unstained ml of medium. Medium was prepared by diluting 7 ml gels were cut into 20 sections of 2 mm length each to Earle’s physiological saline (Grand Island Biological provide eluates for bioassay (see below). Co.) with 3 ml distilled water. Tritiated amino acids Sulfhydryl staining for neurophysins. In order to (protein hydrolysate-yeast profile; Schwartz/Mann) determine whether any of the proteins that were sepa- were added to incubation medium at a concentration of rated by basic PDGE might be neurophysins from the 2 @Ci/ml. Medium was placed in plastic tubes inside 20 pars nervosa, some columns were stained by an adapml screw-cap vials which contained 1 ml of distilled tation of the method used by Adams and Sloper (1956) water to reduce concentration of medium by evapora-
tion. The medium was gassed with a humidified mixture of 95% 0,:5% CO*. Incubations were carried out for 6 hr at 25” in a gyratory metabolic shaker. Medium was removed at 1 hr and replaced with fresh medium. At termination of incubation, medium and tissue were separated and stored on dry ice until processing by PDGE. Regions of PDGE gels containing prominent, stainable bands and the segments between them were cut out and dissolved in 0.3 ml 30% H,O, at 45” overnight in tightly capped vials. Scintillation fluid (dioxane, 730 ml: toluene, 135 ml; methanol, 35 ml; napthalene, 100 g; 2,S-diphenyloxazole, 5 g) was added to vials containing dissolved gels at room temperature. Counting was performed in a Beckman LS-100 scintillation counter at efficiencies ranging from 35 to 46%. Bioassay. Bioactivity of MSH in the samples was estimated by an in vitro light reflectance adaptation (Dickhoff, 1977) of the Anolis skin assay procedure of Bjbrklund et al. (1972). Slices of PDGE columns were immersed in Anolis PSS overnight at 4”. The eluates were adjusted to pH 7.0 with 1 N HCI and 1 N NaOH. For experiments dealing with acid activation of MSH bioactivity, tire eluates from basic PDGE or Sephadex chromatography were separated into two groups; one was adjusted from pH 7.0 to pH 3.0 with 1 N HCl and the volume of the other was adjusted similarly with PSS (pH 7.0). After standing for 30 min at room temperature the pH of the acidified eluates was adjusted to 7.0 with 1 N NaOH and the samples were bioassayed.
RESULTS Electrophoretic Analysis of Pars Inter-media Tissue
Results of bioassays of MSH in eluates from PDGE at basic pH (9.5) of pooled NI-lobe homogenate are shown in Fig. 1. In the eluates that were not acidified, five regions of activity (labeled Bl through B5) were found. They had relative flow (&) rates of 0.16, 0.4, 0.56, 0.67, and 0.8, and contained 10, 10, 20, 50, and lo%, respectively, of the total MSH activity that was eluted from the column. Mammalian /3-MSH had an R, of 0.65 in this PDGE system. No MSH activity was detected in the other segments of the column between these five regions. The effect of acidification of aliquots of these eluates to pH 3 on their MSH bioactivity after subsequent readjustment to pH 7 is also shown in Fig. 1. Regions B2 through B5 did not show any change in MSH bioactivity as a result of acidification,
FIG. 1. Melanocyte-stimulating hormone (MSH) activity of proteins eluted from polyacrylamide disc gel electrophoresis of bullfrog neurointermediate lobe homogenates before and after acidification of the eluates. Acidification involved adjusting the pH of the eluate from 7 to 3 then back to 7. The columns were cut into 20 sections and no MSH activity was detected in the regions which lack vertical bars. Note that the acid treatment has caused activation of the MSH bioactivity of only the most slowly migrating form.
but the slowest migrating region (Bl) showed a fivefold increase in activity. Thus, the percentage distribution of the hormonal activity in regions Bl through BS after acidification became 37,7.4, 1,37, and 7.4, respectively. Testing for sulfydryl groups in the basic PDGE columns with Aician blue after oxidation and rupture of disulfide bonds disclosed that only one band (BS, Fig.1) was stained. Hence, this band may be neurophysin(s), since these proteins are characterized by a high content of cystine (van Dyke et al., 1942). None of the forms of MSH which have been sequenced contains this amino acid. The isoelectric point of a-MSH (10.5; Harris and Lerner, 1957) precludes its migration into the gel at pH 9.5. Hence, it is unlikely that any of the five fractions of the basic system (Fig. I) is the (Y form of the hormone. It was of interest, therefore, to determine the number of MSH-active forms that could be separated and identified on an acidic (pH 4.5) PDGE system and to determine if any of these forms would corre-
was found at this Rf on the acidic gels when NI-homogenate was electrophoresed directly (Fig. 2). Eluates from the B3 region yielded no detectable bands when electrophoresed on acidic gels. This finding indicates that the material in band B3 would not migrate into the acidic gels, or if it did migrate, it was not present in sufficient quantities to appear as clearly stainable band. 0.47 0.97 F The peptide in the eluate of region B4 d Relatwe mobility (Rt) migrated with an Rf of 0.5 on acidic PDGE FIG. 2. Melanocyte-stimulating hormone (MSH) (Fig. 3), which corresponds to band A2 of activity of eluates from acid polyacrylamide disc gel electrophoresis of bullfrog neurointermediate lobe NI tissue homogenate which was subjected homogenates. The gels were cut into 20 regions and directly to acidic PDGE. Thus, the staintested for MSH activity, which was found only in the 4 able protein of band A2 also corresponds to regions with vertical bars above them. mammalian /3-MSH. The eluate of band B2 gave a stainable band on acidic gel with an spond to mammalian a-MSH. Eluates from Rf of 0.2 (Fig. 3) which corresponds to reacidic PDGE of bullfrog NI-lobe homogegion Al of the MSH-active fraction of the nate exhibited four regions of MSH bioac- acidic electrophoresis of the NI homogetivity (Fig. 2) which were designated Al nate . Acidic PDGE of eluates from Bl resulted through A4. The percentage distribution of MSH activity of these regions was 22, 22, in four stainable bands (Fig. 3). Three of 11, and 4.5, respectively. Synthetic a-MSH these bands had Rf values that correhad an R, of 0.97 on the acid gel system. sponded to bands Al, A2, and A4 of the NI Hence, its mobility corresponds to that of homogenate that was directly run on acidic band A4 from the tissue homogenate. PDGE, and the fourth had an Rf of 0.13, To determine the relationships among the corresponding to the band that appeared of region B5 forms of MSH that were separated by the after acidic reelectrophoresis two electrophoretic systems, eluates from and which may be neurophysin (see above). basic PDGE gels were subsequently rerun This finding suggests that band Bl from the on acidic PDGE. Eluates of region B5 re- basic gel is an aggregate of bands B2, B4, sulted in a protein band with an Rf of 0.13 and B5, which is dissociated by elecon the acidic gel (Fig. 3). No MSH activity trophoresis in the acid system, since bands
10.20 ’ ’-ok-5 ’t
run on acid
FIG. 3. Effect of reelectrophore :sing eluates from basic PDGE of bullfrog NI on acid PDGE .
MSH OF BULLFROG
B2 and B4 yielded bands Al and A2, respectively, when they were reelectrophoresed on acidic PDGE. Chromatographic Analysis The molecular size of the different forms of MSH and the presence of acidactivatable forms was further investigated by bioassay of effluent fractions from gel filtration of bullfrog NT homogenate on Sephadex G-50 (Fig. 4). In the fractions that were maintained at pH 7, no MSH activity was detected in those that emerged near the void volume, but the hormonal activity was found in fractions 20 to 65, with peaks in activity at Ve/Vo values of 1.06 and 1.20. Acidification of fractions 20 through 65 (i.e., adjustment from pH 7 to 3, then back to pH 7 after 30 min) did not change their MSH activity. However, the acid treatment resulted in the appearance of a large peak of MSH activity in the previously inactive fractions that emerged just behind the void volume (i.e., fractions 1 through 18). Electrophoresis of aliquots of selected chromatographic fractions that had been main-
tained at pH 7 on the basic pH PDGE system demonstrated that the MSH peaks in fractions 45 and 52 gave rise to bands B4 and B5, respectively. The fractions that emerged behind the void volume gave rise to a band corresponding to B5 on the basic PDGE.
Identification of the Synthesized and Secreted Forms The stained protein patterns obtained after the basic PDGE of the incubated tissue and medium from a 24-hr organ culture of NI lobes are shown in Fig. 5. Bands Bl and B5 were evident in the cultured tissue but in comparison to unincubated tissue (Fig. 1) bands B2-B4 were very faint. In the culture medium only band B4 was present in significant amounts (Fig. 5). Thus, the form of the bullfrog MSH that has electrophoretic mobility similar to mammalian /3-MSH is secreted in significant quantities. Secretion in vitro apparently results in substantial depletion of the hormone from the tissue. Incubation of NI lobes for 6 hr in medium containing 3H-amino acids resulted in incorporation of radioactivity primarily in band B4 in the tissue and the medium (Fig. 6). After 1 hr of incubation the radioactivity in band B4 of the medium was similar to that in the tissue at 6 hr. By the sixth hour of incubation the disintegrations per minute in band B4 of the medium were about threefold higher than those in the tissue at the same time. Since the level of radioactivity in the other regions remained very low, these results indicate that the B4 form 60 t saltpeak of bullfrog MSH is actively synthesized and secreted in vitro. DISCUSSION
FIG. 4. Distribution of MSH activity and protein in chromatographic fractions from a Sephadex G-50 column through which a homogenate of bullfrog neurointermediate lobes was filtered. The effects of aciditication and neutralization of the fractions (i.e.. adjustment of the pH from 7 to 3 then back to 7) are shown. Note that acid activation of MSH bioactivity occurred only in the high molecular weight fractions.
The separation of peptides with MSH activity on acidic and basic acrylamide gel systems demonstrates the multiplicity of forms of the hormone in the bullfrog pars intermedia. Comparison of the relative mobilities of the peptide bands with purified mammalian MSHs allows a tentative iden-
O-l hr nmdiun
FIG. 6. Total radioactivity (less background) of tritiated amino acids incorporated into band B4 during a 6-hr incubation of neurointermediate lobes.
not give rise to an identifiable or corresponding band in the acidic PDGE system. None of these bands (i.e., B2-Al or B3) FIG. 5. Electrophoretic pattern of bullfrog appear to correspond to either mammalian neurointermediate (NI) lobe and medium after 24-hr (Y- or /3-MSHs. The significance of these organ culture of NI lobes. forms of peptide with melanotropic activity in the bullfrog pars intermedia remains to be determined. This stained protein (Al) on tification of two of the bullfrog melanotrothe acid gel may represent a non-MSH pins. The similarity in electrophoretic moprotein (such as neurophysin, see above) bility of band A4 and synthetic (w-MSH which comigrates with a minor component suggests that this form may be the homolog of mammalian (r-MSH. Similarly, the fact having MSH activity in the B5 region of the basic gels. The hormonally active material that band B4 comigrates with a mammalian may have been present on the acid gel in an P-MSH on basic PDGE indicates that the amount that was insufficient to detect as a bullfrog NI may have an MSH comparable stainable band. to this mammalian form of the hormone. The relationships between the electroThe incubation experiments with the NI lobes demonstrated that the presumed /3- phoretically separable forms of MSH and form of bullfrog MSH is actively synthe- those detected in eluates from gel filtration sized and secreted in vitro. Our results do demonstrate that bands B4 and B5 are of not allow any conclusions to be made about relatively low molecular weight and that the acid activatable form of MSH is of large the degree of secretion of the presumed molecular size since it is eluted near the a-form because we have not processed void volume. The low electrophoretic momedium on acidic PDGE. Comparisons of the relative mobilities of bility of the acid activatable form that was on basic PDGE (band Bl) the MSH bands in the two gel systems pro- separated vides additional information on the re- suggests that it may be of high molecular lationships among the various forms of the weight also; thus, it may correspond to the melanotropic peptides. Band B2 appears to large form identified by chromatography. correspond with band Al but band B3 did This suggestions receives support from the
observation that the electrophoresis of Bl eluate on the acidic PDGE system gave rise to several components with MSH activity (Fig. 3). However, basic PDGE of the high molecular weight chromatographic fraction yielded only band B5, which may be neurophysin. Hence, if the high molecular weight chromatographic fraction is similar to or identical with the slow migrating electrophoretic fraction (i.e., Bl) we may not have applied a sufficient amount of the material to the basic PDGE gel columns to allow detection of all of the forms of MSH present in the aggregate. Electrophoresis of the more retarded chromatographic peak also gave rise to band B5 only. This result suggests that if the protein identified in the BS region is indeed neurophysin, it may have the capacity to bind or associate with MSH peptides in both high molecular weight and lower molecular weight forms. The pentapeptide core with MSH activity (Harris, 1960) is contained within the structures of the pituitary hormones (Y- and /3-MSH, ACTH, and P-LPH. Therefore, one would expect that separation and bioassay of pituitary peptides should result in multiple forms with MSH activity. The high resolving power of PDGE makes this technique particularly suitable for separation of these peptides. The five forms of melanotropic peptides detected in the present investigation of bullfrog NI lobes constiiute a greater number of such forms than reported by other workers using PDGE for the leopard frog, Rana pipiens (Preslock and Brinkley, 1970a), or for the laboratory rat. (Baker, 1973). However, Loh and Gainer (1977a) have recently presented evidence indicating I1 melanotropic peptides on acid-urea acrylamide gels of NI lobes of the African clawed toad, Xenopus laevis. Burgers (1963) used starch gel zone electrophoresis to separate four forms of bullfrog MSH. Variations of the forms of peptide and protein hormones have been the subject of several recent reviews (Lowry and Scott,
1975; Lowry et al., 1977; Orth and Nicholson, 1977). The bases of these variations may be summarized into four categories. Primary amino acid sequences may differ, as in the case of (Y- and P-MSH. Larger peptides may represent precursors or prohormones, as has been shown for several peptide hormones (Yalow, 1974). In addition, peptide hormones are sometimes bound to carrier proteins, as is apparent with posterior pituitary nonapeptides and their neurophysins (Sachs et al.. 1969). Finally, hormones may be bound together in the form of aggregates (Steiner, 1973). Variation in amino acid sequences of hormones is most likely a consequence of genetic mutations during protein evolution. Prohormones and hormones bound to themselves (aggregates) or carrier proteins may play central roles in the cellular processes of synthesis, packaging, storage, and secretion. Prohormones or hormones which are aggregated or bound to carrier proteins may have little or no biological activity since active sites of the hormones may not be available to bind with receptors on target tissues. Techniques which would convert prohormones, disperse aggregates, or dissociate the hormone from a carrier would reveal increases in biological activities of test samples containing such forms. Dasgupta et al. (1967) have demonstrated that biological activity of as much as 80% of rat pituitary ACTH is only measurable after treatment with acid. Activation of MSH has been shown to occur in this study by acid treatment of not only band Bl eluates from PDGE, but also the void volume fraction of Sephadex G-50 gel filtration. Presumably, acid treatment may dissociate melanotropic peptides from either a carrier protein or an aggregate form. The probability that MSH may exist in a bound form is suggested by the observation that band Bl on gels of bullfrog NI lobes when run on acid gel is dissociated into multiple bands corresponding to regions of MSH activity. Evidence for binding proteins in pituitary cells
has been presented for ACTH (Nakamura and Tanaka, 1969) and MSH (Namiki et al., 1969). There is insufficient information in the data presented here to distinguish which of several possible mechanisms may be involved in the acid activation of MSH bioactivity. An alternative to the interpretation of acid activation by dissociation of noncovalently bound MSH might involve conversion from ’ a larger peptide prohormone. It is doubtful that the relative mild acid treatment would cleave peptide bonds of a pro-MSH. It is possible, however, that an acid-activatable enzyme capable of converting pro-MSH to an active form may be present in Sephadex G-50 void volume eluates which exhibited acid activation of MSH activity. Scott et af. (1973) have proposed that wMSH is formed by enzymatic cleavage of the 13 residue chain of the amino terminus of ACTH. These workers have succeeded in isolating both a-MSH and an additional peptide fragment, ACTHIs-““, from rat and pig pituitaries. By analogy to this case of ACTH and a-MSH, it is possible that /3-LPH may be a prohormone for P-MSH. Additional evidence for the existence of a precursor storage form of MSH in the anuran pars intermedia has been presented in experiments dealing with synthesis of MSH (Loh and Gainer, 1977b; Jenks et al., 1977). Jenks (1978) has suggested that the storage form of MSH is of high molecular weight. Our observations with the unretarded material from Sephadex chromatography and the slow migrating fraction from basic PDGE, both of which were acid activatable, are consistent with this suggestion. However, the relationships among these different forms of bullfrog MSH, and their physiological significance, remains to be determined. ACKNOWLEDGMENTS We are indebted to Dr. J. Ramachandran for the o-MSH, to Dr. V. F. Thornton for the P-MSH, and to Dr. J. W. Crim for his help in the final preparation of the manuscript. This study was supported by NSF
Grants GB-42687 and PCM 76-14772. by NIH Grant AM-13605, and by funds from the Committee on Research of the University of California at Berkeley.
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