Proc. Natl. Acad. Sci. USA Vol. 81, pp. 578-582, January 1984 Medical Sciences A bombesin immunoreactive peptide in milk (radioinimunoassay/gel filtration/high-performance liquid chromatography) GLORIA D. JAHNKE* AND LAWRENCE H. LAZARUSt Peptide Neurochemistry Workgroup, Laboratory of Behavioral and Neurological Toxicology, National Institute of Environmental Health Sciences, Research Triangle Park, NC 27709 Communicated by George H. Hitchings, September 19, 1983

ABSTRACT Immunoreactivity to the amphibian peptide tains peptide and steroid hormones (32), prostaglandins (33), bombesin was found in instant nonfat dry milk (ca. 0.7 ng/ml) morphine (34), melatonin (35), and immunoglobulins (36) in and in the whey of whole or skim bovine milk (ca. 1.2 ng/ml) addition to a growth factor and enzymes (37, 38). even after ultracentrifugation. The soluble immunoreactivity Moreover, the peptide hormones and the growth factor re- was associated with a peptide exhibiting the following charac- tain their biological activities in milk (37, 39, 40) and, when teristics: (i) parallel displacement in an immunoassay using an ingested by the neonate, appear intact in plasma (35, 39-42). antiserum recognizing bombesin amino acid residues 5-8; (ii) Thus, considering the coincidence between the action of am- separation from both gastrin-releasing peptide and amphibian phibian bombesin and milk on hormone peptide levels, we bombesin by gel filtration-the approximate Mr was 3,200; sought to determine whether milk could be a source of a (iii) denaturation in urea, reduction by dithiothreitol, and bombesin-related peptide. In this communication, we pro- acetylation by iodoacetamide had no effect on its elution profile vide evidence that milk contains bombesin immunoreactivity by gel-filtration chromatography and the aggregation of added that is distinct from both GRP and amphibian bombesin and bombesin to milk proteins or peptides was not observed; (iv) is associated with a heat-stable, protease-sensitive peptide reversed-phase HPLC separated milk immunoreactivity from with a Mr of about 3,200. gastrin-releasing peptide and bombesin; (v) digestion by tryp- sin yielded a smaller immunoreactive peptide fragment, MATERIALS AND METHODS whereas nearly all immunoreactivity was lost by treatment and whole milk from several differ- with a-chymotrypsin; and (vi) the level of immunoreactivity Milk. Pasteurized skim milk is an ent local dairies were purchased 5-7 days before the expira- was unaffected by boiling. These data show that tion date. Instant nonfat dry milk was the product of several exogenous source of bombesin-like immunoreactivity,, which reconstituted according to the manu- may account for the increase of gastric acid and gastrointesti- major companies and of milk. facturer. A portion (200 ml) was acidified to pH 2.3 with for- nal hormone levels after the consumption mic acid and brought to 80% in cold acetone according to Brown et al. (2) for the extraction of bombesin from plasma. Immunoreactivity to bombesin, an amphibian tetradecapep- The extract was clarified by centrifugation (20,000 x g for 30 tide (1), is distributed throughout mammalian tissues (2-5). min at 40C), and the supernatant was decreased in volume Bombesin administered through intraperitoneal or intracis- under vacuum and clarified again by centrifugation. The vol- ternal injection or intravenous infusion elicits diverse phys- ume of this supernatant was then further decreased under iological effects in a variety of animal species, including hu- nitrogen at 500C. mans (6-12). These effects include hypertension (1, 6, 13, Radioimmunoassay (RIA). [Tyr8]Bombesin and GRP were 14), satiety (for review, see ref. 15), change in sugar metabo- iodinated every 4-6 weeks as described (43). The production lism (16, 17), hypothermia (18), modulation of the level of of the antiserum to bombesin has been described (44). Op- many gastrointestinal-associated peptide hormones (6-12, timum RIA conditions, which were systematically deter- 19-24), and increase of gastric acid secretion (7, 10, 13, 14, mined, are as follows: 0.1 M sodium cacodylate, pH 5.6/bo- 19, 20). Bombesin also releases both gastrin and gastric acid in vine serum albumin (5 mg/ml)/125I-labeled [Tyr8]bombesin from isolated fundic mucosa (25) and acts as a spasmogen (or GRP) (2,500 cpm)/antiserum BM-XII-165-4 (final dilu- isolated smooth muscle preparations (1, 6, 13, 14, 26). In tion, 1:4,000)/standard or test solution in a total vol of 100 keeping with these observations, specific bombesin-contain- ,ul. Nonionic (Triton X-100, Nonidet P-40) and ionic (sodium ing cells were identified by immunohistochemistry in the lauryl sulfate) detergents above 0.1% and chaotropic agents gastric mucosa (27). Furthermore, an endogenous bombesin- (urea, guanidine HCl) above 0.1 M substantially decreased like gastrin-releasing peptide (GRP) was isolated from mam- binding of the labeled peptide to the antibody. The antibody- malian non-antral tissue (28), and its pharmacological mode bound ligand was precipitated after a 16-18 hr incubation at of action is similar to bombesin at comparable dosages (17, 40C with 1.0 ml of 15.6% polyethylene glycol 8000 in the 24, 27, 29). presence of 0.05 ml of outdated human plasma (44). Based Infusion of bombesin brings about an increase above basal on the relative crossreactivity of sequence-related peptides plasma levels of the peptide hormones insulin, gastrin-inhib- (Table 1) this antiserum apparently recognizes the mid-re- iting peptide, neurotensin, pancreatic polypeptide, gastrin, gion of bombesin (residues 5-8). The oxidation of trypto- motilin, enteroglucagon, secretin, and growth hormone (6- phan-8 also decreased binding of bombesin to the antibody. 14, 19-23); these are the same peptides that increase after Alytesin, used as the amphibian analogue of chicken proven- the consumption of milk in human neonates (for review, see tricular GRP (45), and porcine GRP produced nonparallel ref. 30). Bombesin also increases the levels of somatostatin displacement curves relative to bombesin (Fig. 1). Other (22) and prolactin (9). Milk, which is a rich nutrient meal, is known peptides, such as physalaemin, neurotensin, and bra- known for its caloric components (protein, carbohydrates, it also con- fat) and micronutrients (vitamins, minerals) (31); Abbreviations: RIA, radioimmunoassay; GRP, gastrin-releasing peptide (bombesin-like heptacosapeptide). The publication costs of this article were defrayed in part by page charge *Present address: School of Veterinary Medicine, North Carolina payment. This article must therefore be hereby marked "advertisement" State University, Raleigh, NC 27650. in accordance with 18 U.S.C. §1734 solely to indicate this fact. tTo whom reprint requests should be addressed. 578 Downloaded by guest on September 24, 2021 Medical Sciences: Jahnke and Lazarus Proc. Natl. Acad Sci. USA 81 (1984) 579

Table 1. Specificity of antiserum to bombesin Relative molar Peptide Structure crossreactivity Bombesin pGlu-Gln-Arg-Leu-Gly-Asn-Gln-Trp-Ala-Val-Gly-His-Leu-Met-NH2 1.00 [Tyr']Bombesin Tyr 1.00 [Tyr8]Bombesin Tyr 0.73 [Tyr5]Bombesin-(5-14) Tyr 0.18* Alytesin Gly Thr 2.62 ± 0.87 (5)* GRPt -Met-Tyr-Pro-Arg His 1.34 ± 0.14 (6)* Ranatensin pGlu-Val-Pro Phe 0.0003 Litorin pGlu Phe 0.0008 Substance P Arg-Pro-Lys-Pro-Gln-Gln-Phe-Phe-Gly 0.000003 Peptide concentrations ranged from 0.1 fmol/ml to 1.3 nmol/ml. Relative molar crossreactivity is based on 50% displacement point from log- logit plots; n value in parentheses when more than two repetitions. *Nonparallel displacement curves. tThe sequence of porcine GRP preceding the bombesin-like region is Ala-Pro-Val-Ser-Val-Gly-Gly-Gly-Thr-Val-Leu-Ala-Lys- (28). dykinin failed to displace the radioligand at concentrations of RESULTS 10 nmol/ml. Quantitation. Whole, skim, and reconstituted dry bovine Chromatography. Gel filtration of milk extracts used Bio- milk, whey, and the acid/acetone extract produced displace- Gel P-4 (100-200 mesh) columns (1.5 x 94 cm) equilibrated ment curves in the RIA parallel to that of bombesin (Fig. 2). with 0.1 M formic acid. The immunoreactive peak from this To rule out the possibility that the bombesin immunoreactiv- column fractionation was concentrated, absorbed onto and ity could be extracted by formic acid from a bacterial con- eluted from a Sep-Pak with methanol, and injected onto a taminant [Escherichia coli has been found to contain insu- reversed-phase HPLC system [Laboratory Data Control unit lin (47)], lipids, or leukocytes (38), milk was centrifuged fitted with a Rheodyne injector with a Spherisorb ODS 5-gm (100,000 x g for 30 min at 4°C) before assaying; the displace- (250 x 4.6 mm) or a Bio-Sil ODS 5-,um (150 x 4 mm, end- ment curve of the resultant whey was superimposable on capped) column]. The samples were eluted with linear gradi- that of the original milk sample (Fig. 2). The level of bombe- ents of either 0-60% or 0-80% acetonitrile (vol/vol)/0.01 M sin immunoreactivity in whole and skim milk was similar, trifluoroacetic acid, pH 2.3. Fractions were collected at 1- about 1.2 ng/ml in bombesin equivalents, and that in recon- min intervals at a flow rate of 1.0 ml/min; a 5-min delay was stituted dry milk was about half of this amount (Table 2). The used before initiating the gradient. To eliminate any possible total amount of immunoreactivity recovered in the acetone contamination of the equipment, blank gradients were as- extract, however, was only 17.5 ± 6.9% (n = 4). The boiling sayed by RIA as described (44). As a further precaution, the of whole milk to inhibit proteases that could interfere with injector and column were flushed with acetonitrile between the RIA (48) gave identical results. The recovery of 6.2 pmol analyses and washed daily with water, acetonitrile, metha- of bombesin added to whole milk was 90.3 ± 20.6% (n = 4). nol, and chloroform/methanol 2:1 (vol/vol). The replacement of labeled bombesin by iodinated GRP in Chemical and Enzymic Treatment. Milk extract was ad- the RIA also resulted in nonparallel displacement curves by justed to 8 M in urea in the presence of 10 mM dithiothreitol the heterologous peptide. In this case, however, whole milk and reduced with iodoacetamide according to Boat et al. (46) caused a concentration-dependent precipitation of labeled before chromatography. The immunoreactive peak from the GRP (data not shown). P-4 columns was neutralized and lyophilized, dissolved in Gel Filtration. Chromatography of the acid/acetone ex- 0.1 M Tris HCl, pH 7.8-8.2/2 mM CaCl2, and digested with tract of milk consistently gave a single symmetric peak of 50 ,ug of a-chymotrypsin or L-1-tosylamido-2-phenylethyl immunoreactivity of Kay 0.07, which corresponded to an ap- chloromethyl ketone-treated trypsin for 16-18 hr at 35°C. parent Mr of 3,200 (Fig. 3). This bombesin-containing mate- Enzymic activity was terminated with a 5-fold excess of soy- rial was completely separated from immunoassayable por- bean trypsin inhibitor and the sample was acidified with for- cine GRP (Ka,, 0.24; radioiodinated GRP was slightly retard- mic acid to a final concentration of 1.0 M. Dry milk, volume (,ul) 1.0 10 100 Whole milk and extract, volume (Al)l 1.0 10 100 90 1 90 80- r__ 70- X60 x 60 X70 ; 50 40 C 30- 40- m 20 ~30- 20- 10- 10-

1 ...... ,, , , , , , ..... 10 100 1,000 10 100 1,000 Peptide, fmol Bombesin, fmol FIG. 1. Radioimmunoassay of bombesin and bombesin ana- FIG. 2. Bombesin immunoassay of milk. The data are expressed logues. The data are presented as a log-logit plot of the ratio of as in Fig. 1. The symbols represent the following: bombesin (o), bound (B) to unbound (BO). The symbols represent the following: whole milk (o), supernatant of centrifuged milk (o), reconstituted bombesin (e), alytesin (A), and porcine GRP (o). dry milk (A), and the acid/acetone extract (o). Downloaded by guest on September 24, 2021 580 Medical Sciences: Jahnke and Lazarus Proc. Natl. Acad Sci. USA 81 (1984)

Table 2. Level of bombesin immunoreactivity in bovine milk Milk Concentration, ng/ml* 2 Whole 1.17 ± 0.89 (6) 0. --84 Skim 1.12 ± 0.37 (3) Instant, reconstitutedt 0.68 ± 0.21 (5) 0 *Equivalents ofbombesin tetradecapeptide (±SD). n value in paren- theses. 0 tReconstituted at 3.2 oz/quart (97.4 g/liter). 0

ed relative to GRP with Kav = 0.29). The trypsin-treated 0 a) immunoreactive material eluted in the region of lower molec- 00 ular weight peptides (Fig. 3). Digestion with a-chymotrypsin 64 0 *a eliminated =95% of the immunoreactivity and no discern- bo ct 1. C) able peak eluted from the P-4 column. Cd

To verify that the immunoreactivity existed in a single Ce peptide chain, rather than as an aggregate or a sulfhydryl 0 0 crosslinked molecule, two experiments were carried out. In one, a portion of the acetone extract was treated with urea, 0. 0 then rechromato- I dithiothreitol, and iodoacetamide (46) and ol0~~~~~~~~~,] -1L , 0 1.5 0 _ C

Cut - 1.0 I 10 0° 0 10 20 30 40 50 E v Retention time, min 04 0.5 O& FIG. 4. Reversed-phase HPLC separation of milk bombesin (Lower) and porcine GRP (Upper). The peptides were absorbed onto a Spherisorb ODS 5 ,um column and eluted with a linear gradi- ent of 0-80% acetonitrile (vol/vol) in 0.01 M trifluoroacetic acid at a I .0Cd o 0 flow rate of 1.0 ml per min and 2% gradient development per min. After removal of acetonitrile, the fractions were lyophilized and dis- solved in the RIA buffer before assaying. , 0.45i- I I I I 0. graphed. The peak of bombesin immunoreactivity eluted with the same Kav as the original untreated sample (Fig. 3). .,O 0.30 In the other, 6.2 pmol of bombesin was added to milk to +-C_ serve as an internal control for possible aggregation during extraction with acid and acetone: milk immunoreactivity and 0 bombesin were clearly separated (data not shown). r. 0. 15 HPLC. Analysis of bombesin immunoreactivity in the milk E extract focused on its separation from porcine GRP and am- phibian bombesin. A single peak of bombesin immunoreac- tivity was found at a retention time of 27.9 min and was com- pletely resolved from GRP (35.8 min) (Fig. 4). Coinjection of concentrations of bombesin immunore- 1.5 approximately equal activity and GRP gave the same resolution. The recovery of bombesin immunoreactivity and GRP from a non-end- capped ODS column (Spherisorb) also differed considerably: 1.0 77.1 ± 19.1% (n = 8) and 10.4 ± 2.4% (n = 5), respectively, using the appropriate standard in the RIA. Under these chro- matographic conditions, the amphibian bombesin elutes be- 0.5 tween the two peptides (44). DISCUSSION Our data indicate that bovine milk contains a bombesin-re- 0 lated peptide with an apparent Mr of 3,200. This peptide is Fraction no. larger than both the amphibian peptide (1) (Mr, 1,620) and endogenous mammalian GRP from non-antral gastric tissue FIG. 3. Gel chromatography of milk extracts. The concentrated (28), and it is not an aggregate of either GRP or bombesin or acid/acetone extract was chromatographed on Bio-Gel P-4 (1.5 x 94 another milk protein. It is clearly separated from both GRP cm) in 0.1 M formic acid. (Upper) Untreated milk extract. o, GRP and bombesin by molecular exclusion chromatography and immunoreactivity. (Middle) L-1-Tosylamido-2-phenylethyl chloro- reversed-phase HPLC. This immunoreactive peptide can be extract. Extract treated methyl ketone-trypsin-digested (Lower) a new that shares homology with bombe- The arrows represent considered peptide with urea/dithiothreitol/iodoacetamide (46). with our which the following molecular weight markers: 1, VO (bovine serum albu- sin based on its crossreactivity antiserum, min); 2, porcine GRP; 3, '25I-labeled [Tyr8]bombesin; 4, I251-labeled recognizes bombesin residues 5-8 (Gly-Asn-Gln-Trp) (Table physalaemin. Vi was fraction 225. 1). The parallel displacement of the labeled peptide by milk Downloaded by guest on September 24, 2021 Medical Sciences: Jahnke and Lazarus Proc. NatL. Acad Sci. USA 81 (1984) 581

bombesin in comparison to amphibian bombesin (Fig. 2) fur- of peptides from amphibian skin also appeared by Erspamer and ther augments this point because neither porcine GRP nor Melchiorri (67). alytesin showed this type of parallel immunodisplacement curve; in fact, both peptides were nonparallel competitors in We appreciate the generous gifts of [Tyr5]bombesin-(5-14) from the RIA (Fig. 1). Thus, the data suggest that bombesin resi- R. de Castiglione (Farmitalia, Milan, Italy), alytesin and [Tyr8]bom- dues 6 and 7 (asparagine and glutamine) exist in milk bombe- besin from J. E. Rivier (Salk Institute, La Jolla, CA), and porcine sin in the appropriate sequence. Furthermore, the recovery GRP from H. Yajima (University of Kyoto, Kyoto, Japan); the ad- of immunoreactivity after boiling milk eliminates the possi- vice and helpful suggestions of R. P. DiAugustine, M. D. Erisman, bility that a proteolytic enzyme was responsible for the par- 0. Hernandez, J.-S. Hong, and W. E. Wilson; the assistance of allel RIA curves (48). B. J. Irons and L. J. Stone; the typing skills of B. J. Highsmith; and The quantity of bombesin immunoreactivity in a half-pint literature retrieval by R. J. Hester. of milk (ca. 240 ng) could be sufficient to cause the release of both gastrin and gastric acid in the stomach. Several of the 1. Erspamer, V. & Melchiorri, P. (1973) Pure Appl. Chem. 35, peptide hormones found in milk-e.g., prolactin (39-41), 463-494. thyrotropin-releasing hormone (42, 49), and luteinizing hor- 2. Brown, M., Allen, R., Villarreal, J., Rivier, J. & Vale, W. mone-releasing hormone (49-51), as well as melatonin (35)- (1978) Life Sci. 22, 2721-2728. are absorbed through the gastrointestinal wall and appear in 3. Track, N. S. & Cutz, E. (1982) Life Sci. 30, 1553-1556. 4. Villarreal, J. A. & Brown, M. R. (1978) Life Sci. 23, 2729- the plasma as bioactive molecules that could presumably in- 2734. teract with receptors. The amount of bombesin used in intra- 5. Moody, T. W. & Pert, C. B. (1979) Biochem. Biophys. Res. venous infusions in human subjects indicates that relatively Commun. 90, 7-14. low concentrations produce significant hormonal changes (6, 6. Erspamer, V. & Melchiorri, P. (1975) in Gastrointestinal Hor- 10-12). Thus, considering the volume of milk consumed dai- mones, ed. Thompson, J. C. (Univ. Texas Press, Austin), pp. ly by neonates (30), the concentration of bombesin immuno- 575-589. reactivity could be sufficient to produce local hormonal ef- 7. Basso, N., Lezoche, E., Giri, S., Percoco, M. & Speranza, V. fects. We speculate that the biological function of this bom- (1977) Digest. Dis. 22, 125-128. besin immunoreactivity may be linked to its release of 8. Delle Fave, G., Kohn, A., deMagistris, L., Manusco, M. & gastric and a Sparvoli, C. (1980) Life Sci. 27, 993-999. acid wide variety of gastrointestinal peptide 9. Pontiroli, A. E., Alberetto, M., Restelli, L. & Facchinetti, A. hormones that could "trigger a cascade of developmental (1980) J. Clin. Endocrinol. Metab. 51, 1303-1305. changes" throughout the gastrointestinal tract in the neonate 10. Varner, A. A., Modlin, I. M. & Walsh, J. H. (1981) Reg. Pep- (30). Furthermore, the therapeutic use of bovine milk in tides 1, 289-296. adult ulcer patients was recently questioned due to the ob- 11. Corazziari, E., Delle Fave, G., Pozzessere, C., Kohn, A., de- served increase in stomach acidity,t because it appears that Magistris, L., Anzini, R. & Torsoli, A. (1982) Gastroenterol- milk released gastric acid to exacerbate the symptoms. ogy 53, 10-14. Thus, one approach for future therapy for ulcer patients may 12. Ghatei, M. A., Jung, R. T., Stevenson, J. C., Hillyard, C. J., be linked to the development of synthetic antagonists of the Adrian, T. E., Lee, Y. C., Christofides, N. D., Sarson, D. L., Mashiter, K., MacIntyre, I. & Bloom, S. R. (1982) J. Clin. bombesin immunoreactive peptide or possibly a hybrid mol- Endocrinol. Metab. 54, 980. ecule containing bombesin sequences and the bioactive se- 13. Melchiorri, P. (1978) in Gut Hormones, ed. Bloom, S. R. quence of urogastrone, a peptide that inhibits gastric acid (Churchill-Livingstone, Edinburgh), pp. 534-540. output. 14. Erspamer, V. (1980) in Gastrointestinal Hormones, ed. Glass, The tissue of origin for the bombesin immunoreactivity G. B. J. (Raven, New York), pp. 343-359. found in milk is unknown at the present time. Most of the 15. Smith, G. P. & Gibbs, J. (1981) in Neurosecretion and Brain peptide hormones found in milk normally occur in plasma Peptides, eds. Martin, J. S., Reichlin, S. & Bick, K. L. (Ra- and usually reach a steady-state level between milk and plas- ven, New York), pp. 389-395. ma soon after birth (52-55). Possible tissue sources for bom- 16. Brown, M. R., Rivier, J. & Vale, W. W. (1977) Life Sci. 21, besin immunoreactivity in milk may be neural or extraneural 1729-1734. 17. Brown, M. R., Marki, W. & Rivier, J. (1980) Life Sci. 27, 125- tissue or both. For example, several known neuropeptides, 128. thyrotropin-releasing hormone (56), somatostatin, cortico- 18. Brown, M., Rivier, J. & Vale, W. (1977) Science 196, 998- tropin-releasing factor (57), and prolactin (58), are also found 1000. in the pancreas. Even GRP (59, 60) and other bombesin-like 19. Erspamer, V., Melchiorri, P. & Sopranzi, N. (1974) Br. J. peptides (60, 61) are apparently associated with nerve tissue. Pharm. 52, 219-225. On the other hand, mammary gland epithelial cells or plasma 20. Hirschowitz, B. I. & Gibson, R. G. (1978) Digestion 18, 227- cells recruited to the gland from the gut, which contain IgA 239. (36), may secrete bombesin immunoreactivity. Whatever the 21. Schusdziarra, V., Rouiller, D., Harris, V., Pfeiffer, E. F. & case may be, the existence of a peptide in mammalian tissue Unger, R. H. (1980) Reg. Peptides 1, 89-96. containing homology to an amphibian peptide is indeed an 22. DuVal, J. W., Saffouri, B., Weir, G. C., Walsh, J. H., Ari- mura, A. & Makhlouf, G. M. (1981) Am. J. Physiol. 241, interesting example of structural conservation in evolution- G242-G247. not only of the peptide itself, but also of genetic components 23. Rokaeus, A. & McDonald, T. (1980) Acta Physiol. Scand. 110, and receptor sites (44, 62). 326. Note Added in Proof. Several new peptides containing sequence ho- 24. McDonald, T. J., Ghatei, M. A., Bloom, S. R., Adrian, T. E., mology to bombesin have been described since the communication Mochizuki, T., Yanaihara, C. & Yanaihara, N. (1983) Reg. of this paper. Reeve et al. (63) isolated a decapeptide and two hepta- Peptides 5, 125-137. cosal peptides from canine small intestine. In porcine spinal cord, 25. Ayalon, A., Yazigi, R., Devitt, P. G., Rayford, P. L. & Minamino et al. (64) purified a decapeptide (neuromedin B), where- Thompson, J. C. (1981) Biochem. Biophys. Res. Commun. 29, as Kangawa et al. (65) describe another peptide (neuromedin C) that 1390-1397. is identical to the decapeptide from canine intestine. The presence of 26. Mazzanti, G., Falconieri Erspamer, G. & Piccinelli, D. (1981) hormones and other neurotransmitters in milk was recently summa- J. Pharm. Pharmacol. 34, 120-121. rized 27. Girod, C., Durand, N. & Gourru, J.-L. (1982) Biomed. Res. 3, by Hazum (66), and a thorough review of the neuronal action 252-260. 28. McDonald, T. J., Jornvall, H., Nilsson, G., Vagne, M., Gha- SK. McArthur (University of California Medical School, San Diego) tei, M., Bloom, S. R. & Mutt, V. (1979) Biochem. Biophys. quoted by Time magazine, July 19, 1982. Res. Commun. 90, 227-233. Downloaded by guest on September 24, 2021 582 Medical Sciences: Jahnke and Lazarus Proc. NatL Acad Sci. USA 81 (1984)

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