Murine monoclonal antibodies (mAbs) were developed to discriminate thymopoietin, a human thymic hormone, and thysplenin, a closely related molecule found in spleen. Three of these recognized both native and synthetic thymopoietin as well as thysplenin. Together they define two non-overlapping epitopes which withstand sodium dodecyl sulfate denaturation and can be detected by western blotting. We used these three mAbs to demonstrate the production of thymopoietin by cultured thymic epithelial cells for up to several weeks. Three additional mAbs were selective for thysplenin. Highly specific mAbs will be useful for characterizing further these physiologically distinct polypeptides.

Neurohypophyseal peptide hormone activity is present in the pineal gland of mammals, and varies over a seasonal cycle. Pineal peptide levels, measured by arginine vasotocin (AVT) radioimmunoassay, increase dramatically for a brief time during August each year. The manner in which this cycle is regulated is as yet unknown. Input to the pineal from sympathetic axons arising in the superior cervical ganglia (SCG) is essential for the generation and regulation of the circadian rhythm in melatonin synthesis, and is the only pathway known to regulate pineal biochemical processes. It was of interest then to determine the impact of the SCG on the seasonal peptide cycle. Levels of pineal arginine vasotocin immunoactivity (iAVT) were monitored during August, 1984, in rats which had been superior cervical ganglionectomized (SCGX), in sham-operated and intact controls (L:D 12:12), and in rats subjected to L:D 22:2. The results indicate that SCGX does not abolish the seasonal cycle, but may influence the timing of the iAVT peak. Inhibition of pineal melatonin synthesis by exposure of rats to L:D 22:2 did not mimic the phase delay seen with SCGX, but did cause a significant increase in the amplitude of the August iAVT activity peak.

Antibodies to the avian B-cell-differentiating hormone bursin (lysyl-histidyl-glycine amide) were raised in mice and rabbits by immunizing with bursin conjugates in Freund's adjuvant. Immunohistochemical staining with these bursin-specific antibodies was restricted to follicular and dendritic reticular epithelial cells of the bursa of Fabricius, and was not found in control avian tissues.

The present study was undertaken to evaluate carefully the influence of age on physiological levels of arginine vasotocin‐like peptide in rat pineal glands. Glands were collected from male and female rats aged 13, 33, 53, and 73 days on August 4, 12, and 19, 1984. Individual extracts were assayed for arginine vasotocin (AVT) by radioimmunoassay. The results confirm our previous observation that rat pineal AVT immunoactivity (iAVT) increases significantly during August each year; and in this study, each group of rats reached the same peak level of iAVT (700–750 pg/gland) regardless of age or gender. Thus we do not confirm a previously reported decrease in AVT activity with age. In our studies thus far, season of the year is the physiological variable with the most significant influence on pineal AVT activity levels.

The thymic hormone, thymopoietin (Tpo), from human (HTpo), bovine (BTpo) and from synthetic (sHTpo) origins bound to the acetylcholine receptor (AChR) solubilized by Triton 1.5% from human muscle. This binding was demonstrated either by inhibition of formation of radiolabeled α bungarotoxin (αBgt)‐AChR complexes measured after precipitation by ammonium sulfate or by a myasthenic serum containing a high concentration of anti‐AChR antibodies, or directly by incubating the human AChR with radiolabeled sHTpo or BTpo. The 125 I‐labeled αBgt‐AChR complexes were totally inhibited by 10 −6 M sHTpo or BTpo. The complexes formed by AChR and the radiolabeled Tpo were recognized specifically by sera containing anti‐AChR antibodies from myasthenic patients. The active pentapeptide derivative of Tpo, thymopentin, another thymic hormone, thymulin, as well as bovine insulin did not interfere with the specific binding of αBgt to human AChR. Tpo and anti‐AChR antibodies could participate together in the inhibition of neuromuscular conduction with Tpo modulating the depressive effect of the antibodies on the neuromuscular junction in myasthenia gravis.

Annals of the New York Academy of SciencesVolume 540, Issue 1 p. 298-300 Thymopoietin: A Marker of the Human Nicotinic Acetylcholine Receptor E. MOREL, E. MOREL INSERM U 25—CNRS UA 122 Hǒpital Necker 161, rue de Sèvres 75730 Paris Cedex 15, FranceSearch for more papers by this authorB. VERNET-DER-GARABEDIAN, B. VERNET-DER-GARABEDIAN INSERM U 25—CNRS UA 122 Hǒpital Necker 161, rue de Sèvres 75730 Paris Cedex 15, FranceSearch for more papers by this authorF. RAIMOND, F. RAIMOND INSERM U 25—CNRS UA 122 Hǒpital Necker 161, rue de Sèvres 75730 Paris Cedex 15, FranceSearch for more papers by this authorT. K. AUDHYA, T. K. AUDHYA Ortho Pharmaceutical Corporation Raritan, New JerseySearch for more papers by this authorG. GOLDSTEIN, G. GOLDSTEIN Ortho Pharmaceutical Corporation Raritan, New JerseySearch for more papers by this authorJ. F. BACH, J. F. BACH INSERM U 25—CNRS UA 122 Hǒpital Necker 161, rue de Sèvres 75730 Paris Cedex 15, FranceSearch for more papers by this author E. MOREL, E. MOREL INSERM U 25—CNRS UA 122 Hǒpital Necker 161, rue de Sèvres 75730 Paris Cedex 15, FranceSearch for more papers by this authorB. VERNET-DER-GARABEDIAN, B. VERNET-DER-GARABEDIAN INSERM U 25—CNRS UA 122 Hǒpital Necker 161, rue de Sèvres 75730 Paris Cedex 15, FranceSearch for more papers by this authorF. RAIMOND, F. RAIMOND INSERM U 25—CNRS UA 122 Hǒpital Necker 161, rue de Sèvres 75730 Paris Cedex 15, FranceSearch for more papers by this authorT. K. AUDHYA, T. K. AUDHYA Ortho Pharmaceutical Corporation Raritan, New JerseySearch for more papers by this authorG. GOLDSTEIN, G. GOLDSTEIN Ortho Pharmaceutical Corporation Raritan, New JerseySearch for more papers by this authorJ. F. BACH, J. F. BACH INSERM U 25—CNRS UA 122 Hǒpital Necker 161, rue de Sèvres 75730 Paris Cedex 15, FranceSearch for more papers by this author First published: November 1988 https://doi.org/10.1111/j.1749-6632.1988.tb27080.xCitations: 3AboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onEmailFacebookTwitterLinkedInRedditWechat No abstract is available for this article. References 1 Goldstein, G. & S. Whittingham. 1966. Lancet ii: 315. 10.1016/S0140-6736(66)92599-2 Google Scholar 2 Audhya, T., D. H. Schlesinger & G. Goldstein. 1981. Biochemistry 20: 6195. 10.1021/bi00524a044 CASPubMedWeb of Science®Google Scholar 3 Venkatasubramanian, K., T. Audhya & G. Goldstein. 1986. Proc. Natl. Acad. Sci. USA 83: 3171. 10.1073/pnas.83.10.3171 CASPubMedWeb of Science®Google Scholar 4 Vernet-der Garabedian, B., E. Morel & J. F. Bach. 1986. J. Neuroimmunol. 12: 65. 10.1016/0165-5728(86)90098-6 CASPubMedWeb of Science®Google Scholar Citing Literature Volume540, Issue1Advances in NeuroimmunologyNovember 1988Pages 298-300 ReferencesRelatedInformation

Thymopentin, the synthetic pentapeptide Arg-Lys-Asp-Val-Tyr corresponding to amino acids 32-36 of thymopoietin, was rapidly degraded in human plasma (T 1/2 = 30 s) and this was shown to be due to proteolytic enzymes in plasma. The biological potency of thymopentin varied greatly with the route of administration, a finding possibly related to its short half-life. Intravenous infusion provided the most potent and bolus intraperitoneal injection the least potent mode of administration. Thus the route and rate of administration are critical factors in determining the effective dose being received by the animal.

A correlative radioimmunological-biochemical-ultrastructural study of the rat pineal gland was undertaken during the summer months when pineal arginine vasotocin (AVT) immunoactivity increases up to 200-fold. RIA confirmed a rapid rise in AVT activity during mid-August regardless of the time of day sampled. Pineal indoles were separated by HPLC and measured using electrochemical detection. Serotonin (5-HT) and 5-hydroxyindoleacetic acid levels were consistently elevated in daytime samples, and there was a significant trend for increased day and nighttime levels of 5-HT from July to September. Mid-dark levels of melatonin also exhibited a significant increase over the sample period. Nighttime levels of N-acetylserotonin mirrored fluctuations in 5-HT in the preceding photoperiod. Ultrastructural components implicated in peptide/protein and/or indole biosynthesis were quantified by stereological morphometry. The greatest amounts of rough endoplasmic reticulum stacks, lipid droplets, and annulate lamellae-like bodies coincided with peak AVT activity. Dense-cored vesicles and synaptic ribbons were consistently more frequent during the dark period. The number of dense-cored vesicles and nucleolar size tended to be greatest before and after the peak in AVT immunoactivity. These observations are consistent with the hypotheses that endoplasmic reticulum and lipid are functionally related to the synthesis and/or storage of peptide/protein factors and that numerical changes in synaptic ribbons and dense-cored vesicles are more closely related to day/night differences in indole metabolism.

In a previous study, measurements of arginine vasotocin‐immunoactivity (iAVT) in immature rats over a period of 14 months, led to the discovery of a significant yearly variation, with peak levels of iAVT in August. In the present study, iAVT was measured in pineals obtained from mature male and female rats and hamsters once or twice weekly from July until early September 1982. For all groups, mean pineal AVT‐immunoactivity was less that 7 pg/gland in early July, but then increased significantly by August 11–13. For hamsters, maximum values of 1,272 ± 49 (mean ± S.E.: n = 3) and 1,065 ± 62 pg/gland were recorded for males and females, respectively. For rats, peak values measured were 940 ± 12 pg/gland for males and 1,040 ± 34 for females. The AVT‐activity levels then decreased to less than 100 pg/gland by early September. Thus, a dramatic August elevation of pineal iAVT is characteristic of hamsters as well as rats, and of mature as well as immature animals.

Groups of male and female laboratory rats, 28-30 days of age, were killed each week from July 1980 to September 1981. Pineal glands were collected, pooled, and extracted. Arginine vasotocin (AVT) activity in the extracts was measured by RIA. For most of the calendar year, pineal AVT immunoactivity ranged between 1.8-7.7 pg/gland. The average (+/- SE) basal AVT activity level was 4.1 +/- 0.3 pg/gland (n = 48). Both years in early August, pineal AVT activity increased several hundred fold. Values of 1720 and 1170 pg/gland were measured in mid-August of 2 successive years. The signal for this dramatic yearly rhythm, and its physiological consequences, are as yet unknown.

It was previously shown that ubiquitin is very similar to the polypeptide cofactor of the ATP-dependent protein degradation system from rabbit reticulocytes (Wilkinson, K. D., Urban, M. K., and Haas, A. L. (1980) J. Biol. Chem. 255, 7529-7532). We have extended this work to show that the peptic peptide maps are identical for bovine ubiquitin and the polypeptide cofactor isolated from human erythrocytes. It was noted however that ubiquitin preparations were less active in stimulating proteolysis than preparations of the polypeptide cofactor. This decreased activity has been shown to be due to the presence of an inactive form of ubiquitin in some preparations. The two forms of ubiquitin are separable by high performance liquid chromatography. The active form of ubiquitin has the COOH-terminal sequence -Arg-Gly-Gly at residues number 74 to 76. The inactive form terminates in -Arg74 as previously reported in the sequence studies of ubiquitin. Limited tryptic digestion of active ubiquitin yields the inactive, later eluting form and the dipeptide glycylglycine. This preteolytic cleavage apparently occurs during purification from most tissues. We thus propose reserving the term ubiquitin for the intact 76-amino acid sequence and designating the 74-amino acid sequence as ubiquitin-t to indicate its derivation by a tryptic-like protease cleavage. This 76-residue sequence is consistent with the covalent structure of protein A-24, a conjugate where carboxyl group of the COOH-terminal glycylglycine of ubiquitin is linked by an amide bond to the epsilon-amino group of Lys-119 of histone H2A. Thus, the structural requirements of the protein and ubiquitin molecules are identical for formation of protein A-24 and for forming the covalent conjugates thought to be intermediates in ATP-dependent protein degradation.

FEBS LettersVolume 128, Issue 2 p. 325-328 Full-length articleFree Access A comparative study of mammalian neurophysin protein sequences David H. Schlesinger, David H. Schlesinger Department of Physiology and Biophysics, University of Illinois Medical Center, Chicago, IL 60612 USA Department of Immunobiology, Ortho Pharmaceutical Corp., Raritan, NJ 08869, USASearch for more papers by this authorT.K. Audhya, T.K. Audhya Department of Physiology and Biophysics, University of Illinois Medical Center, Chicago, IL 60612 USA Department of Immunobiology, Ortho Pharmaceutical Corp., Raritan, NJ 08869, USASearch for more papers by this author David H. Schlesinger, David H. Schlesinger Department of Physiology and Biophysics, University of Illinois Medical Center, Chicago, IL 60612 USA Department of Immunobiology, Ortho Pharmaceutical Corp., Raritan, NJ 08869, USASearch for more papers by this authorT.K. Audhya, T.K. Audhya Department of Physiology and Biophysics, University of Illinois Medical Center, Chicago, IL 60612 USA Department of Immunobiology, Ortho Pharmaceutical Corp., Raritan, NJ 08869, USASearch for more papers by this author First published: June 15, 1981 https://doi.org/10.1016/0014-5793(81)80109-3Citations: 4AboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onEmailFacebookTwitterLinkedInRedditWechat References 1 J.D. Capra, J.M. Kehoe, R. Kotelchuck, R. Walter, E. Breslow, Proc. Natl. Acad. Sci. USA, 69, (1972), 43– 2 J.D. Capra, K.W. Cheng, H.G. Friesen, W.G. North, R. Walter, FEBS Lett., 46, (1974), 71– 74. 3 R. Walter, T.K. Audhya, D.H. Schlesinger, S. Shin, S. Saito, H. Sacks, Endocrinology, 100, (1977), 162– 174. 4 D.H. Schlesinger, R. Walter, T.K. Audhya, A.M. Moses L. Share Conf. Neurohypophysis, International Society of Neuroendocrinology (1976), Karger Basel 53– 57. 5 D.H. Schlesinger, B.T. Pickering, W.B. Watkins, J.C. Peck, L.G. Moore, T.K. Audhya, R. Walter, FEBS Lett., 80, (1977), 371– 373. 6 D.H. Schlesinger, J.D. Capra, R. Walter, Intl. J. Pept. Prot. Res., 6, (1974), 1– 7 T.C. Wuu, S.E. Crumm, Biochem. Biophys. Res. Commun., 68, (1976), 634– 639. 8 D.H. Schlesinger, M. Ernst, A. Nicholas, W.B. Watkins, R. Walter, FEBS Lett., 57, (1975), 55– 9 M.T. Chauvet, J. Chauvet, R. Archer, FEBS Lett., 58, (1975), 234– 237. 10 T.C. Wuu, S.E. Crumm, M. Saffran, J. Biol. Chem., 246, (1971), 6043– 11 T.C. Wuu, S.E. Crumm, J. Biol. Chem., 251, (1976), 2735– 12 D.H. Schlesinger, T.K. Audhya, R. Walter, J. Biol. Chem., 253, (1978), 5019– 5024. 13 M.T. Chauvet, P. Cadagno, J. Chauvet, R. Acher, FEBS Lett., 80, (1977), 374– 376. 14 T.K. Audhya, R. Walter, Arch. Biochem. Biophys., 180, (1977), 130– 139. 15 W.B. Watkins, J. Endocrinol., 59, (1973), 17– 16 G. Schaechtelin, W.G. North, R. Walter, Ann. NY Acad. Sci., 248, (1975), 365– 377. 17 P. Edman, G. Begg, Eur. J. Biochem., 1, (1967), 80– 18 P. Edman, Ann. NY Acad. Sci., 88, (1960), 602– 19 M. Boehnert, D.H. Schlesinger, Anal. Biochem., 96, (1979), 464– 473. 20 J.J. Pisano, T.J. Bronzert, J. Biol. Chem., 244, (1969), 5597– 21 P. Edman, S.B. Needleman Protein Sequence Determination: A Sourcebook of Methods and Techniques (1970), Springer-Verlag New York 211– 255. 22 C.L. Zimmerman, E. Appella, J.J. Pisano, Anal. Biochem., (1977), 569– 573. 23 J. Simmons, D.H. Schlesinger, Anal. Biochem., 30, (1980), 1190– 1206. 24 W.G. North, R. Walter, D.H. Schlesinger, E. Breslow, J.D. Capra, Ann. NY Acad. Sci., 248, (1975), 408– 25 K.W. Cheng, H.G. Friesen, J. Clin. Endocrinol., 34, (1972), 165– 26 W.R. Gray, Nature, 220, (1968), 1300– 1304. Citing Literature Volume128, Issue2June 15, 1981Pages 325-328 ReferencesRelatedInformation

Biological and immunological properties of crude neurophysin, lipid-containing neurophysin and delipidated neurophysin protein mixture isolated from rat pituitary glands have been investigated. Rat uterotonic, avian vasodepressor and rat pressor assays indicate that crude neurophysin and lipid-containing neurophysin also contained 10−5 to 10−6 M neurohypophyseal hormones, with a 15–20-fold higher level of oxytocin as compared to vasopressin. Delipidated neurophysin had barely detectable hormonal levels. Electrolyte excretion studies also seem to indicate higher oxytocin levels in lipid-containing neurophysin. Lipid-containing neurophysin also contained biologically active adrenocorticotrophin (8 ng/μg protein) and immunologically active adrenocorticotrophin-like material (20 ng/μg protein). Excess immunoassayable adrenocorticotrophin-like material is converted to biologically active adrenocorticotrophin by trypsin treatment. Lipids isolated from rat pituitary were reaggregated in vitro with purified rat neurophysin-II to yield a neurophysin lipid complex. The amount of uptake of [2-14C(U) tyrosine]oxytocin by rat neurophysin-II or its neurophysin lipid complex was the same based on radioactivity. The specific avian vasodepressor activity of the oxytocin bound to the rat neurophysin-II lipid complex was enhanced approximately 3-fold.

A lipid-containing neurophysin fraction was isolated and purified from bovine posterior pituitary glands by acid extraction and affinity chromatography on a heparin-Sepharose 4B column. This lipid-rich fraction was found to be composed of noncovalent aggregates of neurophysin proteins and phospholipids such as phosphatidyl choline, phosphatidyl ethanolamine, phosphatidyl serine and sphingomyelin. The lipid-containing neuophysin was delipidated by treatment with choloform-methanol. The resultant apoproteins were characterized as bovine neuroions were developed for the reaggregation of purified bovine neurophysin-I and -II with lipids extracted from bovine posterior pituitary and hypothalamus and with synthetic lecithin. The resultant neurophysin lipid complexes have been shown to band upon isopycnic centrifugation at densities different from those of the respective purified bovine neurophysins.

Sixteen amino acid thiohydantoins encountered during COOH-terminal degradation of peptides and proteins with ammonium thiocyanate have been separated and identified by reverse phase high-performance liquid chromatography using a DuPont Zorbax ODS column and a novel procedure for preparation of thiohydantoin derivative of serine and threonine is described. Three isocratic systems were employed: System 1, buffer A alone (0.01 n sodium acetate, pH 4.5); system 2, 10% acetonitrile in buffer A; system 3, 24% acetonitrile in buffer A. All 16 thiohydantoins could be resolved using a combination of systems 1, 2, and 3. The method is rapid and sensitive (1 nmol) and constitutes a step toward automation of the solid-phase COOH-terminal sequencing method.