Use of monoclonal antibodies to identify thymopoietin in cultured human thymic epithelial cells.

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.

Immunohistochemical localization of bursin in epithelial cells of the avian bursa of Fabricius.

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.

A seasonal pineal peptide rhythm persists in superior cervical ganglionectomized rats.

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.

Influence of age on August levels of pineal immunoreactive arginine vasotocin in rats.

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.

Amino acid sequence of thymopoietin isolated from skin.

The isolation of thymopoietin-reactive material in fetal bovine skin was monitored by means of a radioimmunoassay to thymopoietin. The amino acid sequence of this material was determined to be identical with that of thymopoietin isolated from the thymus. Experimental evidence suggests that thymopoietin in the circulation derives from the thymus and not from the skin, suggesting that the thymopoietin in keratinocytes has a local function, either apocrine and/or immunoregulatory.

Myasthenic sera recognize the human acetylcholine receptor bound to thymopoietin.

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 alpha bungarotoxin (alpha Bgt)-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 125I-labeled alpha Bgt-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 alpha Bgt 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.

Pineal ultrastructure and indole profiles spanning the summer rise in arginine vasotocin immunoactivity.

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.

Thymopoietin to thymopentin: experimental studies.

Thymopoietin is a polypeptide hormone of the thymus consisting of 49 amino acids. The pentapeptide thymopentin (TP-5) Arg-Lys-Asp-Val-Tyr, corresponding to amino acids 32-36 of thymopoietin, appears to represent the active site of thymopoietin in that it has all the biological activities of the native hormone. Thymopoietin is secreted by epithelial cells of the thymus and is pleiotropic in action, affecting neuromuscular transmission, induction of early T cell differentiation and immune regulation. The immuno-regulatory actions of thymopentin on peripheral T cells are mediated by intracellular cyclic GMP elevations in contrast to the intracellular cyclic AMP elevations induced in precursor T cells that trigger their further differentiation to T cells. Thymopoietin and thymopentin have the biological characteristics of being immunonormalizing in a number of animal model systems of immune dysbalance. These include immune dysbalances induced by thymectomy or the thymic involution associated with aging or by other procedures in thymus-intact animals. The normalizing action of thymopentin, whether the immune dysbalance be in the direction of hyper- or hyporesponsiveness, points to its potential utility in human diseases characterized by immune dysbalance.

Thymopentin: stability considerations and potency by various routes of administration.

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.

Pineal arginine vasotocin activity increases 200-fold during August in adult rats and hamsters.

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.

A seasonal variation in arginine vasotocin immunoactivity in rat pineal glands.

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.

Properties of a highly purified antifertility factor from human seminal plasma.

A high molecular weight antifertility factor (AF-1) was obtained in a high degree of purity from human seminal plasma by ultracentrifugation, CM-cellulose and concanavalin A chromatography, and Sepharose 6B gel filtration. A final purification step involving preparative disc electrophoresis was occasionally required. AF-1 showed a dose-dependent inhibition of the in vitro fertilizing ability of capacitated mouse spermatozoa, causing 50% inhibition of fertilization at approximately 27.5 micrograms/10(6) spermatozoa. Removal of the follicle cell layer of the oocyte did not decrease the antifertility effect of AF-1 but inhibition of fertilization was no longer observed after dispersal of the zona pellucida. The effect of AF-1 was on the spermatozoa and not on the oocytes. These results show that AF-1 treatment prevents capacitated spermatozoa from penetrating the zona pellucida and possibly the follicle cell layer. The mechanism by which AF-1 does so is not known because AF-1 did not prevent the in vitro acrosome reaction of guinea pig spermatozoa, nor did it inhibit the activity of human acrosin or bovine testicular hyaluronidase. The antifertility activity of AF-1 is reversed after recapacitation of the AF-1 treated spermatozoa and it can be assumed that AF-1 is either dissipated or loses activity during the transport of the spermatozoa through the female genital tract when capacitation takes place. AF-1 is heat labile. The properties of AF-1 are the same as those found previously for the pellet obtained by high-speed centrifugation of human seminal plasma, indicating that this is the primary, nonparticulate, high molecular weight factor with antifertility activity in human semen.

Stimulation of ATP-dependent proteolysis requires ubiquitin with the COOH-terminal sequence Arg-Gly-Gly.

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.

Bovine neurophysin lipid complex. Their isolation, characterization and reaggregation.

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.

Complete amino acid sequence of bovine neurophysin-I. A major secretory product of the posterior pituitary.

Bovine neurophysin-I (bNP-I) is the first neurophysin protein which contains histidine and possesses an acidic COOH-terminal segment for which the complete amino acid sequence is presented: NH2-Ala-Val-Leu-Asp-Leu-Asp-Val-Arg-Thr-Cys-Leu-Pro-Cys-Gly-Pro-Gly-Gly-Lys-Gly-Arg-Cys-Phe-Gly-Pro-Ser-Ile-Cys-Cys-Gly-Asp-Glu-Leu-Gly-Cys-Phe-Val-Gly-Thr-Ala-Glu-Ala-Leu-Arg- Cys-Gln-Glu-Glu-Asn-Tyr-Leu-Pro-Ser-Pro-Cys-Gln-SerGly-Gln-Lys-Pro-Cys-Gly-Ser- Gly-Gly-Arg-Cys-Ala-Ala-Ala-Gly-Ile-Cys-Cys-Ser-Pro-Asp-Gly-Cys-His-Glu-Asp-Pro-Ala-Cys-Asp-Pro-Glu-Ala-Ala-Phe-Ser-Leu-COOH. Determination of the structure was greatly facilitated by new procedures used for the isolation of bNP-I and of its tryptic peptide fragments. bNP-I isolated from freshly frozen bovine posterior pituitaries is composed of 93 residues, but some preparations contain neurophysin protein with NH2- and COOH-terminal truncated sequences. bNP-I differs from bovine neurophysin-II, the second major neurophysin of cow, in 20 residue positions, and several of the differences cannot be accounted for by single nucleotide replacements in the genes coding for these two neurophysin proteins. The results reported in this study support our earlier hypothesis that neurophysin-gene duplication preceded species divergence.