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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 µg/106spermatozoa. 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.

The identity of the neuronal nicotinic alpha-bungarotoxin (alpha-BGT) site, which appears to be distinct from the functional nicotinic receptor, is unclear. Recent work in our laboratory has shown that the thymus-derived polypeptide thymopoietin potently and specifically interacts at the nicotinic alpha-BGT site in brain. The present results show that thymopoietin also interferes with the binding of 125I-alpha-BGT to chromaffin cells in culture; a dose-dependent inhibition in binding was observed, with an IC50 of 10(-8) M. To assess the long term effect(s) of thymopoietin in nervous tissue, chromaffin cells were exposed to the polypeptide for varying periods of time. Incubation of the cells in culture with thymopoietin (10(-9) to 3 x 10(-7) M) for 2 to 7 days resulted in an approximate 3-fold increase in alpha-BGT binding. Saturation analysis indicated this was due to an increase in the Bmax. The thymopoietin-induced increase in binding could be reversed with nicotine: thus, the sites can be regulated by a nicotinic receptor ligand. Although thymopoietin potently interacted at the nicotinic alpha-BGT receptor, nicotinic receptor responsiveness was not affected after short or long term exposure to the peptide. Neither basal nor nicotinic receptor-stimulated tyrosine hydroxylase activity was altered by thymopoietin. As well, resting and acetylcholine-evoked noradrenaline release remained similar to control after exposure of the cells to the polypeptide. These results indicate that the thymic polypeptide thymopoietin specifically interacts with the nicotinic alpha-BGT receptor population and, furthermore, can regulate the toxin binding sites in chromaffin cells in culture.

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.

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.

Corticotrophin-releasing factor (CRF) bioactivity has been described in the extra-hypothalamic brain, but its relationship to hypothalamic CRF has remained questionable. Of the seven regions of the mouse brain examined, highest concentrations of CRF-like immunoreactivity (CRF-LI) and bioassayable CRF activity were present in the median eminence and hypothalamus. However, substantial CRF-LI and bioassayable CRF activity were also seen in brain extracts from the amygdala, thalamus, frontal cortex, pons medulla and cerebellum. Bioactivity was largely neutralized by prior incubation with heat-inactivated antiserum to ovine CRF. These findings, in conjunction with previous immunocytochemical evidence, strongly suggest that a substance closely resembling hypothalamic CRF is present in the extrahypothalamic brain of the mouse.J. Endocr.(1986) 111,143–149

Recent work has shown that thymopoietin, a polypeptide with actions in the immune and nervous systems, potently binds to the alpha-bungarotoxin (alpha-BGT) receptor. The present study was done to characterize the interaction of thymopoietin at the nicotinic alpha-BGT binding site in cultured muscle cells and to correlate these findings with the effects of the polypeptide on nicotinic receptor-mediated function. Inhibition studies showed that thymopoietin potently inhibited 125I-alpha-BGT binding in C2 muscle cells in culture, with an IC50 of 1.1 nM, a value similar to that for alpha-BGT. Thymopoietin bound to the alpha-BGT receptor in the cells in culture relatively slowly; at 10(-8) M thymopoietin, maximal inhibition occurred after 45 to 75 min of exposure to the polypeptide. Dissociation of thymopoietin from the receptor exhibited a much longer time course; recovery of alpha-BGT binding to control values after exposure to 10(-8) M thymopoietin occurred approximately 16 hr after removal of the polypeptide. The effects of thymopoietin on 125I-alpha-BGT binding correlated well with those on nicotinic function. Thymopoietin potently inhibited nicotinic receptor-mediated 22Na uptake in muscle cells in culture, with an IC50 of 2 nM. This effect was dependent on the length of the preincubation period with thymopoietin, with maximal inhibition occurring after 60 min of exposure to the polypeptide. Recovery of the functional response after thymopoietin (10(-8) M) exposure required about 16 hr. The mode of inhibition of receptor-mediated ion flux by thymopoietin was similar to that observed with alpha-BGT but distinct from that obtained with d-tubocurarine and gallamine. To conclude, thymopoietin, a thymic polypeptide associated with the immune system, potently inhibited both 125I-alpha-BGT binding and nicotinic receptor-mediated function in C2 muscle cells. These findings may have implications for myasthenia gravis and/or other neuromuscular disorders.

Studies were conducted to assess the ability of the thymic polypeptide hormone thymopoietin (TPO) to interact with proto-typical ganglia-type or muscle-type nicotinic acetylcholine receptor ion channels (nAcChoR). Also investigated were interactions of TPO with neuronal nicotinic alpha-bungarotoxin binding sites (nBgtS), which share many features with nAcChoR and may belong to an extended nAcChoR family but do not appear to function as simple ligand-gated ion channels. TPO and alpha-bungarotoxin (Bgt) share the capacity for high affinity (IC50 values in the nanomolar range) interaction with nBgtS, which are expressed as high affinity radioiodinated Bgt binding sites by cells of the SH-SY5Y or IMR-32 human neuroblastomas. TPO and Bgt also share the capacity for high affinity interaction with muscle-type nAcChoR, which are expressed as high affinity binding sites for radioiodinated Bgt or tritium-labeled acetylcholine by cells of the TE671/RD human clone or the BC3H-1 mouse muscle cell line or on membrane preparations from Torpedo electroplax. TPO and Bgt act acutely as high affinity antagonists of muscle-type nAcChoR functional responses, which are measured using an isotopic rubidium ion efflux assay, on TE671/RD or BC3H-1 cells. In contrast, neither TPO nor Bgt are effective, at doses of up to 1 microM, as antagonists of ganglia-type nAcChoR function on SH-SY5Y or IMR-32 cells, nor are they potent as inhibitors of high affinity tritium-labeled acetylcholine binding to sites on putative ganglia-type nAcChoR expressed by SH-SY5Y or IMR-32 cells. These data indicate that some members of the extended nAcChoR family, including nBgtS and functional muscle-type nAcChoR but not ganglia-type nAcChoR, can interact with either Bgt or TPO. The results suggest that TPO may be an endogenous ligand active in both the nervous and immune systems and that some of its actions may be mediated via nBgtS or via functional blockade of muscle-type nAcChoR.

Thymopoietin is a polypeptide hormone of the thymus with physiological effects on the immune system and on acetylcholine-mediated transmission at the neuromuscular synapse. Elucidation of the structure and function of the nicotinic acetylcholine receptor has been facilitated by the use of the electric organs of Torpedo ray or Electrophorus eel as rich sources of the receptor and by the use of snake polypeptide toxins such as alpha-bungarotoxin as highly selective labels of the acetylcholine binding site. We now show that thymopoietin binds with high affinity (Ka approximately equal to 2.5 X 10(9) M-1) to the acetylcholine binding region of the acetylcholine receptor of Torpedo californica, as evidenced by similar and complete inhibition of the binding of radiolabeled thymopoietin or alpha-bungarotoxin by either of these polypeptides. These findings raise intriguing questions concerning the mechanisms whereby alpha-bungarotoxin and the thymopoietin affect acetylcholine receptor function, since these two polypeptides with such similar binding properties have very different functional effects.

Polyclonal antibodies to synthetic human pancreatic growth hormone-releasing factor [hpGRF(1-44)NH2] and rat hypothalamic growth hormone-releasing factor [rhGRF(1-43)OH] were produced in rabbits by injecting these weak immunogens, coupled to thyroglobulin and emulsified with complete Freund's adjuvant in the presence of activated charcoal, directly into the spleen. A subsequent booster injection by the conventional intramuscular route resulted in high-titer antibodies, which at a 1:20,000 dilution were used to develop highly sensitive and specific radioimmunoassays for these peptides. By using antibodies with an apparent Ka of 3.3 X 10(-12) (human) and 7.7 X 10(-11) (rat), the sensitivity of these assays in both human and rat was found to be less than 1 fmol. The antibody to hpGRF(1-44)NH2 is directed against the COOH-terminal region of the molecule, as shown by its crossreactivity with various hpGRF analogues: 140% with hpGRF(30-44)NH2; 1%-2% with hpGRF(1-37)OH, hpGRF(1-40)OH, and hpGRF(1-40)NH2; and none with hpGRF(1-29)NH2. Serial dilutions of human and rat hypothalamic extracts demonstrated parallelism with the corresponding species-specific standard and 125I-labeled tracer. There was no crossreactivity with other neuropeptides, gastrointestinal peptides, or hypothalamic extracts of other species. The hypothalamic content in fmol/mg (wet weight) of tissue was 3.6 +/- 0.2 for the human and 11.1 +/- 5.5 for the rat. Age-related changes in hypothalamic GRF content were present in rats, with a gradual increase from 2 to 16 weeks and a correlation between increasing body weight and GRF content. These radioimmunoassays will serve as important tools for understanding the regulation of growth hormone secretion in both human and rat.

Human thymopoietin and splenin were isolated from human thymus and spleen, respectively, by monitoring tissue fractionation with a bovine thymopoietin RIA cross-reactive with human thymopoietin and splenin. Bovine thymopoietin and splenin are 49-amino acid polypeptides that differ by only 2 amino acids at positions 34 and 43; the change at position 34 in the active-site region changes the receptor specificities and biological activities. The complete amino acid sequences of purified human thymopoietin and splenin were determined and shown to be 48-amino acid polypeptides differing at four positions. Ten amino acids, constant within each species for thymopoietin and splenin, differ between the human and bovine polypeptides. The pentapeptide active site of thymopoietin (residues 32-36) is constant between the human and bovine thymopoietins, but position 34 in the active site of splenin has changed from glutamic acid in bovine splenin to alanine in human splenin, accounting for the biological activity of the human but not the bovine splenin on the human T-cell line MOLT-4.

Thymopoietin, a polypeptide hormone of the thymus that has pleiotropic actions on the immune, endocrine, and nervous systems, potently interacts with the neuromuscular nicotinic acetylcholine receptor. Thymopoietin binds to the nicotinic alpha-bungarotoxin (alpha-BGT) receptor in muscle and, like alpha-BGT, inhibits cholinergic transmission at this site. Evidence is given that radiolabeled thymopoietin similarly binds to a nicotinic alpha-BGT-binding site within the brain and does so with the characteristics of a specific receptor ligand. Thus specific binding to neuronal membranes was saturable, of high affinity (Kd = 8 nM), linear with increased tissue concentration, and readily reversible; half-time was approximately 5 min for association and 10 min for dissociation. Binding of 125I-labeled thymopoietin was displaced not only by unlabeled thymopoietin but also by alpha-BGT and the nicotinic receptor ligands d-tubocurarine and nicotine; various other receptor ligands (muscarinic, adrenergic, and dopaminergic) did not affect binding of 125I-labeled thymopoietin. Thymopoietin was shown by ELISA to be present in brain extracts, displacement curves of thymus and brain extracts being parallel to the standard thymopoietin curve, and Western (immuno) blot identified in brain and thymus extracts a thymopoietin-immunoreactive polypeptide of the same molecular mass as purified thymopoietin polypeptide. We conclude that thymopoietin and thymopoietin-binding sites are present within the brain and that the receptor for thymopoietin is the previously identified nicotinic alpha-BGT-binding site of neuronal tissue.

Incorporation of [35S]]sulphate, [3H]glucose and [3H]serine into glycosaminoglycans and proteoglycans of embryonic-chicken sternum was measured in vitro in incubation medium containing 4-methylumbelliferyl beta-D-xyloside or p-nitrophenyl beta-D-xyloside at low concentrations, and in the absence of inhibitors of protein synthesis. Incorporation of sulphate was decreased by 80% in incubations in which 1mM-4-methylumbelliferyl beta-xyloside or 2.5 mM-p-nitrophenyl beta-xyloside was present; under these conditions, serum factors stimulated incorporation to only a small extent. When the concentration of the xyloside was decreased tenfold, incorporation of sulphate was inhibited by 60-70%, but when normal human serum or L-3,3′,5-tri-iodothyronine or both were also added to the incubation medium, incorporation was markedly stimulated. Experiments in which [35S]sulphate and [3H]glucose were incorporated simultaneously, and enzymic analysis of glycosaminoglycans formed in such experiments, indicated that chondroitin sulphate formed in the presence of 0.1 mM-4-methylumbelliferyl beta-xyloside contained 30-40% less sulphate than did chondrotin sulphate synthesized in the absence of xylosides. Similar experiments, with [3H]serine instead of [3H]glucose, suggested also a 20-30% decrease in chain length of the chondroitin sulphate; this was confirmed by direct gel filtration of labelled glycosaminoglycans on a calibrated column. Incorporation of [3H]glucose or [3H]serine was stimulated by serum and tri-iodothyronine in parallel with incorporation of sulphate. The changes seen in the total chondroitin sulphate were mirrored in the major proteoglycan fraction, purified by isopycnic centrifugation of salt-extracted proteoglycans. The labelling pattern of chondroitin sulphate from this proteoglycan indicated that decreased sulphation of chondroitin sulphate was largely due to the inferior ability of short polysaccharide chains to accept sulphate, with some direct interference with transfer of sulphate to all chains. The results also suggested that the action of serum factors on synthesis of proteochondroitin sulphate is exercised at the level of either protein synthesis or transport to the sites of initiation of polysaccharide synthesis.

Thymopoietin is a 48 to 49 amino acid polypeptide hormone of the thymus, which regulates immune function. The present experiments show that the polypeptide can cause a complete block of transmission of the phrenic nerve diaphragm junction of the rat in vitro; contractile responses evoked by phrenic nerve stimulation were blocked by concentrations of thymopoietin as low as 10(-8) M. The thymopoietin-induced inhibition of indirectly evoked muscle contractions was dose- and time-dependent, with the polypeptide being only slightly less potent than alpha-bungarotoxin (alpha-BGT). Twitch responses to direct electrical stimulation of the muscle were not affected by thymopoietin, indicating that it did not inhibit muscle tension by an action on the muscle contractile mechanism per se. Furthermore, thymopoietin did not alter resting or stimulated release of acetylcholine from the phrenic nerve, suggesting that it did not interact at a presynaptic level. On the other hand, thymopoietin inhibited the binding of [125I]alpha-BGT to the nicotinic receptor of rat hemidiaphragm. In intact muscle tissue, the IC50 value for inhibition of [125I]alpha-BGT binding by thymopoietin was 2.1 x 10(-7) M, a value similar to the concentration of polypeptide required to inhibit phrenic nerve-induced muscle contraction (IC50 value, 0.75-1.6 x 10(-7) M). In a muscle membrane preparation, the potency of thymopoietin to affect [125I]alpha-BGT binding was increased (IC50 value, 0.35 nM); thus thymopoietin has the potential to interact at the nicotinic receptor in the nM range. To conclude, the present results show that thymopoietin inhibits neuromuscular activity by an effect that appears to be a specific interaction at the nicotinic receptor.(ABSTRACT TRUNCATED AT 250 WORDS)

The effects of the thymic polypeptide thymopoietin (Tpo) on the properties of the nicotinic acetylcholine receptor (AcChoR) were investigated by patch clamp techniques on mouse C2 myotubes and by biochemical assays on AcChoR-rich membrane fragments purified from the Torpedo marmorata electric organ. At high concentrations (greater than 100 nM), Tpo inhibits the binding of cholinergic agonists to the AcChoR in a Ca2+-insensitive manner. At lower concentrations (2 nM), Tpo applied on C2 myotubes simultaneously with nondesensitizing concentrations of acetylcholine results in the appearance of long closed times separating groups of openings. This effect depends on the presence of Ca2+ in the external medium. Outside-out recordings, performed with various concentrations of EGTA in the intracellular medium, suggest that Ca2+ acts on the cytoplasmic face of the membrane after entry through acetylcholine-activated channels. Parallel studies with T. marmorata AcChoR-rich membranes show that in the presence of Ca2+ Tpo causes a decrease in the apparent equilibrium dissociation constant of the noncompetitive blocker [3H]phencyclidine, enhances, at low concentrations, the binding of [3H]acetylcholine, and also alters the binding kinetics of the fluorescent agonist 6-(5-dimethylamino-1-naphthalenesulfonamido)-n-hexanoic acid beta-(N-trimethylammonium bromide) ethyl ester to the AcChoR. It was concluded that, in the presence of Ca2+, Tpo displaces the conformational equilibrium of the AcChoR towards a high-affinity desensitized state and increases the transition rate towards the same state.

The role of corticotropin-releasing factor (CRF) in four model stresses (cold, ether, immobilization, and trauma) was examined in the guinea pig by using passive immunoneutralization with anti-CRF antiserum. Plasma corticotropin levels were measured at various times after exposure to stress, and groups treated with CRF antiserum were compared with those treated with normal rabbit serum. Of the four stresses tested, ether had the most pronounced effect on corticotropin secretion. Treatment with anti-CRF inhibited most of the ether-induced corticotropin secretory response, the difference between the normal serum- and the anti-CRF antiserum-treated groups being significant at 5 and 10 min (P less than 0.01). Corticotropin responses to cold stress in the two groups differed at the 0.05 level of significance at 10 and 20 min. After administration of trauma (leg fracture), a statistically significant difference (P less than 0.01) between the two groups also was evident, albeit only at 20 min. During immobilization, corticotropin levels differed significantly from control only in the normal serum-treated group but not in the anti-CRF-treated group. These findings show that CRF antiserum was effective in reducing corticotropin levels, indicating that CRF has an important role in mediating corticotropin response to stress. The fact that neutralization was incomplete might be due to an inability of the antiserum to sufficiently neutralize the endogenous CRF or, more likely, reflects the contribution of additional mediators, notably catecholamines and vasopressin, of corticotropin release upon stress.