Effects of medium composition and metabolic inhibitors on glycosaminoglycan synthesis in chick embryo cartilage and its stimulation by serum and triiodothyronine.

Incorporation of inorganic sulfate into glycosaminoglycans of chick embryo sternum is stimulated by serum and triiodothyronine. Variations in the amino acid content of the medium, and in particular in the concentration of glutamine, changed the incorportion in control and stimulated sterna to the same degree. Omission of Na+ from the medium greatly reduced incorporation in both control and stimulated sterna; incorporation, and its stimulation by triiodothyronine, was restored by raising the concentration of Na+. Ouabain and valinomycin inhibited incorporation more than 90%, and triiodothyronine did not stimulate under these conditions. Puromycin and cycloheximide also inhibited incorporation almost completely, and abolished the stimulation by triiodothyronine and serum. Addition of p-nitrophenyl-beta-xyloside, in the presence of of puromycin ir cycloheximide, restored sulfation to a level of 5-10% of the control value; however, this level of incorporation was not increased by addition of serum or triiodothyronine. Actinomycin D, colchicine and vinblastine inhibited incorporation by 40% or less at the highest concentrations tested; however, these three agents completely abolished the ability of triiodothyronine to stimulate incorporation. Lumicolchicine and cytochalasin B decreased incorporation in controls slightly but did not affect the stimulation by serum or triiodothyronine. The results indicate that thyroid hormones stimulate glycosaminoglycan synthesis only under conditions which support efficient synthesis in control incubations, and suggest that microtubule formation may be essential to the mode of action of thyroid hormones in this system.

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.

Biosynthesis of neurophysin proteins in the dog and their isolation.

Neurophysin (Np) is generally found in close association with vasopressin and oxytocin in the hypothalamo-neurohypophyseal complex. Dog neurophysin I and II have been isolated from fresh and frozen posterior pituitaries. The proteins were characterized on the basis of disc electrophoresis, immunological properties, amino acid composition and partial sequence determination. The amino terminal sequence of dog Np I is Ala-Ala-Leu-Asp-Leu-Asp-Val-Arg-Gln-Cys-Leu-Pro-Cys-Gly-Pro-Gly-Gly-Gln-Gly-while that of dog Np-II is Ala-Met-Ser-Asp-Leu-Glu-Leu. The dog Np I appears to be metabolically less stable than Np II. Isotope experiments with [35S]cystine or 3H-labeled amino acids using a design of "in vitro pulse and in vitro chase" as well as "in vivo pulse and in vivo chase," added further confirmation of the capability of the hypothalamic neurosecretory cells to synthesize concomitantly precursors of Np and vasopressin. The radioactively labeled precursors were converted to Np-like protein and vasopressin, both of which were isolated.

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.

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.

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.

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.

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.

Enniatin production by Fusarium sambucinum: primary, secondary, and unitary metabolism.

Fusarium sambucinum liquid surface cultures on semi-defined medium with glucose as carbon source passed through well-defined phases corresponding to trophophase, idiophase (enniatin production), and a degenerative phase. All the glucose was consumed by day 6, at which time the mycelial dry weight had reached only half its maximum. When glucose was replaced by lactose, there was no separation of trophophase and idiophase. Enniatin production, dry weight, and sugar and nitrogen consumption were in approximate balance throughout the growth period (25 days), after which slow degeneration began. The term 'unitary metabolism' is proposed for this type of unphased behaviour. Unitary metabolism may approximate more closely to that occurring under natural conditions than does the metabolic phase separation observed when rapidly utilized carbon sources are used in laboratory cultures.

Enhancement of somatomedin titers of normal and hypopituitary sera by addition of L-triiodothyronone in vitro at physiological concentrations.

Somatomedin potencies of sera were assayed by following sulfation of mucopolysaccharides in chick embryo sterna in vitro. Apparent potencies of sera from hypophysectomized rats, maintained on a low-iodine diet, were restored to levels above normal by addition to the incubation medium of L-triiodothyronine at 10-7 mol/liter of serum. The potencies of normal rat, human, and fetal calf sera were raised 1.3- to 3-fold by addition of triiodothyronine at 10-9-10-7 mol/liter of serum. L-Thyroxine was about 10 times less effective than triiodothyronine. Triiodothyronine alone did not stimulate sulfation to nearly the same extent as triiodothyronine plus serum, even at higher concentrations. Serum could not be substituted in this system by any of six purified hormones, nor by trace metals or amino acids not included in the incubation mixture. It is concluded that triiodothyronine, in combination with a factor in serum, causes a rapid stimulation of sulfation in chick embryo cartilage, and that thyroid hormones may be involved in the action of normal serum on this tissue.

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.