Tripeptide structure of bursin, a selective B-cell-differentiating hormone of the bursa of fabricius.

Differentiation of lymphoid precursor cells in a variety of species is induced by polypeptide hormones such as thymopoietin for T cells and bursin for B cells. In the present experiments, bursin isolated from the bursa of Fabricius of chicken was found to induce the phenotypic differentiation of mammalian and avian B precursor cells but not of T precursor cells in vitro. Similarly, bursin increased cyclic guanosine monophosphate in cells of the human B-cell line Daudi but not in cells of the human T-cell line CEM. These inducing properties of bursin are the reverse of the inducing properties of thymopoietin produced by the thymus and are appropriate to a physiological B-cell-inducing hormone. A tripeptide sequence (lysyl-histidyl-glycyl-amide) was determined for bursin and confirmed by synthesizing this proposed structure and demonstrating chemical identity of the natural and synthetic peptides. Similarity of biological action was indicated in induction assays by elevation of cyclic adenosine monophosphate and guanosine monophosphate in Daudi B cells but not in CEM T cells.

Abnormally high plasma levels of vitamin B6 in children with autism not taking supplements compared to controls not taking supplements.

BACKGROUND: There have been many studies of the effect of high-dose supplementation of vitamin B6 on children and adults with autism, with all but one reporting benefits. OBJECTIVE: The aim of this study was to investigate the biochemical basis for vitamin B6 therapy by measuring the level of total vitamin B6 in the plasma of unsupplemented children with autism spectrum disorder compared to unsupplemented control subjects. PARTICIPANTS: Children with autism spectrum disorders (n = 35, age 3-9 years) and unrelated typical children (n = 11, age 6-9 years), all from Arizona, were studied. (This includes the data from 24 children with autism from our previous study.) METHODOLOGY: A microbiologic assay was used to measure the level of total vitamin B6 (including phosphorylated and unphosphorylated forms), in a blinded fashion. RESULTS: Children with autism had a 75% higher level of total vitamin B6 than the controls (medians of 56 versus 32 ng/mL, respectively, p = 0.00002). Most of the autistic children (77%) had levels that were more than 2 standard deviations above the median value of the controls. The autistic girls (n = 5) also had elevated levels (mean of 54.6 ng/mL, median of 60 ng/mL). DISCUSSION: These results are consistent with previous studies that found that: (1) pyridoxal kinase had a very low activity in children with autism and (2) pyridoxal 5 phosphate (PLP) levels are unusually low in children with autism. Thus, it appears that the low conversion of pyridoxal and pyridoxine to PLP results in low levels of PLP, which is the active cofactor for 113 known enzymatic reactions, including the formation of many key neurotransmitters. CONCLUSIONS: Total vitamin B6 is abnormally high in autism, consistent with previous reports of an impaired pyridoxal kinase for the conversion of pyridoxine and pyridoxal to PLP. This may explain the many published studies of benefits of high-dose vitamin B6 supplementation in some children and adults with autism.

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.

Structural characterization and localization of corticotropin-releasing factor in testis.

To sequence and thereby definitively characterize corticotropin-releasing factor (CRF)-like material from a representative peripheral tissue, CRF was obtained from 76 ovine testes. The novel extraction procedure involved use of an immunoaffinity column to which a high-affinity CRF monoclonal antibody was attached as well as fast protein liquid chromatography. The complete sequence was elucidated by gas-phase sequencing, carboxyamidopeptidase digestion and cyanogen bromide cleavage. Aside from microheterogeneity at position 39, all the other amino acids were identical to ovine hypothalamic CRF. Additionally, in immunohistochemical studies in the rat, CRF was localized to the Leydig cell. These findings along with related observations by ourselves and others are compatible with the hypothesis that CRF plays a significant local role, possibly by paracrine or autocrine mechanisms.

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.

Interleukin 1 beta mediates stress-induced immunosuppression via corticotropin-releasing factor.

Intracerebroventricular (icv) infusion of human interleukin 1 beta (IL-1) into intact and adrenalectomized rats impairs immune function. Using antibody to IL-1 as well as an inhibitor of IL-1 action, we sought to determine if endogenous IL-1 in the central nervous system has a physiological role in mediating the immunosuppressive effects of stress. Compared with freely moving controls, rats given intermittent electric shock to the tail for 40 min exhibited a fall in T lymphocyte proliferation and natural killer (NK) cell cytotoxicity of 33% and 38%, respectively; however, when pretreated with icv human IL-1 monoclonal antibody, which significantly crossreacts with rat IL-1, the decrement was attenuated to 14.6% and 15%, respectively. When rats were pretreated with icv alpha-MSH, which blocks many IL-1 effects, shock-induced suppression of 42% in both T lymphocyte proliferation and NK cytotoxicity were blunted to 33% and 31%, respectively. Similar results were found in adrenalectomized rats. These findings suggest that endogenous IL-1 is a physiologically relevant mediator of the immune response to stress. As IL-1 has been reported to release CRF, which we have shown always plays a significant role in stress-induced immunomodulation, we then assessed the relationship of IL-1 and CRF in immunosuppression. Infusion of icv IL-1 caused a decrease of 35% in T lymphocyte proliferation and 34% in NK activity, but pretreatment with CRF antibody icv attenuated IL-1 suppression of T lymphocyte proliferation and NK activity to 10% and 8%, respectively. Comparable results were observed in adrenalectomized rats. These findings suggest that CRF antibody is able to block the immunosuppressive effects of IL-1. To further examine the interaction of CRF in mediating stress-induced immunosuppression, we found that animals pretreated with icv CRF antibody, shocked and then given icv IL-1, had a decrement in T lymphocyte proliferation and NK cytotoxicity of 24% and 21%, respectively, demonstrating that the immunosuppressive effect of icv IL-1 is blocked when central CRF has been neutralized by prior administration of icv CRF antibody. In contrast, animals pretreated with icv IL-1 antibody, shocked and then given icv CRF, had decrements of 38% and 40%, respectively, showing that icv CRF does act even when central IL-1 has been neutralized by prior administration of icv IL-1 antibody. Thus, we conclude there is a sequential relationship between two of the known mediators of stress-induced immunosuppression, with release of central IL-1 followed by that of CRF.

Corticotropin-releasing factor levels in the peripheral plasma and hypothalamus of the rat vary in parallel with changes in the pituitary-adrenal axis.

Impressive evidence has emerged indicating that immunoassayable and bioassayable CRF, which is immunoneutralizable, is present not only in the hypothalamus but in many peripheral tissues as well. Using highly specific and sensitive RIAs and immunoaffinity chromatography to investigate whether this extrabrain CRF circulates in the rat, we found low but clearly measurable levels in peripheral plasma (mean, 11.4 +/- 0.8 pg/ml). Immunological findings were corroborated by fast protein liquid chromatography, which resolves peptides by both hydrophobicity and ionic charge. With this approach the major immunoreactive peak was eluted at the position of synthetic rat CRF standard. To assess whether levels of peripheral plasma CRF-like immunoreactivity (CRF-LI) vary in parallel with those of hypothalamic CRF-LI, we performed studies with low and high dose dexamethasone administration and withdrawal, adrenalectomy, and hypophysectomy. Seven days after oral administration of dexamethasone, there was a decrement in the levels of peripheral plasma and hypothalamic CRF-LI. Depending on the dose, recovery was also found 7 days after cessation of the treatment. After either adrenalectomy or hypophysectomy, there were increments in the levels of CRF-LI in both peripheral plasma and hypothalamus. Thus, concentrations of CRF-LI in the peripheral plasma and in the hypothalamus vary in parallel in response to alterations in the pituitary-adrenal axis.

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.

Levels of corticotropin releasing factor-like immunoreactivity in mammalian hypothalamic and extrahypothalamic brain tissue as determined with a monoclonal antibody to the ovine material.

A monoclonal antibody to ovine corticotropin releasing factor (CRF) has been produced by fusion of a non-producing plasmacytoma cell line P3U1 with spleen cells of Balb/c mice immunized with the synthetic 41 amino acid peptide coupled covalently with rabbit myosin by a heterobifunctional reagent, N-succinimidyl 3-(2-pyridyldithio) propionate. A total immunizing dose of 500 micrograms resulted in a highly specific, high-affinity antibody with a Ka of 0.15 x 10(12) M-1, which was used to establish a specific RIA with a sensitivity of 10 pg/tube. Levels of corticotropin releasing factor-like immunoreactivity (CRF-LI) in a pg/mg of hypothalamic tissue ranged from 4-10 in ovine, 2.5-8 in bovine, 47.5-67.5 in mouse and 2.3-20 in human tissue. Moreover, CRF-LI was widely distributed in extrahypothalamic mouse brain at concentrations approximately one half those seen in hypothalamus.

Corticotropin-releasing factor modulates the immune response to stress in the rat.

We examined the role of CRF, a key mediator of the endocrine response to stress, in modulating immunosuppression during the subacute stress of intermittent electrical shock over 1 h. Administration of shock to intact rats resulted in a 74% decrement in T-lymphocyte proliferation and a 59% decrease in natural killer cytotoxicity. Similar suppression of these two parameters of immune function in response to shock was noted in adrenalectomized rats as well. The immunosuppressive effects of this shock were significantly and comparably blunted when both intact and adrenalectomized animals were pretreated 1) iv with either a highly potent polyclonal CRF antibody or a specific CRF antagonist or 2) intracerebroventricularly with either a high affinity monoclonal antibody to CRF or a specific CRF antagonist. An immunomodulatory role for CRF is further supported by the findings that administration of exogenous CRF, either iv (10 micrograms/animal) or intracerebroventricularly (1 microgram/animal), resulted in significant decrements in lymphocyte proliferation and natural killer cytotoxicity, similar to those seen with the stress paradigm. Our observations indicate that CRF plays a significant role in modulating the immune response to subacute stress, largely by adrenal-independent mechanisms.

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.

Antibodies to thymopoietin following implantation of paper disks derivatized with synthetic Cys-thymopoietin.

A synthetic peptide corresponding to Cys-thymopoietin 28-39 was synthesized and coupled by diazo linkages to aminophenyl thioether-derivatized paper disks. Disks were implanted in the peritoneal cavities of mice, initially after soaking in complete Freund's adjuvant and subsequently, at 3 week intervals, without further treatment. After four implantations, 6/6 mice developed antibodies reacting with the synthetic peptide and with native thymopoietin. In contrast, mice conventionally immunized with peptide alone (six mice) or with peptide complexed with thyroglobulin (six mice) all failed to develop antibodies. Mice immunized with disks derivatized with Cys-thymopoietin 9-20, corresponding to the other hydrophilic region of thymopoietin, also failed to develop antibodies. Thymopoietin 28-39 corresponds to an antigenic hydrophilic region of thymopoietin that contains the pentapeptide active site (thymopoietin 32-36, Arg-Lys-Asp-Val-Tyr).

Thymopoietin interacts at the alpha-bungarotoxin site of and induces process formation in PC12 pheochromocytoma cells.

Thymopoietin, a polypeptide isolated from thymus and involved in immune regulation, potently inhibited [125I]alpha-bungarotoxin binding in both pheochromocytoma (PC12) cells in culture (IC50 of 3.9 nM) and in PC12 cell membranes (IC50 of 2.2 nM). The degree of inhibition produced by thymopoietin was similar to that observed with alpha-bungarotoxin; in contrast, nicotinic receptor ligands affected alpha-bungarotoxin binding only at micromolar concentrations, in agreement with previous work. Binding of thymopoietin was reversible. Studies with PC12 cell membranes suggested that the interaction between alpha-bungarotoxin and thymopoietin at the receptor was competitive. The effect of thymopoietin was subsequently assessed on various morphological characteristics of PC12 cells in culture. Exposure of the cells to the polypeptide resulted in neurite extension, which was evident as early as 1-2 days in culture and was maximal after 4-6 days; this response was observed with concentrations of thymopoietin as low as 10(-8) M. Nerve growth factor also induced neurite extension in PC12 cells; however, the effects of nerve growth factor were qualitatively and quantitatively distinct from those which occurred with thymopoietin. Moreover, a monoclonal antibody to nerve growth factor completely prevented the nerve growth factor-induced process formation without affecting the thymopoietin-induced response. On the other hand, alpha-bungarotoxin resulted in the formation of processes which appeared morphologically similar to those induced by thymopoietin, although alpha-bungarotoxin appeared less potent than the thymic polypeptide. The effect of thymopoietin appeared to be specific; thysplenin, a polypeptide with approximately 80% homology with thymopoietin, did not elicit process formation. The thymopoietin-induced effect was reversed upon removal of the polypeptide from the culture medium. These results show that thymopoietin, a polypeptide endogenous to mammalian systems, potently interacted at the alpha-bungarotoxin site in a neuronal cell line. Furthermore, thymopoietin could elicit process formation in PC12 cells, suggesting that it may be a neuronotrophic factor.

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.

Corticotrophin-releasing factor in extra-hypothalamic brain of the mouse: demonstration by immunoassay and immunoneutralization of bioassayable activity.

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.

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.

Biologically active analogs of thymopentin with enhanced enzymatic stability.

Thymopentin, a synthetic pentapeptide fragment of thymopoietin (residues 32-36, Arg-Lys-Asp-Val-Tyr) is biologically active but susceptible to proteolytic digestion. Analogs were synthesized and studied for biological activity and susceptibility to peptidases. Amino acid changes were incorporated at positions known to not affect activity adversely and N-terminal acetylation and C-terminal amidation were used to increase resistance to proteolytic degradation by exopeptidases. Ac-Pro2-TP5-NH2 and Aib2-TP5-NH2 retained activity and were shown to exhibit a high degree of stability when incubated in human serum.

Peptide analogs of thymopentin distinguish distinct thymopoietin receptor specificities on two human T cell lines.

Thymopoietin, a polypeptide hormone of the thymus, and the synthetic pentapeptide thymopentin, corresponding to thymopoietin32-36, both induced elevations of intracellular cyclic GMP in two human T cell lines, CEM and MOLT-4. In contrast, the closely related polypeptide thysplenin, which differs from thymopoietin at position 34, induced intracellular cyclic GMP elevation in MOLT-4 but not in CEM. We synthesized a series of penta- and tetrapeptide analogs of amino acids 32-36 of human thymopoietin and thysplenin, and now show that distinct patterns of activity can be obtained in these small peptides, with selectivity for cyclic GMP elevation in MOLT-4 alone or CEM alone. This suggests that the thymopoietin receptors (TPR) on these two human T cell lines are distinguishable by their differing ligand specificities, and we have termed them alpha TPR and beta TPR for CEM and MOLT-4 receptors, respectively.

Cooperativity of thymopoietin 32-36 (the active site) and thymopoietin 38-45 in receptor binding.

Thymopoietin is a 49 amino acid polypeptide hormone of the thymus whose biological activity is reproduced by the synthetic pentapeptide thymopentin, corresponding to amino acids 32-36. Thymopentin requires the addition of an octapeptide corresponding to thymopoietin 38-45 for full competition with native thymopoietin in a radioreceptor assay with receptor derived from the human T-cell line CEM. Thus thymopoietin appears to bind to its receptor on T-cells by two regions (32-36 and 38-45). Thymopentin alone is biologically active and induces elevations of intracellular cyclic GMP. Whilst occupancy of the adjacent site by thymopoietin 37-45 does not of itself cause an elevation of intracellular cyclic GMP this peptide is not biologically silent as it does enhance the potency of thymopentin.

Immunoreactive thymopoietin in the mouse central nervous system.

A thymopoietin-immunoreactive substance (TP-IRS) has been detected in homogenates of mouse spinal cord and brain using a radioimmunoassay; levels were maximal at birth. TP-IRS was also detected in supernatants of mouse neuroblastoma (NIE-115) and primary spinal cord cultures but not human astrocytic and meningeal tumors or mouse primary astrocyte cultures. With affinity purified rabbit anti-TP globulin, immunofluorescent staining was seen in mouse spinal cord cultures in association with nuclear membranes of neurons and, to a lesser degree, flat background cells. From supernatants of NIE-115 cells grown in tritiated leucine and lysine, proteins of approximately 8000 and 4500 Da were isolated by TP affinity chromatography (compared with 5562 Da for thymic thymopoietin). When injected into mice, these neural proteins partially blocked neuromuscular transmission in a manner similar to thymic thymopoietin.