Vasopressin affects the behavior of rats in a positively-rewarded discrimination task

The effects of subcutaneous injections of vasopressin were investigated in a study utilizing 72 male Long-Evans rats trained in an appetitive black-white discrimination T-maze task. Animals which were reinforced for choosing the black goal arm demonstrated prolonged extinction if they received vasopressin prior to daily extinction sessions. This effect was not observed in animals reinforced for choosing the white goal arm. Prolonged extinction was not found in animals which received vasopresson only during acquisition or in control animals which received saline. Speed and activity scores did not differentiate the groups. These results demonstrate that vasopressin can affect the behavior of rats on a positively-reinforced task.     

Regulation of high- and low-affinity epidermal growth factor receptors by glucocorticoids

It is reported that receptors for epidermal growth factor (EGF) in HeLa S₃ cells exist in two forms, which differ in both affinity and capacity. Both the number of receptors and their distribution into low- and high-affinity forms are modulated by glucocorticoids. Scatchard analysis of saturation binding assays performed at 0 °C indicates that there is a low-affinity class of receptors ($\text{K}{}_{\mathrm{d}}\text{? 1.5}\text{nm}$), which contains approximately 6 × 10⁴ binding sites per cell, and a second, high-affinity class of receptors ($\text{K}{}_{\mathrm{d}}\text{? 0.16}\text{nm}$) containing approximately 5 × 10³ binding sites per cell. Exposure of HeLa S₃ cells to 10^?7m dexamethasone for 24 h increased EGF binding to whole cells by increasing the numbers of low- and high-affinity receptors by 20 and 114%, respectively. The increase in EGF binding depends upon the dose of dexamethasone, being raised from 10^?11 to 10^?6m. EGF binding is half-maximal near 2–4 × 10^?9m, a concentration equal to the K_d of dexamethasone for the glucocorticoid receptor in these cells. The increase in EGF binding is specific for glucocorticoids, occurring when the HeLa S₃ cells are exposed to 10^?7m cortisol or dexamethasone for 24 h, but not when the cells are similarly treated with testosterone, 5α-dihydroxytestosterone, 17β-estradiol, or progesterone. The effect on EGF binding appears to be biphasic; the initial rapid increase occurs between 8 and 12 h, is blocked by both 10^?6m cyclohexamide and 0.1 μg/ml actinomycin D, and is followed by a more gradual increase thereafter. These data indicate that glucocorticoids are able to regulate both the number of EGF receptors and their distribution into high- and low-affinity components. Press, Inc.     

Pituitary desensitization and the regulation of pituitary gonadotropin-releasing hormone (GnRH) receptors following chronic administration of a superactive GnRH analog and testosterone

We recently demonstrated that chronic daily administration of a superactive GnRH analog to intact rats resulted in an initial stimulation of serum LH levels with a subsequent return of LH levels to baseline at a time when testosterone levels were marked decreased. These data demonstrated pituatary desensitization following chronic GnRH analog treatment. Administration of GnRH analog with a dose of testosterone which did not markedly lower serum LH levels when administered alone prevented the stimulation of LH secretion by analog. The present studies were undertaken to determine the effects of GnRH analog and testosterone administration on the regulation of pituitary GnRH receptors. Pituitary GnRH receptor binding was increased by analog treatment alone at 20 days and returned to control levels at 40 and 60 days of treatment in parallel to the observed changes in serum LH, demonstrating that one mechanism by which chronic GnRH analog treatment leads to pituitary desensitization is down-regulation of pituitary GnRH receptors. Testosterone administration alone decreased pituitary GnRH receptor binding. Combined GnRH analog and testosterone administration prevented the increase in pituitary GnRH receptors observed with analog administration alone. These studies demonstrate that changes in pituitary GnRH receptor binding correlate with changes in serum LH and that the stimulatory effects of analog administration on LH are sensitive to inhibition by small doses of testosterone.     

Differences in the stress response of prolactin in young and aged female rats

The drawing of blood by orbital sinus puncture (OSP) under ether anesthesia is known to produce a marked increase in serum prolactin (PRL levels in young cycling female rats. The effect of this stressful procedure on PRL release was compared in young and aged female rats. Nonstressed PRL levels were obtained from blood drawn by decapitation. Whereas OSP with a one-minute ether exposure induced a marked increase in PRL levels in young rats on all days of the estrus cycle, older cycling female rats on the day of diestrus -1 and aged rats exhibiting prolonged diestrus (PD) showed virtually no increase above nonstressed levels. However, increasing the ether exposure time to five minutes did produce a rise in PRL levels. Old cycling female rats on the day of estrus and aged rats exhibiting constant estrus (CE) did show a PRL increase comparable to that seen in young animals. Ovariectomy (OVX) completely abolished the stress response seen in aged CE rats. The response, though markedly decreased, was still present in young ovariectomized rats. These experiments show that the stress-induced rise in PRL promoted by OSP under either anesthesia is markedly diminished in aged rats exhibiting a diestrus state. The attenuated response seen in these rats is believed due to factors characteristic of the diestrous state of aging.     

Prevention by bombesin of cold-restraint stress induced hemorrhagic lesions in rats

Several neuropeptides, injected intraventricularly (ivt), were assessed for their effects on cold-restraint-induced hypothermia and hemorrhagic gastric lesions in 24 hr fasted rats. Bombesin (5-1 μg) further enhanced the drop in body temperature following stress and markedly prevented the gastric erosions in a dose-dependent fashion (5-0.1 μg). β-endorphin exerted a similar effect, but only at the 5 μg dose level. Other peptides (neurotensin, substance P, somatostatin and TRH: 5 μg) did not influence susceptibility to the gastric mucosal damage. Somatostatin and TRH reduced the hypothermic effect of stress. Bombesin is 250 times less potent when injected systemically than ivt and its actions are not reversed by nalaxone. The prevention of gastric erosions by bombesin could initially involve a central mechanism of action, independent of opiate receptors and possibly related to the sustained and marked hyperglycemia observed in bombesin treated rats exposed to stress.     

Male copulatory behavior triggers nightly prolactin surges resulting in successful pregnancy in rats

A positive correlation between the number of preejaculatory intromissions that a female receives during copulation and the probability of successful pregnancy has been demonstrated previously. In the present investigation the nocturnal secretion of prolactin (PRL) was followed for 4 days after mating in female rats receiving either 3–5 intromissions before ejaculation (low intromission group) or 15–18 intromissions (high intromission group). Nightly PRL surges occurred in most of the females (9/12) in the high intromission group and the same 9 females became pregnant. Only 2/9 females in the low intromission group exhibited nightly PRL surges and again only these 2 females became pregnant. This study demonstrates that the stimulation which the female receives from multiple intromissions during mating is effective in setting off nightly PRL surges. We propose that the so-called pregnancy-inducing neuroendocrine reflex which is triggered in this manner is expressed in a characteristic pattern of nightly surges of prolactin, the hormone known to be essential for the activation of the corpora lutea and their secretion of progesterone.     

Analysis of developmentally homogeneous neural crest cell populations in vitro: I. Formation,morphology and differentiative behavior

When early embryonic quail neural tubes are dissected free from surrounding tissues and placed in culture, small stellate neural crest cells usually migrate from the explant onto the substratum. This outgrowth has been reported to consist of a mixture of cells, some of which undergo melanogenesis, while the rest remain unpigmented. We have, in contrast to earlier observations, obtained a spatial separation of the two phenotypes. In these cultures the primary outgrowth of migrating cells remained almost free of pigment-forming cells, whereas small spherical clusters containing several hundred pigment-forming cells appeared on the explanted neural tubes. Whether the clusters remained with the tube explants or were subcultured, all cluster cells differentiated into melanocytes. Prior to melanogenesis, the appearance of the cultured cells from a cluster was indistinguishable from the cells in the outgrowth. The clusters provide a source of neural crest cells, that (1) can be easily obtained in comparatively large numbers, (2) is not contaminated with any other cell type, (3) can be isolated before the onset of differentiation, and (4) is developmentally homogeneous. Thus, the cluster population is well suited for many types of experiments, such as the identification of specific environmental factors that might control neural crest cell differentiation.     

Follitropin receptors in rat testis characterization with enzymatically 125I-labeled human follitropin

The interaction between enzymatically radioiodinated human follitropin and the follitropin receptors in testis homogenate was investigated in immature and adult rats. The ¹²⁵I-labeled human follitropin exhibited high binding activity, with specific binding of up to 17% in the presence of an excess of testis homogenate.Approx. 50% of the bound hormone could be eluted at pH 5, and the receptor purified tracer exhibited a 3.6-fold increase in binding activity when compared with the original tracer preparation. Quantitative analysis of equilibrium binding data was performed with corrections for the measured specific activity and maximum binding activity of the tracer hormone. The equilibrium association constants (K_a) determined at 24°C were not significantly different in immature and adult rat testis, and the mean value for K_a was 3.9 · 10⁹ M^?1. At 37°C, the K_a value obtained using immature rat testis was 1.3 · 10¹⁰ M^?1. The association of ¹²⁵I-labeled human follitropin with immature rat testis homogenate was time and temperature dependent. In the presence of an excess of unlabeled hormone, 30–60% of the preformed hormone · receptor complex was dissociated after 24 h incubation. A specific and sensitive radioligand-receptor assay for follitropin was developed using immature rat testis homogenate. The minimum detectable dose of purified human follitropin was 0.6 ng, and human urinary and pituitary follitropin, ovine follitropin and pregnant mare serum gonadotropin reacted in the assay with equivalent slopes. The potencies of highly purified pregnant mare serum gonadotropin and highly purified human follitropin were similar in the radioligand-receptor assay, consistent with the follitropin bioactivity of the equine gonadotropin.     

Effects of castration and androgen replacement on male courtship behavior in the red-sided garter snake (Thamnophis sirtalis parietalis)

Following artificial hibernation, sexually mature male garter snakes (Thamnophis sirtalis parietalis) exhibited a decline in courtship behavior irrespective of castration, sham operation, or castration with testosterone replacement therapy. Behavior declined more rapidly in castrated animals with testosterone replacement than in castrated or sham-operated animals. In sham-operated animals, the decline in courtship was accompanied by changes in testicular weight and spermatogenic state from small spermatogenically inactive testes to large spermatogenically active testes. Serum androgen levels were more than fourfold greater in sham-operated animals than in castrated animals; cell height of the androgensensitive renal sex segment was greatest in castrated animals with testosterone replacement and least in castrated animals. These findings indicate that following artificial hibernation, male courtship behavior of T.s. parietalis is independent of the presence of the testes.     

Ethanol reduces hepatic estrogen-2-hydroxylase activity in the male rat

Brief exposure to intoxicating levels of ethanol in the male rat produced a marked reduction in a major hepatic enzyme responsible for estrogen metabolism (estrogen-2-hydroxylase). After 4 days of ethanol administration the specific activity of this enzyme decreased by 70% and remained decreased for 6 days following alcohol withdrawal. Enzyme activity returned to control levels by two weeks. However, if animals were retreated with ethanol for one day each week the enzyme activity remained low. Kinetic analysis of the enzymatic activity from ethanol-treated rats showed a decrease in specific activity (Vmax) with no alteration in substrate affinity (apparent Km). The decrease in enzyme activity persisted long after ethanol disappeared from the blood and concentrations of ethanol from 20–100 mM had no effect on enzyme activity when added $\text{in vitro}$. A similar effect of ethanol on hepatic estrogen metabolism in humans may partially explain the elevated serum estrogen levels and the signs of hyperestrogenization observed in male alcoholic patients.     

Configurations of a polymeric antigen adsorbed to a B-cell membrane

For an unsynchronized cell population with several phases of DNA synthesis, the population distribution of tracer (thymidine) incorporation is derived. Compounding this with the Poisson probability for ³H disintegration, the distribution of autoradiographic grains over nuclei is obtained. The changes in distribution after various numbers of cell divisions are obtained on the assumption that the tracer is (approximately) equally partitioned between daughter cells during division. The parameters are expressed in terms of the number of phases of synthesis, specific activity of tracer, rate of incorporation, and autoradiographic exposure time. Application of the theory to experimental material is illustrated.     

Comparative studies on myosin ATPase of a flying and nonflying bird.

Myosin was purified from the flight muscles of a flying (pigeon) and a nonflying (fowl) bird. Ki (ADP) of myosin ATPase of pigeon is higher, but the Km (ATP) is lower than that of fowl. The specific activity (mumole of Pi liberated/min/mg protein) is higher for the fowl. A0.5 (CaCl2) of myosin of both pigeon and fowl is similar. However, the two proteins differ in their interactions with ADP, ATP and p-chloromercuribenzoate. The two proteins have the same tyrosine, tryptophan and sulfhydryl contents. The electrophoretic patterns of the two myosins on SDS-polyacrylamide gels are different. These studies show significant molecular differences in the myosin derived from the flight muscles of a flying (pigeon) and a nonflying (fowl) bird.     

Accumulation of a metallo-carboxypeptidase inhibitor in leaves of wounded potato plants

Mechanical wounding of young potato plants induces over a two fold increase in inhibitory activity against the bovine pancreatic metalloexopeptidase carboxypeptidase A. This increase in inhibitory activity in both wounded and unwounded leaves parallels the increases of two inhibitors of bovine serine endopeptidases, trypsin and chymotrypsin. This suggests that the Proteinase Inhibitor Inducing Factor is regulating the synthesis and accumulation of inhibitors of two different mechanistic classes of proteases found in animals and microorganisms. These increases in antiproteolytic activities due to wounding support the hypothesis that this response is part of a defense mechanism directed against plant pests.     

Preparation and use of α-maltosyl fluoride as a substrate by beta amylase

The preparation of pure (amorphous) α-maltosyl fluoride is described. A modification of the procedure of Brauns was used to obtain analytically pure, crystalline hepta-O-acetyl-α-maltosyl fluoride, the structure of which was assigned by¹⁹F-and¹H-n.m.r. spectroscopy. α-Maltosyl fluoride was obtained by deacetylating the heptaacetate. It behaved as a single compound on thin-layer and paper chromatography, and was essentially completely hydrolyzed to maltose and hydrogen fluoride by 0.01M sulfuric acid in 10 min at 100°. Crystalline beta amylase, likewise, catalyzed essentially complete hydrolysis of α-maltosyl fluoride to give maltose and hydrogen fluoride. The rates of hydrolysis catalyzed by beta amylase preparations from sweet potatoes and soybeans acting on a range of concentrations of the substrate produced linear curves for the relationship, 1/v vs 1/S; reaction constants for crystalline, sweet-potato enzyme were K_m 3.6 mM and V_max ~ 2 μ mol/min/mg. The finding that α-maltosyl fluoride is hydrolyzed 30–60 times faster than maltotriose demonstrates for the first time that beta amylase is capable of effecting hydrolysis at an appreciable rate of a substrate having only two d-glucose residues.     

Interactions of cadmium and selenium in rat plasma in vivo and in vitro

⁷⁵Se and ¹⁰⁹Cd tracers were used to study the binding of Se and Cd to plasma proteins at various SeO₃^2? doses and times up to 24 h after the simultaneous subcutaneous administration of SeO₃^2? and CdCl₂ to adult male rats. The simultaneous injection of CdCl₂ and SeO₃^2? markedly increased both Se and Cd plasma levels over that in control animals. Gel permeation chromatography of plasma indicated that at all times up to 24 h Cd and Se were bound in an atomic ratio of approx. 1 : 1 in 330 000 and 130 000 dalton fractions. From 4 to 24 h, Cd and Se appeared in the 420 000 dalton fraction, also with an atomic ratio of approx. 1 : 1. The 330 000 dalton molecules appeared to have a maximal binding capacity for the Cd-Se complex at a concentration of approx. 30 μmol/ml of plasma, while the 130 000 and 420 000 dalton molecules show a higher binding capacity. Studies in vitro revealed that SeO₃^2? does not interact directly with Cd and plasma proteins. It is metabolized by erythrocytes to a form that interacts in an atomic ratio of 1 : 1 with Cd to form a protein-bound complex of 130 000 daltons.     

Subunit structure of anthranilate synthase from Neurospora crassa: Preparation and characterization of a protease-free form

The multifunctional enzyme complex anthranilate synthase from Neurospora crassa has been purified to homogeneity by a new procedure which yields a stable preparation of the enzyme. Unlike earlier preparations of the enzyme, anthranilate synthase prepared by this technique is not degraded during incubation at 37 °C or during freeze-thaw treatment. Purified anthranilate synthase contains two subunits of M_r 84,000 (β-subunit) and 76,000 (α-subunit), which are shown, by partial proteolysis, to be unrelated in sequence. Immunoprecipitation studies demonstrate that freshly prepared crude extracts of Neurospora contain anthranilate synthase subunits identical in size with those of the purified enzyme. The β-subunit is shown to be the product of the trp1 gene, and the a-subunit, of the trp2 gene.