Thyrotropin-releasing hormone-degrading enzyme in human serum is classified as type II of pyroglutamyl aminopeptidase: influence of thyroid status

The characteristics of thyrotropin-releasing hormone (TRH)-degrading enzyme in human serum were studied. Serum was incubated in 0.1 M phosphate buffer containing [proline-3H]TRH at 37 degrees C. A thin layer chromatography analysis of TRH degradation did not show any radioactive peak located in an acid TRH position, but apparent radioactive peaks corresponding to His-Pro and His-ProNH2 occurred in the presence of p-hydroxymercuriphenyl sulfonic acid, an inhibitor of proline dipeptidase. With ion exchange paper chromatography, the formation of 3H-labeled His-Pro and His-ProNH2 was estimated as an end point in the measurement of pyroglutamyl aminopeptidase (pGlu-peptidase) activity. An assay using p-hydroxymercuriphenyl sulfonic acid was developed to sensitively quantitate the pGlu-peptidase. Neither bacitracin nor p-chloromercuribenzoic acid increased the activity of pGlu-peptidase. The addition of EDTA, dithiothreitol, and o-phenanthroline significantly inhibited pGlu-peptidase activity, but neither iodoacetamide nor ethylmaleimide altered its activity. The pGlu-peptidase had a stereotypic specificity for the tripeptide, pGlu-His-ProNH2 of TRH, and its Km was 44.9 microM. The pGlu-peptidase activity was not changed by either hyper- or hypothyroidism. The present data indicate that a TRH-degrading enzyme in human serum possesses a nature identical to type II of pGlu-peptidase which is not altered by thyroid status.     

Nef genes of SIV

Molecular clones of SIVmac were constructed that differed only in sequences within the nef gene. DEAE-transfection of viral DNA containing an open from of nef yielded virus that replicated with similar kinetics and to a similar extent in macaque peripheral blood lymphocyte (PBL) cultures as virus with a deletion or stop codon within nef. Rhesus monkeys that received each kind of molecularly cloned virus became infected. Our results additionally suggest that mutant forms of virus are selected in vitro while open, functional forms are selected in vivo. In animals infected with virus containing a stop codon within nef, reversion of the stop codon to a coding codon was demonstrated in five of five clones analyzed. These results indicate that nef is playing some role crucial to the virus life cycle in vivo.     

Characterization of human thyrotropin receptor-related peptide-like immunoreactivity in peripheral blood of Graves' disease.

An antiserum raised against an alignment of amino acid-(32-56), termed TSHRP-1, in the extracellular domain of human thyrotropin (TSH) receptor was used to identify the TSH receptor-like substance in plasma of Graves' disease. The dilution curve of plasma TSHRP-1-like immunoreactivity was observed in a manner parallel to the standard synthetic peptide curve in radioimmunoassay, and its molecular weight estimated approximately 60 kDa. The amounts of TSHRP-1-like immunoreactivity were significantly higher in Graves' plasma than those in plasma of normal and hypothyroid patients due to Hashimoto's thyroiditis. The present results indicate that human peripheral blood possesses a soluble form of the extracellular domain of TSH receptor which may contribute to the pathophysiology of Graves' disease.     

Effect of fructose on glycogen synthesis in the perfused rat liver

The effect of fructose on glycogen synthesis was examined in the perfused liver of starved rats. With increasing fructose concentration in the perfusate, glycogen synthesis and the % a form of glycogen synthase increased to a maximum at 2 mM and then decreased, progressively. The glucose 6-P level increased with the increase in fructose concentration. On the other hand, the ATP content was unchanged at a concentration of 2 mM or less and decreased at 3 mM or more. We also showed that the stimulation of glycogen synthesis by fructose at a concentration of 2 mM or less was due to activation of glycogen synthase by accumulated glucose 6-P and that ATP depletion at a concentration of 3 mM or more caused an increase in phosphorylase a and a decrease in glycogen synthase activity even in the presence of a high concentration of glucose 6-P.     

Influence of bile salts on β-glucuronidase activity of intestinal bacteria

T. FUJISAWA AND M. MORI. 1996. The β-glucuronidase activity of intact cells of Escherichia coli and Clostridium perfringens was increased in the presence of bile salts. In contrast, bile salts had inhibitory effects on the activity of β-glucuronidase extracted from the lysed cells. These results suggest that the permeability of the bacterial cells is increased by the presence of bile salts, and that bile salts may significantly enhance bacterial β-glucuronidase activity in the intestinal tract.     

Nest site choice by the three-spined stickleback, Gasterosteus aculeatus (form leiurus), in spring-fed waters

The nest site characteristics of the freshwater three-spined stickleback, Gasterosteus aculeatus (form leiurus ), were quantitatively investigated in springs and the main stream of the Yamayoke and the Tsuya River system, central Japan. Most nests (93·4%) were on a muddy or sandy substratum, at depths of 10–40 cm (84·3%), in water velocities less than 15 cm s⁻¹ (76·2%) and in the temperature range of 14 to 16° C (82·7%), Spring-fed localities provided more of these conditions than the main stream channel and hence contained more potential nesting areas. Thus, they were utilized by male sticklebacks both temporally (prolonged breeding season) and spatially (more nest sites).     

Effects of visible light and other environmental factors on the production of oxygen radicals by hamster embryos

Previous studies have demonstrated that developing hamster embryos are very sensitive to visible light. In order to elucidate why visible light exerts a toxic effect on hamster embryos, we examined the effect of visible light on the production of hydrogen peroxide (H(2)O(2)) within individual embryos, using a fluorimetric method. In addition, we examined the H(2)O(2) generating capacity of other factors which are known to be related to the in vitro developmental capacity of hamster embryos. One-cell hamster embryos were cultured with 2',7'-dichlorodihydrofluorescin diacetate, and the fluorescence emissions of the H(2)O(2)-dependent oxidative product in the embryos were measured using an Olympus microscopic photometry system. When embryos were exposed to visible light (14,000 lux) for a specified period (0, 0.5, 1, 2 or 3 min) prior to measurement, the fluorescence emissions from embryos increased with the time of exposure to visible light. An exposure of even 0.5 min resulted in a significant increase in hydrogen peroxide. This increase was more rapid in embryos cultured under 20% O(2) than in those cultured under 5% O(2), and the response was quicker than that observed in mouse embryos. The fluorescence emissions from embryos cultured under 5% O(2) were significantly (P<0.001) lower than those from embryos cultured under 20% O(2) in TLP medium. However, the effects of different oxygen tensions on fluorescence emissions were medium-dependent, and were not significant in embryos cultured in HECM-1 medium. The addition of L-cysteine to or elimination of phenol red from the media decreased the fluorescence emissions from embryos (P<0.001), but glucose and phosphate did not affect them. These results suggest that the toxic effect of visible light on the in vitro development of hamster embryos might be due to increased generation of reactive oxygen species, induced by the visible light. This could be one of the explanations for the strict conditions required for overcoming the in vitro developmental block. It is also suggested that the promotive effects of low oxygen culture and L-cysteine on embryo development seem to be derived from their ability to reduce reactive oxygen species.