Effects of phenobarbital, 3-methylcholanthrene and polychlorinated biphenyls on sex-specific forms of cytochrome P-450 in liver microsomes of rats

The effects of treatment with phenobarbital, 3-methylcholanthrene or polychlorinated biphenyls (PCB) on the amounts of sex-specific forms of cytochrome P-450, namely P-450-male and P-450-female, in male and female rats were studied. Although treatment with phenobarbital, 3-methylcholanthrene or PCB markedly increased the total amount of hepatic cytochrome P-450, P-450-male and P-450-female were rather decreased or not significantly changed. Thus, the percentages of P-450-male and P-450-female in the total cytochrome P-450 were decreased in liver microsomes from the treated rats. The increases in specific cytochrome P-450, such as P-448-H, P-448-L, and P-450I-c accounted for the increase in the total amount of cytochrome P-450 in the treated rats. The treatment with phenobarbital or PCB increased the activities of testosterone 16 alpha-hydroxylase, benzo(a)pyrene hydroxylase and aminopyrine N-demethylase more markedly in female rats than in male rats. Similarly, the treatment with 3-methylcholanthrene increased benzo(a)pyrene hydroxylase more markedly in female rats. Therefore, the sex-differences in testosterone 16 alpha-hydroxylase, benzo(a)pyrene hydroxylase, and aminopyrine N-demethylase activities became smaller after the drug treatment. These results indicate that sex-specific P-450-male and P-450-female were unaffected, or even depressed by the agents in some cases.     

Immunohistochemical studies on the localization of cellular retinol-binding protein in rat testis and epididymis

The immunohistochemical localization of cellular retinol-binding protein (CRBP) was studied in rat testis and epididymis. Parallel studies were also carried out on the localization of plasma retinol-binding protein (RBP) and transthyretin (TTR) in testis. The studies employed antibodies purified by immunosorbent affinity chromatography, permitting the specific staining and localization of each antigen by the unlabeled peroxidase-antiperoxidase method. For RBP and TTR, specific immune staining was found in the interstitial spaces between the seminiferous tubules, and not in the tubules themselves. In contrast, strong specific immune staining for CRBP was found in the seminiferous tubules, with a striking localization within Sertoli cells. Moreover, a distinct cyclic variation of specific staining for CRBP within Sertoli cells was observed during the spermatogenic cycle. This cyclic variation was seen with regard to both the intensity of staining and to the anatomic distribution of CRBP within the Sertoli cells. Within the epididymis CRBP was selectively localized to the proximal portion of the caput epididymidis, with variations in intensity of the staining of the epithelium of the ducts in different histological zones. Specific immune staining for CRBP was very weak or absent in the other portions of the epididymis. These results were confirmed by radioimmunoassay. Vitamin A-deficient rats showed markedly reduced specific immune staining for CRBP in both testes and epididymides, and greatly reduced levels of CRBP in these tissues on radioimmunoassay. These studies on the localization of CRBP provide information concerning the specific cells and anatomic loci within the testis and epididymis where retinol may be playing an important role in sperm formation and maturation.     

Occurrence of Yersinia enterocolitica in wild-living birds and Japanese serows.

Yersinia spp. were isolated from 34 of 500 birds representing nine species. The highest isolation rate, 5 of 21 (23.8%), was found in blue magpies (Cyanopia cyanus), followed by pheasants (Phasianus colchicus tohkaidi), 5 of 33 (15.2%); gray starlings (Sturnus cineraceus), 6 of 57 (10.5%); tree sparrows (Passer montanus), 1 of 14 (7.1%); bulbuls (Hypsipetes amaurotis), 4 of 57 (7.0%); crows (Corvus levailantii or Corvus corone), 7 of 117 (6.0%); eastern turtledoves (Streptopelia orientalis), 4 of 118 (3.4%); Chinese bamboo pheasants (Bumbusicola thoracica thoracica), 1 of 36 (2.8%); and domestic pigeons (Columba livia domestica), 1 of 47 (2.1%). The isolates were identified as Yersinia enterocolitica O:3, O:4, O:4,32, O:5A, O:6,30, O:7,8, and O:14, Yersinia frederiksenii, Yersinia intermedia, and Yersinia kristensenii. Yersinia spp. were isolated from 35 of 157 wild-living Japanese serows (Capricornis cripus). The isolates were identified as Y. enterocolitica O:4, O:4,32, O:5A, O:7, O:7,8, O:9, O:14, O:18, and O:34, Y. frederiksenii, Y. intermedia, and Y. kristensenii.     

Isolation of two forms of basement membrane proteoglycans

Sequential extractions of the basement membrane producing Engelbreth-Holm-Swarm tumor yielded heparan sulfate proteoglycans with different size core proteins, but the same size heparan sulfate side chains. Saline, a nondenaturing solvent, extracted a small high density proteoglycan with a heterodisperse core protein of Mr = 95,000-130,000 whereas subsequent extraction with 7 M urea, a denaturing solvent, removed a large, low density proteoglycan with a Mr = 350,000-400,000 protein core. The denaturing conditions required for extraction of the large proteoglycan suggest that it interacts strongly with other basement membrane components. Antibodies to these proteoglycans cross-react with both proteoglycans, but the large proteoglycan has additional antigenic sites not present on the small proteoglycan. These proteoglycans may be derived from the same or similar gene products.     

Sulfated proteoglycan synthesis by confluent cultures of rabbit costal chondrocytes grown in the presence of fibroblast growth factor

We examined the effect of fibroblast growth factor (FGF) on proteoglycan synthesis by rabbit costal chondrocyte cultures maintained on plastic tissue culture dishes. Low density rabbit costal chondrocyte cultures grown in the absence of FGF gave rise at confluency to a heterogeneous cell population composed of fibroblastic cells and poorly differentiated chondrocytes. When similar cultures were grown in the presence of FGF, the confluent cultures organized into a homogenous cartilage-like tissue composed of rounded cells surrounded by a refractile matrix. The cell ultrastructure and that of the pericellular matrix were similar to those seen in vivo. The expression of the cartilage phenotype in confluent chondrocyte cultures grown from the sparse stage in the presence vs. absence of FGF was reflected by a fivefold increase in the rate of incorporation of [35S]sulfate into proteoglycans. These FGF effects were only observed when FGF was present during the cell logarithmic growth phase, but not when it was added after chondrocyte cultures became confluent. High molecular weight, chondroitin sulfate proteoglycans synthesized by confluent chondrocyte cultures grown in the presence of FGF were slightly larger in size than that produced by confluent cultures grown in the absence of FGF. The major sulfated glycosaminoglycans associated with low molecular weight proteoglycan in FGF-exposed cultures were chondroitin sulfate, while in cultures not exposed to FGF they were chondroitin sulfate and dermatan sulfate. Regardless of whether or not cells were grown in the presence or absence of FGF, the 6S/4S disaccharide ratio of chondroitin sulfate chains associated with high and low molecular weight proteoglycans synthesized by confluent cultures was the same. These results provide evidence that when low density chondrocyte cultures maintained on plastic tissue culture dishes are grown in the presence of FGF, it results in a stimulation of the expression and stabilization of the chondrocyte phenotype once cultures become confluent.     

Nitroarenes in Suimon River sediment

Mutagenic activity was observed in sediments of the Suimon River bed with and without S9 mix. The direct-acting mutagens in the sediment were investigated. The sediment was extracted with methanol and fractionated on a Silica gel column. The benzene fraction from the Silica gel column exhibited mutagenic activity without S9 mix in strain TA98, while it failed to show mutagenic activity in nitroreductase-deficient strain TA98NR. This observation led to the suspicion that nitro compounds were the direct-acting mutagens of these samples. The benzene fraction was treated by heptafluorobutyric anhydride (HFBA) and investigated with gas chromatography equipped with an electron capture detector (GC-ECD). 2-Nitrofluorene, 4,4'-dinitrobiphenyl, 2,7-dinitrofluorene and 1-nitropyrene were detected and measured quantitatively. The mutagenic activity of a mixture of these compounds was compared with that of the original fraction and the direct-acting mutagenicity of Suimon River sediment can be explained by these nitroarenes, especially 1-nitropyrene.     

Xyloglucan in the Cell Walls of Suspension-Cultured Rice Cells

The xyloglucan present in the 24% KOH extract of the cell wallsof suspension-cultured rice cells was characterized by fragmentationanalysis with Trichoderma viride cellulase and Aspergillus oryzaeß-D-glucosidase. The xyloglucan is composed mainlyof the following oligosaccharide units: Results showed that the xyloglucan of suspension-cultured ricecells is more extensively branched than is that of rice seedlings.Another structural characteristic of the former xyloglucan isthe presence of D-galactosyl-D-xylosyl side chains that arenot found in the latter. (Received June 15, 1984; Accepted January 11, 1985)     

Distribution of vasopressin and oxytocin in rat brain

Arginine-vasopressin and oxytocin in various portions of rat brain were determined by radioimmunoassays. The hormones were extracted from tissue samples into 0.1 N HCl and then purified partially with acetone-petroleum ether extraction. The non-equilibration method was used for the assays. In this method recovery rates of arginine-vasopressin and oxytocin were 73.0 +/- 4.4% and 75.0 +/- 3.8%, respectively. Sensitivities of the assays were 1 pg of arginine-vasopressin and 0.75 pg (0.3 microU) of oxytocin per assay tube. The higher concentrations of arginine-vasopressin and oxytocin were confirmed in the hypothalamo-neurohypophyseal system, where these hormones are synthesized, transported and stored. Relatively high concentrations of these hormones, especially oxytocin, were detected in spinal cord. Amygdala, hippocampus, limbic forebrain and pineal body contained a certain amount of arginine-vasopressin (2-20 pg/mg protein). Oxytocin (1-7 pg/mg protein) was also detected in amygdala, pons and medulla oblongata, pineal body and midbrain. The low concentrations of these hormones were also found in cerebral cortex and cerebellum.     

Kinetic studies on surface-mediated activation of bovine factor XII and prekallikrein. Effects of kaolin and high-Mr kininogen on the activation reactions

The kaolin-mediated reciprocal activation of bovine factor XII and prekallikrein was divided into the following two reactions: the activation of factor XII by plasma kallikrein (reaction 1) and the activation of prekallikrein by factor XIIa (reaction 2). The effects of high-Mr kininogen and kaolin surface on the kinetics of these activation reactions were studied. High-Mr kininogen markedly enhanced the rate of reactions 1 and 2 in the presence of kaolin, and the enhancements were highly dependent on the concentrations of the protein cofactor and amount of kaolin surface. For the activation of factor XII by plasma kallikrein (reaction 1), high-Mr kininogen was required when a low concentration of factor XII and kaolin was used. The molar ratio of the protein cofactor to factor XII for optimal activation was found to be approximately 1:1. The apparent Km value and the kcat/Km value for plasma kallikrein on factor XII were calculated to be 4 nM and 5.2 X 10(7) s-1 X M-1, respectively. The activation of prekallikrein by factor XIIa, (reaction 2) proceeded even in the absence of high-Mr kininogen and kaolin. The addition of the protein cofactor and surface to the reaction mixture remarkably accelerated the reaction, and the apparent Km value for factor XIIa on prekallikrein was reduced from 1 microM to 40 nM. Moreover, the kcat/Km value was altered from 7.3 X 10(4) to 1.1 X 10(6) s-1 X M-1). These results suggest that high-Mr kininogen accelerates the surface-mediated activation of factor XII and prekallikrein by enhancing the susceptibility of factor XII to plasma kallikrein, on the one hand, and the affinity of factor XIIa for prekallikrein, on the other hand. Kaolin may play an important role in the concentration and organization of these components on the negatively charged surface.     

Ovine prolactin potentiates the action of adrenocorticotropic hormone on the secretion of dehydroepiandrosterone sulfate and dehydroepiandrosterone from cultured bovine adrenal cells

To determine the direct effect of prolactin on adrenal androgen secretion, the daily secretions of dehydroepiandrosterone sulfate (DHEA-S), dehydroepiandrosterone (DHEA), androstenedione and cortisol were determined in monolayer culture of bovine adrenal cells in the presence or absence of adrenocorticotropic hormone (ACTH) and/or prolactin. In the absence of ACTH ovine prolactin alone had no effect on steroid secretion during seven-day culture. Ovine prolactin, when administered in combination with ACTH, significantly potentiated the stimulatory effect of ACTH on DHEA-S and DHEA but not androstenedione secretion on the seventh day in culture. On the first day in culture prolactin showed no synergistic effect with ACTH on DHEA and DHEA-S secretion, although ACTH significantly increased DHEA and cortisol secretion. DHEA-S secretion increased as a function of prolactin concentration in the presence of ACTH. These results indicated that long-term treatment by ovine prolactin with ACTH caused the increase in adrenal androgen secretion from bovine adrenal cells. The site of action of prolactin was suggested to be the partial inhibition of adrenal 3 beta-hydroxysteroid dehydrogenase by the result of increases in DHEA-S and DHEA but not androstenedione secretion.