Preparation and antigenic property of methyl 3 beta-hydroxy-19-oxo-5-cholen-24-oate 19-(O-carboxymethyl) oxime-bovine serum albumin conjugate

The preparation and antigenic property of 3 beta-hydroxy-5-cholen-24-oic acid-bovine serum albumin (BSA) conjugate in which the hapten is linked to the carrier protein through an (O-carboxymethyl) oxime bridge at the C-19 position on the steroid nucleus is described. Antibody raised against antigen in the rabbit possessed high titer and specificity to 3 beta-hydroxy-5-cholen-24-oic acid, exhibiting no significant cross-reactions with various bile acids.     

Identification of bile alcohols in rat bile

Bile alcohols in rat bile were analyzed by gas-liquid chromatography-mass spectrometry. Six bile alcohols were newly identified as minor constituents in addition to 5 beta-cholestane-3 alpha,7 alpha,12 alpha,26-tetrol, major bile alcohol of rat bile. The bile alcohols newly identified were 27-nor-5 beta-cholestane-3 alpha,7 alpha,12 alpha,24,25-pentol, 5 alpha-cholestane-3 alpha,7 alpha,12 alpha,26-tetrol, 5 beta-cholestane-3 alpha,7 alpha,12 alpha,24,26-pentol, 5 alpha-cholestane-3 alpha,7 alpha,12 alpha,25,26-pentol, 5 beta-cholestane-3 alpha,7 alpha,12 alpha,25,26-pentol, and 5 beta-cholestane-3 alpha,6 beta,7 beta,25,26-pentol. The biliary bile alcohols of the rat occurred mainly as the sulfuric acid esters and, in lesser amounts, as glucuronoconjugated and unconjugated forms. The amount of total bile alcohols was about 27.9 nmol in 1 ml of bile.     

The role of 5'-methylthioadenosine on rat calvaria cell differentiation.

The role of 5'-methylthioadenosine (MTA), formed during the process of polyamine biosynthesis, on differentiation of osteoprogenitor cells was assessed by its effects on alkaline phosphatase (ALP) activity, bone nodule formation and osteopontin contents of cultured rat calvaria (RC) cells. These three markers were stimulated by exogenous MTA and were depressed by 5'-difluoromethylthioadenosine (DFMTA), a synthetic inhibitor of MTA phosphorylase, which cleaves MTA to adenine and 5-methylthioribose-1-phosphate. 5-Methylthioribose and 2-keto-4-methylthiobutyrate, metabolites of 5-methylthioribose-1-phosphate, had no effects on ALP activity and bone nodule formation in the presence or absence of DFMTA. On the other hand, adenine enhanced ALP activity, bone nodule formation and osteopontin contents in mineralized nodules and also partially reversed DFMTA-induced inhibition of these three markers. MTA, its metabolites and DFMTA did not affect the growth of RC cells under these culture conditions. These results suggest that adenine formed from MTA is important in the differentiation of RC cells.     

Testicular development and serum sex steroid profiles during the annual sexual cycle of the male sturgeon hybrid the bester

Testicular development in the adult male F₁ sturgeon hybrid, the bester ( Huso huso L. female x Acipenser ruthenus L. male), was examined monthly in relation to serum sex steroid levels. Spermatogenesis lasted for 1 year, with meiosis generally starting in September and spermiogenesis in November, although there was considerable variation in testicular developmental stages between fish sampled monthly. Testicular development continued, slowly, during the winter months until April. Fish did not exhibit spontaneous spermiation, and phagocytotic activity of Sertoli cells became prominent from May onwards. Androgen levels increased during Spermatogenesis and remained high throughout the pre-spermiation period. In the degeneration stage, 11-ketotestosterone concentrations declined to low levels, while testoster- one levels remained high. The serum concentration of 17,20β-dihydroxy-4-pregnen-3-one was low throughout the reproductive cycle. Based on these results, it is suggested that the time appropriate for induction of final maturation would be from November–December to April when the testes are in the late stage of development.     

Hormonal induction of all stages of spermatogenesis in germ-somatic cell coculture from immature Japanese eel testis

In cultivated male eel, spermatogonia are the only germ cells present in testis. Our previous studies using an organ culture system have shown that gonadotropin and 11-ketotestosterone (11-KT, a potent androgen in teleost fishes) can induce all stages of spermatogenesis in vitro. for detailed investigation of the control mechanisms of spermatogenesis, especially of the interaction between germ cells and testicular somatic cells during 11-KT-induced spermatogenesis in vitro, we have established a new culture system in which germ cells and somatic cells are cocultured after they are aggregated into pellets by centrifugation. Germ cells (spermatogonia) and somatic cells (mainly Sertoli cells) were isolated from immature eel testis. Coculture of the isolated germ cells and somatic cells without forming aggregation did not induce spermatogenesis, even in the presence of 11-KT. In contrast, when isolated germ cells and somatic cells were formed into pellets by centrifugation and were then cultured with 11-KT for 30 days, the entire process of spermatogenesis from premitotic spermatogonia to spermatozoa was induced. However, in the absence of 11-KT in the culture medium spermatogenesis was not induced, even when germ cell and somatic cells were aggregated. These results demonstrate that physical contact of germ cells to Sertoli cells is required for inducing spermatogenesis in response to 11-KT.     

Demonstration of Antigenic and Genotypic Variation in Orientia tsutsugamushi Which Were Isolated in Japan,and Their Classification into Type and Subtype

A total of 40 strains of Orientia tsutsugamushi (34 isolates from patients and trombiculid mites in Japan, and 6 prototype strains of antigenic variants) were examined for classification based on the reactivities with type-specific monoclonal antibodies in indirect immunofluorescence tests, and on the restriction fragment length polymorphism of a polymerase chain reaction (PCR)-amplified 56-kilodalton type-specific antigenic protein gene. By these methods, several antigenic and genotypic variants were found among the strains, and these variants were classified into types and further into subtypes. These results suggest that there are many variants in O. tsutsugamushi, and the methods used here seem to be useful for the systematic classification of the numerous variants. A strain which may be a new type distinguishable from those identified previously was also found in this study. Furthermore, variety in the degree of pathogenicity in mice related to type and/or subtype classification were observed.     

Promoter regions of cysteine endopeptidase genes from legumes confer germination-specific expression in transgenic tobacco seeds

Cysteine endopeptidases, SH-EP from Vigna mungo and EP-C1 from Phaseolus vulgaris, act to degrade seed storage protein during seed germination. Using transgenic tobacco plants, expression of SH-EP and promoter activity of the EP-C1 gene were analyzed in transgenic tobacco plants. The promoters of the two genes in tobacco seeds showed germination-specific activation, although post-translational processing of SH-EP and regulatory regions of promoter of the gene for EP-C1 were found to differ between leguminous seeds and transgenic tobacco seeds.     

The function of a ribosomal frameshifting signal from human immunodeficiency virus-1 in Escherichia coli

A 15-17 nucleotide sequence from the gag-pol ribosome frameshift site of HIV-1 directs analogous ribosomal frameshifting in Escherichia coli. Limitation for leucine, which is encoded precisely at the frameshift site, dramatically increased the frequency of leftward frameshifting. Limitation for phenylaianine or arginine, which are encoded just before and just after the frameshift, did not significantly affect frameshifting. Protein sequence analysis demonstrated the occurrence of two closeiy related frameshift mechanisms. In the first, ribosomes appear to bind leucyl-tRNA at the frameshift site and then slip leftward. This is the 'simultaneous slippage’mechanism. In the second, ribosomes appear to slip before binding amlnoacyl-tRNA, and then bind phenylaianyl-tRNA, which is encoded in the left-shifted reading frame. This mechanism is identicai to the‘overlapping reading’we have demonstrated at other bacterial frameshift sites. The HIV-1 sequence is prone to frame-shifting by both mechanisms in E. coli.     

Lysophosphoinositide-specific phospholipase C in rat brain synaptic plasma membranes

A membrane preparation from rat brain catalyzed the hydrolysis of [2-³H]glycerol-labeled lysophosphatidylinositol (lysoPI) to yield monoacylglycerol (MG) and inositolphosphates. This phospholipase C activity had an optimal pH of 8.2. The membrane preparation did not require the addition of Ca²⁺ for its maximum activity, but the activity was inhibited by addition of 0.1 mM EDTA to the assay mixture and was restored by simultaneous addition of 0.2 mM Ca²⁺. The activity was found to be localized in synaptic plasma membranes prepared by Ficoll and Percoll density gradients. The phospholipase C was highly specific for lysoPI; diacylglycerol formation from phosphatidylinositol, and MG formation from lysophosphatidylcholine, lysophosphatidylethanolamine, and lysophosphatidylserine were below 5% of that observed with lysoPI under the conditions used. We concluded that there is a pathway for phosphatidylinositol metabolism in brain synaptic membranes which is different from the well-characterized phosphoinositide-specific phospholipase C pathway.Abbreviations PI phosphatidylinositol - lysoPI lysophosphatidylinositol - lysoPI-PLC lysophosphoinositide-specific phospholipase C - PI-PLC phosphoinositide-specific phospholipase C - MG monoacylglycerol - PLC phospholipase C To whom to address reprint requests.