Formation of 5 alpha-products as major C19-steroids in estrogen-responsive mouse Leydig cell tumor lines

Homogenates of estrogen-responsive mouse Leydig cell tumors (T 124958-R and T 22137) or 28- and 120-day-old mouse testes were incubated with [3H]progesterone or [14C]4-androstene-3,17-dione in the presence of NADPH, and progesterone metabolism and enzyme activities were estimated. The growth of T 124958-R tumor transplanted in BALB/c mice was markedly stimulated by estrogenization of host mice, but the growth of T 22137 tumor was evidently suppressed by the estrogenization. The major C21-17-OH-steroids and C19-steroids formed from progesterone by both tumors and the testes of immature mice were 5 alpha-steroids, such as 3 alpha,17-dihydroxy-5 alpha-pregnan-20-one, 5 alpha-androstane-3,17-dione, androsterone, 3 beta-hydroxy-5 alpha-androstan-17-one and 5 alpha-androstane-3 alpha,17 beta-diol. In contrast, the major steroids formed by the testes of adult mice were testosterone and 4-androstene-3,17-dione, and no or little 5 alpha-steroids were produced. 5 alpha-Reductase activities in both tumor cells (40-50 nmol/l X 10(8) cells per h) were also found to be approx. 5-6 times higher than that in Leydig cells of adult mouse testes (8 nmol/l X 10(8) Leydig cells per h), though 17-hydroxylase activity was much higher in the Leydig cells of adult testes (730 nmol/l X 10(8) Leydig cells per h) than in both tumor cells (1-7 nmol/l X 10(8) cells per h). Furthermore, the presence of significant amounts of endogenous androsterone and/or 5 alpha-androstane-3 alpha,17 beta-diol was demonstrated in both tumors by radioimmunoassay. The present results demonstrate for the first time that C19-5 alpha-steroids are major C19-steroid products (immature type of testicular androgen production) in Leydig cell tumor lines.     

Controlled release of adeno-associated virus from alginate hydrogel microbeads with enhanced sensitivity to ultrasound

Adeno-associated virus (AAV)-based gene therapy holds promise as a fundamental treatment for genetic disorders. For clinical applications, it is necessary to control AAV release timing to avoid an immune response to AAV. Here we propose an ultrasound (US)-triggered on-demand AAV release system using alginate hydrogel microbeads (AHMs) with a release enhancer. By using a centrifuge-based microdroplet shooting device, the AHMs encapsulating AAV with tungsten microparticles (W-MPs) are fabricated. Since W-MPs work as release enhancers, the AHMs have high sensitivity to the US with localized variation in acoustic impedance for improving the release of AAV. Furthermore, AHMs were coated with poly-l -lysine (PLL) to adjust the release of AAV. By applying US to the AAV encapsulating AHMs with W-MPs, the AAV was released on demand, and gene transfection to cells by AAV was confirmed without loss of AAV activity. This proposed US-triggered AAV release system expands methodological possibilities in gene therapy.     

Isolation and characterization of different-sized nucleoli from Ehrlich ascites tumor cells : Evidence of different nucleolar ribosomal cistrons

A procedure was developed for isolation of variously sized nucleoli in order to study the mechanism of nucleolar formation from multiple nucleolar organizers and to compare the compositions of different-sized nucleoli from Ehrlich ascites tumor cells. Relatively small nucleoli and large nucleoli from Ehrlich ascites tumor cells were separated by centrifugation at 400 g for 5 min in a layer of 0.34 M sucrose over 0.88 M sucrose. Small nucleoli remained in the 0.34 M sucrose layer while the large nucleoli accumulated in the 0.88 M sucrose.Three fractions, provisionally named small, intermediate and large nucleoli, containing 0.33, 0.41 and 0.84 pg DNA/nucleolus, respectively, were separated. Unfractionated nucleoli contained 0.59 pg DNA/nucleolus. The RNA content also increased with the size of the nucleolus and no significant difference was observed in the RNA/DNA ratios in the three fractions. Large nucleoli incorporated more [³H]uridine and [³²P]orthophosphate into RNA than did small nucleoli, but the base compositions of the RNAs extracted from the different-sized nucleoli were similar. No significant fragmentation occurred on sonication of large nucleoli for 3 min, so the observed difference in the DNA contents was not due to mechanical damage of the nucleoli.The DNAs of these different-sized nucleoli were analysed on CsCl gradients. The nucleoli contained similar percentages of satellite DNA (20–22%) which were also similar to those of total, unfractionated nucleoli. Approx. 10% of the extranucleolar DNA is satellite DNA—thus the nucleolar fractions were probably not appreciably contaminated with extranucleolar DNA. The DNA of small nucleoli contained a slightly lower percentage (0.058%) of ribosomal cistrons than large nucleoli (0.081%). This means that the higher content of DNA in the large nucleoli is not merely due to longer sized chromatin with extra regions of the vicinity of nucleolar organizers. Thus these results suggest that the total content of ribosomal cistrons/nucleolus is roughly proportional to the DNA content of the nucleoli, at least in Ehrlich ascites tumor cells. Namely, the number of ribosomal cistrons per nucleolus for small, intermediate and large nucleoli is 40, 60 and 130, respectively.     

Unique T Cells with Unconventional Cytokine Profiles Induced in the Livers of Mice during Schistosoma mansoni Infection

During infection with Schistosoma, serious hepatic disorders are induced in the host. The liver possesses unique immune systems composed of specialized cells that differ from those of other immune competent organs or tissues. Host immune responses change dramatically during Schistosoma mansoni infection; in the early phase, Th1-related responses are induced, whereas during the late phase Th2 reactions dominate. Here, we describe unique T cell populations induced in the liver of mice during the period between Th1- and Th2-phases, which we term the transition phase. During this phase, varieties of immune cells including T lymphocytes increase in the liver. Subsets of CD4⁺ T cells exhibit unique cytokine production profiles, simultaneously producing both IFN-γ and IL-13 or both IFN-γ and IL-4. Furthermore, cells triply positive for IFN-γ, IL-13 and IL-4 also expand in the S. mansoni-infected liver. The induction of these unique cell populations does not occur in the spleen, indicating it is a phenomenon specific to the liver. In single hepatic CD4⁺ T cells showing the unique cytokine profiles, both T-bet and GATA-3 are expressed. Thus, our studies show that S. mansoni infection triggers the induction of hepatic T cell subsets with unique cytokine profiles.     

Regulation of active and quiescent somatic stem cells by Notch signaling

Somatic stem/progenitor cells actively proliferate and give rise to different types of mature cells (active state) in embryonic tissues while they are mostly dormant (quiescent state) in many adult tissues. Notch signaling is known to regulate both active and quiescent states of somatic stem cells, but how it regulates these different states is unknown. Recent studies revealed that the Notch effector Hes1 is expressed differently during the active and quiescent states during neurogenesis and myogenesis: high in the quiescent state and oscillatory in the active state. When the Hes1 expression level is high, both Ascl1 and MyoD expression are continuously suppressed. By contrast, when Hes1 expression oscillates, it periodically represses expression of the neurogenic factor Ascl1 and the myogenic factor MyoD, thereby driving Ascl1 and MyoD oscillations. High levels of Hes1 and the resultant Ascl1 suppression promote the quiescent state of neural stem cells, while Hes1 oscillation-dependent Ascl1 oscillations regulate their active state. Similarly, in satellite cells of muscles, known adult muscle stem cells, high levels of Hes1 and the resultant MyoD suppression seem to promote their quiescent state, while Hes1 oscillation-dependent MyoD oscillations activate their proliferation and differentiation. Therefore, the expression dynamics of Hes1 is a key regulatory mechanism of generating and maintaining active/quiescent stem cell states.     

Identification of the amino acid residues conferring substrate specificity upon Selenomonas ruminantium lysine decarboxylase

Lysine decarboxylase (LDC, EC 4.1.1.18) from Selenomonas ruminantium has decarboxylating activities towards both L-lysine and L-ornithine with similar K(m) and Vmax. Here, we identified four amino acid residues that confer substrate specificity upon S. ruminantium LDC and that are located in its catalytic domain. We have succeeded in converting S. ruminantium LDC to an enzyme with a preference in decarboxylating activity for L-ornithine when the four-residue of LDC were replaced by the corresponding residues of mouse ornithine decarboxylase (EC 4.1.1.17).