Disposition and metabolism of 16 beta-ethyl-17 beta-hydroxy-4-estern-3-one (TSAA-291), a new antiandrogen, in rats.

Matabolic fate of a new antiandrogen, 16 beta-ethyl-17 beta-hydroxy-4-estren-3-one (TSAA-291), was studied in rats. 14C-TSAA-291 intramuscularly injected as an aqueous suspension was absorbed gradually to give an increase in the plasma level which attained a plateau at 0.5 h, persisted till 8 h and then declined with an approx. half-life of 3.6 days. The drug was widely distributed in tissues, with the concns. almost equal to or higher than that in the plasma. The 14C-drug was eliminated mostly as metabolites within 10 days after dosing with higher activities found in the feces than in urine. Biliary 14C effectively underwent enterohepatic cycling. Biliary metabolites of TSAA-291 were characterized by the combined use of deuterium labeling and GLC-MS analysis. The metabolites identified were as follows: the parent drug, monohydroxy TSAA-291 having the additional hydroxy function in the steroid skeleton, 17 beta-hydroxy-16 beta-(1 xi-hydroxyethyl)-4-estren-3-one, 16 beta-ethyl-17 beta-hydroxy-5 beta-estran-3-one, 16 beta-ethyl-17 beta-hydroxy-5 alpha-estran-3-one, 16 beta-ethyl-5 beta-estrane-3 alpha, 17 beta-diol, 16 beta-ethyl-5 alpha-estrane-3 alpha, 17 beta-diol, 16 beta-ethyl-3 alpha-hydroxy-5 beta-estran-17-one and 16 beta-ethyl-3 alpha-hydroxy-5 alpha-estran-17-one. Monoketodihydroxy and/or trihydroxy metabolites were also detected in the bile.     

Real-time monitoring of antibody secretion from hybridomas on a microchip by time-resolved luminescence anisotropy analysis

This article presents a real-time monitoring system for cellular analysis using micro total analysis systems technology. Time-resolved luminescence anisotropy analysis was adopted for real-time detection of small amounts of a target protein produced by a small number of cells. The system was tested by real-time monitoring of the antibody secretion by hybridomas. The cells were successfully cultivated in a micro-incubation chamber (240 nl) fabricated on a microchip. The quantification of the antibody was achieved using the Ru(II) complex-labeled Staphylococcus aureus protein A probe, which can bind specifically to the Fc region of the antibody. Using this system, we detected as little as 24 fmol of immunoglobulin G under physiological conditions without the bound/free separation protocol. We successfully achieved real-time and quantitative monitoring of small amounts of antibody production by approximately 200 hybridoma cells. This method could be applied to various cellular analyses using small numbers of cells.     

Ontogenetic and seasonal changes in the diet of the halfbeak <Emphasis Type="Italic">Zenarchopterus dunckeri</Emphasis> at Iriomote Island,southern Japan

Ontogenetic and seasonal changes in the diet of the halfbeak Zenarchopterus dunckeri were examined in the Urauchi River estuary, Iriomote Island, southern Japan, using gut content analysis. The diet of this species changed dramatically with growth. Smaller juveniles fed mostly on small zooplankton and small gastropods, with a shift to the consumption of terrestrial insects, such as ants and flies, by larger individuals. Seasonal dietary differences were also apparent in adults. Ants were the most dominant prey item in the spring to fall months, whereas flies were abundant in the diet in the winter and early summer months.     

Repression of fatty-acyl-CoA oxidase-encoding gene expression is not necessarily a determinant of high-level production of dicarboxylic acids in industrial dicarboxylic-acid-producing Candida tropicalis

The synthesis of dicarboxylic acids (DCAs) in Candida tropicalis is thought to be induced by a decrease in fatty acyl-CoA-oxidase activity. However, in the present study we demonstrate that repression of the POX4 gene, encoding fatty acyl-CoA oxidase, does not directly lead to high-level production of DCAs. No fatty acyl-CoA-oxidase activity was detected if the POX4 gene of C. tropicalis strain 1098 (wild-type strain) was disrupted. Furthermore, introduction of the POX4 gene from C. tropicalis strain M1210A3, which is a mutant derived from strain 1098 and is used as an industrial DCA-producing strain, still exhibited low-level fatty acyl-CoA-oxidase activity. Nevertheless, production of DCA was not observed in either case. Furthermore, the increase in acyl-CoA-oxidase activity by expression of the POX4 gene in strain M1210A3 did not reduce high-level production of DCA. These results suggest that alterations in acyl-CoA-oxidase activity are not necessarily related to production of DCA in industrial DCA-producing C. tropicalis M1210A3.     

Transport properties of a system y+L neutral and basic amino acid transporter. Insights into the mechanisms of substrate recognition

The properties of system y(+)L-mediated transport were investigated on rat system y(+)L transporter, ry(+)LAT1, coexpressed with the heavy chain of cell surface antigen 4F2 in Xenopus oocytes. ry(+)LAT1-mediated transport of basic amino acids was Na(+)-independent, whereas that of neutral amino acids, although not completely, was dependent on Na(+), as is typical of system y(+)L-mediated transport. In the absence of Na(+), lowering of pH increased leucine transport, without affecting lysine transport. Therefore, it is proposed that H(+), besides Na(+) and Li(+), is capable of supporting neutral amino acid transport. Na(+) and H(+) augmented leucine transport by decreasing the apparent K(m) values, without affecting the V(max) values. We demonstrate that although ry(+)LAT1-mediated transport of [(14)C]l-leucine was accompanied by the cotransport of (22)Na(+), that of [(14)C]l-lysine was not. The Na(+) to leucine coupling ratio was determined to be 1:1 in the presence of high concentrations of Na(+). ry(+)LAT1-mediated leucine transport, but not lysine transport, induced intracellular acidification in Chinese hamster ovary cells coexpressing ry(+)LAT1 and 4F2 heavy chain in the absence of Na(+), but not in the presence of physiological concentrations of Na(+), indicating that cotransport of H(+) with leucine occurred in the absence of Na(+). Therefore, for the substrate recognition by ry(+)LAT1, the positive charge on basic amino acid side chains or that conferred by inorganic monovalent cations such as Na(+) and H(+), which are cotransported with neutral amino acids, is presumed to be required. We further demonstrate that ry(+)LAT1, due to its peculiar cation dependence, mediates a heteroexchange, wherein the influx of substrate amino acids is accompanied by the efflux of basic amino acids.     

Generating Porcine Chimeras Using Inner Cell Mass Cells and Parthenogenetic Preimplantation Embryos

Background

The development and validation of stem cell therapies using induced pluripotent stem (iPS) cells can be optimized through translational research using pigs as large animal models, because pigs have the closest characteristics to humans among non-primate animals. As the recent investigations have been heading for establishment of the human iPS cells with naïve type characteristics, it is an indispensable challenge to develop naïve type porcine iPS cells. The pluripotency of the porcine iPS cells can be evaluated using their abilities to form chimeras. Here, we describe a simple aggregation method using parthenogenetic host embryos that offers a reliable and effective means of determining the chimera formation ability of pluripotent porcine cells.

Methodology/Significant Principal Findings

In this study, we show that a high yield of chimeric blastocysts can be achieved by aggregating the inner cell mass (ICM) from porcine blastocysts with parthenogenetic porcine embryos. ICMs cultured with morulae or 4–8 cell-stage parthenogenetic embryos derived from in vitro-matured (IVM) oocytes can aggregate to form chimeric blastocysts that can develop into chimeric fetuses after transfer. The rate of production of chimeric blastocysts after aggregation with host morulae (20/24, 83.3%) was similar to that after the injection of ICMs into morulae (24/29, 82.8%). We also found that 4–8 cell-stage embryos could be used; chimeric blastocysts were produced with a similar efficiency (17/26, 65.4%). After transfer into recipients, these blastocysts yielded chimeric fetuses at frequencies of 36.0% and 13.6%, respectively.

Conclusion/Significance

Our findings indicate that the aggregation method using parthenogenetic morulae or 4–8 cell-stage embryos offers a highly reproducible approach for producing chimeric fetuses from porcine pluripotent cells. This method provides a practical and highly accurate system for evaluating pluripotency of undifferentiated cells, such as iPS cells, based on their ability to form chimeras.     

The bvg‐repressed gene brtA,encoding biofilm‐associated surface adhesin,is expressed during host infection by Bordetella bronchiseptica

Bordetella species display phase modulation between Bvg⁺ and Bvg^? phases. Because expression of known virulence factors is up‐regulated in the Bvg⁺ phase, bacteria in this phase are considered competent for infection. However, the Bvg^? phase is of negligible importance for infection. No studies have shown that bacterial factors specific to the Bvg^? phase (bvg‐repressed factors) are expressed in the course of Bordetella infection. In the present study, the gene brtA (B ordetella RT X‐family A dhesin), which is a typical bvg‐repressed gene but is expressed in B. bronchiseptica infecting hosts, was characterized. BrtA is composed of repeated pairs of the VCBS unit and dystroglycan‐type cadherin‐like unit, the von Willebrand Factor A domain, RTX motif and type I secretion target signal. It is herein demonstrated that BrtA is secreted by the type I secretion system and is essential for Ca²⁺‐dependent bacteria‐to‐substrate adherence, followed by biofilm formation. Although the contribution of BrtA to bacterial colonization of the rat trachea currently remains unclear, this is the first study to present concrete evidence for the expression of a bvg‐repressed gene during infection, which may provide a novel aspect for analyses of Bordetella pathogenesis.