Effect of coal particle size on the coal hydrogenation reaction — In relation to the plasticity of coal

The effect of coal particle size on the catalytic hydrogenation of dry coal was investigated for three Hokkaido coals (Japan) of different ranks. It was found that the effect of coal particle size on conversion is dependent on coal rank. A greater difference in conversion with respect to coal particle size is noticed for coals of lower carbon content. The physical appearance of the products in the autoclave after the reaction suggests that the effect of particle size on conversion is dependent on the plastic properties of the heated coal sample. When the reaction proceeds with coal in the plastic state, the effect of particle size is small. As indicated by the product distribution, the plastic properties of a coal sample are related to the yield of asphaltene (hexane insoluble/benzene solubles) and/or the structural parts of original coal which yield asphaltene.     

Saccharomyces cerevisiae mata mutant cells defective in pointed projection formation in response to α-factor at high concentrations

We have isolated Saccharomyces cerevisiae MAT a mutant cells that do not form a pointed projection but elongate in response to α-factor at high concentrations. Complementation tests defined three genes, PPF1, PPF2, and PPF3 (for pointed projection formation), necessary for pointed projection formation. Allelism tests with genes known to be needed for projection formation revealed that PPF1 is identical to SPA2, while PPF2 and PPF3 are not allelic to SST2, STE2, SPA2, BEM1 or SLK1/SSP31/BCK1. The morphology of MAT a ppf mutants treated with high concentrations of α-factor is similar to that of MAT a PPF cells treated with α-factor at low concentrations. Quantitative mating tests showed that PPF2 and PPF3 are not essential for mating in either MAT a or MATα background. Monitoring of division arrest and expression of an α-factor-inducible gene revealed that mutations in the PPF genes do not affect the responses of MAT a cells to low concentrations of α-factor. Unlike wild-type cells, the ppf mutants exhibited early recovery from α-factor-induced division arrest. Furthermore, vegetatively growing ppf3-1 cells are slightly defective in cell separation of mother and daughter cells and in selection of the correct bud sites in all cell types. These results indicate that PPF2 and PPF3 are involved in the response to α-factor at high concentrations and that PPF3 is also required for proper establishment of polarity in vegetative growth.     

Production of recombinant tumor necrosis factor receptor/Fc fusion protein by genetically manipulated chickens

We previously reported the production of recombinant proteins using genetically manipulated chickens and quails. In this study, we constructed a retroviral vector encoding an expression cassette for a fusion protein of the extracellular domain of the human tumor necrosis factor (TNF) receptor 2 and Fc region of human IgG1 (TNFR/Fc), which is expected as an effective drug for inflammatory diseases such as rheumatoid arthritis. The concentrated viral vector was injected into developing chicken embryos. The chickens that hatched stably produced TNFR/Fc in the serum and egg yolk for six months. It appears that the fused protein is transported and accumulated into yolk from the serum, which is mediated by the Fc receptor. The protein purified from the yolk and serum inhibited the cytotoxic activity of TNF-* toward L929 cells, indicating that the protein produced by the chickens is biologically active. These results indicate the effectiveness of the recovery of Fc-fused proteins from the yolk of genetically manipulated chickens.     

A new approach to species determination for yeast strains: DNA microarray-based comparative genomic hybridization using a yeast DNA microarray with 6000 genes

DNA-DNA hybridization is known as the superior method in the elucidation of relationships between closely related taxa, such as species and strain. For species determination we propose a new DNA-DNA hybridization method: the DNA microarray-based comparative genomic hybridization (CGH) method, using a yeast DNA microarray with approximately 6000 genes. The genome from a yeast strain as a sample strain (Sample) was labelled with Cy3-dye and hybridized to a single DNA microarray, together with the Cy5-labelled genome of S. cerevisiae S288C as a reference strain (Reference). The log2 ratio values [log2[Cy3(Sample)/Cy5(Reference)]: Ratio] of signal intensities of all the gene spots were estimated and divided into the following groups: Ratio < or = -1; -1 < Ratio < 1; 1 < or = Ratio. The hybridization profiles of the genomes of type strains belonging to the genus Saccharomyces were significantly different from that of S. cerevisiae S288C. The Ratio-based grouping allowed us to discriminate between some species from S. cerevisiae more clearly. Furthermore, cluster analysis discriminated between closely related species and strains. Using this method, we were able to not only perform species determination but also to obtain information on alternation in gene copy number of such gene amplifications and deletions with single-gene resolution. These observations indicated that DNA microarray-based CGH is a powerful system for species determination and comparative genome analysis.     

Metabolic Activation of Heterocyclic Amines and Expression of Xenobiotic‐Metabolizing Enzymes in the Gastrointestinal Tract of Rats

Heterocyclic amines get entry into human body mainly through ingestion of pan‐fried meats cooked at high temperatures. Exposure of the gastrointestinal tract (GIT) to ingested xenobiotics prior to delivery to the liver may lead to metabolic activation, which may explain the high incidence of GIT carcinogenesis. Therefore, this study investigated the mutagenic activation of 2 heterocyclic amines, 2‐aminoanthracene (2‐AA) and 3‐amino‐1‐methyl‐5H‐prydo[4,3‐b]indole (Trp‐P‐2), in the GIT of rats. In addition, the constitutive mRNA expression profiles of xenobiotic‐metabolizing enzymes (XMEs) in the GIT of rats were examined. Metabolic activation of 2‐AA was detected in all GIT tissues except the duodenum and rectum, and it was detected at high levels in the ileum and cecum. Furthermore, we revealed high metabolic activation of 2‐AA and Trp‐P‐2 in the jejunum. The mRNA expression of phase I and II enzymes in rat GIT corresponded with their mutagenic activation ability. In conclusion, our results suggest that different expression levels of XME among GIT tissues may contribute to the tissue‐specific differences in metabolic activation of xenobiotics such as heterocyclic amines in rats.     

Purification and characterization of an extracellular alpha-neoagarooligosaccharide hydrolase from Bacillus sp. MK03

An agarolytic bacterium was isolated from soil in Gifu prefecture, Japan, and identified as Bacillus sp. strain MK03. The strain secreted neoagarooligosaccharide hydroluse into the culture medium. The enzyme was purified 49.7-fold from the culture fluid by ammonium sulfate precipitation and anion-exchange and gel-filtration column chromatographic methods. The purified enzyme appeared as a single band on polyacrylamide gel electrophoresis (PAGE) and sodium dodecyl sulfate (SDS)-PAGE. Estimations of the molecular mass by gel filtration and SDS-PAGE gave values of 320 kDa and 42 kDa, respectively, indicating that the enzyme is octametric. The enzyme cleaved the alpha-1,3 linkage in neoagarobiose to produce 3,6-anhydro-L-galactose and D-galactose. It also selectively cleaved the alpha-1,3 linkage at the nonreducing end in neoagarotetraose or neoagarohexaose to give 3,6-anhydro-L-galactose and agarotriose or agaropentaose. The optimum temperature and pH for the enzyme were 30 degrees C and 6.1, respectively. The N-terminal amino acid sequence showed no homology to sequences of other known neoagarooligosaccharide hydrolases and agarases.     

Effect of direct ovarian injection of vascular endothelial growth factor gene fragments on follicular development in immature female rats

Vascular endothelial growth factor (VEGF) expression in granulosa cells is associated with the thecal vasculature growth during ovarian follicular development. We hypothesized that injection of VEGF gene fragments directly into the rat ovary would induce production of a large number of ovulatory follicles and that these follicles would ovulate. To test this hypothesis, we treated immature female rats with combinations of hormones and VEGF gene fragments. The animals were divided into two groups: one group received solution containing transfection reagents as a control (n = 5), while the other group received direct ovarian injection of VEGF gene fragments at 19 (n = 5), 21 (n = 5), 23 (n = 5), or 25 (n = 5) days after birth followed by i.p. administration of 20 IU equine chorionic gonadotropin (eCG) at the age of 26 days. Forty-eight hours after eCG injection, animals were given 20 IU human chorionic gonadotropin (hCG) i.p. and then the oocytes in both groups were counted. The maximum number of ovulated oocytes was obtained when the VEGF gene fragments were injected into the rat ovary at 21 days after birth. Histological examination revealed that the injection of VEGF gene fragments markedly increased the vascular density around the preovulatory follicles and also the number of these follicles. Our data provide the first reported evidence that most ovulatory follicles generated by injection of VEGF gene fragments are able to ovulate upon hCG treatment. These results demonstrate that injection of VEGF gene fragments directly into the ovary stimulates the development of antral follicles by inducing the formation of thecal vasculature in immature female rats.     

Constitutive Effects of Lead on Aryl Hydrocarbon Receptor Gene Battery and Protection by β‐carotene and Ascorbic Acid in Human HepG2 Cells

Lead (Pb) is an environmental pollutant that can get entry into human body through contaminated foods, drinks, and inhaled air leading to severe biological consequences, and has been responsible for many deaths worldwide. The objectives of this study were 1st to investigate the modulatory effects of environmentally relevant concentrations of Pb on AhR gene battery, which is controlling xenobiotics metabolism. 2nd, trials to reduce Pb‐induced adverse effects were done using some phytochemicals like β‐carotene or ascorbic acid. Human hepatoma (HepG2) cell lines were exposed to a wide range of Pb concentrations varying from physiological to toxic levels (0 to 10 mg/L) for 24 h. High Pb concentrations (1 to 10 mg/L) significantly reduced phase I (CYP1A1 and 1A2) and phase II (UGT1A6 and NQO1) xenobiotic metabolizing enzyme mRNA expression in a mechanistic manner through the AhR regulation pathway. Additionally, these Pb concentrations induced oxidative stress in HepG2 cells in terms of production of reactive oxygen species (ROS) and induced heme oxygenase‐1 mRNA expression in a concentration‐dependent phenomenon. Coexposure of HepG2 cells to physiological concentrations of some micronutrients, like β‐carotene (10 μM) or ascorbic acid (0.1 mM), along with Pb (1 mg/L) for 24 h significantly reduced the levels of ROS production and recovered AhR mRNA expression into the normal levels. Thus, consumption of foods rich in these micronutrients may help to reduce the adverse effects of lead in areas with high levels of pollution.     

E-cadherin gene-engineered feeder systems for supporting undifferentiated growth of mouse embryonic stem cells

Conventionally, embryonic stem (ES) cells are cultured on a cell layer of mouse embryonic fibroblasts (MEFs) as feeder cells to support undifferentiated growth of ES cells. In this study, cell–cell interactions between mouse ES and feeder cells were artificially engineered via an epithelial cell adhesion molecule, E-cadherin, whose expression is considerable in ES cells. Mouse mesenchymal STO and NIH3T3 cells that were genetically engineered to express E-cadherin were used in ES cell cultures as feeder cells. ES cells cultured on the E-cadherin-expressing feeder cells maintained the expression of stem cell markers, alkaline phosphatase (AP), Oct3/4, Nanog and Sox2, and the efficiency of AP-positive colony formation was comparable to MEFs, and much better than parental STO and NIH3T3 cells. Furthermore, ES cells maintained on the E-cadherin-expressing feeder cells possessed the ability to differentiate into the three germ layers both in vitro and in vivo. The results indicated that E-cadherin expression in feeder cells could improve the performance of feeder cells, which may be further applicable to create new artificial feeder cell lines.