Anti-Prelog Reduction of Prochiral Carbonyl Compounds by Oenococcus oeni in a Biphasic System

An aqueous-organic biphasic system was established and used with whole cells of Oenococcus oeni to reduce 2-octanone to (R)-2-octanol. The conversion reached 99% when the Tris/borate buffer was increased from 50 mM to 300 mM in the aqueous phase. In addition, the conversion increased as the log P value of the organic solvent changed from 0.5 to 6.6. Under optimized conditions, the conversion of (R)-2-octanol reached 99% from 0.5 M 2-octanone with an optical purity of 99% e.e. The biphasic system allows the anti-Prelog reduction of aliphatic and aromatic ketones to furnish (R)-configurated alcohols in high optical purity as well.     

Staining with Fluorescein Diacetate Correlates with Hepatocyte Function

To establish the importance of fluorescein diacetate (FDA) as a viability stain for cultured hepatocytes. we hypothesized that FDA staining would correlate positively with hepatocyte viability and function. Mixtures of live and dead cells were stained with FDA and scanned by flow cytometry. A close correlation was observed between the live cell fraction and percent viability as determined by FDA staining (R² = 0.962). Hepatocytes were also sorted into low fluorescence and high fluorescence groups. Both albumin production and lidocaine metabolism (P-450 activity) were significantly increased in the high fluorescence group compared to the low fluorescence group. An automated, fluorescence-activated assay was useful for rapid assessment of hepatocyte viability. In addition. the intensity of green fluorescence following staining with FDA correlated well with two specific measures of hepatocyte function.     

Functional analyses of a putative plasma membrane Na+/H+ antiporter gene isolated from salt tolerant Helianthus tuberosus

Jerusalem artichokes (Helianthus tuberosus L.) can tolerate relatively higher salinity, drought and heat stress. In this paper, we report the cloning of a Salt Overly Sensitive 1 (SOS1) gene encoding a plasma membrane Na⁺/H⁺ antiporter from a highly salt-tolerant genotype of H. tuberosus, NY1, named HtSOS1 and characterization of its function in yeast and rice. The amino acid sequence of HtSOS1 showed 83.4 % identity with the previously isolated SOS1 gene from the Chrysanthemum crassum. The mRNA level in the leaves of H. tuberosus was significantly up-regulated by presence of high concentrations of NaCl. Localization analysis using rice protoplast expression showed that the protein encoded by HtSOS1 was located in the plasma membrane. HtSOS1 partially suppressed the salt sensitive phenotypes of a salt sensitive yeast strain. In comparison with wild type (Oryza sativa L., ssp. Japonica. cv. Nipponbare), the transgenic rice expressed with HtSOS1 could exclude more Na⁺ and accumulate more K⁺. Expression of HtSOS1 decreased Na⁺ content much larger in the shoot than in the roots, resulting in more water content in the transgenic rice than WT. These data suggested that HtSOS1 may be useful in transgenic approaches to improving the salinity tolerance of glycophyte.     

Insights into the fluoride-resistant regulation mechanism of <Emphasis Type="Italic">Acidithiobacillus ferrooxidans</Emphasis> ATCC 23270 based on whole genome microarrays

Acidophilic microorganisms involved in uranium bioleaching are usually suppressed by dissolved fluoride ions, eventually leading to reduced leaching efficiency. However, little is known about the regulation mechanisms of microbial resistance to fluoride. In this study, the resistance of Acidithiobacillus ferrooxidans ATCC 23270 to fluoride was investigated by detecting bacterial growth fluctuations and ferrous or sulfur oxidation. To explore the regulation mechanism, a whole genome microarray was used to profile the genome-wide expression. The fluoride tolerance of A. ferrooxidans cultured in the presence of FeSO₄ was better than that cultured with the S⁰ substrate. The differentially expressed gene categories closely related to fluoride tolerance included those involved in energy metabolism, cellular processes, protein synthesis, transport, the cell envelope, and binding proteins. This study highlights that the cellular ferrous oxidation ability was enhanced at the lower fluoride concentrations. An overview of the cellular regulation mechanisms of extremophiles to fluoride resistance is discussed.     

生物信息学在发现新基因方面的应用

自生物信息学作为一门交叉学科诞生以来,其在计算机、农业和生命科学等各方面发挥了重要的作用,在后基因组时代,更是成为发现新基因的重要手段。对生物信息学的概况做了回顾与展望,并简述了生物信息学近年在发现新基因方面所取得的成果。     

Elements in the N-terminal signaling sequence that determine cytosolic topology of short-chain dehydrogenases/reductases. Studies with retinol dehydrogenase type 1 and cis-retinol/androgen dehydrogenase type 1

High affinity, retinoid-specific binding proteins chaperone retinoids to manage their transport and metabolism. Proposing mechanisms of retinoid transfer between these binding proteins and membrane-associated retinoid-metabolizing enzymes requires insight into enzyme topology. We therefore determined the topology of mouse retinol dehydrogenase type 1 (Rdh1) and cis-retinoid androgen dehydrogenase type 1 (Crad1) in the endoplasmic reticulum of intact mammalian cells. The properties of Rdh1 were compared with a chimera with a luminal signaling sequence (11beta-hydroxysteroid dehydrogenase (11beta-HSD1)(1-41)/Rdh1(23-317); the green fluorescent protein (GFP) fusion proteins Rdh1(1-22)/GFP, Crad1(1-22)/GFP, and 11beta-HSD1(1-41)/GFP; and signaling sequence charge difference mutants using confocal immunofluorescence, antibody access, proteinase K sensitivity, and deglycosylation assays. An N-terminal signaling sequence of 22 residues, consisting of a hydrophobic helix ending in a net positive charge, anchors Rdh1 and Crad1 in the endoplasmic reticulum facing the cytoplasm. Mutating arginine to glutamine in the signaling sequence did not affect topology. Inserting one or two arginine residues near the N terminus of the signaling sequence caused 28-95% inversion from cytoplasmic to luminal, depending on the net positive charge remaining at the C terminus of the signaling sequence; e.g. the mutant L3R,L5R,R16Q,R19Q,R21Q faced the lumen. Experiments with N- and C-terminal epitope-tagged Rdh1 and molecular modeling indicated that a hydrophobic helix-turn-helix near the C terminus of Rdh1 (residues 289-311) projects into the cytoplasm. These data provide insight into the features necessary to orient type III (reverse signal-anchor) proteins and demonstrate that Rdh1, Crad1, and other short-chain dehydrogenases/reductases, which share similar N-terminal signaling sequences such as human Rdh5 and mouse Rdh4, orient with their catalytic domains facing the cytoplasm.