外消旋薄荷醇对映选择性酶促酯化中酸酐与羧酸的比较研究

利用脂肪酶在有机溶剂中催化对映选择性酯化反应对外消旋薄荷醇进行了有效的光学拆分。对分别使用酸酐和相应的游离羧酸作酰基给体时的反应性能进行了比较。发现酸酐的反应性远高于对应的游离羧酸,但在酶的催化作用下酸酐易水解成为游离羧酸;在微水系统中使用过高浓度的酸酐会导致酶缺水而失活,同时会促进手性醇的非选择性酯化,从而降低产物的光学纯度。然而,在连续流加丙酸酐的半批式反应系统中,所有这些缺点均可有效地克服。与使用游离丙酸的批式反应系统相比,ｄｌ-薄荷醇的反应时间缩短了一半,酶的稳定性大幅度提高,而产物ｌ薄荷醇酯的光学纯度不相上下（＞９８％ｅ）。     

A Mitosis-specific Phosphorylation of the Gap Junction Protein Connexin43 in Human Vascular Cells: Biochemical Characterization and Localization

Western blotting studies revealed that connexin43 (Cx43), one of the major gap junction proteins in human vascular endothelial cells, is posttranslationally modified during mitosis. This mitosis-specific modification results in a Cx43 species that migrates as a single protein band and was designated Cx43_m. Cx43_m was shown to be the result of additional Ser/Thr phosphorylation as indicated by: (a) the increased gel mobility induced by both alkaline phosphatase and the Ser/ Thr-specific protein phosphatase-2A (PP2A) and (b) the removal of virtually all ³²P_i from Cx43_m by PP2A. Immunofluorescent confocal microscopy of mitotic cells revealed that Cx43 is intracellularly located, while in nonmitotic cells Cx43 is located at regions of cell–cell contact. Dye coupling studies revealed that mitotic endothelial cells were uncoupled from each other and from nonmitotic cells. After cytokinesis, sister cells resumed cell coupling independent of de novo protein synthesis. The mitosis-specific phosphorylation of Cx43 correlates with the transient loss of gap junction intercellular communication and redistribution of Cx43, suggesting that a protein kinase that regulates gap junctions is active in M-phase.     

干旱和湿润生境下辽东栎群体遗传结构及其适应意义的初步研究

应用同工酶分析方法,测定北京市东灵山区两个分别代表干旱和湿润生境的辽东栋（ＱｕｅｒｃｕｓｌｉａｏｔｕｎｇｅｎｓｉｓＫｏｉｚ．）群体的遗传结构。共分析统计了１３ 个酶系统３０ 个位点。结果表明：辽东栎群体内部存在丰富的遗传变异（多态位点百分率为８６．６％ ,等位基因平均数为２．２５）。两群体遗传结构的相似性程度很高（Ｄ＝ ０．０２９, ＧＳＴ＝ ０．０４８）;但在个别位点上仍存在较大差异,这些差异的产生可能与对小生境的适应有关。对不同年龄段的初步分析结果显示,基因频率的动态变化可能有其适应意义     

Alcohol and aldehyde dehydrogenase genotypes and drinking behavior of Chinese living in Shanghai

Alcohol dehydrogenase (ADH) and aldehyde dehydrogenase (ALDH), the principal enzymes responsible for oxidative metabolism of ethanol, exist in multiple, genetically determined molecular forms. Widely different kinetic properties in some of these isozymes account for the individual differences in alcohol sensitivity. In this study we used the polymerase chain reaction/restriction fragment length polymorphism method to determine the genotypes of the ADH2 and ALDH2 loci of alcoholic and nonalcoholic Chinese living in Shanghai. We also investigated the subjects' drinking patterns by means of semistructured interviews. The alcoholics had significantly lower frequencies of the ADH2² and ALDH2² alleles than did the nonalcoholics, suggesting the inhibitory effects of these alleles for the development of alcoholism. In the nonalcoholic subjects, ADH2² had little, if any, effect, despite the significant effect of the ALDH2² allele in decreasing the alcohol consumption of the individual. Taken together, these results fit the proposed hypothesis for the development of alcoholism, i.e., drinking behavior is greatly influenced by the individual's gentoypes of alcohol-metabolizing enzymes, and the risk of becoming alcoholic is proportionate with the ethanol consumption of the individual.     

Regulation of neuronal nitric oxide synthase by histone,protamine, and myelin basic protein

We examined the effects of endogenous basic proteins rich in the amino acidL-arginine on neuronal NO synthase activity by monitoring cyclic GMP formation in intact neuron-like neuroblastoma N1E-115 cells. Histone, protamine and myelin basic protein significantly stimulated cyclic GMP formation, both in a time- and concentration-dependent manner. These effects were blocked by hemoglobin and NO synthase inhibitors. Removal of the extracellular/intracellular Ca²⁺ gradient by a Ca²⁺ chelator completely abolished the cyclic GMP responses elicited by histone and protamine, suggesting that influex of extracellular Ca²⁺ might be involved in their activation of NO synthase. The effects of myelin basic protein on cyclic GMP formation, however, appeared to be due to Ca²⁺ release from intracellular stores. In cytosolic preparations of rat cerebellum, these basic proteins inhibited the metabolism ofL-arginine intoL-citrulline by NO synthase. We conclude from our findings that endogenous basic proteins might be involved in the regulation of neuronal NO synthase activity. Their effects on the enzyme could be either stimulatory or inhibitory, depending on whether the basic proteins exert their effects extracellularly or intracellularly, respectively.     

Three-dimensional structure prediction of the NAD binding site of proton-pumping transhydrogenase from Escherichia coli

A three-dimensional structure of the NAD site of Escerichia coli transhydrogenase has been predicted. The model is based on analysis of conserved residues among the transhydrogenases from five different sources, homologies with enzymes using NAD as cofactors or substrates, hydrophilicity profiles, and secondary structure predictions. The present model supports the hypothesis that there is one binding site, located relatively close to the N-terminus of the α-subunit. The proposed structure spans residues α145 to α287, and it includes five β-strands and five α-helices oriented in a typical open twisted α/β conformation. The amino acid sequence following the GXGXXG dinucleotide binding consensus sequence (residues α172 to α177) correlates exactly to a typical fingerprint region for ADP binding βαβ folds in dinucleotide binding enzymes. In the model, aspartic acid α195 forms hydrogen bonds to one or both hydroxyl groups on the adenosine ribose sugar moiety. Threonine α196 and alanine α256, located at the end of βB and βD, respectively, create a hydrophobic sandwich with the adenine part of NAD buried inside. The nicotinamide part is located in a hydrophobic cleft between αA and βE. Mutagenesis work has been carried out in order to test the predicted model and to determine whether residues within this domain are important for proton pumping directly. All data support the predicted structure, and no residue crucial for proton pumping Was detected. Since no three-dimensional structure of transhydrogenase has been solved, a well based tertiary structure prediction is of great value for further experimental design in trying to elucidate the mechanism of the energy-linked proton pump. © 1995 Wiley-Liss, Inc.     

The type IV pre-pilin leader peptidase of Xanthomonas campestris pv. campestris is functional without conserved cysteine residues

Type IV pre-pilin leader peptidase was demonstrated to be required for protein secretion, in addition to its involvement in biogenesis of type IV pili. The type IV pre-pilin leader peptidase gene of Xanthomonas campestris pv. campestris was located on a 3 kb Acc l fragment on account of its hybridization with the DNA fragment containing the type IV pre-pilin leader-peptidase gene pilD/xcpA of Pseudomonas aeruginosa . Sequencing of the cloned fragment revealed an open reading frame (ORF) (designated xpsO ) of 287 amino acid residues. A protein with an apparent molecular mass of approximately 32.5 kDa was synthesized in vitro from a DNA fragment containing the xpsO gene. The amino acid sequence shares 50% identity with that of PilD throughout the entire sequence. Among other type IV pre-pilin leader peptidases, XpsO is unique in not having the two conserved -CXXC- motifs in a cytoplasmic domain. Instead, new motifs were noted when the protein was compared with XpsE, which is another member of the extracellular protein-secretion machinery. When the xpsO gene was introduced into the pilD mutant of P. aeruginosa , both the sensitivity against infection with the pilus-specific phage PO4 and the ability to secrete extracellular protein were recovered. Furthermore, immunoblot analysis indicated that the P. aeruginosa pilin was apparently processed in vivo by the xpsO gene product.