Biocatalytic properties of native and immobilized subtilisin 72 in aqueous-organic and low water media

Subtilisin 72 was immobilized on cryogel of poly(vinyl alcohol), the macroporous carrier prepared by the freeze-thaw-treatment of concentrated aqueous solution of the polymer. The obtained biocatalyst was active and stable in aqueous, aqueous-organic, as well as in low water media. The stability of immobilized biocatalyst was substantially higher than that of native enzyme in all mixtures especially in aqueous buffer containing 5–8 M Urea and in acetonitrile/60–90%DMF mixtures. The ability of native and immobilized subtilisin to catalyze peptide bond formation between Z-Ala-Ala-Leu-OMe and Phe-pNA was studied in non-aqueous media. Considerable enzyme stabilization in acetonitrile/90%DMF mixture, induced by the immobilization, resulted in higher product yield (57%) than in case of native subtilisin suspension (32%). Detailed study of synthesis reaction revealed that notable increase in product yield could be reached using increase in both substrate concentrations up to 200 mM.     

Role of PYK2 in the development of obesity and insulin resistance

Non-receptor proline-rich tyrosine kinase-2 (PYK2), which is activated by phosphorylation of one or more of its tyrosine residues, has been implicated in the regulation of GLUT4 glucose transporter translocation and glucose transport. Some data favor a positive role of PYK2 in stimulating glucose transport, whereas other studies suggest that PYK2 may participate in the induction of insulin resistance. To ascertain the importance of PYK2 in the setting of obesity and insulin resistance, we (1) evaluated the regulation of PYK2 in mice fed a high-fat diet and (2) characterized body and glucose homeostasis in wild type (WT) and PYK2(-/-) mice on different diets. We found that both PYK2 expression and phosphorylation were significantly increased in liver and adipose tissues harvested from high-fat diet fed mice. Wild type and PYK2(-/-) mice were fed a high-fat diet for 8 weeks to induce insulin resistance/obesity. Surprisingly, in response to this diet PYK2(-/-) mice gained significantly more weight than WT mice (18.7+/-1.2g vs. 9.5+/-0.6g). Fasting serum leptin and insulin and blood glucose levels were significantly increased in high-fat diet fed mice irrespective of the presence of PYK2 protein. There was a close correlation between serum leptin and body weight. Intraperitoneal glucose tolerance tests revealed that as expected, the high-fat diet resulted in increased blood glucose levels following glucose administration in wild type mice compared to those fed normal chow. An even greater increase in blood glucose levels was observed in PYK2(-/-) mice compared to wild type mice. These results demonstrate that a lack of PYK2 exacerbates weight gain and development of glucose intolerance/insulin resistance induced by a high-fat diet, suggesting that PYK2 may play a role in slowing the development of obesity, insulin resistance, and/or frank diabetes.     

Characterization of acetohydroxyacid synthase from Mycobacterium tuberculosis and the identification of its new inhibitor from the screening of a chemical library

Acetohydroxyacid synthase (AHAS) is a thiamin diphosphate- (ThDP-) and FAD-dependent enzyme that catalyzes the first common step in the biosynthetic pathway of the branched-amino acids such as leucine, isoleucine, and valine. The genes of AHAS from Mycobacterium tuberculosis were cloned, and overexpressed in E. coli and purified to homogeneity. The purified AHAS from M. tuberculosis is effectively inhibited by pyrazosulfuron ethyl (PSE), an inhibitor of plant AHAS enzyme, with the IC(50) (inhibitory concentration 50%) of 0.87 microM. The kinetic parameters of M. tuberculosis AHAS were determined, and an enzyme activity assay system using 96-well microplate was designed. After screening of a chemical library composed of 5600 compounds using the assay system, a new class of AHAS inhibitor was identified with the IC(50) in the range of 1.8-2.6 microM. One of the identified compounds (KHG20612) further showed growth inhibition activity against various strains of M. tuberculosis. The correlation of the inhibitory activity of the identified compound against AHAS to the cell growth inhibition activity suggested that AHAS might be served as a target protein for the development of novel anti-tuberculosis therapeutics.     

Insecticidal activity of proteinous venom from tentacle of jellyfish Rhopilema esculentum Kishinouye

Insecticidal activity of proteinous venom from tentacle of jellyfish Rhopilema esculentum Kishinouye was determined against three pest species, Stephanitis pyri Fabriciusa, Aphis medicaginis Koch, and Myzus persicae Sulzer. R. esculentum full proteinous venom had different insecticidal activity against S. pyri Fabriciusa, A. medicaginis Koch, and M. persicae Sulzer. The 48 h LC50 values were 123.1, 581.6, and 716.3 microg/mL, respectively. Of the three pests, R. esculentum full proteinous venom had the most potent toxicity against S. pyri Fabriciusa, and the corrected mortality recorded at 48 h was 97.86%. So, S. pyri Fabriciusa could be a potential target pest of R. esculentum full proteinous venom.     

Mapping peptides correlated with transmission of intrasteric inhibition and allosteric activation in human cystathionine beta-synthase

Cystathionine beta-synthase plays a key role in the intracellular disposal of homocysteine and is the single most common locus of mutations associated with homocystinuria. Elevated levels of homocysteine are correlated with heart disease, Alzheimer's and Parkinson's diseases, and neural tube defects. Cystathionine beta-synthase is modular and subjected to complex regulation, but insights into the structural basis of this regulation are lacking. We have employed hydrogen exchange mass spectrometry to map peptides whose motions are correlated with transmission of intrasteric inhibition and allosteric activation. The mass spectrometric data provide an excellent correlation between kinetically and conformationally distinguishable states of the enzyme. We also demonstrate that a pathogenic regulatory domain mutant, D444N, is conformationally locked in one of two states sampled by the wild type enzyme. Our hydrogen exchange data identify surfaces that are potentially involved in the juxtaposition of the regulatory and catalytic domains and form the basis of a docked structural model for the full-length enzyme.     

Integration of Genetic and Cytological Maps and Development of a Pachytene Chromosome-based Karyotype in Papaya

A significant amount of genetic and genomic resources have been developed in papaya (Carica papaya, $ {\hbox{2n = 2}} \times { = 18} $ ), including genetic linkage maps consisting of nine major and three minor linkage groups. However, the 12 genetic linkage groups have not been integrated with the nine chromosomes of papaya. Bacterial artificial chromosome (BAC) clones associated with each linkage group were recently isolated. These linkage group-specific BACs were mapped to meiotic pachytene chromosomes of papaya using fluorescence in situ hybridization (FISH). The FISH mapping results integrated the 12 linkage groups into the nine papaya chromosomes. We developed a pachytene chromosome-based high resolution karyotype for the hermaphrodite plant genome of papaya cultivar SunUp. The chromosomal distribution of heterochromatin in the papaya genome is provided in the karyotype with the X chromosome representing the most euchromatic chromosome in the papaya genome. FISH mapping also revealed a significant amplification of sequences related to the 5S ribosomal RNA genes, which was detected in the male-specific region of the Y chromosome, but not in the corresponding region in the X chromosome.     

High-throughput antibody generation using multiplexed immunization and immunogen array analysis

In this work, the authors developed a new screening approach using multiplexed immunization and immunogen array analysis to improve the efficiency of antibody screening for high-throughput antibody generation. The immunogen array is based on a 96-well format in which different immunogens and negative as well as positive controls are immobilized in each well, thus making it possible to screen hundreds of antibody candidates simultaneously. To demonstrate this approach, a model of 4 mixed immunogens immunization was employed. In total, 675 antibody candidates were screened before and after established antibody hybridomas in parallel with immunogen arrays and enzyme-linked immunosorbent assay. The signal intensity, specificity, and cross-reactivity of produced antibody candidates were analyzed using a hierarchical cluster algorithm to track the characteristics of antibody candidates during antibody generation, which might reduce the number of false-positive and false-negative binding of antibodies. Moreover, 4 monoclonal antibodies that were produced successfully recognized their corresponding target antigens.     

基于按摩的电刺激器的设计

目的设计一种基于中医按摩的电刺激仪器——将产生的电刺激信号通过中医的按摩手法给患者按摩。方法研究采用STC89C58RD＋单片机作为控制器,采用LCD点阵液晶显示作为可视化的提示工具,采用4＊4矩阵键盘作为输入控制接口,采用变压器作为电刺激信号升压器件。结果研究实现了预期目标,用户可通过LCD提示输入控制点刺激信号强度的按键,单片机处理后输出相应的脉冲波形,进而触发生成用于按摩的电刺激信号。结论对51单片机开发应用,实现了输入不同的频率信息,产生不同强度的电刺激信号,使人感觉不同程度的麻刺激,能够用于治疗和保健。     

洗必泰与新洁尔灭的协同杀菌效果

采用悬液定量杀菌试验,以协同系数T/E值作为评价指标,对洗必泰与新洁尔灭的协同杀菌效果进行了实验室研究。发现0.1%洗必泰与0.1%新洁尔灭协同作用2 min,对金黄色葡萄球菌、大肠埃希菌和白色念珠菌均起到了显著协同杀菌效果。     

海南岛翼手类地理分布格局的聚类分析

2007年7月～2008年7月,对海南岛翼手类动物进行了物种多样性调查。按照《世界哺乳动物名典》和Simmons的分类系统进行分类,结合考察标本记录,整理调查结果和文献资料。统计海南岛翼手类动物计37种,隶属于2亚目7科（包括采集标本,计16种,隶属于2亚目4科）。依据分布型对其组成成分进行分析,结果表明海南岛翼手类动物东洋界种类相对较多,反映出强烈的华南区的特征;岛屿的影响表现在亚种分化和缺乏某些大陆类群。依物种组成对分布区进行聚类分析,结果呈现明显的四阶现象。根据物种多样性组成和聚类分析结果,可以认为以乐东、东方和陵水为主要区域范围的尖峰岭和吊罗山两大山系是目前统计海南岛翼手类物种分布的热点区。