High-level cryIVD and cytA gene expression in Bacillus thuringiensis does not require the 20-kilodalton protein, and the coexpressed gene products are synergistic in their toxicity to mosquitoes.

Interactions among the 20-kDa protein gene and the cytA and cryIVD genes located in a 9.4-kb HindIII fragment were studied. A series of plasmids containing a combination of these different genes was constructed by using the Escherichia coli/Bacillus thuringiensis shuttle vector pHT3101. The plasmids were then used to transform an acrystalliferous strain, cryB, derived from B. thuringiensis subsp. kurstaki. The results from sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblot analyses suggest that although the 20-kDa protein is required for the efficient CytA protein production in E. coli, it is not required in B. thuringiensis. With or without the truncated 20-kDa protein gene, the CtyA and/or CryIVD proteins are produced and form parasporal inclusions in B. thuringiensis cells. However, more-efficient expression is obtained when a second protein, probably acting as a chaperonin, is present. In addition, the time course studies show that the CytA and CryIVD proteins are coordinately produced. Both the crude B. thuringiensis culture and purified inclusions from each recombinant B. thuringiensis strain are toxic to Culex quinquefasciatus larvae. The parasporal inclusions formed in B. thuringiensis cells are mosquitocidal, with CytA synergizing CryIVD toxicity.     

Reagentless amperometric immunosensor for human chorionic gonadotrophin based on direct electrochemistry of horseradish peroxidase

A novel amperometric immunosensor for determination of human serum chorionic gonadotrophin (HCG) was constructed by immobilization of HCG with titania sol-gel on a glassy carbon electrode and the direct electrochemistry of horseradish peroxidase (HRP) labeled to HCG antibody (HRP-anti-HCG). The morphologies of the HCG membrane were characterized to be chemically clean, porous and homogeneous. HRP-anti-HCG was functionally conjugated with the immobilized HCG after incubation in phosphate buffer (PBS) containing HRP-anti-HCG. A direct electron transfer of HRP with a rate constant of 1.35+/-0.40 s(-1) was observed at the HRP-anti-HCG-HCG/titania sol-gel membrane modified electrode in 0.1 M PBS pH 7.0. With a competitive mechanism the differential pulse voltammetric peak current of the immobilized HRP decreased linearly with an increasing HCG concentration from 2.5 to 12.5 mIU/ml in the incubation solution. The HCG immunosensor showed a detection limit of 1.4 mIU/ml, a good accuracy and acceptable precision and reproducibility with an intra-assay CV of 4.7% at 5.0 mIU/ml and an inter-assay precision of 8.1% obtained at 10 mIU/ml. The biosensor displayed a good stability in a storage period of 30 days.     

C4-dicarboxylates sensing mechanism revealed by the crystal structures of DctB sensor domain

C₄-dicarboxylates are the major carbon and energy sources during the symbiotic growth of rhizobia. Responses to C₄-dicarboxylates depend on typical two-component systems (TCS) consisting of a transmembrane sensor histidine kinase and a cytoplasmic response regulator. The DctB-DctD system is the first identified TCS for C₄-dicarboxylates sensing. Direct ligand binding to the sensor domain of DctB is believed to be the first step of the sensing events. In this report, the water-soluble periplasmic sensor domain of Sinorhizobium meliloti DctB (DctBp) was studied, and three crystal structures were solved: the apo protein, a complex with C₄ succinate, and a complex with C₃ malonate. Different from the two structurally known CitA family of carboxylate sensor proteins CitA and DcuS, the structure of DctBp consists of two tandem Per-Arnt-Sim (PAS) domains and one N-terminal helical region. Only the membrane-distal PAS domain was found to bind the ligands, whereas the proximal PAS domain was empty. Comparison of DctB, CitA, and DcuS suggests a detailed stereochemistry of C₄-dicarboxylates ligand perception. The structures of the different ligand binding states of DctBp also revealed a series of conformational changes initiated upon ligand binding and propagated to the N-terminal domain responsible for dimerization, providing insights into understanding the detailed mechanism of the signal transduction of TCS histidine kinases.     

The stimulatory effect of ROCK inhibitor on bovine corneal endothelial cells

Reagents which can promote the proliferation, adhesion and migration of cultured corneal endothelial cells (CECs) will be helpful for the treatment of reduced visual acuity due to CECs deficiency. The objectives of this study were to investigate the potential use of an inhibitor of Rho-associated protein kinase (ROCK), Y-27632, to cultured bovine corneal endothelial cells (B-CECs) and evaluated its effects on the proliferation, adhesion and migration of B-CECs. The proliferation of cultured B-CECs was moderately enhanced by 10 μM Y-27632. Y-27632 induced fibroblast-like morphological changes in the cultured B-CECs and normal cell morphology could recover after Y-27632 removal. In addition, Y-27632 was found to significantly enhance the adhesion and migration of B-CECs. Furthermore, the hanging drop aggregation assay showed that Y-27632 promoted B-CECs to form cellular networks and sheets, which proliferated along the liquid–air interface and migrated to the surface of the lid of dish. Our study demonstrated that Y-27632 is a potentially powerful reagent which can enhance the proliferation of cultured B-CECs. Y-27632 will be useful in CEC injection therapy and topical application for CEC deficiency.     

ApCl基因在毕赤酵母中的表达

为了研究ApCl基因的功能,应用基因重组技术将ApCl基因片段克隆入大肠埃希菌—酵母穿梭质拉pGAPZαA,构建重组真核表达质拉pGAPZαA-ApCl,电转化巴斯德单赤酵母GS115,Zeocin筛选出阳性克隆转化子.通过比较转化空载体pGAPZaA和重组载体pGAPZαA-ApCl的不同菌株分别在含有高盐和高山梨醇浓度的液体培养基中生长情况,发现毕赤酵母GS115在转化ApCl基因后其抗旱、抗盐能力显著提高(约3倍),进一步验证了ApCl基因对提高生物抗逆能力有显著作用,为将来分离该蛋白及进一步研究奠定基础.     

克鲁维酵母Y-85菊粉酶的纯化和性质

克鲁维酵母(Kluyveromyces sp.)Y-85产生的胞内菊粉酶(endocellular inulinase)和胞外菊粉酶(exocellular inulinase)粗酶液分别经PEG6000-磷酸盐缓冲液双水相抽提得部分纯化酶液。前者进一步用硫酸铵分级沉淀、Protein-PAK DEAE离子交换、Protein-PAK200SW凝胶过滤后得到两个菊粉酶组分EⅠ和EⅡ;后者采用DEAE-Sephacel离子交换、Sephadex G150凝胶过滤后得到菊粉酶Eexo。经Waters 650E蛋白纯化系统鉴定,三者均呈单一的对称峰;EⅠ和EⅡ达聚丙烯酰胺盘状凝胶电泳纯。EⅠ、EⅡ和Eexo的分子量分别为42kD、65kD和57kD;三者均为糖蛋白,多糖含量分别为30％、35％和25％;I/S(Inulinaseactivity/Sucrase activity)比值分别为0.086、0.078和0.072;三者均属外切菊粉酶。EⅠ、EⅡ和Eexo酶反应最适pH分别为4.6、4.5和4.6,最适温度分别为52℃、52℃和55℃;Ag^+、Hg^(2+)和PCMB对酶活性有强烈的抑制作用;三者水解菊芋粉糖液的产物均为果糖(86.5％)和葡萄糖(13.5％)。     

A novel spliced variant of the type 1 corticotropin-releasing hormone receptor with a deletion in the seventh transmembrane domain present in the human pregnant term myometrium and fetal membranes

CRH exerts its actions via activation of specific G protein-coupled receptors, which exist in two types, CRH-R1 and CRH-R2, and arise from different genes with multiple spliced variants. RT-PCR amplification of CRH receptor sequences from human myometrium and fetal membranes yielded cDNAs that encode a novel CRH-R type 1 spliced variant. This variant (CRH-R1d) is present in the human pregnant myometrium at term only, which suggests a physiologically important role at the end of human pregnancy and labor. The amino acid sequence of CRH-R1d is identical to the CRH-R1alpha receptor except that it contains an exon deletion resulting in the absence of 14 amino acids in the predicted seventh transmembrane domain. Binding studies in HEK-293 cells stably expressing the CRH-R1d or CRH-R1alpha receptors revealed that the deletion does not change the binding characteristics of the variant receptor. In contrast, studies on the G protein activation demonstrated that CRH-R1d is not well coupled to the four subtypes of G proteins (G(s), G(i), G(o), G(q)) that CRH-R1alpha can activate. These data suggest that although the deleted segment is not important for CRH binding, it plays a crucial role in CRH receptor signal transduction. Second messenger studies of the variant receptor showed that CRH and CRH-like peptides can stimulate the adenylate cyclase system, with reduced sensitivity and potency by 10-fold compared with the CRH-R1alpha. Furthermore, CRH failed to stimulate inositol trisphosphate production. Coexpression studies between the CRH-R1d or CRH-R1alpha showed that this receptor does not play a role as a dominant negative receptor for CRH.     

The extracellular domain of p185(c-neu) induces density-dependent inhibition of cell growth in malignant mesothelioma cells and reduces growth of mesothelioma in vivo

EGFR is involved in the density-dependent inhibition of cell growth, while coexpression of EGFR with erbB2 can render normal cells transformed. In this study, we have examined the effect of a species of p185 that contains the transmembrane domain and the extracellular domain of p185(c-neu), on growth properties of a human malignant mesothelioma cell line that coexpresses EGFR and erbB2. The ectodomain form of p185(c-neu) enhanced density-dependent inhibition of cell growth and we found that p21 induction appeared to be responsible for this inhibitory effect. Previously, the extracellular domain species was shown to suppress the transforming abilities of EGFR and p185(c-neu/erbB2) in a dominant-negative manner. The ability of this subdomain to affect tumor growth is significant, as it reduced in vivo tumor growth. Unexpectedly, we found that the domain did not abrogate all of EGFR functions. We noted that EGFR-induced density-dependent inhibition of cell growth was retained. Tyrosine kinase inhibitors of EGFR did not cause density-dependent inhibition of cell growth of malignant mesothelioma cells. Therefore, simultaneously inhibiting the malignant phenotype and inducing density-dependent inhibition of cell growth in malignant mesothelioma cells by the extracellular domain of p185(c-neu) may represent an important therapeutic advance.     

寄生革翅目蠼螋的被毛孢一新种

在湖北省神农架林区采集到一种寄生于蠼螋成虫的真菌标本snj121022,分离并获得菌株 GZUIFR-snj121022,经形态学、系统发育和拆分网络综合分析,鉴定其为被毛孢属的一个新种,命名为神农架被毛孢Hirsutella shennongjiaensis。本文对其进行了详细的形态特征描述、图解和系统发育分析。     

中心体蛋白Cenexin在大鼠精子发生过程中的表达特征

中心体蛋白Cenexin是成熟中心粒的唯一标志分子。为阐明中心粒在大鼠精子发生中的成熟以及功能,我们首先通过RT－PCR技术从大鼠睾丸组织中扩增出了Cenexin cDNA片段,原核表达重组蛋白后,用其免疫小鼠制备了高滴度的抗Cenexin的多克隆抗体,然后利用免疫荧光染色、Western Blot和半定量RT－PCR方法,研究了大鼠精子发生过程中Cenexin蛋白和基因的表达特征。结果显示Cenexin mRNA水平在精原细胞和精母细胞中较高,随后表达水平下降,而蛋白质分子在精原细胞到精子细胞中都定位于细胞的一个中心粒上,表示有成熟中心粒的存在,在长形精子细胞中该蛋白位于鞭毛的基体部。附睾的绝大多数成熟精子中Cenexin免疫染色消失。中心体蛋白Cenexin在精子变态期的表达变化可能与精子鞭毛形成的起始有关。