水葫芦肌动蛋白基因片段的克隆及序列分析

利用植物肌动蛋白保守片段设计简并引物,通过RT-PCR 扩增出水葫芦肌动蛋白基因片段并连接到 pMD19-T 载体,转化E. coli,测序。序列分析结果表明,该序列与油棕、小“果蕉等植物的相似性程度达到85%以上, 分子进化分析水葫芦肌动蛋白基因序列与油棕、小“果蕉等单子叶植物亲缘关系较近,但并没有与某些植物的肌动 蛋白很好地聚成一类。本研究所克隆得到的水葫芦肌动蛋白基因序列能够为进一步研究水葫芦其他基因的表达提 供内标。     

牛骨骼肌G-肌动蛋白的分离纯化及超高压处理对G-肌动蛋白的影响

对破碎后的冷却牛肌肉,经匀浆、过滤、丙酮抽提,制成粗肌动蛋白干粉,并将其多次超速离心、透析、DEAE—52离子交换层析而进一步纯化进行研究。经SDS—PAGE检测得到高纯度的G-肌动蛋白,其分子质量约为42kDa,分别采用100,200,300,400MPa超高压处理,并保压5min。SDS—PAGE检测结果表明,随着压力的升高,肌动蛋白质条带下面出现暗带,颜色逐渐加深。暗带的出现可能与超高压处理后蛋白质降解成小分子量的蛋白质或多肽等物质有关,这为超高压对蛋白质结构的影响提供了理论依据。     

灰飞虱actin基因的克隆及原核表达

通过生物信息学分析获得了灰飞虱actin基因的序列信息,采用RT-PCR方法从灰飞虱中克隆了actin基因的开放阅读框,并将其在大肠杆菌BL21(DE3)中进行原核表达。序列分析结果表明,该基因开放阅读框长1 131 bp,编码377个氨基酸,蛋白质理论分子量为4.17×104,理论等电点为5.28。将此序列登录GenBank数据库,登录号为KC683802。序列比对发现,该基因序列在多个物种间保守,与褐飞虱actin基因的同源性高达95%。构建的原核表达载体pET32a-actin,在大肠杆菌BL21(DE3)中进行获得表达,SDS-PAGE分析结果表明,表达的蛋白质主要存在于包涵体中。     

胞间连丝结构与组分的研究进展

胞间连丝是植物体内连接相邻细胞的一种跨细胞的细胞器,是细胞间物质运输和信息传递的通道,为相邻细胞间小分子、病毒颗粒及一些特殊大分子的运输提供途径。细胞间的共质通道就在质膜和其内部的压缩内质网间形成,压缩内质网又称连丝微管。由于胞间连丝结构的复杂性,使得对其结构组分的研究受到很大限制,但近几年随着电子显微镜、免疫标记、显微注射技术的广泛应用,已有证据表明肌动蛋白、肌球蛋白、中心蛋白和钙网蛋白等是胞间连丝的组分,这些蛋白之间相互作用调节胞间连丝的通透性,控制物质运输。     

蛋白激酶C抑制剂Staurosporine对血小板聚集、蛋白磷酸化和肌动蛋白聚合的影响

目的：进一步研究蛋白激酶C抑制剂Staurosporine在血小板聚集、蛋白磷酸化、肌动蛋白聚合中的作用。方法：以32P－Na2HPO4标记血小板；以血小板聚集仪测定血小板聚集；以SDS－PAGE分离蛋白质，进行放射自显影；以TritonX－100抽提法沉淀骨架蛋白。结果：（1）1μmol／LStaurosporine完全抑制0．5U／ml凝血酶或10μmol／LPMA诱导的血小板聚集，大部分抑制20μmol／LA23187诱导的血小板聚集。（2）1μmol／LStaurosporine几乎完全抑制0．5U／ml凝血酶、或10μmol／LPMA、或20μmol／LA23187诱导的血小板40kD和20kD蛋白磷酸化。（3）1μmol／LStaurosporine完全抑制0．5U／ml凝血酶或10μmol／LPMA诱导的血小板肌动蛋白聚合。结论：Staurosporine抑制蛋白激酶C的活性．从而抑制血小板的聚集和肌动蛋白聚合；蛋白激酶C在血小板聚集和肌动蛋白聚合中起着重要调控作用。     

白羊草肌动蛋白基因片段的克隆及序列分析

根据几种禾本科植物肌动蛋白基因的保守序列设计一对简并引物,以白羊草(Bothriochloa ischaemum)幼叶总RNA为模板,采用RT-PCR方法克隆获得一个肌动蛋白基因cDNA片段,命名为BiACT,该基因片段长度为720bp,编码240个氨基酸残基。氨基酸比对分析结果表明,BiACT基因编码的氨基酸与其他植物肌动蛋白的氨基酸序列具有较高的同源性。     

Characterization of fast skeletal myosin from white croaker in comparison with that from walleye pollack

ABSTRACT:   Enzymatic and structural properties of white croaker fast skeletal muscle myosin were determined and compared with those of walleye pollack counterpart. Ca²⁺-ATPase activity of white croaker myosin was decreased to approximately 70% of the original activity during 1 day of storage at 0°C and pH 7.0 in 0.5 M KCl and 0.1 mM dithiothreitol, whereas that of walleye pollack was decreased to approximately 20% under the same condition. The activation energy ( E _a) for inactivation of white croaker myosin calculated by the Arrhenius plot for inactivation rate constant (K_D) was 1.2-fold higher than that of walleye pollack. While Ca²⁺-ATPase showed a similar KCl-dependency for the two species, the maximal activity was observed at pH 6.2 and 6.3 for white croaker and walleye pollack, respectively. Actin-activated myosin Mg²⁺-ATPase activity of white croaker was approximately half that of walleye pollack at 0.05 M KCl and pH 7.0, although the two myosins showed a similar affinity to F-actin with K _m of 1.7 and 1.4, respectively. Limited proteolysis with α-chymotrypsin cleaved heat-denatured white croaker myosin mainly at heavy meromyosin/light meromyosin (HMM/LMM) junction, whereas walleye pollack myosin was cleaved at several sites in LMM as well as at the HMM/LMM junction.     

Inhibition of plasminogen activator inhibitor-1 by small interfering RNA attenuates pulmonary fibrosis in rats

AIM: To examine the effects of silencing of plasminogen activator inhibitor-1 (PAI-1) expression by small interfering RNA (siRNA) on bleomycin (BLM)-induced rat pulmonary fibrosis. METHODS: Total 72 Wistar rats were divided into 4 groups: control, BLM, BLM+non-specific siRNA (BLM+N), and BLM+ PAI-1 siRNA (BLM+P). Pulmonary fibrosis was induced by intratracheal injection of BLM (5 mg/kg), whereas equal volume of normal saline was used in control group. After the administration of BLM or normal saline, the rats were treated with tracheal injection of PAI-1-siRNA (7.5 nmol/0.2 mL per rat) in BLM+P group, non-specific siRNA (7.5 nmol/0.2 mL per rat) in BLM+N group, and 0.2 mL normal saline in BLM group and control group, twice a week, 8 times in 28 d. On day 7, 14, and 28, the rats (n=6 at each time point) were sacrificed. The bronchoalveolar lavage fluid (BALF) from the left lung was harvested to examine the activity of PAI-1. The mRNA expression of collagen type Ⅲ, α-smooth muscle actin (α-SMA) and tissue inhibitor of metalloproteinase-1 (TIMP-1) in the middle lobe of the right lung was detected by RT-PCR. RESULTS: PAI-1 activity and the expression of collagen type Ⅲ, α-SMA and TIMP-1 were increased in BLM group on day 7, 14 and 28. Intratracheal injection of PAI-1 siRNA twice a week continuously reduced PAI-1 activity in the BALF (P<0.05),and decreased the expression of collagen type Ⅲ, α-SMA and TIMP-1 in the fibrotic lung tissues on day 7, 14 and 28. Statistical differences in the expression of collagen type Ⅲ, α-SMA and TIMP-1 between BLM+P group and BLM group at the same time point were observed. CONCLUSION: Intratracheal injection of PAI-1 siRNA twice a week continuously inhibits the expression of PAI-1. PAI-1 siRNA ameliorates BLM-induced pulmonary fibrosis by down-regulation of TIMP-1 expression.