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。     

Identification and Expression of a β-actin Gene from Liposcelis bostrychophila Badonnel （Psocoptera： Liposcelididae）

A β-actin gene, Libβ-actinl, from the psocid, Liposcelis bostrychophila, was isolated, sequenced, and expressed in Escherichia coli. The cDNA sequence was 1 281 bp in length and contained an open reading frame of 1 131 bp encoding 376 amino acids with a predicted molecular weight of 41.82 kDa. According to a BlastN search, the coding region shared the highest identity (97%) with Pediculus humanus actin 5C, while the deduced amino acid sequence was completely identical to a mutant of Drosophila melanogaster actin 5C. Comparison of the nucleotide and deduced amino acid sequences confirmed the high similarity between Libβ-actinl and homologs in other insect species. The 3′ untranslated region (3′ UTR) of the Libβ-actinl mRNA had a high A+U content (approximately 75%) and contained three repeats of the AUUUUUA and AUUUA motifs, which may play a role in regulating mRNA decay. The expression of Libβ-actinl was further analyzed in insecticide induced and control psocids. The results indicated that there was no significant difference in expression of Libβ-actinl between the induced and control groups, suggesting that Libβ-actinl may be an appropriate internal control for the gene expression profiling in this insect. Furthermore, Libβ-actinl was also heterologously expressed in Escherichia coli, which provided a basis to investigate the physiological functions of actin genes in the psocid.     

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。     

VPA alleviates bleomycin-induced pulmonary fibrosis in rats

AIM: To investigate the effect of sodium valproate (VPA) on bleomycin-induced pulmonary fibrosis (PF) and its mechanism. METHODS: SD rats (n=42) were randomly divided into model group and treatment group. Bleomycin at dose of 5 mg/kg was intratracheally injected to establish a rat PF model. The rats in treatment group were given normal saline (NS, 0.5 mL/d), VPA (300 mg·kg^-1·d^-1) or dexamethasone (DEX, 0.6 mg·kg^-1·d^-1) via intraperitoneal injection from the 14th day to the 28th day after modeling. The rats in model group were sacrificed on 0 d, 14 day and 28 d after modeling . The rats in treatment group were killed at 14th day after treatment. The effects of VPA on PF were evaluated by HE and Masson staining. The hydroxyproline (HYP) content in the rat lung tissues was detected, and the expression of α-smooth muscle actin (α-SMA) and E-cadherin was determined by Western blotting. RESULTS: HE staining showed that the alveolar structure and interstitial morphology in VPA group were better than those in NS group and DEX group. The level of collagen in VPA group was significantly lower than that in DEX group and NS group by determining the HYP content and Masson staining. VPA reduced the expression of α-SMA, a mesenchymal marker protein of PF, while increased the expression of epithelial marker protein E-cadherin. CONCLUSION: VPA reduces collagen content and distribution, and up-regulates the expression of the epithelial marker protein E-cadherin, while down-regulates mesenchymal marker protein α-SMA, thereby preventing the rat lung tissues from bleomycin-induced PF.     

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。     

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.     

绿色荧光蛋白在家蚕细胞中的表达

利用家蚕细胞质肌动蛋白基因（A3）启动子和NPV病毒即刻早期蛋白IE启动子在家蚕细胞中表达GFP，实验结果表明是可行与有效的。     

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。     

Gelation of low salt soluble proteins from scallop adductor muscle in relation to its freshness

ABSTRACT: The low salt extract from scallop striated adductor muscle contained extra proteins in addition to sarcoplasmic proteins and was turned into a gel upon standing or immediately by the addition of Ca²⁺. The amount of extra protein, which was composed of a large amount of actin and a small amount of myosin, decreased with a decrease in adenosine triphosphate (ATP) content in the muscle during storage at 5°C. Actin was easily extracted from scallop myofibrils in low salt solution with ATP at 1 mM or above. Furthermore, ATP was required to induce the gelation of the low salt extract. A visual observation of the gelation of low salt extract is therefore a simple and easy method to inspect prerigor state of scallop adductor muscle.     

The Process and Development Mechanism of Age-related Fibrosis in the Pancreatic Islets of Sprague-Dawley Rats: Immunohistochemical Detection of Myofibroblasts and Suppression Effect by Estrogen Treatment

The mechanism of spontaneous islet fibrosis in Sprague-Dawley rats was investigated. Using sections of the pancreas in naive males aged 26 to 102 weeks old and 26-week-old males injected with β-estradiol 3-benzoate (EB), the incidence of lesions and histological scores of fibrosis were examined in conjunction with immunohistochemistry for α-smooth muscle actin (α-SMA), platelet-derived growth factor receptor-α (PDGFRα) and estrogen receptor-α (ERα). The incidence of islet fibrosis increased in 78-week-old animals compared to the 26-week-old animals, and the incidence of atrophy in the fibrotic islet increased in animals over 52 weeks old. α-SMA and PDGFRα were positively stained mainly in fibrotic/inflammatory islets, and the histological score of α-SMA in the fibrotic islet decreased age-dependently. Notably, α-SMA and PDGFRα were co-expressed in inflammatory islets with a high score at all ages. The positive index of ERα in the EB-treated group increased when compared with that of the naive group. However, it was independent of the existence of fibrosis. In contrast, the score of α-SMA and PDGFRα decreased in the EB-treated group. In conclusion, it was clarified that a part of age-related fibrosis in islets became atrophy with age, and α-SMA-positive myofibroblasts were considered to contribute to the development of fibrosis. Strong PDGFRα stainability in fibrotic/inflammatory islets may imply that myofibroblasts were stimulated by PDGF to produce an extracellular matrix. Although estradiol has been known to suppress fibrosis/inflammation in the islet, nuclear-located ER-dependent signaling was considered not to be involved in the suppression mechanism. EB possibly affected the inhibition of the appearance of myofibroblasts.