Localization of smooth muscle actin in the iridocorneal angle of normal and spontaneous glaucomatous beagle dogs

Purpose To date, our knowledge of the canine trabecular meshwork (TM) with regard to contractility is incomplete. It is important to understand the potential contractile capability within the TM and possible changes associated with spontaneous hypertensive glaucoma. To that end we have examined the presence of actin, including smooth muscle (SM) actin, in the normal and glaucomatous canine iridocorneal angle (ICA) morphologically and immunohistochemically. Methods Sections from the ICAs of 12 Beagles with inherited glaucoma (3 months to 6 years old) and age‐matched normal Beagles were treated with target retrieval, protein and power blocked and sequentially incubated with the primary antibody (rat anticanine SM actin) and the secondary antibody (rabbit antirat immunoglobulin), followed by peroxidase labeled streptavidin and incubation with substrate‐chromogen solution (AEC). Smooth muscle fibers that lined an artery within canine heart tissue were used as positive controls. Separate specimens were prepared for ultrastructual observation. Results Ultrastructurally, cells within the inner, posterior region of the corneoscleral TM and outer, posterior region of the uveal TM contained many microfilaments, 6 nm in diameter (i.e. actin). Immunohistochemistry demonstrated that cells within these regions possessed SM actin, having been greatest posteriorly, but extended anteriorly to a lesser extent. In the preglaucomatous affected dog the localization pattern for SM actin was identical to that seen in the normal dogs. With the progression of the disease the pattern disappeared. Conclusions The interior presence of myofibroblastic cells within the canine ICA suggests that these cells and the smooth muscle cells of the ciliary body along the same plane of orientation function to facilitate the removal of aqueous humor and are likely to be influenced by vascular mediators. The contractile apparatus for the ICA in the dog with inherited glaucoma appeared identical to that of the normal dog prior to expression of the disease, but weakened as the disease progressed.     

豌豆肌动蛋白异型体PEAc3的原核表达及其聚合特性分析

植物肌动蛋白在植物顶端生长、细胞分裂分化和细胞信号转导等多种生命活动中发挥着重要作用.豌豆中存在多种肌动蛋白异型体.研究豌豆肌动蛋白异型体PEAc3与组氨酸标签(His-tag)及绿色荧光蛋白(GFP)的融合表达,并分析了融合蛋白的聚合特性.采用RT-PCR的方法克隆PEAc3基因,构建原核表达载体pET30a-His-PEAc3-GFP.用DNAman生物学软件分析表明,PEAc3融合蛋白全长675个氨基酸,分子量74.74 ku,等电点5.81.将pET30a-His-PEAc3-GFP转入大肠杆菌BL21中,优化的诱导表达条件为:25℃,当菌液OD600达到0.8时加入IPTG(浓度0.1 mmol/L),诱导表达4 h.采用尿素变性复性、镍柱亲和层析的方法从包涵体中纯化获得高纯度融合蛋白.融合蛋白His-PEAc3一GFP能够体外聚合,聚合临界浓度为0.5 μmol/L.单体His-PEAc3-GFP对DnaseⅠ有抑制作用,聚合后对肌球蛋白Mg-ATPase活性有一定的激活作用.上述结果表明,原核表达的His-PEAc3-GFP可能具有类似于一般肌动蛋白的聚合特性.     

Characterization of myofibrillar adenosine triphosphatase activity and liberation of actin from myofibrils upon heating chicken breast meat

Denaturation of actin and myosin in myofibrils induced by heating at 50°C was investigated to reveal the mechanism of irreversible liberation of actin from myofibrils on heating at lower temperatures than conventional cooking. Denaturation of these proteins was determined by Mg²⁺‐ATPase (adenosine triphosphatase) and Ca²⁺‐ATPase activities. When minced meat was heated for 20 min, actin was liberated accompanying denaturation of 80% of actin and 50% of myosin. Heating of the myofibrillar fraction (MFF) isolated from meat homogenate induced much slower denaturation of actin than myosin. When MFF was heated with sarcoplasmic fractions, denaturation of actin was facilitated, suggesting that sarcoplasmic fractions contain factors to facilitate actin denaturation. Inosine‐5′‐monophosphate, a component of sarcoplasmic fractions, was shown to have no effect on actin and myosin denaturation. These results suggest that heating meat at 50°C dissociates binding (‘Bond A’) between actin and myosin participating in ATPase activities, resulting in denaturation of both proteins under influence of sarcoplasmic components. Although denaturation of actin and myosin disrupted Bond A, actin was not liberated simultaneously, suggesting the presence of another bond (‘Bond B’, more heat‐stable than Bond A) between both proteins and necessity of disruption of Bond B for actin release from myofibrils.     

斯氏艾美尔球虫肌动蛋白解聚因子在HeLa细胞中的表达

为真核表达斯氏艾美尔球虫(Eimeria stiedae)肌动蛋白解聚因子(ADF)基因,本研究采用RT-PCR技术扩增ADF基因,构建pVAX- 1-ADF真核重组质粒,并转染HeLa细胞.采用RT-PCR方法检测ADF mRNA转录水平、western blot检测ADF蛋白表达水平.琼脂糖凝胶电泳结果表明PCR扩增出一条大小为350 bp的特异性片段,与预期值相符.Western blot检测显示,转染pVAX- 1-ADF的HeLa细胞在大约13ku处出现特异性表达条带,与预期值相符.本研究构建了E.stiedae ADF基因真核表达质粒pVAX- 1-ADF,并且在体外真核细胞中表达,为进一步寻找防治E.stiedae的疫苗候选基因奠定了基础.     

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。     

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.     

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.     

Fungal and plant gene expression during synchronized infection of tomato leaves by Botrytis cinerea

An inoculation procedure was developed to obtain efficient and synchronous infection on detached tomato leaves by Botrytis cinerea. In spray-inoculated leaves incubated at 20 °C, the infection process consisted of three phases: the formation of primary necrotic lesions (until 20 hpi), a quiescent phase (20-72 hpi), and the expansion of a proportion of the primary lesions (from 72 hpi onwards), resulting in full tissue maceration. At 4 °C, the infection progressed slowly but steadily without inducing necrotic responses in the host. The actin and -tubulin genes of B. cinerea were cloned, characterized and used as probes on blots containing RNAs from leaves at various stages of the infection. The genes displayed a similar expression pattern throughout the infection and the hybridization signal reflected the amount of fungal biomass. The actin mRNA accumulated to higher levels than the -tubulin mRNA. Tomato PR protein mRNAs (chitinase, -1,3-glucanase and PR-1) were induced during the infection, albeit with different kinetics and to different levels. At 20 °C, -1,3-glucanase and PR-1 mRNAs were induced more rapidly than chitinase mRNAs. At 4 °C, mRNAs encoding extracellular -1,3-glucanase and intracellular, as well as extracellular chitinase were hardly induced.     

Study of Defense-related Gene Expression in Grapevine Leaves and Berries Infected with Botrytis cinerea

Defense responses of grapevine towards Botrytis cinerea were investigated. The expression of genes coding for proteins involved in defense were studied: (a) phenylalanine ammonia-lyase (PAL) and stilbene synthase (StSy), (b) an acidic chitinase (VCH3) and a basic chitinase (VCHIT1b), and (c) a polygalacturonase inhibitor protein (PGIP). Since no PGIP was known in grapevine, a complete cDNA sequence was first characterized by PCR and RACE-PCR amplifications. RNAs isolated from infected leaves and infected berries were analysed by semi-quantitative and real-time RT-PCRs. In infected leaves, the expression of PAL, StSy, PGIP and VCH3 genes occurred 6hours post inoculation (hpi). Increase of VCHIT1b gene expression was delayed (24hpi). Maximum levels of induction of these genes were observed at 48hpi, except for the VCH3 gene (24hpi). Activation of these defense responses was not sufficient to stop B. cinerea spread. In berries, no VCH3 gene expression was detected. Maximum levels of induction were observed in stage 3 (loss of berry colour and abundant production of conidia) for the PAL and PGIP genes, and in stage 4 (shrivelled berry) for the StSy and VCHIT1b genes.     

利用荧光定量PCR检测禾谷丝核菌的相对生物量

小麦纹枯病是以禾谷丝核菌(Rhizoctonia cerealis)侵染为主的小麦土传病害。为建立检测禾谷丝核菌在寄主小麦(Triticum aestivum)中的相对生物量的可靠方法,促进小麦抗纹枯病机制的研究,本研究克隆了禾谷丝核菌肌动蛋白基因RcActin的部分(3′端)cDNA,并设计了RcActin的特异引物。该引物不仅能区分禾谷丝核菌与寄主小麦,还能区分全蚀病菌(Gaeumannomyces graminis)、根腐病菌(Bipolaris sorokiniana)和立枯丝核菌(Rhizoctonia solani)等常见小麦土传病害的病原菌,表明该引物能用于小麦纹枯病的分子检测,也能用于相对表达量的测定。利用相对定量法,以RcActin相对于寄主管家基因的相对表达量作为禾谷丝核菌相对生物量的指标,结果表明,此方法能准确反映禾谷丝核菌在寄主中的相对生物量和对小麦纹枯病抗性程度进行快速鉴定。