TAZ Is a Negative Regulator of PPARγ Activity in Adipocytes and TAZ Deletion Improves Insulin Sensitivity and Glucose Tolerance

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热应激对大鼠睾丸HSPs mRNA表达的影响

目的:观察和分析大鼠睾丸局部短暂热应激对HSP70、HSP90、HSP105 mRNA表达的影响。方法:Wistar雄性大鼠随机分为5组:正常对照(N)组、热应激0天(H0)组、热应激5天(H5)组、热应激10天(H10)组、热应激15天(H15)组。N组睾丸局部22℃水浴20 min,其余各组均睾丸局部43℃水浴20 min。采用HE染色观察组织形态学变化,采用Real Time PCR的方法检测HSP70、HSP90、HSP105 mRNA的表达量。结果:HE染色镜下观察结果显示:与N组相比,H5组部分曲细精管萎缩,生精细胞明显消失,H10组、H15组大部分曲细精管萎缩,生精细胞大量消失。HE染色形态计量结果显示:与N组相比,H5组、H10组、H15组睾丸实质体积比明显减小(p0.05),睾丸间质体积比明显增加(P0.05)。RT-PCR结果显示:与N组相比,H0组HSP70 m RNA的表达H0组明显增高(p0.05),H5组、H10组、H15组HSP90 m RNA的表达明显降低(P0.05),H5组HSP105 m RNA的表达明显降低(P0.05)。结论:HSP70、HSP90、HSP105 m RNA的表达在热应激后都会发生变化,变化的具体情况不尽相同,推测它们热应激后在生殖细胞凋亡过程中发挥不同的作用有关,并且和组织的损伤有密切的联系。     

The use of a quantitative real-time polymerase chain reaction assay for identification and enumeration of Lactobacillus buchneri in silage

Aims:  To detect and quantify Lactobacillus buchneri in plant samples with the aid of polymerase chain reaction (PCR) methods.
Methods and Results:  DNA from silage samples spiked with different amounts of L. buchneri cells was isolated using a lysozyme/sodium dodecyl sulfate lysis and phenol/chloroform extraction method. The DNA served as a template for PCR amplification with primers specific for the bacterium. The primers were developed by comparison of 16S rDNA sequences from different lactic acid bacteria (LAB) and testing for specificity with 11 different strains of LAB. As few as 100 L. buchneri colony-forming units per gram of silage could be detected. Additionally, the technique was successfully applied to quantify the population of L. buchneri in two cultivars of corn with or without inoculation.
Conclusions:  The PCR assay provided a specific and rapid tool for identifying and enumerating L. buchneri in silage samples.
Significance and Impact of the Study:  The use of microbial inoculants for silage production is a safe and environment friendly practice, but the full potential of such additives can only be achieved with a better understanding of the fate and activity of the microbes involved. The current study describes a methodology to detect and enumerate L. buchneri , a micro-organism used as an inoculant.     

Species-specific detection and quantification of common barnacle larvae from the Japanese coast using quantitative real-time PCR

Species-specific detection and quantification methods for barnacle larvae using quantitative real-time polymerase chain reaction (qPCR) were developed. Species-specific primers for qPCR were designed for 13 barnacle species in the mitochondrial 12S ribosomal RNA gene region. Primer specificity was examined by PCR using template DNA extracted from each of the 13 barnacle species, other unidentified barnacle species, and field collected zooplankton samples. The resulting PCR products comprised single bands following agarose gel electrophoresis when the templates corresponded to primers. The amplifications were highly species-specific even for the field plankton samples. The field plankton samples were subjected to qPCR assay. The calculated DNA contents for each barnacle species were closely correlated with the number of larvae measured by microscopic examination. The method could be applied to quantify barnacle larvae in natural plankton samples.     

Biphasic and Dually Coordinated Expression of the Genes Encoding Major Shell Matrix Proteins in the Pearl Oyster Pinctada fucata

Regional expression patterns of shell matrix protein genes of Pinctada fucata were investigated using real-time quantitative polymerase chain reaction (PCR) and in situ hybridization. Six shell matrix proteins examined in this study indicated a distinct biphasic pattern of expression, falling into one of the following three groups: (1) expressed only in the more dorsal region of the mantle (MSI60 and N16); (2) expressed only in the more ventral region (MSI31, Prismalin-14, and Aspein); and (3) expressed in both regions (nacrein). The ubiquity of the last protein probably reflects its general role as a carbonate-producing enzyme, while the other groups are interpreted as corresponding to the distinction between the two varieties of shell layers, the aragonitic nacreous layer and the calcitic prismatic layer. In addition, the constituent genes of each of these two groups indicated similar levels of relative expression among different sites even among different individuals, suggesting that the genes of each group share a single upstream regulatory factor, respectively, and that these genes are expressed in a dually coordinated fashion.     

Cloning and characterization of genes specifically expressed during infection stages in the rice blast fungus

We isolated promoters of 12 genes from the rice blast fungus based on the sequences of randomly selected expressed sequence tags (ESTs) (appressorium formation stage cDNA library of Magnaporthe available from GenBank). These promoters (and the 5' coding regions if any) were fused in frame with egfp, and their expression patterns were examined under the epifluorescence microscope. Among them, two turned out to be specifically active in structures necessary for infection, viz. a promoter of adenylate cyclase interacting protein 1-like gene expressed in conidia, germ tubes, and appressoria, and a promoter of putative membrane-associated or secreted protein gene specifically expressed in appressoria. Although targeted knockout mutants of either gene failed to show detectable phenotypic alterations under laboratory conditions, these ESTs should be useful for identification of genes expressed during infection stages.     

Chibby functions in Xenopus ciliary assembly,embryonic development,and the regulation of gene expression

Wnt signaling and ciliogenesis are core features of embryonic development in a range of metazoans. Chibby (Cby), a basal-body associated protein, regulates β-catenin-mediated Wnt signaling in the mouse but not Drosophila. Here we present an analysis of Cby?s embryonic expression and morphant phenotypes in Xenopus laevis. Cby RNA is supplied maternally, negatively regulated by Snail2 but not Twist1, preferentially expressed in the neuroectoderm, and regulates β-catenin-mediated gene expression. Reducing Cby levels reduced the density of multiciliated cells, the number of basal bodies per multiciliated cell, and the numbers of neural tube primary cilia; it also led to abnormal development of the neural crest, central nervous system, and pronephros, all defects that were rescued by a Cby-GFP chimera. Reduction of Cby led to an increase in Wnt8a and decreases in Gli2, Gli3, and Shh RNA levels. Many, but not all, morphant phenotypes were significantly reversed by the Wnt inhibitor SFRP2. These observations extend our understanding of Cby?s role in mediating the network of interactions between ciliogenesis, signaling systems and tissue patterning.     

Development of real-time PCR methods for the rapid detection of low concentrations of Gluconobacter and Gluconacetobacter species in an electrolyte replacement drink

AIMS: To develop a rapid real-time polymerase chain reaction (PCR) method to detect Gluconobacter and Gluconacetobacter species in electrolyte replacement drinks. METHODS AND RESULTS: Samples of electrolyte replacement drinks were artificially contaminated with Gluconobacter species and then filtered to collect cells. DNA was extracted from the filters and analysed by real-time PCR on the ABI Prism 7000 system, using commercial detection kits for lactic and acetic acid bacteria. In addition, specific primers and Taqman probe were designed and used for the detection of seven Gluconobacter and Gluconacetobacter species. All the assays tested demonstrated a linear range of quantification over four orders of magnitude, suggesting detection levels down to 1 CFU ml(-1) in the original drink. CONCLUSIONS: A real-time PCR method was developed to detect low concentrations of Gluconobacter and Gluconacetobacter sp. in an electrolyte replacement drink. SIGNIFICANCE AND IMPACT OF THE STUDY: Real-time PCR methods allow a rapid, high throughput and automated procedure for the detection of food spoilage organisms. The real-time PCR assay described is as sensitive as the conventional method that involves pre-enrichment, enumeration on a selective agar (typically malt extract agar) and identification with a differential medium (typically Wallerstein nutrient agar). The real-time PCR assay also provides a more rapid rate of detection, with results in less than 24 h following enrichment for Gluconobacter and Gluconacetobacter species.     

PTEN在早孕小鼠子宫内膜的表达及其对胚泡着床的影响

本研究旨存检测肿瘤抑制基因PTEN(phosphatase andtensinhomologdeletedonchromosometen)在早孕小鼠子宫内膜中的表达规律,探讨PTEN在小鼠胚胎着床过程中的作用.采用实时荧光定量聚合酶联反应(real.time fluorescent quantitative PCR.FQ.PCR)和免疫组织化学方法分别检测未孕及孕1、3、4、5、7 d小鼠子宫内膜PTEN mRNA和蛋白的表达;子宫角注射PTEN反义寡核苷酸观察胚泡着床数.FQ-PCR结果显示,妊娠小鼠子宫内膜组织PTENmRNA的表达高于未妊娠小鼠,且随着妊娠天数的增加表达逐渐增强,到妊娠第5天达最高.免疫组织化学分析显示,PTEN蛋白在子宫内膜的表达规律与mRNA结果一致.子宫角注射PTEN反义寡核苷酸后胚泡着床数明显减少.结果提示,PTEN在妊娠早期子宫内膜持续表达,可能参与了胚泡着床.     

Expression patterns of microRNAs 155 and 451 during normal human erythropoiesis

To clarify the roles of microRNAs (miRNAs) in erythropoiesis, the expression of miR-155, miR-221, miR-223, and miR-451 were analyzed during the differentiation of purified normal human erythroid progenitors in a liquid culture system. Cells increased almost 500-fold in a number, and differentiated to benzidine-positive mature erythroblasts. Analyses of miRNA expression using the quantitative real-time polymerase chain reaction showed that the expression level of miR-155 decreased about 200-fold, and that the expression of miR-451 increased about 270-fold during 12 days of cultures. A moderate down-regulation of miR-221 and miR-223 was observed. MiR-451 was expressed in red blood cells about 10⁴-fold more than in granulocytes, obtained from normal human peripheral blood. These observations suggest that miR-155 and miR-451 are key molecules for normal erythroid differentiation, and that quantitative assays of the two miRNAs may be a relevant method for analyzing pathological erythropoiesis.