Proteomic Characterization of Equine Cerebrospinal Fluid

Cerebrospinal fluid (CSF) is a biofluid that is reflective of overall health. Although proteomic profiling of human CSF has been performed in the context of a variety of disease states, this report represents the first comprehensive proteomic analysis of equine CSF. A total of 320 proteins were confidently identified across six healthy horses, and these proteins were further characterized by gene ontology terms mapped in UniProt, and normalized spectral abundance factors were calculated as a measure of relative abundance. Theses results provide an optimized protocol for analysis of equine CSF and lay the groundwork for future studies involving the study of equine CSF in the context of pathogenic disease states.     

A gene expression estimator of intramuscular fat percentage for use in both cattle and sheep

Background

The expression of genes encoding proteins involved in triacyglyceride and fatty acid synthesis and storage in cattle muscle are correlated with intramuscular fat (IMF)%. Are the same genes also correlated with IMF% in sheep muscle, and can the same set of genes be used to estimate IMF% in both species?

Results

The correlation between gene expression (microarray) and IMF% in the longissimus muscle (LM) of twenty sheep was calculated. An integrated analysis of this dataset with an equivalent cattle correlation dataset and a cattle differential expression dataset was undertaken. A total of 30 genes were identified to be strongly correlated with IMF% in both cattle and sheep. The overlap of genes was highly significant, 8 of the 13 genes in the TAG gene set and 8 of the 13 genes in the FA gene set were in the top 100 and 500 genes respectively most correlated with IMF% in sheep, P-value = 0. Of the 30 genes, CIDEA, THRSP, ACSM1, DGAT2 and FABP4 had the highest average rank in both species. Using the data from two small groups of Brahman cattle (control and Hormone growth promotant-treated [known to decrease IMF% in muscle]) and 22 animals in total, the utility of a direct measure and different estimators of IMF% (ultrasound and gene expression) to differentiate between the two groups were examined. Directly measured IMF% and IMF% estimated from ultrasound scanning could not discriminate between the two groups. However, using gene expression to estimate IMF% discriminated between the two groups. Increasing the number of genes used to estimate IMF% from one to five significantly increased the discrimination power; but increasing the number of genes to 15 resulted in little further improvement.

Conclusion

We have demonstrated the utility of a comparative approach to identify robust estimators of IMF% in the LM in cattle and sheep. We have also demonstrated a number of approaches (potentially applicable to much smaller groups of animals than conventional methods) to using gene expression to rank animals for IMF% within a single farm/treatment, or to estimate differences in IMF% between two farms/treatments.     

家蚕泛素结合酶新基因的克隆与基因组结构及5′调控区分析

在家蚕丝腺cDNA文库测序过程中,发现一个编码家蚕泛素结合酶的EST序列,利用3′RACE方法克隆了一个新的家蚕泛素结合酶基因cDNA全长序列,命名为BmUCE2 I(GenBank登录号为DQ219874)。家蚕BmUCE2 I基因全长cDNA由465 bp的开放阅读框序列(ORF)、97 bp的5′端非翻译区序列(5-′UTR)和237 bp的3′端非编码区序列(3-′UTR)组成,其编码的154个氨基酸与其他真核生物间具有较高的同源性。利用BmUCE2 I的EST片断作探针,通过筛选家蚕噬菌体基因组文库,获得了家蚕BmUCE2 I基因组序列和5′调控序列。BmUCE2 I基因由4个外显子和3个内含子组成,在5′端上游调控区域没有类似TATA盒元件,但在-219~-268 bp的区域存在一个50bp的启动子序列,此外还存在CF2-Ⅱ、FTZ、DFD、BRCZ2、DL、STAT、PRD-HD等多个转录因子结合位点。家蚕泛素结合酶新基因的克隆、基因结构及5′调控区的分析为进一步研究泛素蛋白水解酶复合通路相关基因的调控规律提供了重要依据。     

Influence of hot water treatment on brown rot of peach and rapid fruit response to heat stress

Recent results on hot water as an alternative treatment open a new perspective in disease incidence reduction. In the present work peach fruit were wounded, inoculated with conidia of Monilinia laxa and 15 min, 3, 6, 12, 24 and 48 h after inoculation treated by dipping in hot water (HT) at 60 °C for 20 s. The effect of heat treatment on some cell wall genes involved in ripening such as β-galactosidase (β-GAL), pectin lyase (PL), polygalacturonase (PG) and pectin methyl esterase (PME), was analyzed by qRT-PCR. The expression levels of defense related genes, phenylalanine ammonia lyase (PAL) and chitinase (CHI), heat stress-related genes such as heat shock proteins 70 and 90 (HSP70, HSP90), and reactive oxygen species (ROS) scavenging genes were also evaluated by qRT-PCR. A 100% disease incidence reduction, as compared to untreated fruit, was obtained by treating 6 and 12 h after inoculation. Moreover, brown rot was inhibited by 85.7% when fruit were heat-treated 48 h after inoculation. The expression levels of cell wall genes (β-GAL, PL, PG and PME) showed a general decrease in HT fruit as compared to the control, whereas PAL, CHI, HSP70 and ROS-scavenging genes increased their expression level in HT samples with respect to the untreated ones. Our results show a curative activity of heat on peach inoculated with M. laxa 48 h before treatment. Each analyzed gene proved to be differentially expressed following heat treatment.     

甲状腺素对猪小肠上皮细胞miR-let-7a基因表达及细胞增殖的影响

miR-let-7a在动物细胞的分化、增殖与凋亡等方面发挥越来越重要的作用。甲状腺激素(TH)作用非常广泛,机体的每个细胞几乎都是TH作用的靶细胞,其可以促进组织分化、生长和成熟。本实验用甲状腺素(T4)浓度分别为(0、0.02、0.03、0.05、0.075、0.1、0.2μmol/L)在体外培养猪的小肠上皮细胞。结果表明:T4处理组的细胞体积形态相对于空白对照组没有明显变化;当T4添加浓度为0.03μmol/L时,细胞的增殖率显著低于其他组(P0.05);当T4浓度为0~0.03μmol/L时,let-7a的表达随着添加剂量的增加而升高,浓度从0.03~0.2μmol/L变化时,let-7a的表达呈现降低趋势,浓度为0.03μmol/L时表达量极显著高于其他组(P0.01)。let-7a的表达量与细胞增殖呈负相关。     

Cortical gene expression pattern in rat focal cerebral ischemia

AIM: To investigate the genes differential expression in cortex during rat focal cerebral ischemia.METHODS: cDNA microarray chips containing numerous cDNAs were used to investigate the gene expression pattern between samples of focal cerebral ischemia and sham-control operation rats. RESULTS: Two hundred and eleven genes differentially expressed were screened out, among these genes, up-and down-regulated genes were 199 and 12, respectively. CONCLUSIONS: The analysis of gene expression pattern of focal cerebral ischemia based on cDNA microarray can realize high-throughput screening of the genes associated with the focal cerebral ischemia. The differential expression of genes may be related to the pathogenesis of focal cerebral ischemic diseases.     

Transplantation of bcl-2 gene-modified bone marrow mesenchymal stem cells improves cardiac function and angiogenesis in rabbit ischemic car-diac insufficiency model

AIM: To investigate the effects of transplantation of bone marrow mesenchymal stem cells (BMSCs) modified by bcl-2 gene on myocardial cell apoptosis, angiogenesis and cardiac function in the rabbit after acute myocardial infarction (MI).METHODS: The rabbit BMSCs were isolated, cultured and purified in vitro. The BMSCs were transfected with adenovirus or adenovirus-Bcl-2. The rabbit model of MI was established by ligation of left anterior descending branch. The rabbits were injected with Ad-Bcl-2-BMSCs (MI+Bcl-2-BMSCs group), Ad-BMSCs (MI+BMSCs group) and DMEM (MI group) in infarction marginal zone 2 weeks after ligation. The cardiac function was evaluated by echocardiography.The apoptosis of myocardial cells was measured by TUNEL. The mRNA expression of VEGF was detected by real-time PCR. The expression of CD31 was examined by immunohistochemical staining, and new blood capillaries were counted at 4 weeks after BMSCs transplantation. The correlation of the above values with cardiac function was analyzed.RESULTS: The cardiac function was better, the apoptotic rate was lower, the mRNA expression of VEGF and the capillary density were higher in both MI+Bcl-2-BMSCs group and the MI+BMSCs group than those in MI group, and those in MI+Bcl-2-BMSCs group increased more obviously.The left ventricular ejection fraction (LVEF) had a negative correlation with the myocardial cell apoptosis rate. A positive correlation with the mRNA expression level of VEGF and the capillary density was also observed.CONCLUSION: The transplantation of BMSCs modified by bcl-2 gene significantly reduces the myocardial cell apoptosis, promotes angiogenesis, improves heart function of the rabbits with MI.     

Construction and identification of recombinant adenovirus vector containing CTLA4Ig gene

AIM:To construct a recombinant adenovirus expression vector containing CTLA4Ig gene.METHODS:The CTLA4Ig gene derived from the plasmid PCDNA3.0/CTLA4Ig by using polymerase chain reaction (PCR) was inserted into the backward position of cytomegalovirus (CMV) immediate early promoter of the shuttle plasmid (pAdTrack-CMV). After being identified by endonuclease, PCR and sequencing, the recombinant shuttle plasmid pAdTrack-CTLA4Ig was co-transformed into E.coli. BJ5183 cells with the adeoviral backbone plasmid pAdEasyl-1 to obtain the homologous recombination. The adenovirus was generated in 293 cells. A series methods such as PCR and fluorescence microscope was employed to identify the generated recombinant adenovirus.RESULTS:Recombinant CTLA4Ig adenoviruses were constructed and the titer of virus was generally up to 1.65×10¹² phaque forming units per liter (PFU/L).CONCLUSION:Success in constructing recombinant pAdTrack-CTLA4Ig will be the base of the further research on its expression in the mammalian cells, and be potenially used in the prevention of transplant rejection and autoimmunity diseases.     

君子兰转化酶基因片段(CMCW1)的克隆和序列分析

 分析植物酸性转化酶基因的保守区序列，设计一对PCR引物，以君子兰基因组DNA为模板，采用PCR方法扩增出长约500 bp的DNA片段，克隆入pGEM-TEasy载体，测序结果表明获得君子兰酸性转化酶基因家族的一个成员CMCW1，该基因片段长518 bp，不含内含子，编码172个氨基酸。其序列已在GenBank中登记(登记号为AY151269)。在GenBank中进行同源性检索的结果表明，该成员编码的氨基酸与其它植物细胞壁酸性转化酶编码的氨基酸同源性较高。     

冷胁迫对黄瓜CBF1基因沉默植株相关耐冷指标的影响

以黄瓜CBF1基因沉默植株为试材,研究了冷胁迫下黄瓜CBF1基因的沉默对植株相关耐冷指标的影响。结果表明,与冷胁迫和常温条件下的正常植株相比,冷胁迫后的基因沉默植株的相对电导率和丙二醛含量均显著增加,叶绿素含量显著降低;冷胁迫后的基因沉默植株体内脯氨酸和可溶性蛋白含量增加的幅度远远低于冷胁迫后的正常植株增加的幅度。