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.     

应用RT-PCR制备登革病毒诊断基因芯片探针

根据GenBank数据库中的生物信息,利用BLAST免费分析软件找出4种型别登革病毒的保守序列及各型特异性序列,针对上述序列设计引物经RT-PCR扩增登革病毒的特异片段,利用此RT-PCR法收集探针是一种快速、简便制备基因芯片探针的实用方法。     

cDNA-AFLP analysis of salt-inducible genes expression in Chrysanthemum lavandulifolium under salt treatment

Chrysanthemum lavandulifolium (Fisch. ex Trautv.) Makino is a halophyte species that belongs to the Asteraceae family, and the genus Chrysanthemum. It is one of the ancestors of C. × morifolium Ramatella. Understanding the tolerance mechanism associated with salt stress in C. lavandulifolium could provide important information for explaining the salt tolerance of higher plants and could also help enhancing breeding programs of cultivated Chrysanthemum. In this study, cDNA amplified fragment length polymorphism (cDNA-AFLP) was used to detect differential gene expression in leaves of C. lavandulifolium in response to NaCl treatment. The determination of membrane permeablility, peroxidase activity (POD), malon-dialdehyde (MDA), as well as proline and leaf chlorophyll contents under different NaCl concentrations showed that a 200 mM NaCl treatment was an optimal condition for the cDNA-AFLP experiment. Using this concentration during different times (0, 3 h, 12 h, 24 h and 48 h), we obtained 1930 cDNA fragments using 64 primers. After sequencing 234 randomly chosen cDNA clones and BLASTx analyzing, we got 129 expressed sequence tags (ESTs) which had no significant homology with other sequences, 85 ESTs were homologous to genes with known functions, whereas the rest of ESTs showed homology to unclassified or putative proteins. 25 ESTs that were similar to known functional genes involved in several abiotic and biotic stresses were confirmed by semi-quantitative RT-PCR and qRT-PCR. The expression patterns of these salt-responsive genes not only responded to salt stress but also to plant hormones, such as abscisic acid (ABA), and to other abiotic stresses such as drought and cold. These results indicate an extensive cross-talk among several stresses. Our results provide interesting information for further understanding the molecular mechanisms of salt tolerance in C. lavandulifolium.     

Molecular cloning and characterization of the sheep α-TTP gene and its expression in response to different vitamin E status

The α-tocopherol transfer protein (α-TTP) is a ~ 32 kDa protein that exhibits a marked ligand specificity and selectively recognizes of α-tocopherol, which is the most active form of vitamin E. The α-TTP gene has been cloned and its physiological functions have been studied in numbers of species, however, the understanding of sheep α-TTP is still in his infancy. In this study, the full-length cDNA of sheep α-TTP gene was cloned from sheep liver by using of rapid amplification of complementary DNA ends (RACE). As a result, the sheep α-TTP gene was 1098 bp in nucleotide which contained 23 bp 5'-untranslated region (UTR), 226 bp 3'-UTR and 849 bp open reading frame (ORF) that encoded a basic protein of 282 amino acids. Further bioinformatic analysis indicated that the sheep α-TTP gene had a high homologous of both nucleotide and amino acid sequences compared with that of other species and had a Sec14p-like lipid-binding domain which called the CRAL-TRIO domain. Moreover, the expression of sheep α-TTP mRNA and protein in response to different vitamin E supplemented levels were observed according to quantitative real-time PCR (qRT-PCR) and Western blotting analysis. The results showed that dietary vitamin E levels did not affect α-TTP mRNA expression significantly while the low vitamin E supplemented level groups of sheep had significantly higher α-TTP protein compared to high-vitamin E groups.     

Differential expression of microRNAs in mouse embryonic bladder

MicroRNAs (miRNAs) are involved in several biological processes including development, differentiation and proliferation. Analysis of miRNA expression patterns in the process of embryogenesis may have substantial value in determining the mechanism of embryonic bladder development as well as for eventual therapeutic intervention. The miRNA expression profiles are distinct among the cellular types and embryonic stages as demonstrated by microarray technology and validated by quantitative real-time RT-PCR approach. Remarkably, the miRNA expression patterns suggested that unique miRNAs from epithelial and submucosal areas are responsible for mesenchymal cellular differentiation, especially regarding bladder smooth muscle cells. Our data show that miRNA expression patterns are unique in particular cell types of mouse bladder at specific developmental stages, reflecting the apparent lineage and differentiation status within the embryonic bladder. The identification of unique miRNAs expression before and after smooth muscle differentiation in site-specific area of the bladder indicates their roles in embryogenesis and may aid in future clinical intervention.     

反义trxs基因对转基因小麦种子内源trxh基因表达的影响

以转反义硫氧还蛋白基因(anti-trxs)株系01TY70-1-17-5及其对照小麦品种‘豫麦70’为试验材料,以小麦中稳定表达的肌动蛋白基因actin为内标,用半定量反转录聚合酶链式反应(semi-QRT-PCR)方法,对转基因株系及其对照种子中trxh基因时空表达情况进行了检测。检测结果表明,花后15~30d转基因株系trxh基因转录量平均比对照下降了20.1%,花后25d显著低于对照(P<0.05);胚乳trxh基因转录量最低,平均比对照低19.4%;种子吸涨24h时间内,转基因株系trxh基因转录量较对照均略低,但差异不显著。表明,外源trxs基因的导入直接干扰了内源基因的表达。     

Nm23-M5 mediates round and elongated spermatid survival by regulating GPX-5 levels

Nucleoside diphosphate (NDP) kinases are involved in numerous regulatory processes associated with proliferation, development, and differentiation. Previously, we cloned a new member of the NDPK family from mouse, Nm23-M5, which encodes a 211-amino acid protein and has 86% identity to the human Nm23-H5 [Hwang, K.C., Ok, D.W., Hong, J.C., Kim, M.O. and Kim, J.H. (2003) Cloning, sequencing, and characterization of the murine Nm23-M5 gene during mouse spermatogenesis and spermiogenesis. Biochem. Biophys. Res. Commun. 306, 198-207]. To better understand Nm23-M5 function, we generated transgenic mice with reduced Nm23-M5 levels in vivo using a short hairpin RNA (shRNA) knock-down system. Nm23-M5 expression was markedly reduced, as indicated by Northern and Western blot analysis. Nm23-M5 shRNA transgenic mice exhibited reduced numbers of haploid cells. Furthermore, the antioxidant enzyme glutathione peroxidase 5 (GPX-5) is regulated by Nm23-M5 at the level of both expression and activity. These results reveal that expression of Nm23-M5 plays a critical role in spermiogenesis by increasing the cellular levels of GPX-5 to eliminate reactive oxygen species.     

Four members of the HSP101 gene family are differently regulated in Triticum durum Desf

Heat shock proteins play an essential role in preventing deleterious effects of high temperatures. In many plants, HSP101 has a central role in heat stress survival. We report the isolation and characterization of four cDNAs corresponding to different members of the durum wheat HSP101 gene family. Expression analysis revealed differences in their induction. Accordingly, durum wheat HSP101 genes are differently regulated, therefore having distinct roles in stress response and thermotolerance acquisition. These findings are important for further dissection of the molecular mechanisms underlying the stress response and for understanding the functions of the HSP101 family members. This information could be important for the exploitation of specific alleles in marker assisted selection for abiotic stress resistance.     

Stress induces tRNA cleavage by angiogenin in mammalian cells

tRNAs play a central role in protein translation, acting as the carrier of amino acids. By cloning microRNAs, we unexpectedly obtained some tRNA fragments generated by tRNA cleavage in the anticodon loop. These tRNA fragments are present in many cell lines and different mouse tissues. In addition, various stress conditions can induce this tRNA cleavage event in mammalian cells. More importantly, angiogenin (ANG), a member of RNase A superfamily, appears to be the nuclease which cleaves tRNAs into tRNA halves in vitro and in vivo. These results imply that angiogenin plays an important physiological role in cell stress response, except for the known function of inducing angiogenesis.     

Upregulation of DNA repair genes in active cirrhosis associated with hepatocellular carcinoma

Phenotypic changes in injured livers involve complex network of genes whose interplays may lead to fibrosis and cirrhosis, a major risk of hepatocellular carcinoma. Gene expression profiles in fibrotic livers were analyzed by using cDNA microarray, hierarchical clustering and gene ontology. Analyses of a major cluster of upregulated genes in cirrhosis identified a new set of genes involved in DNA repair and damage. The upregulation of DNA repair genes was confirmed by real-time quantitative polymerase chain reaction and associated with necroinflammatory activity (P<0.001). Increased DNA repair activity in cirrhosis with inflammatory activity may reflect increased DNA damages as a consequence of chronic liver injury.     

逆转录-聚合酶链反应在乙型脑炎减毒活疫苗生物反应罐清洁验证中的应用

为了对乙型脑炎减毒活疫苗生物反应罐清洁后乙型脑炎病毒(JEV)检测方法进行探讨,从GenBank中收录的乙型脑炎病毒的E蛋白基因序列设计一对引物,以乙型脑炎减毒株SA14-14-2培养物提取RNA作为模板,进行逆转录和PCR扩增。结果表明乙型脑炎减毒株SA14-14-2扩增出预期的特异性条带,阴性对照没有扩增出任何条带。聚合酶链反应与血吸附试验比较,有灵敏、快速、稳定性的特点,可用于生物反应罐清洁后乙型脑炎残留病毒的检测。