逆境相关植物锌指蛋白的研究进展

锌是植物必需的营养元素,锌指蛋白因其具有指状结构特征且能结合Zn2 而得名,植物锌指蛋白包含特有的QALGGH保守结构,可能涉及调控植物特有的生物学功能。人们已经从拟南芥(Arabidopsis thaliana)、矮牵牛(Petunia hybrida Vilm)、水稻(Oryza sativa)、大豆(Glycine max)、棉花(Gossypium hirsutum)等植物中克隆了许多编码锌指蛋白的基因,并对其结构及功能进行了研究。利用转基因技术,将一些与逆境胁迫相关的锌指蛋白基因在目标植物中过量表达后,能对植物起到增强抗逆性的作用,说明锌指蛋白在增强植物逆境抗性方面有着广阔的应用前景。     

The cell agglutination agent, phytohemagglutinin-L, improves the efficiency of somatic nuclear transfer cloning in cattle (Bos taurus)

One of the several factors that contribute to the low efficiency of mammalian somatic cloning is poor fusion between the small somatic donor cell and the large recipient oocyte. This study was designed to test phytohemagglutinin (PHA) agglutination activity on fusion rate, and subsequent developmental potential of cloned bovine embryos. The toxicity of PHA was established by examining its effects on the development of parthenogenetic bovine oocytes treated with different doses (Experiment 1), and for different durations (Experiment 2). The effective dose and duration of PHA treatment (150 microg/mL, 20 min incubation) was selected and used to compare membrane fusion efficiency and embryo development following somatic cell nuclear transfer (Experiment 3). Cloning with somatic donor fibroblasts versus cumulus cells was also compared, both with and without PHA treatment (150 microg/mL, 20 min). Fusion rate of nuclear donor fibroblasts, after phytohemagglutinin treatment, was increased from 33 to 61% (P < 0.05), and from 59 to 88% (P < 0.05) with cumulus cell nuclear donors. The nuclear transfer (NT) efficiency per oocyte used was improved following PHA treatment, for both fibroblast (13% versus 22%) as well as cumulus cells (17% versus 34%; P < 0.05). The cloned embryos, both with and without PHA treatment, were subjected to vitrification and embryo transfer testing, and resulted in similar survival (approximately 90% hatching) and pregnancy rates (17-25%). Three calves were born following vitrification and embryo transfer of these embryos; two from the PHA-treated group, and one from non-PHA control group. We concluded that PHA treatment significantly improved the fusion efficiency of somatic NT in cattle, and therefore, increased the development of cloned blastocysts. Furthermore, within a determined range of dose and duration, PHA had no detrimental effect on embryo survival post-vitrification, nor on pregnancy or calving rates following embryo transfer.     

PTP1B inhibitor improves both insulin resistance and lipid abnormalities in vivo and in vitro

PTP1B is a negative regulator of insulin signaling pathway. This study investigated the effects of compound CCF06240, a PTP1B inhibitor, on insulin sensitivity and lipid metabolic abnormalities in vivo and in vitro, respectively. The insulin resistant IRM mouse model was induced by HFD. The responses to insulin were determined by OGTT, ITT, and hyperinsulinemic-euglycemic clamp test. The body weight and the levels of serum TC and TG were measured to estimate the lipid metabolism in vivo. Recombinant human GST-PTP1B protein was used to measure the inhibition of CCF06240 on PTP1B activity. The hepatocyte lipid accumulation was induced by high concentrations of FFA and insulin in HepG(2) cells, and evaluated by the Oil Red O method. In IRM mice, the insulin resistance was improved; the body weight and the levels of TC and TG were also reduced by oral CCF06240 administration. In lipid accumulated model cells, CCF06240 was found to reverse the increased PTP1B activity, enhance the insulin-induced tyrosine phosphorylation in insulin signaling pathway, attenuate the FFA-insulin-induced cellular lipid accumulation, and down-regulate the expressions of genes related fatty acid synthesis. These results demonstrated that the PTP1B inhibitor, compound CCF06240, could increase insulin sensitivity through the regulation of insulin signaling pathway, and decrease FFA-insulin-induced hepatocytes lipid accumulation by reducing fatty acid syntheses.     

Effects of endogenous beta-amyloid overproduction on tau phosphorylation in cell culture

Alzheimer's disease is characterized by beta-amyloid (Abeta) overproduction and tau hyperphosphorylation. Recent studies have shown that synthetic Abeta promotes tau phosphorylation in vitro. However, whether endogenously overproduced Abeta promotes tau phosphorylation and the underlying mechanisms remain unknown. Here, we used mouse neuroblastoma N2a stably expressing wild-type amyloid precursor protein (APPwt) or the Swedish mutant APP (APPswe) to determine the alterations of phosphorylated tau and the related protein kinases. We found that phosphorylation of tau at paired helical filament (PHF)-1, pSer396 and pThr231 epitopes was significantly increased in cells transfected with APPwt and APPswe, which produced higher levels of Abeta than cells transfected with vector or amyloid precursor-like protein 1. The activity of glycogen synthase kinase-3 (GSK-3) was up-regulated with a concomitant reduction in the inhibitory phosphorylation of GSK-3 at its N-terminal Ser9 residue. In contrast, the activity of cyclin-dependent kinase-5 (CDK-5) and protein kinase C (PKC) was down-regulated. Inhibition of GSK-3 by LiCl, but not inhibition of CDK-5 by roscovitine, arrested Abeta secretion and tau phosphorylation. Inhibition of PKC by GF-109203X activated GSK-3, whereas activation of PKC by phorbol-12,13-dibutyrate inhibited GSK-3. These results suggest that endogenously overproduced Abeta induces increased tau phosphorylation through activation of GSK-3, and that inactivation of PKC is at least one of the mechanisms involved in GSK-3 activation.     

Diallyl trisulfide induces apoptosis and inhibits proliferation of A549 cells in vitro and in vivo

Lung cancer is the leading cause of cancer-related mortality all over the world. In recent years, pulmonary adenocarcinoma has surpassed squamous cell carcinoma in frequency and is the predominant form of lung cancer in many countries. Epidemiological investigations have shown an inverse relationship between garlic (Allium sativum) consumption and death rate from many cancers. Diallyl trisulfide (DATS) is one of the garlic-derived compounds (also known as: organosulfer compounds, OSC). DATS can induce apoptosis and inhibit the growth of many cancer cell lines. Our study demonstrated that the apoptotic incidents induced by DATS were a mitochondria-dependent caspase cascade through a significant decrease of the anti-apoptotic Bcl-2 that resulted in up-regulation of the ratio of Bax/Bcl-2 and the activity of caspase-3, -8, and -9. Eventually, DATS induced the apoptosis and inhibited the proliferation in a concentration- and time-dependent manner. Furthermore, by establishing an animal model of female BALB/c nude mice with A549 xenografts, we found that oral gavage of DATS significantly retarded growth of A549 xenografts in nude mice without causing weight loss or any other side effects compared with the control group. All the evidence both in vitro and in vivo suggested that DATS could be an ideal anti-cancer drug.     

A combined approach for improving alkaline acetyl xylan esterase production in Pichia pastoris, and effects of glycosylation on enzyme secretion, activity and stability

High level expression of axe1, a gene previously cloned from Volvariella volvacea that encodes an acetyl xylan esterase with two potential N-linked glycosylation sites, has been achieved in Pichia pastoris using a codon-optimized axe1 synthesized by the primer extension PCR procedure. The GC content of the codon-optimized axe1 was 48.62% compared with 55.49% in the native gene. Using the codon-optimized construct, AXE1 expression in P. pastoris was increased from an undetectable level to 136.45U/ml six days after induction of yeast cultures grown in BMMY medium. A further increase (to 463U/ml) was achieved when conditions for yeast culture were optimized as follows: 2.8% methanol, 0.63% casamino acids, and pH 8.0. This latter value represented a 3.4-fold and 246-fold increase in the enzyme levels recorded in non-optimized P. pastoris cultures and in rice straw-grown cultures of V. volvacea, respectively. N-linked glycosylation played an essential role in AXE1 secretion but had only a slight effect on the catalytic activity and stability of the recombinant enzyme.     

党参染色体核型分析

对党参细胞染色体进行了核型分析。研究表明,党参染色体数为2 n=16。染色体的核型为2n=12m 4 sm=16。第1、2、3、4、5、8对是中间着丝粒染色体(m);第6对和第7对染色体属于近中着丝点染色体,未观察到有携带随体的染色体存在。