甘肃不同矿区样品中的细菌多样性

甘肃矿产资源丰富,矿区酸性矿坑水(acid mine drainage,AMD)及周边土壤中嗜酸微生物种类丰富,具有相当大的研究价值.本研究通过Illumina高通量测序方法,对甘肃省4个矿区AMD及周边土壤中的细菌群落组成、相似性及功能组成进行分析.结果 表明,煤矿样品GNM细菌物种组成最为丰富,金矿样品LNJ1与紫金矿样品LNZJK细菌组成最相似,与属于铜矿的BYT物种较相近,与LNJ1同地区的LNJ2样品细菌组成相比较远.所有样品细菌主要分为放线菌门(Actinobacteria)、拟杆菌门(Bacteriodetes)、厚壁菌门(Firmicutes)和变形菌门(Proteobacteria)四大门,其中变形菌门(Proteobacteria)丰度最高.放线菌门(Actinobacteria)中不动杆菌属(Acinetobacter)、类诺卡氏属(Nocardioides)和芽球菌属(Blastococcus)表现出较高丰度;厚壁菌门(Firmicutes)中微小杆菌属(Exiguobacterium)和芽孢杆菌属(Bacillus)表现出较高丰度,且微小杆菌属(Exiguobacterium)丰度极高.通过功能预测发现,与煤矿样品GNM相比,LNJ1、LNJ2、BYT和LNZJK4个金属矿区样品中的微生物具有较强的氨基酸与碳源的转运与代谢、转录等功能以及较低的能量生产与转运、无机离子转运与代谢能力.通过探究AMD中的细菌多样性及其与重金属之间的相互关系,了解AMD采样区微生物群落结构,以期挖掘出更为丰富的具有重金属抗性的菌株资源,以应用于生物浸矿和环境修复等领域.     

贺兰山马鹿冬春季生境的选择

2007年12月～2008年1月和2008年4～5月,在贺兰山利用痕迹检验法和直接观察法对马鹿阿拉善亚种冬春季生境选择进行研究.通过在选定的15条沟段里进行调查,冬春季各测定了131个和181个样方的18种生态因子.结果表明,冬春季贺兰山马鹿在植被类型、优势乔木、灌木密度、灌木高度、灌木距离、坡向、坡位、海拔高度、距裸岩距离和隐蔽级10种生态因子上差异极显著(P ＜ 0.001),其余生态因子无显著差异(P ＜ 0.05),相对于冬季生境而言,马鹿春季更偏爱选择以油松和青海云杉为优势乔木的山地针叶林带,灌木密度大、高度高、距离近,半阴半阳坡的中坡位,海拔较高,距裸岩较近,隐蔽程度高的生境.典则判别系数显示冬春季马鹿在生境选择上存在一定程度的重叠,但春季马鹿的分布范围要比冬季更广一些,Wilk's λ值显示冬春季马鹿的生境选择具有较高的差异性(Wilk's λ = 0.683,x2 = 116.995,df = 13,P ＜ 0.001).逐步判别分析表明在区分冬春季马鹿生境方面有一系列生态因子发挥作用,依照贡献值的大小依次为:乔木密度、灌木距离、坡度、海拔高度、距水源距离、距裸岩距离和隐蔽级,由这7个变量构成的方程对冬春季马鹿生境的正确区分率达到79.9%,其中对春季的误判率为22.1%,对冬季的误判率为20.6%.     

Single chain antibody displays glutathione S-transferase activity

Substrate binding and the subsequent reaction are the two principal phenomena that underlie the activity of enzymes, and many enzyme-like catalysts were generated based on the phenomena. The single chain variable region fragment of antibody 2F3 (scFv2F3) was elicited against hapten GSH-S-DN2phBu, a conjugate of glutathione (GSH), butyl alcohol, and 1-chloro-2,4-dinitrobenzene (CDNB); it can therefore bind both GSH and CDNB, the substrates of native glutathione S-transferases (GSTs). It was shown previously that there is a serine residue that is the catalytic group of GST in the CDR regions of scFv2F3 close to the sulfhydryl of GSH. Thus, we anticipated that scFv2F3 will display GST activity. The experimental results showed that scFv2F3 indeed displayed GST activity that is equivalent to the rat-class GST T-2-2 and exhibited pH- and temperature-dependent catalytic activity. Steady-state kinetic studies showed that the Km values for the substrates are close to those of native GSTs, indicating that scFv2F3 has strong affinities for the substrates. Compared with some other GSTs, its kcat value was found to be low, which could be caused by the similarity between the GSH-S-DN2phBu and the reaction product of GSH and CDNB. These results showed that our approach to imitating enzymes is correct, which is that an active site may catalyze a chemical reaction when a catalytic group locates beside a substrate-binding site of a receptor. It is important to consider product inhibition in hapten design in order to obtain a mimic with a high catalytic efficiency.     

Effects of macrophage-specific adiponectin expression on lipid metabolism in vivo

Epidemiological studies have associated low circulating levels of the adipokine adiponectin with multiple metabolic disorders, including metabolic syndrome, obesity, insulin resistance, type II diabetes, and cardiovascular disease. Recently, we reported that adiponectin selectively overexpressed in mouse macrophages can improve insulin sensitivity and protect against inflammation and atherosclerosis. To further investigate the role of adiponectin and macrophages on lipid and lipometabolism in vivo, we engineered the expression of adiponectin in mouse macrophages (Ad-TG mice) and examined effects on plasma lipoproteins and on the expression levels of genes involved in lipoprotein metabolism in tissues. Compared with the wild-type (WT) mice, Ad-TG mice exhibited significantly lower levels of plasma total cholesterol (-21%, P < 0.05) due to significantly decreased LDL (-34%, P < 0.05) and VLDL (-32%, P < 0.05) cholesterol concentrations together with a significant increase in HDL cholesterol (+41%, P < 0.05). Further studies investigating potential mechanisms responsible for the change in lipoprotein cholesterol profile revealed that adiponectin-producing macrophages altered expression of key genes in liver tissue, including apoA1, apoB, apoE, the LDL receptor, (P < 0.05), and ATP-binding cassette G1 (P < 0.01). In addition, Ad-TG mice also exhibited higher total and high-molecular-weight adipnection levels in plasma and increased expression of the anti-inflammatory cytokine IL-10 as well as a decrease in the proinflammatory cytokine IL-6 in adipose tissue. These results indicate that macrophages engineered to produce adiponectin can influence in vivo gene expression in adipose tissue in a manner that reduces inflammation and macrophage infiltration and in liver tissue in a manner that alters the circulating lipoprotein profile, resulting in a decrease in VLDL and LDL and an increase in HDL cholesterol. The data support further study addressing the use of genetically manipulated macrophages as a novel therapeutic approach for treatment of cardiometabolic disease.     

芒果APl同源基因的克隆及其生物信息学分析

我们采用RT-PCR方法克隆了2个APl同源基因全长cDNA,分别命名为MAPl-1(GenBank accession No.FJ529206)和MAPl-2(GenBank accession No.FJ529207).MAPl-1编码247个氨基酸,开放阅读框长度为741 bp,蛋白质分子量为28.54kD,等电点为8.31;MAPl-2编码248个氨基酸,开放阅读框长度为744 bp,蛋白质分子量为28.78 kD,等电点为8.70.同源性分析表明,它们的核苷酸序列与其它木本植物APl同源基因的一致性为72%～81%.实验分析表明,MAPl-1和MAPl-2第1至第61个氨基酸含有一个MADS盒结构域,第88至第178个为K盒结构域;两个基因均定位于细胞核,且功能位点分布存在着不同,推测这两个基因在花器官发育过程中的功能存在差异.蛋白二级结构预测显示,MAPl-1蛋白有12个a-螺旋,4个β折叠区,14个β-转角;而MAPl-2蛋白有11个a-螺旋,5个β折叠区,15个β-转角:其大多数氨基酸具有亲水性.本研究有助于进一步了解芒果的开花分子机理及成花的生物学发育阶段.     

含铬重组液激活部分缺失金属原子簇的钼铁蛋白的研究

棕色固氮菌（Ａｚｏｔｏｂａｃｔｅｒ ｖｉｎｅｌａｎｄｉｉ）固氮酶钼铁蛋白经邻菲口罗啉和Ｏ２ 处理后,变为部分缺失ＦｅＭｏｃｏ 和Ｐ－ｃｌｕｓｔｅｒ的失活蛋白。与由Ｋ２ＣｒＯ４、高柠檬酸铁、Ｎａ２Ｓ和二硫苏糖醇组成的重组液保温后,处理蛋白对乙炔和质子还原的活性都得以显著恢复;然而,它的吸收光谱和圆二色谱虽有明显恢复,但仍与还原钼铁蛋白有所不同。这表明,激活蛋白中也许存在功能与钼铁蛋白相似,而结构则有所差异的含铬（ＣｒＦｅ）蛋白     

松口蘑菌丝体的分离和RAPD-PCR分析

针对松口蘑 [Tricholomamatsutake(S .ItoetImai)Sing .]菌丝体分离培养困难和各种相关分离物目前难以用出菇试验鉴定的现实 ,采用 8种培养基配方 ,对 9个不同来源的松口蘑子实体的不同部位及菌根、菌土进行组织分离 ,计接种试管 81 0多支 ,结果从菌褶部位获得 94支慢生型的菌丝体分离菌株 ,从菌柄部位仅获得 1支快生型的菌丝体分离菌株。以马铃薯葡萄糖土壤滤液培养基 (PDAS)、马铃薯葡萄糖麦麸滤液培养基 (PDAW )、BM培养基、马铃薯葡萄糖琼脂培养基 (PDA)对菌褶进行组织分离 ,获慢生型菌丝体的成功率依次为 74.4%、35.5%、156%和 8.9%。以各分离菌株的来源松口蘑子实体和中日两国松口蘑研究者提供的分离菌株作为DNA参照样品 ,对从供试子实体、菌根、菌土进行组织分离获得的各种相关纯培养物进行亲菌鉴定。采用筛选的 1 7个随机引物介导 2 5个供试松口蘑子实体及其分离菌体的RAPD(RandomAmplifiedPolymorphicDNA) PCR反应 ,全部获得了清晰而稳定的DNA指纹图谱 ,结果一致表明 :每个松口蘑子实体的菌盖 (含菌褶…     

Expression and purification of the catalytic domain of human vascular endothelial growth factor receptor 2 for inhibitor screening

Vascular endothelial growth factor (VEGF), an endothelial cell-specific mitogen, can act in tumor-induced angiogenesis by binding to specific receptors on the surface of endothelial cells. One such receptor, VEGFR-2/KDR, plays a key role in VEGF-induced angiogenesis. Here, we expressed the catalytic domain of VEGFR-2 as a soluble active kinase using Bac-to-Bac expression system, and investigated correlations between VEGFR-2 activity and enzyme concentration, ATP concentration, substrate concentration and divalent cation type. We used these data to establish a convenient, effective and non-radioactive ELISA screening technique for the identification and evaluation of potential inhibitors for VEGFR-2 kinase. We screened 200 RTK target-based compounds and identified one (TKI-31) that potently inhibited VEGFR-2 kinase activity (IC50=0.596 microM). Treatment of NIH3T3/KDR cells with TKI-31 blocked VEGF-induced phosphorylation of KDR in a dose-dependent manner. Moreover, TKI-31 dose-dependently suppressed HUVEC tube formation. Thus, we herein report a novel, efficient method for identifying VEGFR-2 kinase inhibitors and introduce one, TKI-31, that may prove to be a useful new angiogenesis inhibitor.     

Colonization of endophyte Acremonium sp. D212 in Panax notoginseng and rice mediated by auxin and jasmonic acid

Endophytic fungi can be beneficial to plant growth. However, the molecular mechanisms underlying colonization of Acremonium spp. remain unclear.In this study, a novel endophytic Acremonium strain was isolated from the buds of Panax notoginseng and named Acremonium sp. D212. The Acremonium sp. D212 could colonize the roots of P. notoginseng,enhance the resistance of P. notoginseng to root rot disease, and promote root growth and saponin biosynthesis in P. notoginseng. Acremonium sp. D212 could secrete indole-3-acetic acid(IAA) and jasmonic acid(JA), and inoculation with the fungus increased the endogenous levels of IAA and JA in P. notoginseng. Colonization of the Acremonium sp. D212 in the roots of the rice line Nipponbare was dependent on the concentration of methyl jasmonate(Me JA)(2–15 μmol/L) and 1-naphthalenacetic acid(NAA)(10–20 μmol/L). Moreover, the roots of the JA signaling-defective coi1-18 mutant were colonized by Acremonium sp. D212 to a lesser degree than those of the wild-type Nipponbare and mi R393 boverexpressing lines, and the colonization was rescued by Me JA but not by NAA. It suggests that the cross-talk between JA signaling and the auxin biosynthetic pathway plays a crucial role in the colonization of Acremonium sp. D212 in host plants.     

Purification and characterization of a novel chlorpyrifos hydrolase from Cladosporium cladosporioides Hu-01

Chlorpyrifos is of great environmental concern due to its widespread use in the past several decades and its potential toxic effects on human health. Thus, the degradation study of chlorpyrifos has become increasing important in recent years. A fungus capable of using chlorpyrifos as the sole carbon source was isolated from organophosphate-contaminated soil and characterized as Cladosporium cladosporioides Hu-01 (collection number: CCTCC M 20711). A novel chlorpyrifos hydrolase from cell extract was purified 35.6-fold to apparent homogeneity with 38.5% overall recovery by ammoniumsulfate precipitation, gel filtration chromatography and anion-exchange chromatography. It is a monomeric structure with a molecular mass of 38.3 kDa. The pI value was estimated to be 5.2. The optimal pH and temperature of the purified enzyme were 6.5 and 40°C, respectively. No cofactors were required for the chlorpyrifos-hydrolysis activity. The enzyme was strongly inhibited by Hg2?, Fe3?, DTT, β-mercaptoethanol and SDS, whereas slight inhibitory effects (5-10% inhibition) were observed in the presence of Mn2?, Zn2?, Cu2?, Mg2?, and EDTA. The purified enzyme hydrolyzed various organophosphorus insecticides with P-O and P-S bond. Chlorpyrifos was the preferred substrate. The Km and Vmax values of the enzyme for chlorpyrifos were 6.7974 μM and 2.6473 μmol·min?1, respectively. Both NH2-terminal sequencing and matrix-assisted laser desorption/ionization time-of-flight/time-of-flight mass spectrometer (MALDI-TOF-MS) identified an amino acid sequence MEPDGELSALTQGANS, which shared no similarity with any reported organophosphate-hydrolyzing enzymes. These results suggested that the purified enzyme was a novel hydrolase and might conceivably be developed to fulfill the practical requirements to enable its use in situ for detoxification of chlorpyrifos. Finally, this is the first described chlorpyrifos hydrolase from fungus.