含PR启动子的原核高效表达载体的构建及应用

组建了一个仅含P_R启动子的原核高效表达载体pRC,它同时含有cⅠ调控基因、多酶切位点和两个强的转录终止序列.现已成功地用于表达重组人肿瘤坏死因子-α（hTNF-α）、重组人白细胞介素-3（hIL-3）和抗溶菌酶（HEL）抗体Fd基因,表达量均占菌体总蛋白的36％以上.同时还研究了不同的宿主菌和原核增强子序列等因素对P_R启动子载体表达的影响.此外,还比较了分别以P_R、P_L或P_RP_L作为启动子时表达hTNF-α的情况,结果表明,单用P_R或P_L启动子可获得与使用P_RP_L串联启动子一样的高效表达.     

重寄生真菌盾壳霉pH信号通路中Pal相关基因的功能鉴定

【目的】研究pH信号通路(Pal)在重寄生真菌盾壳霉与寄主核盘菌互作过程中的作用。【方法】从盾壳霉全基因组信息中分析获得了6个Pal相关基因CmpalA、CmpalB、CmpalC、CmpalF、CmpalH和CmpalI的全编码序列和氨基酸序列,通过PEG介导的原生质转化技术获得了CmpalA、CmpalB、CmpalC、CmpalF和CmpalH等5个基因的敲除突变体,分析这些敲除突变体与野生型在菌落培养性状、重寄生能力、降解草酸能力、产生抗真菌物质能力等方面的差异。【结果】与野生型相比,在pH 6–8的条件下,5个Pal相关基因敲除突变体的菌丝生长受到显著抑制,这说明缺失Pal相关基因使盾壳霉对高pH值环境更加敏感。菌核重寄生试验发现5个Pal相关基因敲除突变体的重寄生能力均显著低于野生型。qRT-PCR试验结果表明,敲除Pal相关基因之后导致重寄生相关酶基因Cmch1、Cmg1和Cmsp1的表达量显著降低,而且pH信号通路下游的CmpacC基因的表达量也显著降低。Pal相关基因敲除突变体在pH 6条件下对草酸盐的降解能力显著高于野生型,同时这5个突变体在pH 8条件下产生抗真菌物质能力也显著高于野生型。【结论】pH信号通路相关基因的缺失影响盾壳霉对环境pH的响应。pH信号通路在盾壳霉与核盘菌互作中发挥重要作用,不仅影响盾壳霉的重寄生作用,而且还影响盾壳霉的草酸降解作用和抗真菌作用。     

Spindlin1, a novel nuclear protein with a role in the transformation of NIH3T3 cells

spindlin1, a novel human gene recently isolated by our laboratory, is highly homologous to mouse spindlin gene. In this study, we cloned cDNA full-length of this novel gene and send it to GenBank database as spindlin1 (Homo sapiens spindlin1) with Accession No. AF317228. In order to investigate the function of spindlin1, we studied further the subcellular localization of Spindlin1 protein and the effects of spindlin1 overexpression in NIH3T3 cells. The results showed that the fusion protein pEGFP-N1-spindlin1 was located in the nucleus and the C-terminal is correlated with nuclear localization of Spindlin1 protein. NIH3T3 cells which could stably express spindlin1 as a result of RT-PCR analysis compared with the control cells displayed a complete morphological change; made cell growth faster; and increased the percentage of cells in G2/M and S phase. Furthermore, overexpressed spindlin1 cells formed colonies in soft agar in vitro and formed tumors in nude mice. Our findings provide direct evidence that spindlin1 gene may contribute to tumorigenesis.     

The molecular basis of spinocerebellar ataxia type 48 caused by a de novo mutation in the ubiquitin ligase CHIP

The spinocerebellar ataxias (SCAs) are a class of incurable diseases characterized by degeneration of the cerebellum that results in movement disorder. Recently, a new heritable form of SCA, spinocerebellar ataxia type 48 (SCA48), was attributed to dominant mutations in STIP1 homology and U box-containing 1 (STUB1); however, little is known about how these mutations cause SCA48. STUB1 encodes for the protein C terminus of Hsc70 interacting protein (CHIP), an E3 ubiquitin ligase. CHIP is known to regulate proteostasis by recruiting chaperones via a N-terminal tetratricopeptide repeat domain and recruiting E2 ubiquitin-conjugating enzymes via a C-terminal U-box domain. These interactions allow CHIP to mediate the ubiquitination of chaperone-bound, misfolded proteins to promote their degradation via the proteasome. Here we have identified a novel, de novo mutation in STUB1 in a patient with SCA48 encoding for an A52G point mutation in the tetratricopeptide repeat domain of CHIP. Utilizing an array of biophysical, biochemical, and cellular assays, we demonstrate that the CHIP^A52G point mutant retains E3-ligase activity but has decreased affinity for chaperones. We further show that this mutant decreases cellular fitness in response to certain cellular stressors and induces neurodegeneration in a transgenic Caenorhabditis elegans model of SCA48. Together, our data identify the A52G mutant as a cause of SCA48 and provide molecular insight into how mutations in STUB1 cause SCA48.     

Targeting UPR branches,a potential strategy for enhancing efficacy of cancer chemotherapy

Cancer cells are often exposed to cell intrinsic stresses and environmental perturbations that may lead to accumulation of unfolded and/or misfolded proteins in...     

铅锌矿渣场植被自然演替与基质的交互效应

矿业废弃地生态系统自然恢复的植被演替过程与机理是生态恢复研究的重要内容之一.以空间代替时间的方法,选择立地条件基本一致的4个不同自然恢复年限铅锌矿区为对象,研究黔西北土法炼锌渣场废弃地植被自然演替与矿渣基质理化性质的交互效应.结果表明： 随着堆置时间的增加,矿渣基质的营养条件明显得到改善,全氮、全磷和全钾含量极显著增加, pH上升,电导率下降,容重降低,有效铅和镉显著降低. 同时,随着恢复时间的增长,植物群落的物种丰富度、多样性指数和均匀度也相应提高.植物群落组成以多年生草本植物为主,植物群落演替在前20年较为缓慢,30年后植被群落盖度可达到53%,超过40年盖度可达87%.矿渣理化性质与物种多样性显著相关,典型变量分别是全氮、全磷和全钾;物种多样性指数与有效铅和镉呈显著负相关.土法炼锌渣场废弃地植被自然演替过程在30年后速度加快,植被生长的限制因子是营养供给不足和重金属的有效性高.     

A highly selective and colorimetric assay of lysine by molecular-driven gold nanorods assembly

In this contribution, a simple, rapid, colorimeteric and selective assay for lysine was achieved by a controllable end-to-end assembly of gold nanorods (AuNRs) in the presence of Eu(3+) and lysine. This one-pot end-to-end assembly of 11-mercaptoundecanoic acid (MUA) modified AuNRs was occurred in Britton-Robinson buffer of pH 6.0, which involves the coordination binding between Eu(3+) and COO(-) groups as well as the electrostatic interaction of the COO(-) groups of MUA with the -NH(3)(+) group of lysine. As monitored by absorption spectra, scanning electron microscopic (SEM) images and dynamic light scattering (DLS) measurement, the end-to-end chain assembly results in large red-shift in the longitudinal plasmon resonance absorption (LPRA), giving red-to-blue color change of AuNRs. Importantly, it was found that the red-shift of LPRA is linearly proportional to the concentrations of lysine in the range of 5.0×10(-6)-1.0×10(-3)M with the limit of detection (LOD) being 1.6×10(-6)M (3σ/k). This red-shift of LPRA is highly selective, making it possible to develop a rapid, selective and visual assay for lysine in food samples.