Requirement of Cysteines and Length of the Human Respiratory Syncytial Virus M2-1 Protein for Protein Function and Virus Viability

The M2-1 protein of human respiratory syncytial virus (hRSV) promotes processive RNA synthesis and readthrough at RSV gene junctions. It contains four highly conserved cysteines, three of which are located in the Cys(3)-His(1) motif at the N terminus of M2-1. Each of the four cysteines, at positions 7, 15, 21, and 96, in the M2-1 protein of hRSV A2 strain was individually replaced by glycines. When tested in an RSV minigenome replicon system using beta-galactosidase as a reporter gene, C7G, C15G, and C21G located in the Cys(3)-His(1) motif showed a significant reduction in processive RNA synthesis compared to wild-type (wt) M2-1. C96G, which lies outside the Cys(3)-His(1) motif, was fully functional in supporting processive RNA synthesis in vitro. Each of these cysteine substitutions was introduced into an infectious antigenomic cDNA clone derived from hRSV A2 strain. Except for C96G, which resulted in a viable virus, no viruses were recovered with mutations in the Cys(3)-His(1) motif. This indicates that the Cys(3)-His(1) motif is critical for M2-1 function and for RSV replication. The functional requirement of the C terminus of the M2-1 protein was examined by engineering premature stop codons that caused truncations of 17, 46, or 67 amino acids from the C terminus. A deletion of 46 or 67 amino acids abolished the synthesis of full-length beta-galactosidase mRNA and did not result in the recovery of viable viruses. However, a deletion of 17 amino acids from the C terminus of M2-1 reduced processive RNA synthesis in vitro and was well tolerated by RSV. Relocation of the M2-1 termination codon upstream of the M2-2 initiation codons did not significantly affect the expression of the M2-2 protein. Both rA2-Tr17 and rA2-C96G did not replicate as efficiently as wt rA2 in HEp-2 cells and was restricted in replication in the respiratory tracts of cotton rats.     

磷脂转移蛋白基因在畜禽生产中的应用

磷脂转移蛋白（PLTP）是脂代谢中重要的转运蛋白,并且推测PLTP可能也在机体免疫和胚胎发育中起重要作用,与疾病抵抗力和胚胎发育程度相关。PLTP基因可能是影响畜禽经济性状的主效基因或与主基因相连锁,PLTP基因的HaeⅢ遗传标记可能是影响畜禽初生重和生长性状的重要标记基因。     

Both Decrease in ACL1 Gene Expression and Increase in ICL1 Gene Expression in Marine-Derived Yeast Yarrowia lipolytica Expressing INU1 Gene Enhance Citric Acid Production from Inulin

In this study, some of the ATP-citrate lyase genes (ACL1) were deleted and the copy number of the iso-citrate lyase gene (ICL1) was increased in the marine-derived yeast Yarrowia lipolytica SWJ-1b displaying the recombinant inulinase. It was found that lipid content and iso-citric acid in the transformant 30 obtained were greatly reduced and citric acid production was greatly enhanced. It was also found that the ACL1 gene expression and ATP-citrate lyase activity in the transformant 30 were declined and the ICL1 gene expression and iso-citrate lyase activity were promoted. During the 2-l fermentation, 84.0 g/l of citric acid and 1.8 g/l of iso-citric acid in the fermented medium were attained from 10.0 % of inulin by the transformant 30 within 214 h. The results showed that only 0.36 % of the residual reducing sugar and 1.0 % of the residual total sugar were left in the fermented medium, suggesting that 89.6 % of the total sugar was used for citric acid production and cell growth by the transformant 30.     

酵母豆蔻酰辅酶A：蛋白质N端转酰基酶基因的克隆与表达

利用ＰＣＲ技术,从酵母染色体中扩增得到酵母豆蔻酰－ＣｏＡ：蛋白质Ｎ端转酰基酶（ＹＳＣＮＭＴ）基因,并克隆到ｐＢｌｕｅｓｃｒｉｐｔＫＳ＋载体中。由ＤＮＡ全序测定表明,获得了ＹＳＣＮＭＴ编码基因。进一步构建了Ｔ７Ｐｒｏｍｏｔｅｒ控制下的含上述完整ＹＳＣＮＭＴ编码基因的表达质粒ｐＭＦＴ７－５－ＮＭＴ,转化大肠杆菌ＢＬ２１（ＤＥ３）,进行ＩＰＴＧ诱导表达研究。通过ＳＤＳ－ＰＡＧＥ分析,观察到一与理论分子量一致的诱导条带（约５３ｋＤ）,占全菌蛋白的３９％左右,且可溶性部分约占上清液中全部蛋白的３４％。经一步Ｐ１１磷酸纤维素阳离子交换柱层析,将其纯化到纯度达９７％以上．纯化的表达产物经Ｎ端氨基酸序列分析,所测定的Ｎ端５个氨基酸的序列,与从克隆的ＹＳＣＮＭＴ基因推出的氨基酸序列完全一致（不含Ｎ端Ｍｅｔ）。对所得的ＹＳＣＮＭＴ进行酶活力鉴定,观察到了明显的活力。     

酵母蛋白质相互作用与基因表达谱和亚细胞定位的相关性

研究酵母（yeast）蛋白质相互作用与基因表达谱和蛋白质亚细胞定位的关系.首先,构建了蛋白质相互作用正样本集、负样本集、随机组对负样本集和混合样本集.然后,对于4个数据集中的所有蛋白质对,通过比较它们的基于距离的基因共表达的分布以及它们中具有已知亚细胞定位的蛋白质对的共定位出现率,实现了这些高通量数据的交叉量化分析.结果揭示,与非相互作用蛋白质对相比,相互作用蛋白质对的基因表达谱具有较高的相似性;相互作用蛋白质对更倾向于具有相同的亚细胞定位.结果还揭示出这些蛋白质特征相关的总体趋势.     

胃癌中APC基因I1307K突变及蛋白质表达

为探讨肿瘤抑制基因APC结构及表达异常与胃癌发生、发展的关系,采用ARMS PCR检测胃癌中APC基因I1307K突变存在与否,免疫组织化学方法分析胃癌中APC蛋白表达水平。结果表明,在 62例胃癌高发区易感人群血液标本及45例胃癌中未检测到I1307K突变;胃癌（早期、进展期）中APC蛋白表达阳性率显著低于正常黏膜,进展期胃癌中APC蛋白表达阳性率显著低于早期胃癌,淋巴结转移阳性的胃癌中APC蛋白表达阳性率显著低于淋巴结转移阴性者。因此认为I1307K突变可能与国人胃癌发生无明显相关;APC蛋白低表达与胃癌发生、进展及淋巴结转移密切相关。 Abstract:In order to explore the correlation of the abnormalities of tumor suppressor gene APC with the carcinogenesis and progression of gastric cancer.The I1307K mutation of APC gene in gastric cancer was analysed using Amplification Refractory Mutation System PCR(ARMS ,PCR),also the expression of APC protein in gastric cancer of different stages was detected by immunohistochemical method.We found that there wasn't I1307K mutation of APC gene in 62 cases of blood samples of susceptible population in high incidence areas of gastric cancer and 45 cases of gastric cancer tissues.The positive rates of APC protein in gastric cancer (both early and progressive gastric cancer) were significantly lower than that in normal mucosa,the positive rates of APC protein in progressive gastric cancer were significantly lower than that in early gastric cancer,the positive rates of APC protein in gastric cancer with lymph node metastasis were significantly lower than that in gastric cancer without lymph node metastasis.So it was thought that there might be no correlation between the I1307K mutation of APC gene and carcinogenesis of gastric cancer in China,but the decreased expression of APC protein was closely related to the carcinogenesis,progression and lymph node metastasisof gastric cancer.     

黄曲霉素合成相关基因表达与环境因素的关系

简单介绍了黄曲霉毒素的发现、分布、危害和范围,详细叙述了黄曲霉毒素生物合成中相关基因的表达与调控,概述了黄曲霉毒素合成中的关键基因、酶和调控因子重要性,分析了影响黄曲霉毒素合成的环境因素。不仅在基础理论上对黄曲霉毒素合成机理进行了深入探讨,而且在应用研究上为减少粮食和食品受到重金属污染和黄曲霉毒素危害提供了新的思路。     

Requirement of NOX2 Expression in Both Retina and Bone Marrow for Diabetes-Induced Retinal Vascular Injury

Objective

Diabetic retinopathy, a major cause of blindness, is characterized by increased expression of vascular endothelial growth factor (VEGF), leukocyte attachment to the vessel walls and increased vascular permeability. Previous work has shown that reactive oxygen species (ROS) produced by the superoxide generating enzyme NOX2/NADPH oxidase play a crucial role in the vascular pathology. The aim of this work was to identify the cellular sources of the damaging NOX2 activity by studies using bone marrow chimera mice.

Methods

Bone marrow cells were collected from the femurs and tibias of wild type and NOX2 deficient (NOX2^-/-) donor mice and injected intravenously into lethally irradiated NOX2^-/- and wild type recipients. Following recovery from radiation, mice were rendered diabetic by streptozotocin injections. The following groups of bone marrow chimeras were studied: non-diabetic WT→WT, diabetic WT→WT, diabetic WT→NOX2^-/-, diabetic NOX2^-/-→WT. After 4 weeks of diabetes, early signs of retinopathy were examined by measuring ROS, expression of VEGF and ICAM-1, leukocyte attachment to the vessel wall and vascular permeability.

Results

The retinas of the diabetic WT→WT chimeras showed significant increases in ROS as compared with the non-diabetic chimeras. These diabetes-induced alterations were correlated with increases in expression of VEGF and ICAM-1, leukocyte adhesion and vascular permeability. Each of these diabetes-induced alterations were significantly attenuated in the diabetic WT→NOX2^-/- and NOX2^-/-→WT chimera groups (p<0.05).

Conclusion

NOX2-generated ROS produced by both bone marrow-derived cells and resident retinal cells contribute importantly to retinal vascular injury in the diabetic retina. Targeting NOX2 in bone marrow and/or retinal cells may represent a novel therapeutic strategy for the treatment/prevention of vascular injury in the diabetic retina.     

酵母PHO80基因的克隆,表达及功能分析

利用原位杂交方法从野生型酵母菌染色体ＤＮＡ中克隆了ＰＨＯ８０基因,全长约４．２ｋｂ,包括１．１ｋｂ的上游序列和８７９ｂｐ的编码序列。以ＵＲＡ３基因为筛选标记,通过体内同源重组和营养互补筛选获得了ＰＨＯ８０基因缺失突变株。进一步研究了ＰＨＯ８０在细胞内的功能,它是酵母阻遏型酸性磷酸酯酶结构基因以及调控基因ＰＨＯ８１表达的负调控因子,但不影响ＰＨＯ４和ＰＨＯ８５的表达。还构建了ＰＨＯ８０－ＬａｃＺ融合基因,探索它在细胞内的表达规律。β－半乳糖苷酶活力测定结果表明,ＰＨＯ８０基因在细胞内呈低水平表达,并受自身产物和ＰＨＯ８５的抑制,推测它与ＰＨＯ５和ＰＨＯ８１基因的调控模式可能相似。     

The Role of Cytokinin Biosynthetic Gene in Regulating the Expression of a Class of Pathogenesis-Related Protein Genes in Tobacco Plants

The expression characteristics of a class of pathogenesis-related protein (PR) genes, namely basic chitinase, β-1, 3-glucanase, osmotin and extensin, were studied in tobacco (Nicotiana tabacum cv. Wisconsin 38) plants. RNA blot hybridization showed that these four genes were regulated in a developmental and organ-specific manner in tobacco. In the transgenic fascicular shoots which contained the active cytokinin biosynthetic gene (ipt gene) from Agrobacterium tumefaciens, the expressions of these four genes were co-regulated by overproduction of endogenous cytokinins and vector effect. Cytokinins reduced the expressions while vector effect induced the expressions of these four genes. Heat shock also de creased the steady-state levels of the four RNAs. These data suggest a complex regulation of PR genes.     

The 14-3-3 Proteins: Gene,Gene Expression,and Function

14-3-3 Proteins were discovered by Moore and Perez in the soluble extract of bovine brain. These proteins are highly abundant in the brain. In this review 14-3-3 cDNA cloning, nucleotide sequence of 14-3-3 cDNA, the structure of 14-3-3 gene and 14-3-3 gene expression, in situ hybridization of 14-3-3 mRNA in the brain, the function and regulation of 14-3-3 protein, the binding of 14-3-3 protein to other proteins, the effects of 14-3-3 protein on the binding of a protein to other proteins, and the effect on protein kinase, etc., are concisely described. From the recent rapid development of proteom technology, markedly more target proteins of 14-3-3 protein should be discovered.     

水稻非特异性脂质转移蛋白的原核表达、纯化及抑菌功能

将编码水稻非特异性脂质转移蛋白 (nonspecificlipidtransferprotein ,nsLTP)基因 (LTP110 )的克隆到硫氧还蛋白融合表达载体PET32a( )中 ,在BL2 1(DE3)trxB-宿主菌中实现了融合蛋白的高表达。通过Ni2 chelatingSepharosefastflow柱纯化融合蛋白后 ,通过肠激酶酶切再过该亲和柱得到了重组LTP110。CD谱扫描表明重组蛋白质与体内提取的nsLTP二级结构相似 ;荧光脂质结合实验表明该蛋白质具有结合脂肪酸分子的活性。对该蛋白质的抑菌功能进行研究后表明 ,LTP110具有抑制稻瘟病菌孢子萌发的功能 ,在较低浓度即能发挥活性     

Phospholipid Association Is Essential for Dynamin-related Protein Mgm1 to Function in Mitochondrial Membrane Fusion

Mgm1, the yeast ortholog of mammalian OPA1, is a key component in mitochondrial membrane fusion and is required for maintaining mitochondrial dynamics and morphology. We showed recently that the purified short isoform of Mgm1 (s-Mgm1) possesses GTPase activity, self-assembles into low order oligomers, and interacts specifically with negatively charged phospholipids (Meglei, G., and McQuibban, G. A. (2009) Biochemistry 48, 1774–1784). Here, we demonstrate that s-Mgm1 binds to a mixture of phospholipids characteristic of the mitochondrial inner membrane. Binding to physiologically representative lipids results in ∼50-fold stimulation of s-Mgm1 GTPase activity. s-Mgm1 point mutants that are defective in oligomerization and lipid binding do not exhibit such stimulation and do not function in vivo. Electron microscopy and lipid turbidity assays demonstrate that s-Mgm1 promotes liposome interaction. Furthermore, s-Mgm1 assembles onto liposomes as oligomeric rings with 3-fold symmetry. The projection map of negatively stained s-Mgm1 shows six monomers, consistent with two stacked trimers. Taken together, our data identify a lipid-binding domain in Mgm1, and the structural analysis suggests a model of how Mgm1 promotes the fusion of opposing mitochondrial inner membranes.Mitochondrial dynamics have been implicated in neurodegenerative diseases such as dominant optic atrophy and Parkinson disease (1, 2). Mitochondrial morphology is regulated by balanced membrane fusion and fission reactions that are orchestrated by members of the highly conserved dynamin-related protein family (3). Dynamin-related proteins are large GTPases that can self-assemble and promote membrane remodeling (4, 5). We have shown previously that the dynamin-related protein Mgm1 has GTPase activity, self-assembles into low order oligomers, and binds to negatively charged phospholipids (6). Mgm1 exists as two isoforms in the mitochondria; l-Mgm1² is anchored to the IM via a transmembrane domain, and s-Mgm1 is peripherally associated with the IM and also found in the intermembrane space. s-Mgm1 results from the regulated cleavage by the mitochondrial rhomboid protease (7, 8). It was shown recently that both isoforms are essential but have distinct roles in mitochondrial membrane fusion whereby only s-Mgm1 requires its GTPase activity (9). It is proposed that l-Mgm1 serves as a receptor for s-Mgm1 to mediate fusion of opposing membranes upon GTP hydrolysis. Here, we provide molecular data indicating that lipid binding of s-Mgm1 is required for proper membrane fusion. Furthermore, structural analysis of s-Mgm1 assembled onto liposomes suggests a model whereby stacked trimers of s-Mgm1 on opposing membranes would facilitate fusion.     

Expression of 135-kDa Insecticidal Protein Gene from Bacillus thuringiensis in the Yeast Saccharomyces cerevisiae

Bacillus thuringiensis subsp. aizawai produces 130-kDa and 135-kDa (CrylA(a)) insecticidal proteins. When Saccharomyces cerevisiae was transformed by the vector carrying a cryIA(a) gene, the gene expression could not be observed. When the 5′-upstream region from the initiation codon was removed using a synthetic oligonucleotide, the CryIA(a) protein was successfully synthesized in yeast. The yeast extract containing CryIA(a) protein had insecticidal activity against Plutella xylostella larvae.     

人脂素基因LIPIN1在酵母中的异源表达及细胞功能分析

脂类代谢调控是维持生物体能量平衡的重要环节,脂类代谢调控的紊乱与肥胖症、糖尿病和高血压等疾病密切相关。脂素基因三LIPIN1是诱导脂肪细胞分化、调控脂类合成的关键基因．其编码的磷脂磷酸酶（phosphatidate phosphatase,PAP）在人体三酰甘油合成中起关键作用,是维持人体脂类平衡的重要保障。此外,该基因还作为重要的转录辅激活因子参与多种生长及营养代谢调控。多种生物中均有类似功能的基因被发现,暗示了其功能的多样性及物种间的保守性。该文利用酿酒酵母在脂类代谢研究中性状易于表征、同源基因剧刖功能明确的优势,通过同源重组技术构建脂素缺陷型酵母,探索脂素基因在维持酵母正常生长及脂类合成中的重要作用,并通过功能互补及生物信息学技术对比分析了人源LIPIN1基因与酵母PdH1基因编码蛋白在结构和功能上的保守性,为脂素基因LIPIN1的细胞功能研究提供基础数据。