Possible effect of 30K proteins in embryonic development of silkworm Bombyx mori

The silkworm Bombyx mori possesses a 30K protein family of 3×10~4 Da,the biologicalfunctions of which have not been fully identified.The relationship between the 30K protein family and theembryonic development of temperature sensitive sex-linked mutant strain of silkworm was investigated bytwo dimensional polyacrylamide gel electrophoresis(2D-PAGE)and Matrix assisted laser desorption ionization-time of flight mass spectrometry(MALDI-TOF MS).The results show that protein spots 1-5 of the 30Kprotein family,mainly existing in normal strain,are possibly related to embryonic development.The earlyconsumption of a 30K protein named 6G1-30K-1 and the accumulation of 30K proteins named 6G1-30K-3and 6G1-30K-4 are likely caused by the destruction of physiological balance in normal embryonic development,which may lead to lower hatchability of the temperature sensitive strain.The results suggest that reasonablemetabolism of 30K proteins is a prerequisite for the embryo's normal development.     

Heat shock protein 90 indirectly regulates ERK activity by affecting Raf protein metabolism

Extracellular signal-regulated protein kinase (ERK) has been implicated in the pathogenesis of several nerve system diseases. As more and more kinases have been discovered to be the client proteins of the molecular chaperone Hsp90, the use of Hsp90 inhibitors to reduce abnormal kinase activity is a new treatment strategy for nerve system diseases. This study investigated the regulation of the ERK pathway by Hsp90. We showed that Hsp90 inhibitors reduce ERK phosphorylation without affecting the total ERK protein level. Further investigation showed that Raf, the UPstream kinase in the Ras-Raf-MEK-ERK pathway, forms a complex with Hsp90 and Hsp70. Treating cells with Hsp90 inhibitors facilitates Raf degradation,thereby down-regulating the activity of ERK.     

Sequence analysis and structural prediction of the severe acute respiratory syndrome coronavirus nsp5

The non-structural proteins (nsp or replicase proteins) of coronaviruses are relatively conserved and can be effective targets for drugs. Few studies have been conducted into the function of the severe acute respiratory syndrome coronavirus (SARS-CoV) nsp5. In this study, bioinformatics methods were employed to predict the secondary structure and construct 3-D models of the SARS-CoV GD strain nsp5. Sequencing and sequential comparison was performed to analyze the mutation trend of the polymerase nsp5 gene during the epidemic process using a nucleotide-nucleotide basic local alignment search tool (BLASTN) and a protein-protein basic local alignment search tool (BLASTP). The results indicated that the nsp5 gene was steady during the epidemic process and the protein was homologous with other coronavirus nsp5 proteins. The protein encoded by the nsp5 gene was expressed in COS-7 cells and analyzed by sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE). This study provided the foundation for further exploration of the protein‘s biological function, and contributed to the search for anti-SARS-CoV drugs.     

Positive Charges on Lysine Residues of the Extrinsic 18 kDa Protein Are Important to Its Electrostatic Interaction with Spinach Photosystem Ⅱ Membranes

To determine the contribution of charged amino acids to binding with the photosystem II complex （PSII）, the amino or carboxyl groups of the extrinsic 18 kDa protein were modified with N- succinimidyl propionate （NSP） or glycine methyl ester （GME） in the presence of a water-soluble carbodiimide, respectively. Based on isoelectric point shift, 4-10 and 10-14 amino groups were modified in the presence of 2 and 4 mM NSP, respectively. Similarly, 3-4 carboxyl groups were modified by reaction with 100 mM GME. Neutralization of negatively charged carboxyl groups with GME did not alter the binding activity of the extrinsic 18 kDa protein. However, the NSP-modified 18 kDa protein, in which the positively charged amino groups had been modified to uncharged methyl esters, failed to bind with the PSII membrane in the presence of the extrinsic 23 kDa protein. This defect can not be attributed to structural or conformational alterations imposed by chemical modification, as the fluorescence and circular dichroism spectra among native, GME- and NSP-modified extrinsic 18 kDa proteins were similar. Thus, we have concluded that the positive charges of lysyl residues in the extrinsic 18 kDa protein are important for its interaction with PSII membranes in the presence of the extrinsic 23 kDa protein. Furthermore, it was found that the negative charges of carboxyl groups of this protein did not participate in binding with the extrinsic 23 kDa protein associated with PSII membranes.     

猪繁殖与呼吸综合症病毒GP5蛋白原核表达初步研究

猪繁殖与呼吸综合症是由猪繁殖与呼吸综合症病毒(Porcine reprodutive and respiratory syndrome virus,PRRSV)引发的病毒性传染病,病症主要表现为怀孕母猪早产、流产、木乃伊胎以及仔猪呼吸症状和高死亡率[1,2].该病于1987年首先在美国爆发[3],1990年德国亦发现此病,并迅速传播到欧洲其他国家.我国在1996年报道有该病流行[4],由于该病的迅速蔓延使整个世界养猪业遭受了巨大的经济损失.     

猪繁殖与呼吸综合征病毒N蛋白的原核表达、纯化与结构分析

将猪繁殖与呼吸综合征病毒 (PRRSV)BJ 4毒株N基因克隆至原核表达载体pET2 8a中 ,得到重组表达载体pET2 8 N ,转化EscherichiacoliBL2 1(DE3)细胞 ,获得可溶性表达 ,表达量占菌体蛋白的 2 8%。经ProbandNi2 亲和层析获得重组蛋白P2 8 N ,圆二色谱 (CD)测定结果表明 ,P2 8 N重组蛋白螺旋占 2 6 1% ,折叠占 2 3 7% ,转角 19 8% ,卷曲占 30 3%。并进一步绘制出PRRSVN蛋白的二级结构图     

枯草芽孢杆菌ccpA基因敲除及对其核黄素产量的影响

CcpA蛋白是介导枯草芽孢杆菌碳分解代谢物阻遏(CCR)的全局调控因子,由ccpA基因编码。CCR效应的存在影响B.subtilis对葡萄糖的利用,降低B.subtilis生产发酵产品的效率。采用基因重组技术敲除了核黄素发酵菌株B.subtilis24/pMX45的ccpA基因,构建了CcpA缺陷株B.subtilis24A1/pMX45。发酵结果显示:B.subtilis24A1/pMX45能够在70h内基本耗尽10%的葡萄糖,生物量达到1.5×109个细胞/mL,溢流代谢产物积累量减少,在8%和10%葡萄糖浓度下,B.subtilis24A1/pMX45核黄素产量分别比B.subtilis24/pMX45提高了62%和95%。CcpA的缺陷,可以缓解葡萄糖引起的CCR效应,显著提高菌株的核黄素产量。     

Sinorhizobium morelens S-5中N-氨甲酰基水解酶基因的克隆、表达和纯化

N-氨甲酰基水解酶是一种非常具有工业应用价值的水解酶,可用于制备光学纯氨基酸。通过LA PCR从Sinorhizobium morelensS-5菌中克隆到1.3kb的DNA片段,测序表明该片段上含有一个完整的N-氨甲酰基水解酶的基因(hyuC)序列。将hyuC基因克隆到表达载体pET30a上,重组质粒pET30a-HyuC在大肠杆菌中获得了高水平表达。重组的N-氨甲酰基水解酶经过热处理和三步柱色谱分离而纯化。纯化倍数为16.1倍,收率21.2%。该酶为同源四聚体,亚基分子量是38kDa。最适温度是60℃,最适pH为7.0。该酶有较高的热稳定性和氧化稳定性。Fe2 和Ca2 对酶的活性有一定的促进作用,而金属螯合剂和巯基试剂对酶活无明显影响。     

乙肝患者肝纤维化血清标志物表达水平及其临床意义

目的探讨乙肝患者肝纤维化血清标志物表达水平及其与病情、病程的关系。方法用R IA方法对135例乙肝患者进行血清透明质酸酶(HA)、层粘连蛋白(LN)、Ⅲ型胶原前体(PⅢC)、Ⅳ型胶原(ⅣC)检测。结果乙肝患者血清中HA、LN、PⅢC和ⅣC水平分别为(338.7±122.6)、(226.2±43.3)、(152.8±33.7)和(132.6±41.1)μg/L。其中,急性黄疸肝炎、慢性肝炎轻度、慢性肝炎中度、慢性肝炎重度、肝硬变代偿期、肝硬变失代偿期、肝癌和肝硬变+肝癌患者HA、LN、PⅢC和ⅣC水平分别为(134.2±101.4)、(185.1±104.5)、(373.5±147.1)、(445.5±123.6)、(477.3±186.0)、(653.7±98.5)、(597.5±91.3)和(723.6±110.4)μg/L;(106.7±31.3)、(179.5±28.5)、(234.6±66.1)、(287.3±57.0)、(294.8±66.4)、(345.6±61.2)、(322.1±71.0)和(357.8±88.3)μg/L;(103.5±19.2)、(108.6±21.4)、(145.3±37.3)、(194.5±41.7)、(188.8±39.5)、(247.6±48.0)、(251.3±37.3)和(286.7±35.8)μg/L;(49.6±14.7)、(83.4±15.3)、(134.5±50.5)、(178.7±61.4)、(183.2±61.3)、(256.8±72.4)、(217.7±64.5)和(281.1±57.0)μg/L。结论肝癌、肝硬化、肝硬变+肝癌和慢性肝炎重度患者存在明显的肝纤维化征象,急性黄疸肝炎、慢性肝炎轻度患者肝纤维化程度较轻,在一定范围内肝纤维化程度与肝病的病情、病程关系密切。     

热休克蛋白质70对大鼠肝脏缺血再灌注后NOS活力的影响

目的为探讨热应激预处理对肝脏缺血再灌注损伤的保护作用的机制,采用局部热应激处理诱导热休克蛋白质(HSP70)的表达,检测了HSP70对肝脏缺血再灌注时NOS活力的影响。方法将实验大鼠随机分为热应激预处理组与非预处理组,对比观察两组动物肝脏缺血再灌注后0、4、8、12、24h期间内肝脏HSP70的表达、NOS活力及血清乳酸脱氢酶(lactate dehydrogenase,LDH)的活性与肝脏组织学改变。结果热应激预处理组HSP70的表达水平均比非预处理组同一时间点高,而NOS活力及血清LDH的活性较非预处理组低。与非预处理组比较,经热应激预处理肝组织损伤较轻。结论热应激预处理诱导产生的热休克蛋白70保护肝脏缺血再灌注损伤的作用途径之一可能是通过抑制NO的产生,从而降低大量自由基对肝脏的损害。