Biochemical characterization of human enteropeptidase light chain

The synthetic gene encoding human enteropeptidase light chain (L-HEP) was cloned into plasmid pET-32a downstream from the gene of fusion partner thioredoxin immediately after the DNA sequence encoding the enteropeptidase recognition site. The fusion protein thioredoxin (Trx)/L-HEP was expressed in Escherichia coli BL21(DE3). Autocatalytic cleavage of the fusion protein and activation of recombinant L-HEP were achieved by solubilization of inclusion bodies and refolding of Trx/L-HEP fusion protein. The kinetic parameters of human and bovine enteropeptidases in the presence of different concentrations of Ca2+ and Na+ for cleavage of the specific substrate GD4K-na and nonspecific substrates such as small ester Z-Lys-SBzl and chromogenic substrates Z-Ala-X-Arg-pNA have been comparatively analyzed. It is demonstrated that positively charged ions increased the Michaelis constant (Km) for cleavage of specific substrate GD4K-na, while the catalytic constant (k(cat)) remained practically unchanged. L-HEP demonstrated secondary specificity to the chromogenic substrate Z-Ala-Phe-Arg-pNA with k(cat)/Km 260 mM(-1) x sec(-1). Enzymatic activity of L-HEP was suppressed by inhibitors of trypsin-like and cysteine (E-64), but not metallo-, amino-, or chymotrypsin-like proteinases. L-HEP was active over a broad range of pH (6-9) with optimum activity at pH 7.5, and it demonstrated high stability to different denaturing agents.     

Effects of bovine parathyroid hormone and 1,25-dihydroxyvitamin D3 on the production of prostaglandins by cells derived from human bone

Local production of prostaglandins by osteoblasts may be important in controlling the bone resorbing activity of some hormones which have receptors on osteoblasts. We have demonstrated that osteoblast-like cells derived from human bone can incorporate [14C]arachidonic acid into phospholipids and synthesise immunoreactive PGE. Parathyroid hormone increases both the release of incorporated arachidonic acid and the synthesis of PGE. This is the first demonstration of modulation of bone cell prostaglandin synthesis by a bone resorbing hormone.     

5种光合细菌种间原生质体融合及优良农用融合子的筛选鉴定

处于对数生长期的光合细菌球形红假单胞菌(Rhodopseudomonassphaeroides)、沼泽红假单胞菌(Rhodopseudomonaspalustris)、嗜酸红假单胞菌(Rhodopseudomdnasacidophila)、深红红螺菌(Rhodospirarubrum)、万尼氏红微菌(Rhodomocrobiumvannielii),经溶菌酶(3mg/L)处理50min后,获得了它们的菌体形成的原生质体,其再生率分别为80%、71%、82%、61%、74%.取等量的亲本菌株在35%的PEG(MW6000)诱导下两两融合5min,共10种组合.其融合率为球×沼2.5×10-4、球×嗜2.1×10-4、球×深2.0×10-4、球×万2.1×10-4、沼×嗜2.8×10-4、沼×深2.4×10-4、沼×万2.6×10-4、嗜×深2.0×10-4、嗜×万2.3×10-4、深×万2.4×10-4.经影印法鉴定:形成的融合子可以分别生长于以相应的有机物为唯一碳源的培养基上,所有融合子体积均相当于两亲本株体积之和,融合子菌落形态特征介于两亲本株之间.从中随机挑选100个融合子,以辣椒苗作为靶标植物,从上述融合子中筛选到了1株具有显著促进作物生长、提高抗病性的融合子.     

Lysosomal fusion and SNARE function are impaired by cholesterol accumulation in lysosomal storage disorders

The function of lysosomes relies on the ability of the lysosomal membrane to fuse with several target membranes in the cell. It is known that in lysosomal storage disorders (LSDs), lysosomal accumulation of several types of substrates is associated with lysosomal dysfunction and impairment of endocytic membrane traffic. By analysing cells from two severe neurodegenerative LSDs, we observed that cholesterol abnormally accumulates in the endolysosomal membrane of LSD cells, thereby reducing the ability of lysosomes to efficiently fuse with endocytic and autophagic vesicles. Furthermore, we discovered that soluble N‐ethylmaleimide‐sensitive factor attachment protein (SNAP) receptors (SNAREs), which are key components of the cellular membrane fusion machinery are aberrantly sequestered in cholesterol‐enriched regions of LSD endolysosomal membranes. This abnormal spatial organization locks SNAREs in complexes and impairs their sorting and recycling. Importantly, reducing membrane cholesterol levels in LSD cells restores normal SNARE function and efficient lysosomal fusion. Our results support a model by which cholesterol abnormalities determine lysosomal dysfunction and endocytic traffic jam in LSDs by impairing the membrane fusion machinery, thus suggesting new therapeutic targets for the treatment of these disorders.     

Improvement of the cell volume ofCandida blankii through protoplast fusion

Summary The yeastCandida blankii ESP-94, capable of utilizing xylose as substrate, was isolated for the production of single-cell protein (SCP) on bagasse hydrolysates. However, the small cell volume of strain ESP-94 would complicate harvesting of the cells during a continuous fermentation process. Auxotrophic mutants of strain ESP-94 were generated and intraspecific protoplast fusion experiments performed in an attempt to increase the cell volume of strain ESP-94. The fusion products were characterised with respect to cell volume, DNA content and genetic stability. Six genetically stable fusants with bigger cell volumes and higher DNA contents were obtained. One such fusant, fusant F17, had a cell volume 3-times that of strain ESP-94, while exhibiting similar growth rates to strain ESP-94 ond-xylose as carbon source.     

可溶性肿瘤坏死因子受体II-脂联素球部融合蛋白的真核表达及生物学活性检测

本研究设计和构建了一种人肿瘤坏死因子受体II胞外区与人脂联素球部的融合基因sTNFRII-gAD,且相应的融合蛋白在哺乳动物细胞BHK-21S的无血清培养体系中实现了表达,并对该融合蛋白进行了初步鉴定。首先,用RT-PCR方法从人的外周血淋巴细胞总RNA中扩增人肿瘤坏死因子II型受体胞外区基因片段,与脂联素球部基因片段融合,克隆至pAAV2neo表达载体中,构建成pAAV2neo-sTNFRII-gAD。随后,用pAAV2neo-sTNFRII-gAD转染BHK-21S细胞获得G418抗性细胞BHK-21S/pAAV2neo-sTNFRII-gAD;然后,将原来含有血清的培养液换成无血清的化学成分限定的培养液,细胞从贴壁培养方式转换成悬浮培养方式;最后,收集BHK-21S/pAAV2neo-sTNFRII-gAD无血清悬浮培养24h后的培养上清,进行sTNFRII-gAD融合蛋白的鉴定分析。酶切鉴定和测序结果显示,所构建的pAAV2neo-sTNFRII-gAD质粒结构正确,sTNFRII-gAD序列与预期一致;分别用抗人肿瘤坏死因子受体II和抗人脂联素球部的单克隆抗体检测pAAV2neo-sTNFRII-gAD瞬时转染的BHK-21S细胞,免疫荧光呈现阳性;免疫印迹分析在pAAV2neo-sTNFRII-gAD稳定转染的BHK-21S细胞上清中检测到sTNFRII-gAD融合蛋白的表达,并以单体、三聚体和三聚体以上的多聚体形式存在。活性测定结果表明,sTNFRII-gAD融合蛋白具有显著抑制TNFα杀伤L929细胞的活性。因此,本研究为下一步大量制备sTNFRII-gAD融合蛋白用于体内外功能研究提供了良好基础。     

三重融合PCR法构建感染性辛德毕斯嵌合病毒cDNA克隆

利用三重融合PCR法,即将3个DNA片段放在同一个反应里进行长片段PCR扩增法,融合得到了包含辛德毕斯病毒XJ-160株结构基因E3、E2、6K、3′UTR和辛德毕斯病毒YN87448株结构基因E1的融合DNA片段E3E26KE13′UTR。通过此融合片段两端引入的XbaI和XhoI酶切位点将其连接到XJ-160株的感染性全基因组cDNA克隆骨架上,成功构建了辛德毕斯病毒株XJ-160与YN87448外膜糖蛋白基因E1相互替换的嵌合病毒cD-NA克隆,命名为pBR-XJ160YE1。该克隆线性化后经体外转录,RNA转录体脂质体法转染BHK-21细胞,36h后细胞发生病变。间接免疫荧光检测到病毒蛋白的表达。提取5次传代后细胞上清中病毒RNA,RT-PCR法检测证明病毒来源于嵌合病毒cDNA克隆。此感染性辛德毕斯嵌合病毒cDNA克隆可以作为研究辛德毕斯病毒E1糖蛋白基因相关功能及XJ-160病毒和YN87448病毒存在单方向血清学反应的分子机理的分子生物学工具。     

杆状病毒SpltMNPVSl136基因的克隆、表达及其产物功能

计算机分析斜纹夜蛾核多角体病毒（SpltMNPV)基因组序列,发现第136个读码框基因表达产物具有病毒囊膜蛋白的基本特征,计算机预测的SL136蛋白氨基酸序列N端具有信号肽,C端具有跨膜区,N端区还有一个许多病毒融合蛋白共有的卷曲螺旋结构。通过PCR扩增,我们克隆了Sl136基因并分别构建了原核表达载体pBVSl136及重组病毒表达载体,SDS-PAGE结果表明Sl136基因在大肠杆菌和昆虫细胞中均获得了较高的表达,另外,我们还构建了一个瞬时表达载体pUCSl136。单独转染Sl-zsu-1细胞后,低pH值环境可诱导细胞发生膜融合并形成合胞体,这些实验结果表明,Sl136基因表达的产物是一个病毒膜融合蛋白。     

脑啡肽-干扰素α-m融合蛋白外用治疗单纯疱疹病毒感染

为探讨脑啡肽-干扰素α-m融合蛋白(EI)外用治疗单纯疱疹病毒感染的作用,分别用HSV-1感染兔子角膜、HSV-2感染豚鼠阴道建立动物感染模型.兔子角膜感染24h后用融合蛋白滴眼液治疗,每天3次,每次0.5ml,共14天.豚鼠阴道感染48h后,用融合蛋白涂剂抹外阴病灶,每天3次,每次10mg,共14天.用IFNα-m和生理盐水/赋形剂作为对照.采用记录病损程度分级法进行临床症消减观察,并于治疗前后测定实验动物病灶病毒滴度、HSV抗体滴度及NK细胞活性.结果显示:与IFNα-m相比,EI治疗组实验动物病毒感染症状大幅减轻且病程缩短,动物病灶中病毒滴度下降,抗HSV抗体滴度升高,NK细胞活性增强.说明脑啡肽干扰素融合蛋白具有较强的消除炎症和局部抗病毒作用,可用于治疗HSV感染引起的疾病.     

Yeast homotypic vacuole fusion requires the Ccz1-Mon1 complex during the tethering/docking stage

The function of the yeast lysosome/vacuole is critically linked with the morphology of the organelle. Accordingly, highly regulated processes control vacuolar fission and fusion events. Analysis of homotypic vacuole fusion demonstrated that vacuoles from strains defective in the CCZ1 and MON1 genes could not fuse. Morphological evidence suggested that these mutant vacuoles could not proceed to the tethering/docking stage. Ccz1 and Mon1 form a stable protein complex that binds the vacuole membrane. In the absence of the Ccz1-Mon1 complex, the integrity of vacuole SNARE pairing and the unpaired SNARE class C Vps/HOPS complex interaction were both impaired. The Ccz1-Mon1 complex colocalized with other fusion components on the vacuole as part of the cis-SNARE complex, and the association of the Ccz1-Mon1 complex with the vacuole appeared to be regulated by the class C Vps/HOPS complex proteins. Accordingly, we propose that the Ccz1-Mon1 complex is critical for the Ypt7-dependent tethering/docking stage leading to the formation of a trans-SNARE complex and subsequent vacuole fusion.