斜面法与橡皮塞法保藏丝状真菌的效果

对利用斜面法和橡皮塞法保藏某些丝状真菌的效果进行了试验,共保藏丝状真菌29属,69种,128株。保藏时间2-17a,通过斜面转接,其存活率达89.8％。存活的菌株仍维持其原有特性。试验结果表明:利用斜面法与橡皮塞法保藏某些丝状真菌是简便可行的。     

磷酸钙法转染哺乳动物细胞SP2／0及其表达产物检测

将真核细胞表达质粒以磷到钙介导法导入小鼠骨髓瘤细胞ＳＰ２／０转染细胞经超声破碎后,分别以显色法及化学发光法检测其目的蛋白（ＨＢｓＡｇ）,证实化学发光法的检测灵敏度高于目前常用的显色法,且其结果易于保存。对化学发光法的实验条件及磷酸钙转染法的有关影响因素进行了初步探讨。     

天然水中重金属化学形态研究进展

本文介绍了天然水中溶解重金属的形态及其分析方法,着重介绍了阳极溶出伏安法、原子吸收光谱法等重金属形态分析方法,评价了各种分析方法的优缺点并对其进行了展望。     

秋水仙素诱导兰属'素心黄'多倍体的方法研究

采用继代培养法和侧芽萌动培养法比较秋水仙素诱导'素心黄'多倍体的效果.结果表明,继代培养法加倍效果较好,其诱导率为16.7%;细胞学鉴定表明,该方法诱导培育的植株具有多倍体表型特征,其体细胞染色体数2n=80,未见嵌合现象,而对照2n=40.     

鄂西北产鱼腥草提取物体外抗氧化性的四种化学方法评价

采用α-脱氧核糖法、邻苯三酚自氧化法、DPPH法、铁氰化钾还原法等4种国内外常用的体外抗氧化活性检测方法,对鄂西北产鱼腥草石油醚提取物、无水乙醇提取物、乙酸乙酯提取物、三氯甲烷提取物的抗氧化性进行化学评价,结果表明鱼腥草提取物具有一定抗氧化性,其浓度与其抗氧化活性强弱顺序一致。     

两种检测抗生素S632A对Vero细胞毒性作用方法的比较

为检测S632A对细胞的毒性作用,比较了CPE法及MTT法检测其效果,结果可见,2种方法均可证明S632A对细胞的毒性较低,MTT法比CPE法敏感性高。     

白假丝酵母菌体外生物膜药敏性不同检测方法的比较

目的比较不同的方法对白假丝酵母菌体外生物膜药敏性检测的差异。方法分别采用菌落计数法(CFU)、AlamarBlue试剂法、XTT减低法、MTT法对白假丝酵母菌体外48h成熟生物膜的药敏性进行检测,并将AlamarBlue试剂法、XTT减低法、MTT法与CFU法进行比较,观察其相关性及差异性。结果 AlamarBlue试剂法、XTT减低法、MTT法与CFU法都有较高的相关性,相关系数分别为r=0.969、r=0.971、r=0.982(P0.01);与CFU法的差异分别为(0.093±0.127)、(0.054±0.113)、(0.013±0.066),其差异无统计学意义(P0.05)。结论 AlamarBlue试剂法、XTT减低法、MTT法均可替代CFU法对白假丝酵母菌生物膜进行药敏检测。MTT法与CFU法的相关性最大,差异性最小;AlamarBlue试剂作为一种新的试剂,操作简单,对细胞及人类无毒害,更加适合高通量检测。     

牙鲆胚胎冷冻损伤的观察

为了观察牙鲆胚胎在冷冻保存过程中的形态受损情况,将其浸入20%PM（20%丙二醇和20%甲醇1∶1的混合液）中平衡,并用程序化法和玻璃化法对其冷冻保存2h后解冻,用摄影显微镜记录其在抗冻剂里平衡时和冷冻保存后的形态。结果显示在平衡过程中胚胎卵膜出现凹陷（称为溶液损伤）,但可以恢复;在冷冻过程出现胞内冰损伤,是致命的;用程序化法冷冻保存的尾芽期胚胎卵膜和卵黄完好,胚体边缘受伤;用玻璃化法保存的尾芽期胚胎,卵膜和卵黄损伤较重,胚体损伤较轻;因此将玻璃化法和程序化法结合可以达到扬长避短的效果。     

担载血管生长因子的乳液法纤维用于血管再生的研究

目的:制备担载血管生长因子(VEGF)的乳液法静电纺丝纤维膜,对其开展一系列表征,从而研究其血管再生的潜能。方法:通过W/O乳液法制备担载VEGF的静电纺丝纤维膜,并对其形态、力学性质进行表征。用VEGF ELISA分析方法对其体外释放动力学进行研究。运用CCK-8法检测乳液法静电纺丝纤维膜中VEGF的活性变化。结果:乳液法静电纺丝纤维膜呈现连通的三维网状结构,平均直径为1μm,模拟了细胞外基质(ECM),最大拉伸应力为3.03±0.66 M Pa,具有良好的抗拉伸能力,能够支持细胞的生长。乳液法纤维膜中VEGF在体外累积释放了14天,总释放量超过20000 pg,达到血管再生的有效浓度。CCK-8结果显示,乳液法纤维膜中的VEGF仍然保持较高的蛋白活性。结论:担载VEGF的乳液法静电纺丝纤维膜能够缓释出活性的蛋白,具有血管再生的潜能。     

黄胸鼠的年龄鉴定和种群年龄组成

本文经晶体干重法鉴定黄胸鼠的年龄,分析其种群年龄组成,并与其他年龄鉴定方法如体重法、体长法、臼齿磨损法进行了比较。     

Purification of Bacteroides amylophilus protease

A protease was released by Bacteroides amylophilus cells in late stationary phase, approximately 12 hr after maximum cell density was reached. The protease was concentrated by adsorption on diethylaminoethyl (DEAE)-Sephadex and was purified 532-fold by DEAE-Sephadex chromatography, by G-200-Sephadex gel filtration, and by isoelectric focusing. The purified protease was active between pH 4.5 and 12.0 with optima at pH 6.0 and 11.5. Evidence against there being a single protease was given by the differential inhibition of esterase and protease activities by some inhibitors. There was some evidence that only a single protease was present as the ratio of protease activity at various pH values did not alter significantly during purification or when the purified protease was partially heat-inactivated or treated with two specific trypsin-type protease inhibitors: N-alpha-tosyl-l-lysylchloromethyl ketone or phenylmethane-sulfonyl fluoride. Two forms of the same protease were found by acrylamide gel electrophoresis. Gel filtration confirmed the presence of protease in 30,000 and 60,000 molecular-weight forms. Treatment with 1 mm ethylenediaminetetraacetic acid or with 4 m urea failed to convert the 60,000-molecular-weight to the 30,000-molecular-weight species.     

极端嗜盐古生菌(Natrinema sp.)R6-5胞外嗜盐蛋白酶的纯化和性质研究

采用bacitracin-Sepharose 4B亲和层析的方法得到凝胶电泳均一的来自极端嗜盐古生菌(Natrinema sp.)R6-5的胞外嗜盐蛋白酶。经SDS-PAGE分析该酶亚基分子量为62kDa。PMSF对它的活性完全抑制,表明它是一种丝氨酸蛋白酶,该酶反应的最适NaCl浓度为3mol/L,最适温度为45℃,最适pH值为8.0。在高盐条件下能维持高活性并十分稳定,具有重要的潜在应用价值。     

Characterization of 73 kDa thiol protease from Serratia marcescens and its effect on plasma proteins

The 73-kDa protease (73K protease) was purified from a clinical isolate of Serratia marcescens kums 3958. The purified protease appeared homogeneous by sodium dodecyl sulfate polyacrylamide gel electrophoresis in the presence or absence of 2-mercaptoethanol. The protease is active in a broad pH range with maximum activity at pH 7.5-8.0. The protease appeared to be a thiol protease, since it was inhibited by sulfhydryl reactive compounds such as p-chloromercuribenzoic acid, fluorescein mercuric acetate (FMA), iodoacetamide, and N-ethylmaleimide, and the protease activity was enhanced by various reducing agents such as cysteine, glutathione, 2-mercaptoethanol, and dithiothreitol. The protease contained 2 mol of free sulfhydryl residues per mol of protease. When the protease was reacted with FMA, a maximum of 2 mol of FMA per mol of enzyme was found reacted, based on fluorescence quenching in which the enzyme inactivation was paralleled linearly with the loss of both SH groups. This indicates possible equal involvement of the two thiol groups for the enzyme activity. The inactivation of the protease by FMA was partially restored by a dialysis in the presence of cysteine or dithiothreitol. The protease was not inhibited by high molecular weight kininogen but was inhibited by alpha 2-macroglobulin. The protease bound stoichiometrically to alpha 2-macroglobulin with 1:1 molar ratio and 25% activity remained constant even after the addition of 4 molar excess of alpha 2-macroglobulin. The protease extensively degraded IgG, IgA, fibronectin, fibrinogen, and alpha 1-protease inhibitor.(ABSTRACT TRUNCATED AT 250 WORDS)     

Purification and characterization of human T-cell leukemia virus type I protease produced in Escherichia coli

Human T-cell leukemia virus type I (HTLV-I) protease has been purified to homogeneity from a strain of recombinant Escherichia coli. The protease was expressed as a larger precursor, which was autoprocessed to form a mature protease. Protein chemical analyses revealed the coding sequence of mature protease, which agreed with the putative sequence predicted from the sequence of bovine leukemia virus protease. The purified protease processed the natural substrate gag precursor (p53) to form gag p19 and gag p24. The protease activity was inhibited by pepstatin A. These results provide direct evidence that this protease belongs to the aspartic protease family and has an activity consistent with the protease in HTLV-I virion.     

Separation of protease activity from acetylcholinesterase of the electric EEL

We examined the protease activity reported to be associated with acetylcholinesterase (AChE) by extensive purification of the electric eel enzyme. Upon edrophonium-Sepharose chromatography of a commercial preparation, a majority of the protease activity was recovered in the effluent with no AChE activity, while a marginal activity was detected in the AChE fraction eluted with edrophonium chloride. Further chromatography of the edrophonium eluate on hydroxyapatite gave partially overlapping peaks of protease and AChE activities. Finally, the protease activity was mostly removed from the AChE fraction by passing through an ovoinhibitor-agarose column. The protease activity in the edrophonium eluate was inhibited by various serine protease inhibitors, but not by AChE inhibitors. These results suggest that the AChE and protease activities are physically separable, and thus that the protease activity, so far reported as intrinsic to AChE, is probably due to contaminants.     

Cloning and expression in Escherichia coli of the Serratia marcescens metalloprotease gene: secretion of the protease from E. coli in the presence of the Erwinia chrysanthemi protease secretion functions.

The Serratia marcescens extracellular protease SM is secreted by a signal peptide-independent pathway. When the prtSM gene was cloned and expressed in Escherichia coli, the cells did not secrete protease SM. The lack of secretion could be very efficiently complemented by the Erwinia chrysanthemi protease B secretion apparatus constituted by the PrtD, PrtE, and PrtF proteins. As with protease B and alpha-hemolysin, the secretion signal was located within the last 80 amino acids of the protease. These results indicate that the mechanism of S. marcescens protease SM secretion is analogous to the mechanisms of protease B and hemolysin secretion.     

Purification and characterization of a serine protease secreted by Brevibacillus sp. KH3 for reducing waste activated sludge and biofilm formation

A novel protease secreted by Brevibacillus sp. KH3 isolated from excess sludge at 50 °C and used as a sludge-lysing strain was investigated in this study. Sludge reduction was minimized by protease inhibitors and a 40-kDa protease, which significantly contributed to this sludge-reducing activity, was purified as the target protein. The final purified protease demonstrated 92-fold higher specific activity than the initial crude extracts. The sludge-reducing efficiency deteriorated relative to decreased protease activity triggered by EDTA; thus, the purified protease was a causative agent in reducing excess sludge. The 40-kDa protease was a serine metalloprotease and showed the highest activity at 50 °C and pH 8.0, and the activity was enhanced in the presence of calcium ions, indicating that the purified protease contained calcium ion. Furthermore, this 40-kDa protease inhibited biofilm formation in excess sludge. These results imply that sludge reduction is because of reduction of biofilm formation in excess sludge.     

Purification and characterization of a serine protease from Cucumis trigonus Roxburghi

Kachri fruit, Cucumis trigonus Roxburghi, contains high protease activity and has been used as meat tenderizer in the Indian subcontinent. A 67 kDa serine protease from Kachri fruit was purified by DEAE-Sepharose and CM-Sepharose chromatography, whose optimum activity was at pH 11 and 70 degrees C. Its activity was strongly inhibited by PMSF, but not by EDTA, pepstatin, or cysteine protease inhibitors. The substrate specificity of the purified protease towards synthetic peptides was comparable to cucumisin, the first characterized subtilisin class plant protease from the sarcocarp of melon fruit (Cucumis melo). These characteristics, along with the N-terminal amino acid sequence, indicated that the isolated protease from Cucumis trigonus Roxburghi is a cucumisin homologue, which belongs to the serine protease family.     

Inhibition of Protease Production of Various Bacteria by Ammonium Salts: Its Effect on Toxin Production and Virulence

Production of protease by many bacteria was found to be inhibited by ammonium salts, and the enzyme production was more sensitive to the salts than was growth of the organisms. Inhibition of protease production by some pathogenic bacteria may result in the recognition of an exotoxin which otherwise would have been digested by the protease. In the case of Pseudomonas aeruginosa, qualitatively different toxicities could be demonstrated in the culture fluids, depending on the presence or absence of protease in such a fluid. The toxicity of the culture in the presence of a high titer of protease may be due primarily to the protease, whereas the toxicity exhibited in the absence of protease could be due to proteinacious exotoxin. Producers of high titers of protease tended to be less virulent in vivo than producers of low titers of the enzyme, which exert their toxicities by a separate exotoxin.     

Regulation of Exocellular Proteases in Neurospora crassa: Role of Neurospora Proteases in Induction

Cells of Neurospora crassa strain 74A, grown on sucrose for 12 h and transferred to a medium containing protein as sole carbon source, would not produce exocellular protease in significant amounts. When a filtrate from a culture induced to make protease by normal growth on a medium containing protein as principal carbon source was added to an exponential-phase culture in protein medium, exocellular protease was made in amounts similar to those made during normal induction. The material in the culture filtrate that participated in the induction process was identified as protease by its heat lability, molecular weight, and the dependence of induction rate on units of proteolytic activity added to the exponential-phase culture. Induction of the formation of exocellular protease by exponential-phase cells appears to require a protein substrate, added proteolytic activity, and protein synthesis. The protease produced by induced exponential-phase cells was as efficient in promoting induction as normally induced enzyme, whereas constitutive intracellular enzyme was only 50% as efficient. The bacterial protease thermolysin was able to induce exocellular protease at 90.7% of the rate observed with added N. crassa exocellular protease.