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Purification and characterization of an arylamine N-acetyltransferase from Lactobacillus acidophilus. 嗜酸乳杆菌中芳胺n -乙酰转移酶的纯化及特性研究。
Pub Date : 1999-01-01
G W Chen, C F Hung, S H Chang, J G Lin, J G Chung

N-acetyltransferase from Lactobacillus acidophilus was purified by ultrafiltration, DEAE-Sephacel, gel filtration chromatography on Sephadex G-100, and DEAE-5pw on high performance liquid chromatography, as judged by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) on a 12% (w/v) slab gel. The purified enzyme was thermostable at 37 degrees C for 1 h with a half-life of 32 min at 37 degrees C, and displayed optimum activity at 37 degrees C and pH 7.0. The K(m) and Vmax values for 2-aminofluorene were 0.842 mM and 2.406 nmol/min/mg protein, respectively. Among a series of divalent cations and salts, Zn2+, Ca2+, Fe2+, Mg2+, and Cu2+ were demonstrated to be the most potent inhibitors. The enzyme had a molecular mass of 44.9 kD. The three chemical modification agents, iodoacetamide, phenylglyoxal, and diethylpyrocarbonate, all exhibited dose-, time-, and temperature-dependent inhibition effects. Preincubation of purified N-acetyltransferase with acetyl coenzyme A (AcCoA) provided significant protection against the inhibition of iodoacetamide and diethylpyrocarbonate, but only partial protection against the inhibition of phenylglyoxal. These results indicate that cysteine, histidine, and arginine residues are essential for this bacterial activity, and the first two are likely to reside on the AcCoA binding site, but the arginine residue may be located close to the AcCoA binding site. This report is the first demonstration of acetyl CoA:arylamine N-acetyltransferase in L. acidophilus.

采用超滤、DEAE-Sephacel、Sephadex G-100凝胶过滤层析、DEAE-5pw高效液相色谱纯化嗜酸乳杆菌n -乙酰转移酶,采用十二烷基硫酸钠-聚丙烯酰胺凝胶电泳(SDS-PAGE)在12% (w/v)平板凝胶上进行鉴定。纯化后的酶在37℃下耐热1 h,在37℃下半衰期为32 min,在37℃和pH 7.0时表现出最佳活性。2-氨基芴的K(m)和Vmax分别为0.842 mM和2.406 nmol/min/mg蛋白。在一系列二价阳离子和盐中,Zn2+、Ca2+、Fe2+、Mg2+和Cu2+被证明是最有效的抑制剂。酶的分子量为44.9 kD。三种化学改性剂碘乙酰胺、苯乙二醛和焦碳酸二乙酯均表现出剂量、时间和温度依赖的抑制作用。纯化的n -乙酰转移酶与乙酰辅酶A (AcCoA)预孵育对碘乙酰胺和焦碳酸二乙酯的抑制有显著的保护作用,但对苯乙二醛的抑制只有部分保护作用。这些结果表明,半胱氨酸、组氨酸和精氨酸残基对这种细菌的活性至关重要,前两种残基可能位于AcCoA结合位点上,而精氨酸残基可能位于AcCoA结合位点附近。这是首次在嗜酸乳杆菌中发现乙酰辅酶a:芳胺n -乙酰转移酶。
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引用次数: 0
Isolation and characterization of the K6 type yeast killer protein. K6型酵母杀伤蛋白的分离与鉴定。
Pub Date : 1999-01-01
F Izgü, D Altinbay, A K Sağiroğlu

The optimum production of K6 type yeast killer protein by Kluyveromyces fragilis NCYC 587 occurred at pH 4.0-4.4 and at 22-24 degrees C in a killer-zone assay test. The K6 killer protein was concentrated by acetone precipitation of the culture supernatant and purified by native polyacrylamide rod gel electrophoresis. The protein migrated as a single band on discontinuous gradient SDS polyacrylamide gel electrophoresis and had a molecular weight of 42,313. The isoelectric point of the K6 type protein was determined at pH 5.97 by high voltage vertical polyacrylamide gel electrofocusing. Western blot analysis revealed that the K6 killer toxin was a nonglycosylated protein.

在杀伤区试验中,脆弱克鲁维菌NCYC 587在pH 4.0 ~ 4.4和22 ~ 24℃条件下产生K6型酵母杀伤蛋白的最佳条件。用丙酮沉淀培养上清浓缩K6杀伤蛋白,用天然聚丙烯酰胺棒凝胶电泳纯化。该蛋白在不连续梯度SDS聚丙烯酰胺凝胶电泳上呈单条带迁移,分子量为42,313。在pH 5.97条件下,用高压垂直聚丙烯酰胺凝胶电聚焦法测定了K6型蛋白的等电点。Western blot分析显示,K6杀伤毒素是一种非糖基化蛋白。
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引用次数: 0
Purification and partial characterization of a toxic serine protease produced by pathogenic Vibrio alginolyticus. 致病性溶藻弧菌产生的一种有毒丝氨酸蛋白酶的纯化和部分特性。
Pub Date : 1999-01-01
F R Chen, P C Liu, K K Lee

An extracellular lethal toxin produced by Vibrio alginolyticus strain Swy originally isolated from diseased kuruma prawn (Penaeus japonicus) was purified using the AKTA purifier system with hydrophobic interaction chromatography, anion exchange and gel filtration columns. The toxin is an alkaline serine protease, inhibited by phenyl methylsulphonyl fluoride (PMSF), antipain and shows maximal activity at pH 8 to 11, having a pI of 4.3 and a molecular weight of approximately 33 kD. The toxin was completely inhibited by FeCl2 but partially inhibited by 3,4-dichloroisocoumarin (3,4-DCI), ethylenediamine tetraacetic acid (EDTA), ethylene glycol-bis(beta-amino-ethyl ether) N,N,N',N'-tetraacetic acid (EGTA), CuCl2 and ZnCl2. The purified protease was lethal for kuruma prawn at an LD50 of 0.29 microgram protein/g body weight. The haemolymph withdrawn from the moribund prawns injected with the toxic protease was unable to clot. The coagulogen in the kuruma prawn plasma showed an increased migration rate after incubation with this serine protease, and a plasma colour change from blue to pink was recorded. The addition of PMSF completely inhibited the lethal toxicity of the purified protease, indicating that this serine protease was a lethal toxin produced by the bacterium. The 33 kD protease was therefore a toxic protease produced by V. alginolyticus strain Swy.

采用疏水相互作用色谱、阴离子交换柱和凝胶过滤柱相结合的AKTA净化系统,对从日本对虾(Penaeus japonicus)中分离得到的溶藻弧菌Swy产生的胞外致死毒素进行了纯化。该毒素是一种碱性丝氨酸蛋白酶,被苯基甲基磺酰氟(PMSF)抑制,具有镇痛作用,在pH 8 ~ 11时显示出最大活性,pI为4.3,分子量约为33 kD。该毒素被FeCl2完全抑制,但被3,4-二氯异香豆素(3,4- dci)、乙二胺四乙酸(EDTA)、乙二醇-双(β -氨基乙醚)N,N,N',N'-四乙酸(EGTA)、CuCl2和ZnCl2部分抑制。纯化后的蛋白酶对黑虾的致死量为0.29微克蛋白/g体重。从注射了有毒蛋白酶的死虾身上提取的血淋巴不能凝结。用该丝氨酸蛋白酶孵育后,黑斑对虾血浆中的凝血原迁移速度增加,血浆颜色由蓝色变为粉红色。PMSF的加入完全抑制了纯化蛋白酶的致死毒性,表明该丝氨酸蛋白酶是由细菌产生的致死毒素。因此,33 kD蛋白酶是由溶藻弧菌菌株Swy产生的一种有毒蛋白酶。
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引用次数: 0
Liver tissue expression of CD80 and CD95 antigens in chronic hepatitis C: relationship with biological and histological disease activities. 慢性丙型肝炎肝组织中CD80和CD95抗原的表达:与生物学和组织学疾病活动的关系
Pub Date : 1999-01-01
G Fiore, G Piazzolla, V Galetta, L Caccetta, O Schiraldi, S Antonaci

The well known discrepancy between cytotoxic T lymphocyte (CTL) infiltration in the liver and disease biological activity, as assessed by alanine aminotransferase (ALT) levels, during the course of chronic hepatitis C virus (HCV) infection, suggests the possible failure of cytotoxic mechanisms devoted to virus clearance. To further investigate the biological events involved in CTL-mediated lysis, i.e. B7/CD28 costimulatory and Fas/Fas-ligand pathways, the CD80 and CD95 antigen expression in liver tissue specimens from chronically HCV-infected patients was evaluated. The results were analysed in relation to serum ALT values and the histological activity (HAI) of liver disease. The data provide evidence for a strong and comparable hepatocyte CD80 and C95 structure expression in chronically HCV-infected livers. CD80- and CD95-carrying liver cells were more frequently distributed at the periportal region of the hepatic lobule, above all near piecemeal necrosis areas, among infiltrating CTL. On the other hand, a negative correlation was found between liver tissue expression of both antigens and serum ALT activity. The relationship with HAI was not statistically significant. The results imply that HCV infection triggers CD80 and CD95 molecule expression on hepatocytes. Further studies are required to clarify the relevance of such a finding in the context of virus-host interactions.

众所周知,在慢性丙型肝炎病毒(HCV)感染过程中,通过丙氨酸转氨酶(ALT)水平评估的肝脏细胞毒性T淋巴细胞(CTL)浸润与疾病生物活性之间存在差异,这表明用于病毒清除的细胞毒性机制可能失败。为了进一步研究ctl介导的裂解过程中涉及的生物学事件,即B7/CD28共刺激途径和Fas/Fas-配体途径,我们评估了慢性hcv感染患者肝组织标本中CD80和CD95抗原的表达。分析结果与血清ALT值和肝脏疾病的组织学活性(HAI)的关系。这些数据为慢性hcv感染的肝脏中CD80和C95结构的强烈表达提供了证据。在浸润的CTL中,携带CD80和cd95的肝细胞更多地分布在肝小叶的门静脉周围区域,尤其是在零星坏死区域附近。另一方面,肝组织中两种抗原的表达与血清ALT活性呈负相关。与HAI的关系无统计学意义。结果表明,HCV感染可触发肝细胞上CD80和CD95分子的表达。需要进一步的研究来澄清这一发现在病毒-宿主相互作用背景下的相关性。
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引用次数: 0
Effects of a recombinant gene product and growth conditions on plasmid stability in pectinolytic Escherichia coli cells. 重组基因产物及生长条件对果胶溶解大肠杆菌细胞质粒稳定性的影响。
Pub Date : 1999-01-01
Y Tierny, C G Hounsa, J P Hornez

The genes encoding pectin methylesterase (pme) and pectate lyase (pel) from Bacteroides thetaiotaomicron were previously cloned in Escherichia coli. In the absence of selective pressure the recombinant vectors harbouring a functional pel gene were rapidly lost. This instability was due to a toxic effect of the pel gene product when overproduced and was closely related (1) to a decrease of the growth rate, and (2) to the impossibility of transforming different strains of E. coli with the recombinant plasmids harbouring a functional pel gene. When the expression level of the pel gene was reduced and the tet gene partially deleted, the stability was greatly improved. The export of pectate lyase in the extracellular medium was significantly enhanced in the presence of glycine with a positive effect on plasmid stability for low concentrations. Furthermore, using a factorial design at two levels, the effects of tetracycline, ampicillin, glucose and magnesium on pBT4 stability were quantified.

编码果胶甲基酯酶(pme)和果胶裂解酶(pel)的基因先前已在大肠杆菌中克隆。在没有选择压力的情况下,含有功能性pel基因的重组载体迅速丢失。这种不稳定性是由于pel基因产物在过量生产时产生毒性作用,并且与:(1)生长速度下降密切相关,(2)与携带功能pel基因的重组质粒不可能转化不同的大肠杆菌菌株密切相关。当pel基因表达量降低,tet基因部分缺失时,稳定性大大提高。在甘氨酸的存在下,果胶裂解酶在细胞外培养基中的出口显著增强,对低浓度质粒的稳定性有积极影响。此外,采用两个水平的析因设计,量化四环素、氨苄西林、葡萄糖和镁对pBT4稳定性的影响。
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引用次数: 0
Physiological consequences of mutations in Escherichia coli heat shock dnaK and dnaJ genes. 大肠杆菌热休克dnaK和dnaJ基因突变的生理后果。
Pub Date : 1999-01-01
K I Wolska, J Paciorek, K Kardyś

Mutations in the heat shock genes, dnaK and dnaJ, cause severe defects of several cellular functions. Null dnaJ and dnaKdnaJ mutations can be transduced in a restricted range of temperature. The efficiency of transformation with three unrelated plasmids, viz pACYC184, pBR322 and pSC101, is two times lower in dnaK mutants while the dnaJ mutant is characterized by slightly impaired transformation with pSC101 only. The lack of DnaJ function negatively influences the stability of pSC101 at 42 degrees C, and this plasmid cannot be stably maintained at 30 degrees C in the delta dnaKdnaJ mutant. The double deletion mutant, delta dbaKdnaJ, is characterized by impaired osmoadaptation. The galactokinase content is lower in both mutants tested compared with wild-type strains even at 30 degrees C. The efficient complementation of some of these defects by the wild-type alleles present on low-copy number plasmid was achieved.

热休克基因dnaK和dnaJ的突变会导致几种细胞功能的严重缺陷。空dnaJ和dnaKdnaJ突变可以在有限的温度范围内转导。dna ak突变体与三个不相关的质粒pACYC184、pBR322和pSC101的转化效率低2倍,而dnaJ突变体仅与pSC101的转化略有受损。缺乏DnaJ功能会对pSC101在42℃下的稳定性产生负面影响,在delta dnaKdnaJ突变体中,该质粒不能在30℃下稳定维持。双缺失突变体dbaKdnaJ的特征是渗透适应受损。即使在30℃的温度下,两种突变体的半乳糖激酶含量也低于野生型菌株。在低拷贝数质粒上存在的野生型等位基因有效地弥补了这些缺陷。
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引用次数: 0
Adherence of Fusobacterium necrophorum subspecies necrophorum to different animal cells. 坏死性梭杆菌亚种坏死性梭杆菌在不同动物细胞上的粘附。
Pub Date : 1999-01-01
Y Okada, M Kanoe, Y Yaguchi, T Watanabe, H Ohmi, K Okamoto

The adherence of Fusobacterium necrophorum subsp. necrophorum to the surfaces of animal cells was studied in order to elucidate the differences between the bacterial appearance in clinical specimens from various animals. The bacterial cells had a strong affinity for murine and rabbit cheek cell surfaces. The bacterium showed a moderate affinity for goat cells, whereas it adhered not so well to canine, feline, human or porcine cells. Treatment of the bacterial cells with haemagglutinin antiserum prior to the binding assay reduced the degree of attachment to murine and rabbit cells. Scanning electron microscopy revealed that the adherent fusobacteria often penetrated into murine and rabbit cell membranes. These observations indicate that the bacterial attachment contributes to the establishment of the infection in mice and rabbits. It is suggested that the weak binding ability resulted in a low incidence of the bacterium in canine, feline and porcine lesions.

坏死梭杆菌亚种的粘附性。为了阐明不同动物临床标本中细菌外观的差异,对动物细胞表面的Necrophorum进行了研究。细菌细胞对小鼠和兔颊细胞表面有很强的亲和力。这种细菌对山羊细胞有中等程度的亲和性,而对犬、猫、人或猪细胞的亲和性不太好。在结合实验之前用血凝素抗血清处理细菌细胞降低了对小鼠和兔细胞的附着程度。扫描电镜显示黏附的梭杆菌经常穿透小鼠和家兔的细胞膜。这些观察结果表明,细菌附着有助于在小鼠和家兔中建立感染。提示该细菌的弱结合能力导致其在犬、猫和猪病变中的发病率较低。
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引用次数: 0
Postantibiotic effect of ampicillin/sulbactam against mycobacteria. 氨苄西林/舒巴坦对分枝杆菌的抗生素后作用。
Pub Date : 1999-01-01
K Prabhakaran, E B Harris, B Randhawa

The postantibiotic effect (PAE) is an important pharmacodynamic property of antibiotics. Most drugs continue to exert a suppressive effect on the growth of bacteria, both in vitro and in vivo, even after the drug concentrations have fallen below detectable levels. Only limited information is available on the PAE of slow-growing organisms like mycobacteria. The PAE of ampicillin/sulbactam (Unasyn) was investigated against six species of mycobacteria, viz Mycobacterium avium, M. africanum, M. bovis BCG, M. simiae, M. scrofulaceum and M. tuberculosis H37Ra, by spectrophotometry. The cell counter method was also used in one set of experiments. The bacteria were exposed to ampicillin/sulbactam for 2 h, 24 h, 72 h or 7-10 days. Five concentrations, 5, 10, 50 or 100 micrograms/ml, of the drug were tested. Afterwards, the bacteria were washed free of Unasyn and allowed to multiply. Treatment of the mycobacteria for 2 h did not produce any PAE, although 100 micrograms/ml of the drug caused slower growth. Exposure to 50, 60, or 100 micrograms/ml, resulted in a prolonged PAE of approximately 3 days. The data on the PAE of Unasyn may be of clinical relevance in determining dosage regimens of the drug.

抗生素后效应(PAE)是抗生素重要的药效学特性。大多数药物在体内和体外对细菌的生长都有抑制作用,即使在药物浓度降到可检测的水平以下。关于分枝杆菌等生长缓慢的生物体的PAE信息有限。用分光光度法研究了氨苄西林舒巴坦(Unasyn)对6种分枝杆菌(即鸟分枝杆菌、非洲分枝杆菌、牛分枝杆菌、猴分枝杆菌、结核分枝杆菌和结核分枝杆菌H37Ra)的抑菌活性。其中一组实验也采用了细胞计数法。细菌分别暴露于氨苄西林/舒巴坦2 h、24 h、72 h或7-10 d。测试了5、10、50、100微克/毫升五种浓度的药物。之后,将细菌洗净,使其不含Unasyn,并允许其繁殖。分枝杆菌处理2小时不产生任何PAE,尽管100微克/毫升的药物导致生长较慢。暴露于50,60或100微克/毫升,导致PAE延长约3天。有关Unasyn PAE的数据可能对确定药物的剂量方案具有临床相关性。
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引用次数: 0
Amidine derivatives are highly effective against Trypanosoma evansi trypomastigotes. 脒类衍生物对伊氏锥虫有较好的防治效果。
Pub Date : 1999-01-01
L Gomes-Cardoso, A Echevarria, F Aguiar-Alves, A M Jansen, L L Leon

The in vitro effect of N,N'-diphenyl-4-R-benzamidine (where R = H, CN, Br, Cl, CH3, OCH3 and NO2) in three isolates of Trypanosoma evansi was studied. The compounds were solubilized in dimethysulphoxide (DMSO) and tested in a concentration range of 5 to 160 micrograms/ml. The parasites were isolated from a horse, a dog and a coati. They were maintained in immunosuppressed rats, since they could not be cultured in vitro, and further purified through a diethylaminoethanol (DEAE) column. The trypomastigotes obtained were mixed with different concentrations of the drugs and after incubation at 26 degrees C for 24 h, the remaining parasites were counted in a Neubauer chamber. The percentage of inhibition was evaluated compared with the control, without the drugs. Most of the amidine derivatives showed high activity against the three T. evansi isolates, but different patterns of sensitivity to the tested compounds were observed. At least four compounds with Br, Cl, OCH3 and NO2 as substituents, were much more effective than Berenil [4,4'-(diazoamine)-dibenzamidine aceturate], the reference drug used, which is included in the same chemical class of amidines.

研究了N,N'-二苯基-4-R-苯并脒(R = H, CN, Br, Cl, CH3, OCH3和NO2)对3株伊氏锥虫的体外作用。将这些化合物溶于二甲基亚砜(DMSO)中,并在5至160微克/毫升的浓度范围内进行测试。这些寄生虫是从一匹马、一条狗和一只长尾浣熊身上分离出来的。由于它们不能在体外培养,因此在免疫抑制的大鼠体内维持它们,并通过二乙胺乙醇(DEAE)柱进一步纯化。将获得的锥乳线虫与不同浓度的药物混合,在26℃孵育24小时后,在Neubauer箱中计数剩余寄生虫。在不给药的情况下,与对照组比较,评估抑制率。多数胺类衍生物对3种伊氏伊文氏菌均表现出较高的活性,但对被试化合物的敏感性存在差异。至少有四种以Br、Cl、OCH3和NO2为取代基的化合物比参比药Berenil[4,4'-(重氮胺)-醋酸二苯并脒]更有效,后者属于同一类胺类。
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引用次数: 0
Bacterial degradation of hydrocarbons as evidenced by respirometric analysis. 细菌降解碳氢化合物的证据,呼吸分析。
Pub Date : 1999-01-01
I Saadoun, M A al-Akhras, J Abu-Ashour

The microbial biodegradability of mineral oil and other hydrocarbons, namely hexane, decane and tetradecane was determined using the Warburg constant volume respirometer. Results of oxygen uptake indicated that hexane and tetradecane were more degradable than mineral oil and decane. Rhodococcus erythropolis and Erwinia cancerogena showed the highest (0.866) and lowest (0.115) oxygen quotient (Qo2) values, respectively, when exposed to mineral oil. Staphylococcus warneri and Enterobacter cloacae showed the highest (2.895) and (2.816) Qo2 values, respectively, when exposed to hexane; whereas E. cloacae and E. cancerogena showed the lowest Qo2 values (1.289 and 1.824), respectively. Both R. erythropolis and E. cloacae had the highest Qo2 values (2.859 and 2.289), respectively, when exposed to tetradecane. More oxygen was consumed by R. erythropolis than the other bacterial cultures when exposed to all hydrocarbons. In contrast, less oxygen was taken by E. cancerogena than the other bacterial cultures when exposed to all hydrocarbons, except for hexane.

用Warburg定容呼吸计测定了矿物油和其他碳氢化合物(即己烷、癸烷和十四烷)的微生物可降解性。吸氧结果表明,己烷和十四烷比矿物油和癸烷更易降解。矿物油处理的红红球菌和癌弧菌氧商(Qo2)值最高(0.866),最低(0.115)。正己烷对瓦纳里葡萄球菌和阴沟肠杆菌的Qo2值最高,分别为2.895和2.816;而阴沟肠杆菌和癌性肠杆菌的Qo2值最低,分别为1.289和1.824。当暴露于十四烷时,红杉和阴沟肠杆菌的Qo2值最高,分别为2.859和2.289。当暴露于所有碳氢化合物时,红孢霉比其他细菌培养物消耗更多的氧气。相比之下,当暴露于除己烷以外的所有碳氢化合物时,癌变杆菌比其他细菌培养物吸收的氧气少。
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引用次数: 0
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