α-Isopropylmalate synthase as a marker for the leucine biosynthetic pathway in several clostridia and in Bacteroides fragilis

-Isopropylmalate synthase (EC 4.1.3.12) is present in extracts of Bacteroides fragilis, Clostridium thermoaceticum, Clostridium formicoacetium, Clostridium pasteurianum, and Clostridium kluyveri with specific activities (mol -isopropylmalate formed per min and g protein) of 8.6, 8.9, 2.4, 1.9, and 0.3, respectively. The product -isopropylmalate was identified by gas chromatography combined with mass spectroscopy. The presence of 5 mM leucine in the growth medium represses the synthesis of -isopropylmalate synthase in C. thermoaceticum by 40 and 70 %. The enzyme from C. pasteurianum was partially purified to a specific activity of 1413. All studied enzyme properties are similar to those of the enzymes from aerobic bacteria. It is suggested that in these anaerobic bacteria the -isopropylmalate pathway is present in addition to the pathway via the ferrodoxin-dependent, reductive carboxylation of branched chain fatty acids.Abbreviations used -KIV -Ketoisovalerate - -IPM -Isopropylmalate - CoA Coenzyme A     

NADH-dependent reduction of d-proline in Clostridium sticklandii. Reconstitution from three fractions containing NADH dehydrogenase,d-proline reductase,and a third protein factor

The enzyme system from Clostridium sticklandii catalyzing the NADH-dependent reduction of d-proline was co-purified by chromatography on DEAE-cellulose at pH 8.2 and ammonium sulfate fractionation, and resolved into fractions containing three different protein components, NADH dehydrogenase, d-proline reductase and a third protein factor, by chromatography on DEAE-cellulose at pH 7.0. Upon recombination of the fractions containing the three different protein components, the NADH-dependent reduction of d-proline was successfully reconstituted. The NADH dehydrogenase fractions oxidized NADH in the presence of artificial electron acceptors, and were inhibited by p-hydroxymercuriphenylsulfonate (50% at 80 nM). They contained 3–4 different enzyme bands as revealed by polyacrylamide-gel electropherograms stained with the NADH-dependent reduction of 2,3,5-triphenyltetrazolium chloride. d-Proline reduction was also coupled to a leuco-methylene blue-generating system containing d-glucose and glucose-oxidase (EC 1.1.3.4). Circumstantial evidence indicated that, among the clostridial proteins, only d-proline reductase and the third protein factor were needed for this reaction.Non-standard abbreviations TTC 2,3,5-triphenyltetrazolium chloride     

Electron microscopic study on the quaternary structure of the isolated particulate alcohol-acetaldehyde dehydrogenase complex and on its identity with the polygonal bodies of Clostridium kluyveri

The alcohol-acetaldehyde dehydrogenase complex of Clostridium kluyveri has been separated from contaminating -hydroxybutyryl-CoA dehydrogenase by repeated precipitation with manganese and ammonium sulfate. Mn⁺⁺ was required for maximum alcohol dehydrogenase activity.The molecular weight of the enzyme complex was 194,000 as determined by sucrose density gradient centrifugation. The enzyme complex has been shown to contain two types of subunits with molecular weights of 55,000±2,600 and 42,000±1,200, respectively which are arranged in H-shaped particles.In solutions with an ionic strength above 25 mM the enzyme complex precipitated in the form of lumps as has been shown with specific ferritin-conjugated antibodies. These lumps are assumed to be aggregated polygonal bodies present in C. kluyveri.     

The improvement of glucose/xylose fermentation by Clostridium acetobutylicum using calcium carbonate

Batch fermentation of 60g/l glucose/xylose mixture by Clostridium acetobutylicum ATCC 824 was investigated on complex culture medium. Different proportions of mixtures, ranged between 10 and 50g of each sugar/l, were fermented during pH control at 4.8 (optimum pH for solventogenesis) or during CaCO₃ addition. Using xylose-pregrown cells and pH control, an important amount of xylose was left over at the end of the fermentation when the glucose concentration was higher than that of xylose. The addition of 10g of CaCO₃/l (to prevent the pH dropping below 4.8) increased xylose uptake: a substantial decrease of residual xylose was observed when xylose-pregrown cells as well as glucose-pregrown cells were used as inoculum for all the mixture proportions studied. MgCO₃ (Mg²⁺-containing compound) and CaCl₂ (Ca²⁺-containing compound) reduced residual xylose only during pH control at 4.8 by NaOH addition. As butanol is the major limiting factor of xylose uptake in C. acetobutylicum, fermentations were carried out with or without CaCO₃ in butanol-containing media or in iron deficient media (under iron limitation, butanol synthesis occurred early and could inhibit xylose uptake). Results showed that an excess of CaCOCaCO₃ could increase butanol tolerance which resulted in an increase in xylose utilization. This positive effect seem to be specific to Ca²⁺- or Mg²⁺-containing compounds, going beyond the buffering effect of carbonate.     

艰难拟梭菌毒素致病机制及毒素基因调控的研究进展

艰难拟梭菌(Clostridioides difficile)是一种产芽孢的革兰氏阳性专性厌氧杆菌,是引起抗生素相关性腹泻的主要致病菌。艰难拟梭菌产生的毒素A和毒素B在其致病过程中发挥关键作用。毒素发挥毒性作用依赖其4个功能结构域：葡萄糖基转移酶结合域、半胱氨酸蛋白酶结合域、易位域和受体结合域。毒素的受体结合域识别并结合细胞表面受体,介导毒素内吞并形成内体。经过自体催化切割,毒素将真正的毒性片段——葡萄糖基转移酶结合域释放到胞浆中,葡萄糖基转移酶能够失活宿主肠上皮细胞内的GTP酶导致细胞骨架解聚和坏死,进而引起腹泻和伪膜性结肠炎等临床症状。艰难拟梭菌毒素产生受致病基因座内及基因座外许多调控因子的调节。tcdR和tcdC基因位于致病基因座内,对毒素基因的表达分别起促进和抑制作用,而基因座外如spo0A、codY等基因则通过抑制tcdR的表达从而间接影响毒素蛋白产生。本文将重点介绍艰难拟梭菌毒素的致病过程和影响毒素基因表达的分子调控机制,以期为开发针对毒素的治疗手段提供新思路。     

艰难梭菌荚膜致病作用的初步研究

本文在我室先前研究的基础上,以3株艰难梭菌的荚膜多糖提取物、全菌悬液以及全菌免疫血清分别在小鼠中进行半数致死量和保护试验等动物实验,以探讨艰难梭菌荚膜的致病作用。结果测得其中一株艰难梭菌的荚膜多糖对小鼠有致死毒性,其LD50为2.83mg,特异性抗血清保护试验证实艰难梭菌免疫血清对动物有保护作用。实验结果可能为进一步研究艰难梭菌荚膜的致病作用提供了参考资料。     

艰难梭菌荚膜多糖的提纯和分析

本文采用改进的丙酮法制备了艰难梭菌荚膜多糖。制备的荚膜多糖纯品含荚膜多糖达72％,在Sepharope-4B凝胶层析中,该英膜多糖的分配系数(Kd)为0.77。经对流免疫电泳法鉴定,纯化后的荚膜多糖仍保持着原性,用制备的艰难梭菌荚膜多糖免疫小白鼠,经间接法ELISA检测,未见抗荚膜多糖抗体明显增高。     

1993年美国疫苗可防性疾病情况及儿童免疫计划

１９９３年美国疫苗可防性疾病情况及儿童免疫计划美国儿童常规接种白喉、ｂ型流感杆菌（Ｈｉｂ）、乙型肝炎、麻疹、腮腺炎、百日咳、脊髓灰质炎、风疹相破伤风这９种疫苗。公共卫生监督和流行病学评估表明儿童接种疫苗对发病率有实质性的影响。１９９３年１２月美国未发...     

牛分枝杆菌32KDa蛋白诱导人细胞毒T细胞的应答

牛分枝杆菌３２ＫＤａ蛋白诱导人细胞毒Ｔ细胞的应答牛分枝杆菌、结核分枝杆菌以及其它分枝杆菌中均存在一高度同源的３２ＫＤ蛋白（Ｐ３２）．人感染结核、麻风病期间及接种ＢＣＧ后均有Ｐ３２表达,提示Ｐ３２是刺激细胞免疫和体液免疫的主要靶抗原,为了验证Ｐ３２在体...     

Toxins A and B of Clostridium difficile

Abstract: The toxins produced by Clostridium difficile share several functional properties with other bacterial toxins, like the heat-labile enterotoxin of Escherichia coli and cholera toxin. However, functional and structural differences also exist. Like cholera toxin, their main target is the disruption of the microfilaments in the cell. However, since these effects are not reversible, as found with cholera toxin, additional mechanisms add to the cytotoxic potential of these toxins. Unlike most bacterial toxins, which are built from two structurally and functionally different small polypeptide chains, the functional and binding properties of the toxins of C. difficile are confined within one large polypeptide chain, making them the largest bacterial toxins known so far.