Soluble Oligomers of the Pore-forming Toxin Cytolysin A from Escherichia coli Are Off-pathway Products of Pore Assembly

The α-pore-forming toxin Cytolysin A (ClyA) is responsible for the hemolytic activity of various Escherichia coli and Salmonella enterica strains. Soluble ClyA monomers spontaneously assemble into annular dodecameric pore complexes upon contact with membranes or detergent. At ClyA monomer concentrations above ∼100 nm, the rate-limiting step in detergent- or membrane- induced pore assembly is the unimolecular reaction from the monomer to the assembly-competent protomer, which then oligomerizes rapidly to active pore complexes. In the absence of detergent, ClyA slowly forms soluble oligomers. Here we show that soluble ClyA oligomers cannot form dodecameric pore complexes after the addition of detergent and are hemolytically inactive. In addition, we demonstrate that the natural cysteine pair Cys-87/Cys-285 of ClyA forms a disulfide bond under oxidizing conditions and that both the oxidized and reduced ClyA monomers assemble to active pores via the same pathway in the presence of detergent, in which an unstructured, monomeric intermediate is transiently populated. The results show that the oxidized ClyA monomer assembles to pore complexes about one order of magnitude faster than the reduced monomer because the unstructured intermediate of oxidized ClyA is less stable and dissolves more rapidly than the reduced intermediate. Moreover, we show that oxidized ClyA forms soluble, inactive oligomers in the absence of detergent much faster than the reduced monomer, providing an explanation for several contradictory reports in which oxidized ClyA had been described as inactive.     

内源荧光法在鉴别细菌表征中的应用

实现对细菌种属快速而高选择性的鉴别和表征在生化检测、临床医学和公共卫生领域变得日趋重要.内源荧光法以其灵敏度高、在线检测时间短、样品前处理简单等优势为微生物鉴别和表征提供了新方法.本文介绍了内源荧光法用于鉴别细菌表征的方法和原理;详细综述了近年来该方法用于食品分析、临床检验、环境监测和防生物恐怖等领域的细菌鉴别、代谢及追踪细菌源的现状和研究进展,并对其应用前景作了展望.     

Tolerance of Ornamental Succulent Plant Crown of Thorns (Euphorbia milli) to Chromium and its Remediation

The potential of an ornamental shrub Crown of thorns (Euphorbia milli) was evaluated for remediation of soil contaminated with Cr. The plant is one of the rare succulent ornamental shrubs with a slow to moderate growth rate and is capable of blooming almost year-round. The plant could tolerate well up to 75 mg of applied Cr and beyond that there was mortality of plants. Though the plant could not be classified as a hyperaccumulator, the plant was still very efficient in translocating Cr from roots to shoots as evident from the data on uptake and translocation efficiency values. The translocation efficiency of over 80% in our study demonstrates that a large proportion of Cr has been translocated to the harvestable biomass of the plant and therefore, this plant could be effectively recommended for the remediation of soils contaminated with low to medium level of contamination i.e., up to 50 mg/kg soil.     

乳酸乳球菌fabZ1和fabZ2基因功能的鉴定

大肠杆菌(Escherichia coli)是Ⅱ型脂肪酸合成系统的模式生物,3-羟基脂酰ACP脱水异构酶(FabA)是不饱和脂肪酸合成中的关键酶.生物信息学分析表明,乳酸乳球菌(Lactococcus lactis)的基因组中没有标注为3-羟基脂酰ACP脱水异构酶的基因,但有两个标注为3-羟基脂酰ACP脱水酶基因LlfabZ1和LlfabZ2,其编码的蛋白质与EcFabZ的相似性分别为41%和45.1%,且都具有3-羟基脂酰ACP脱水酶两个保守的α螺旋结构.用携带LlfabZ1和LlfabZ2的质粒载体遗传互补大肠杆菌fabA温度敏感突变株CY57,在42℃下不能恢复生长,但无细胞抽提物的结果显示LlFabZ1能够使反-2-癸烯酰ACP异构成顺-3-癸烯酰ACP,而LlFabZ2则不能.互补大肠杆菌fabZ突变株HW7显示,在诱导的条件下,含有LlfabZ2的转化子能够恢复生长,而LlfabZ1则不能.体外重建脂肪酸合成反应及蛋白质活性测定表明,LlFabZ1具有3-羟基脂酰ACP脱水异构酶功能,而LlFabZ2只具有3-羟基脂酰ACP脱水酶功能.另外,未得到LlfabZ1和LlfabZ2的突变株,表明LlFabZ1和LlFabZ2可能是乳酸乳球菌脂肪酸合成酶系中的必不可少的关键蛋白.上述结果证实了乳酸乳球菌fabZ1和fabZ2两个基因在脂肪酸合成中的功能.     

Evidence for specificity of cultivable bacteria associated with arbuscular mycorrhizal fungal spores

Bacteria associated with arbuscular mycorrhizal (AM) fungal spores may play functional roles in interactions between AM fungi, plant hosts and defence against plant pathogens. To study AM fungal spore-associated bacteria (AMB) with regard to diversity, source effects (AM fungal species, plant host) and antagonistic properties, we isolated AMB from surface-decontaminated spores of Glomus intraradices and Glomus mosseae extracted from field rhizospheres of Festuca ovina and Leucanthemum vulgare. Analysis of 385 AMB was carried out by fatty acid methyl ester (FAME) profile analysis, and some also identified using 16S rRNA gene sequence analysis. The AMB were tested for capacity to inhibit growth in vitro of Rhizoctonia solani and production of fluorescent siderophores. Half of the AMB isolates could be identified to species (similarity index 0.6) within 16 genera and 36 species. AMB were most abundant in the genera Arthrobacter and Pseudomonas and in a cluster of unidentified isolates related to Stenotrophomonas. The AMB composition was affected by AM fungal species and to some extent by plant species. The occurrence of antagonistic isolates depended on AM fungal species, but not plant host, and originated from G. intraradices spores. AM fungal spores appear to host certain sets of AMB, of which some can contribute to resistance by AM fungi against plant pathogens.     

Snake venom hyaluronidase: An evidence for isoforms and extracellular matrix degradation

The present study attempts to establish the isoforms of hyaluronidase enzyme and their possible role in the spreading of toxins during envenomation. Screening of venoms of 15 snakes belonging to three different families revealed varied hyaluronidase activity in ELISA-like assay, but with relatively similar pH and temperature optima. The zymograms of individual venoms showed varied activity banding patterns and indicated the presence of at least two molecular forms of the enzyme. During envenomation, activity of hyaluronidase is considered crucial for the spreading of toxins and is presumed to distort the integrity of extracellular matrix through the degradation of hyaluronic acid in it. This property has been addressed through localization of hyaluronic acid in human skin and muscle tissue sections using the probe, biotinylated hyaluronic acid binding protein. Faint and discontinuous staining pattern of hyaluronidase treated tissue sections over intense staining of untreated tissue sections confirm the selective degradation of hyaluronic acid in extracellular matrix and thus provide an evidence for the spreading property of the enzyme.     

Effect of bacterial lypopolysaccharide on penicillin-induced seizure activity in rats

In experiments on Wistar rats, we found that, within an early period (2 to 4 h) after injection of bacterial lypopolysaccharide, LPS (0.1 mg/kg, i.p.), the latency of generalized seizures induced by injection of benzylpenicillin (sodium salt; 3.0 million IU/kg, i.p.) became significantly shorter, while the severity of seizure manifestations was higher than in the control group. Within this period, the power of oscillations of the delta and alpha-frequency ranges increased in the frontal cortex and hippocampal structures; fast ECoG components (beta and gamma rhythms) were suppressed, and the power of the theta activity decreased. In the hippocampal structures, these changes were more clearly pronounced, as compared with the neocortex. Within a later period of the action of LPS (12 to 18 h from the moment of injection), the latency of penicillin-induced seizures significantly exceeded the control value, and the severity of such seizures was lower. Under such conditions, we observed a smaller power of the synchronized activity of delta and alpha frequencies combined with intensification of the theta activity (most clearly pronounced in the hippocampal structures), and also an increase in the power of “desynchronized” rhythms (beta and gamma oscillations) in the cortex and hippocampus. Neirofiziologiya/Neurophysiology, Vol. 40, No. 3, pp. 236–241, May–June, 2008.     

B.B.R对严重烧伤大鼠肠道屏障功能的保护作用

目的 :探索 B.B.R(复合生态制剂 )对严重烧伤早期肠道屏障功能的保护作用 ,为防治肠源性感染寻找新途径。方法 :选用健康 Wistar大鼠 13 0只 ,体重 180～ 2 2 0 g,雌雄各半 ,随机分为 B.B.R治疗组 ,BFL(单一双歧杆菌制剂 )治疗组 ,烧伤对照组 (BC)和正常对照组 (NC) ,建立 3 0 % °烫伤肠源性感染的动物模型 ,按时分批活杀取材 ,检测细菌易位率、盲肠膜菌群、血浆内毒素和肠粘膜 s Ig A的含量。结果 :B.B.R和 BFL 组 ,伤后 3 d肠道细菌易位率分别为 10 %、2 0 .7% ,与 BC组 (3 3 % )比较明显降低了细易位率。血浆内毒素 BC组明显高于 NC组 (P>0 .0 5 ) ,而 B.B.R组与 NC组比较 ,差异无显著性 (P<0 .0 1)。BC组盲肠中双歧杆菌数量与 BC组比较明显减少 ,而 B.B.R、BFL 组与 NC组比较差异无显著性 (P>0 .0 5 ) ;NC组肠道中酵母菌和大肠埃希菌与 NC比较明显增加 ,B.B.R组差异无显著性 ,肠粘液 s Ig A水平与上述指标有类似变化。结论 :3 0 % °烫伤大鼠肠道内容物双歧杆菌明显下降 ,大肠埃希菌、酵母菌迅速过度生长 ,导致肠道微生态失衡 ,应用 B.B.R治疗后 ,促进肠粘膜机械屏障功能的恢复 ,减少细菌和内毒素的易位 ,调整肠道微生态平衡 ,提高了肠道局部和全身免疫功能