小黑杨花粉植株过氧化物酶同工酶分析

1989年冬笔者对已自行加倍的80株小黑杨花粉植株的休眠芽进行了过氧化物酶同工酶分析,发现株间有差异,酶谱可截然分成两类。第一类酶谱与小黑杨原株(花药培养供体)不同,在B带区多了一条带纹即B_2带,此类约占78％。第二类酶谱与小黑杨原株完全相同,此类约占22％。无论株间形态分离还是过氧化物酶同工酶酶谱差异都充分说明这些小黑杨单倍体植株来源于花粉,它们是重组花粉在植株水平上的表现。实验还证明,小黑杨花粉植株休眠芽的过氧化物酶同工酶酶谱是稳定的,可用来鉴定小黑杨花粉植株的无性系,相信用同工酶鉴定杨树无性系会对林业生产起重要作用。     

大熊猫生殖道菌群研究

本文对大熊猫的生殖道微生物菌群进行菌体形态、革兰氏染色、芽孢染色、平板计数等方法以及生理生化测试鉴定。结果显示,大熊猫阴道主要细菌为乳杆菌(Lactobacillus)、大肠埃希氏菌(Escherichia coli)、克雷伯氏菌(Klebsiella),其中优势菌为乳杆菌。     

详解罪魁祸首O104:H4

大肠杆菌学名大肠埃希氏菌(Escherichia coli或E.coli),在生物界的分类为细菌界变形菌门γ-变形菌纲肠杆菌目肠杆菌科埃希氏菌属大肠杆菌种。大肠杆菌潜伏在人体内已有数百万年的历史,甚至在人类远祖还不能称作人类的时候就已经开始了。直到1885年一名叫埃舍利希的德国小儿科医生,在健康婴儿的尿     

细菌的酶抗酶及免疫酶染色法的研究

细菌的酶抗酶(PAP)及免疫酶(IES)染色法是测定抗体或抗原的一种新方法。它操作简单,不须荧光显微镜及分光光度等特殊设备,可直接用肉眼判定。目前国内尚未见到在细菌方面应用的报导。作者自已制作了酶抗酶血清及市     

谷子种质资源抗黑穗病鉴定与过氧化物酶研究

对全国2050份谷子种质资源进行了抗黑穗病鉴定,对不同抗、感黑穗病的谷子种质资源进行了过氧化物酶活性、过氧化物酶同工酶的比较研究。结果表明,谷子不同种质资源对黑穗病的抗性存在着明显的差异。谷子种质资源对黑穗病的抗性比较稳定。谷子感染黑穗病后,抗病品种的过氧化物酶活性明显高于感病品种。过氧化物酶同工酶可以作为一种遗传标记。     

异质性荧光染色的巨噬细胞功能研究──Ⅱ．异质性荧光染色的巨噬细胞过氧化物酶活性

细胞化学研究文献报导,巨噬细胞中过氧化物酶的活性有明显的差异,只有部分巨噬细胞过氧化物酶呈阳性反应。本文用淀粉、白喉类毒素和卡介苗分别诱导和活化小鼠腹腔巨噬细胞,进行过氧化物酶反应,并以７６９０－Ｘｕ荧光染液复染后证明,酶反应阳性细胞呈蓝色荧光,而酶反应阴性细胞为淡蓝绿色和黄色荧光。实验表明,过氧化物酶阳性的巨噬细胞是分化程度低的幼稚细胞,因此,过氧化物酶的活性可作为低分化的巨噬细胞的一种标志酶。同时,本文用免疫荧光单克隆抗体间接染色法观察了三种物质诱导和活化的异质性荧光染色的巨噬细胞的分泌功能。     

大乳头水螅基盘再生进程中过氧化物酶的表达

目的观察大乳头水螅(Hydra magnipapillata)基盘再生进程中基盘过氧化物酶的表达情况,探讨水螅基盘过氧化物酶的生理作用。方法通过ABTS细胞化学染色法显示水螅基盘过氧化物酶的表达。结果水螅基盘再生20h后其基盘过氧化物酶开始出现少量表达,其后过氧化物酶表达量逐渐增加;基盘再生52h后该酶表达量趋于稳定。过氧化物酶仅在基盘周边区域外胚层中表达,而在基盘中央区域(反口孔)外胚层中无表达。结论水螅基盘再生进程中过氧化物酶的表达量逐渐增加直接反映了基盘再生时细胞分化过程,基盘表达的过氧化物酶可能在维持基盘结构的稳定上起一定的作用。     

绿脓杆菌的快速鉴定方法

比较了绿脓杆菌、埃希氏大肠杆菌、摩尔根变形杆菌和几株不发酵葡萄糖的革兰氏阴性杆菌的乙酰胺酶产生情况,证明只有绿脓杆菌产生乙酰胺酶。对临床采集的１０８份标本应用乙酰胺酶法进行了检测,表明６～８小时即得出结果。根据乙酰胺酶检测绿脓杆菌,能够达到快速准确的目的。为临床治疗提供了有利条件。     

生物酶催化聚合的研究进展

酶催化聚合反应是近年来的研究热点之一,氧化还原酶是应用广泛的一类催化聚合酶。概括总结了近年来酶促聚合反应的研究进展,重点叙述了辣根过氧化物酶(HRP)、大豆过氧化物酶(SBP)和漆酶(laccase)的结构、催化机理以及它们在酶催化聚合中的应用概况。     

一种新的DyP-type过氧化物酶在大肠杆菌中的重组表达、纯化及鉴定

染料脱色过氧化物酶(DyP-type过氧化物酶)是含有亚铁血红素,能降解各种有毒染料的一类蛋白.为了研究运动发酵单胞菌Zymomonas mobilis ZM4 (ATCC 31821)中一种新的DyP-type过氧化物酶的特点和功能,以Z.mobilis基因组DNA为模板,通过PCR扩增目的基因,克隆到大肠杆菌表达载体pET-21b(+)中.通过ZmDyP与其他DyP-type过氧化物酶的比对,发现它们存在着共同保守氨基酸D149、R239、T254、F256和GXXDG结构基序,说明ZmDyP是Dyp-type过氧化物酶家族的一个新成员.经IPTG诱导大肠杆菌中pET21 b(+)-ZmDyP表达,并将表达的酶进行金属螯合层析纯化.SDS-PAGE分析表明,纯酶分子量为36 kDa,而活性染色显示分子量为108 kDa,表明该酶在活性状态下可能是一个三聚体.光谱扫描显示ZmDyP有一个典型的亚铁血红素吸收峰,说明它是含有亚铁血红素的蛋白.对ZmDyP性质进行了研究,发现以2,2-二氨-双(3-乙基苯并噻唑-6-磺酸)ABTS为底物,ZmDyP表现出更高的转化效率.这些研究结果丰富了DyP-type 过氧化物酶家族信息,并且为ZmDyP的结构功能和反应机制研究奠定了基础.     

Genetic analysis of Escherichia coli O111:B4, a strain of medical and biochemical interest.

Procedures have been worked out which allow, for the first time, the genetic analysis of Escherichia coli O111:K58:H2 (O111:B4). The approximate map position of mutant loci was determined by mating with 15 Hfr strains of E. coli K-12. In addition, P1 transduction procedures were used for establishing relative gene order and linkage for any region of the E. coli O111:B4 chromosome. To obtain these, it was necessary to select for a rare P1 lysogen since E. coli O111:B4 is resistant to phage P1. Finally, genetic homology between E. coli strains K-12 and O111:B4 is suggested since they can form stable haploid hybrids, and several loci have similar map positions in the two strains.     

<Emphasis Type="Italic">In Vitro</Emphasis> antibacterial effects of antilipopolysaccharide DNA aptamer-C1qrs complexes

DNA aptamers were developed against lipopolysaccharide (LPS) from E. coli O111:B4 and shown to bind both LPS and E. coli by a colorimetric enzyme-based microplate assay. The polyclonal aptamers were coupled to human C1qrs protein either directly using a bifunctional linker or indirectly using biotinylated aptamers and a streptavidin-C1qrs complex. Both systems significantly reduced colony counts when applied to E. coli O111:B4 and K12 strains across a series of 10x dilutions of the bacteria in the presence of human serum; it was diluted 1: 10(3) in order to avoid significant bacterial lysis by the competing alternate pathway of complement activation. A number of candidate DNA aptamer sequences were cloned and sequenced from the anti-LPS aptamer library for future screening of antibacterial or "antibiotic" potential and to aid in eventual development of an alternative therapy for antibiotic-resistant bacterial infections.     

Escherichia coli lipopolysaccharides produce serotype-specific hypothermic response in biotelemetered rats

We investigated whether LPS-induced hypothermia develops in a serotype-specific manner in biotelemetered conscious rats. Two different Escherichia coli serotypes of LPSs were injected at a dose of 250 mug/kg ip. E. coli O55:B5 LPS elicited an initial hypothermia and subsequent fever, but E. coli O111:B4 LPS caused more potent monophasic hypothermia. Serum tumor necrosis factor (TNF)-alpha levels were dramatically elevated at the initial phase of the hypothermia induced by both LPSs. This elevation tended to subside at the nadir of E. coli O55:B5 LPS-induced response but progressively increased at the nadir of E. coli O111:B4 LPS hypothermia. Serum IL-10 levels were moderately elevated at the initial phase of the hypothermia and persisted at the same level at the nadir of each LPS-induced response. No change was observed at the serum IL-18 levels. A selective cyclooxygenase (COX)-1 enzyme inhibitor, valeryl salicylate (20 mg/kg sc), abolished the hypothermia without any effect on the elevated cytokine levels. Another COX-1-selective inhibitor, 5-(4-chlorophenyl)-1-(4-methoxyphenyl)-3-(trifluoromethyl)-1H-pyrazole (SC-560; 1 mg/kg sc) inhibited hypothermic responses as well. Meanwhile, cytokine levels were also reduced by SC-560 treatment. These findings suggest that LPS-induced hypothermia may have serotype-specific characteristics in rats. E. coli O111:B4 LPS has more potent hypothermic activity than E. coli O55:B5 LPS; that may presumably be related to its higher or sustained capability to release antipyretic cytokines, such as TNF-alpha. COX-1 enzyme may be involved in the generation of the hypothermia, regardless of the type of LPS administered.     

Evaluation of parameters affecting quantitative detection of Escherichia coli O157 in enriched water samples using immunomagnetic electrochemiluminescence

We report here the use of immunomagnetic (IM) electrochemiluminescence (ECL) for quantitative detection of Esherichia coli O157:H7 in water samples following enrichment in minimal lactose broth (MLB). IM beads prepared in-house with four commercial anti-O157 monoclonal antibodies were compared for efficiency of cell capture. IM-ECL responses for E. coli O157:H7 (strain SEA13B88) were similar for all four commercial anti-O157 LPS monoclonal antibodies. The ECL signal was linearly correlated with E. coli O157:H7 cell concentration, indicating a constant ECL response per cell. Twenty-two strains of E. coli O157:H7 or O157:NM gave comparable ECL signals using IM beads prepared in-house. To assess the potential for interference from background bacteria in MLB-enriched water samples, 10(4) cells of E. coli O157:H7 (strain SEA13B88) were added to enriched samples prior to analysis. There was considerable variability in recovery of E. coli O157:H7 cells; net ECL signals ranged from 1% to 100% of expected values (i.e., percent inhibition from 0% to 99%). Cultures of Klebsiella pneumoniae, Klebsiella oxytoca, and Enterobacter cloacae, subsequently isolated from MLB-enriched water samples via IM separation (IMS), were observed to interfere with the binding of E. coli O157:H7 cells to IM beads. Recoveries of 10(4) E. coli O157:H7 cells were 相似文献   

HeLa cell invasion by a strain of enteropathogenic Escherichia coli that lacks the O-antigenic polysaccharide

The interaction with HeLa cells of an enteropathogenic Escherichia coli (EPEC) strain and its plasmid-cured derivative strain was examined. An O111:NM EPEC strain B171 harbours a 54 megadalton plasmid (pYR111) necessary for the expression of both localized adherence (LA) to HeLa cells and the O-repeating side chain of the lipopolysaccharide. Under light microscopy, the plasmid-cured derivative strain B171-4 was observed to interact with HeLa cells in a pattern distinct from LA. Transmission electron microscopy showed that the bacteria were internalized by HeLa cells. In contrast, strain B171 induced pedestal-like projections and invaginations of the plasma membrane, but was never completely internalized. A quantitative assay to determine the number of internalized bacteria revealed that strain B171-4 was internalized at levels 30-70-fold higher than those of avirulent E. coli strains. Cytochalasin B reduced the levels of internalization of both strain B171-4 and an enteroinvasive E. coli strain (E11), but did not affect LA by strain B171. These results suggest that EPEC strain B171 may carry a specific chromosomally determined surface factor needed to initiate internalization by HeLa cells. However, a plasmid-determined factor alters the nature of this interaction; the combined effects of the chromosomal and plasmid determinants lead to the characteristic attachment of the bacteria in clusters on the surface of the eukaryotic cell.     

Monoclonal antibodies for detection of the H7 antigen of Escherichia coli.

Two murine monoclonal antibodies (MAbs) (2B7 and 46E9-9) reactive with the H7 flagellar antigen of Escherichia coli were produced and characterized. A total of 217 E. coli strains (48 O157:H7, 4 O157:NM, 23 O157:non-H7, 22 H7:non-O157, and 120 non-O157:nonH7), 17 Salmonella serovars, and 29 other gram-negative bacteria were used to evaluate the reactivities of the two MAbs by indirect enzyme-linked immunosorbent assay (ELISA). Both MAbs reacted strongly with all E. coli strains possessing the H7 antigen and with H23- and H24-positive E. coli strains. Indirect ELISA MAb specificity was confirmed by inhibition ELISA and by Western blotting (immunoblotting), using partially purified flagellins from E. coli O157:H7 and other E. coli strains. On a Western blot, MAb 46E9-9 was more reactive against H7 flagellin of E. coli O157:H7 than against H7 flagellin of E. coli O1:K1:H7. Competition ELISA suggested that MAbs 2B7 and 46E9-9 reacted with closely related H7 epitopes. When the ELISA reactivities of the MAbs and two commercially available polyclonal anti-H7 antisera were compared, both polyclonal antisera and MAbs reacted strongly with E. coli H7 bacteria. However, the polyclonal antisera cross-reacted strongly both with non-H7 E. coli and with many non-E. coli bacteria. The polyclonal antisera also reacted strongly with H23 and H24 E. coli isolates. The data suggest the need to define serotype-specific epitopes among H7, H23, and H24 E. coli flagella. The anti-H7 MAbs described in this report have the potential to serve as high-quality diagnostic reagents, used either alone or in combination with O157-specific MAbs, to identify or detect E. coli O157:H7 in food products or in human and veterinary clinical specimens.     

RAPID detection and quantification of E. coli O157/O26/O111 in minced beef by real-time PCR

AIM: To develop a real-time PCR detection procedure for Escherichia coli O111, O26 and O157 from minced meat. METHODS AND RESULTS: Strains (n = 8) of each of E. coli O26, E. coli O111 and E. coli O157 were inoculated at ca 10-20 CFU g(-1) into minced retail meat and enriched for 6 h at 41.5 degrees C as follows: E. coli O26 in tryptone soya broth (TSB) supplemented with cefixime (50 microg l(-1)), vancomycin (40 mg l(-1)) and potassium tellurite (2.5 mg l(-1)); E. coli O111 in TSB supplemented with cefixime (50 microg l(-1)) and vancomycin (40 mg l(-1)); E. coli O157 in E. coli broth supplemented with novobiocin (20 mg l(-1)). DNA was extracted from the enriched cultures, and detected and quantified by real-time PCR using verotoxin (vt1 and vt2) and serogroup (O157 per gene; O26 fliC-fliA genes and O111 wzy gene) specific primers. CONCLUSIONS: The methods outlined were found to be sensitive and specific for the routine detection of E. coli O111, O26 and O157 in minced beef. SIGNIFICANCE AND IMPACT OF THE STUDY: The enrichment, isolation and detection procedures used in this study provide a rapid routine-based molecular method for the detection and differentiation of E. coli O26, O111 and O157 from minced meat.     

Effects of different serotypes of Escherichia coli lipopolysaccharides on body temperature in rats

The effects of Escherichia coli O55:B5, O127:B8, and O111:B4 serotypes' lipopolysaccharides (LPS) on body temperature were investigated in rats. LPSs were injected intraperitoneally at doses of 2, 50, and 250 microg/kg. A multiphasic and no-dose dependent increase in rectal temperature was observed in response to E. coli O55:B5 LPS at all doses, and in response to E. coli O127:B8 LPS at 2 and 50 microg/kg doses. The highest dose of the latter caused a dual change in rectal temperature, in which hypothermia preceded fever. E. coli O111:B4 LPS was either pyrogenic or hypothermic at 2 and 250 microg/kg doses; respectively, whereas a dual response was observed when the 50 microg/kg dose was injected. Although dual responses were observed after administration of all LPSs at 50 microg/kg dose when the body temperature was recorded by biotelemetry, the hypothermia induced by E. coli O55:B5 LPS was significantly smaller. These data suggest that LPSs induce dose and serotype-specific variable changes on body temperature in rats. This variability may be related to the structure of LPSs. The data also indicate that LPS causes hypothermia with or without fever in rats.     

Effects of growth phase and boiling of enteropathogenic Escherichia coli strains on their interaction with Pseudomonas aeruginosa lectins

Escherichia coli strains from' serotypes O86, 0128 and O111 varied in their reactivity with Pseudomonas aeruginose lectins (PA-I with D-galactose specificity and PA-II which binds L-fucose, D-mannose, L-galactose and D-fructose). Generally, cells of O86 strains were agglutinated by PA-I, but not by PA-II, and those of O128 serotype were agglutinated by PA-II, and not by PA-I. Adsorption tests showed that cells of E. coli O86 strains adsorb PA-I to a greater extent than PA-II, while most E. coli O128 strains adsorbed higher amounts of PA-II. Cells of E. coli O111B4 which were not agglutinated by either Pseudomonas lectin could still adsorb both. Boiling of O86 and O128 cells frequently enhanced their agglutinability as well as their lectin adsorption capacity. The agglutinability enhancement was somewhat more prominent in boiled stationary phase cells than in log phase cells probably due to late synthesis of the O antigen components concomitantly with the heat-sensitive components (K antigens) which masked them. PA-I agglutinating activity was inhibited by the lipopolysaccharide (LPS) extracted from E. coli O86 cells, while PA-II was inhibited by the LPS extracted from E. coli O128 cells. These findings indicate that the receptors to the Pseudomonas lectins probably reside in the terminal part of the O-specific-polysaccharide of the LPSs of these bacteria.     

Optimization of enrichment and plating procedures for the recovery of Escherichia coli O111 and O26 from minced beef

AIM: Optimization of enrichment media and selective agars for the detection of Escherichia coli O26 and O111 from minced beef. METHODS AND RESULTS: This study compared a number of different enrichment conditions and plating media for the recovery of E. coli O26 and E. coli O111 from minced beef. The optimum enrichment conditions for E. coli O26 was observed in beef samples enriched at 41.5 degrees C in tryptone soya broth supplemented with cefixime (50 microg l(-1)), vancomycin (40 mg l(-1)) and potassium tellurite (2.5 mg l(-1)). Similar enrichment conditions were optimal for E. coli O111 with the omission of potassium tellurite. The optimum agar for recovery of E. coli O26 and giving the most effective suppression of contaminants was MacConkey agar [lactose replaced by rhamnose (20 g l(-1))] and supplemented with cefixime (50 microg ml(-1)) and potassium tellurite (2.5 mg l(-1)). Optimum recovery of E. coli O111 was on chromocult agar, supplemented with cefixime (50 microg ml(-1)), cefsulodin (5 mg l(-1)) and vancomycin (8 mg l(-1)). Minced beef samples were inoculated with a number of strains of E. coli O26 (n=9) and O111 (n=8), and the developed enrichment and plating methods, used in combination with immunomagnetic separation, were shown to be an effective method for the recovery of all strains. CONCLUSIONS: Routine cultural methods for the recovery of E. coli O26 and O111 from minced beef are described. SIGNIFICANCE AND IMPACT OF THE STUDY: The optimized enrichment and plating procedure described for the recovery of E. coli O111 and O26 from meat can be used to extend research on these emerging pathogens in beef.