低温寡营养条件下副溶血弧菌形成活的非可培养状态及其复苏研究

为研究在低温寡营养条件下副溶血弧菌(Vibrio parahaemolyticus)能否进入活的非可培养状态(VBNC),将浓度为1×1010CFU/mL的副溶血弧菌HW799在陈海水中4℃保存,每隔5天取样分别用吖啶橙染色荧光显微镜直接计数法(AODC)、活菌直接计数法(DVC)和涂布平板法(PC)测定细菌总数、活细菌数和可培养细菌数.在第30天时总细菌数基本不变,仍保持在109CFU/mL,活菌数为106CFU/mL,比总菌数低了约三个数量级,可培养细菌数为零,表明绝大部分副溶血弧菌HW799进入了VBNC状态;用扫描电镜、流式细胞仪对副溶血弧菌HW799活的非可培养状态、正常状态以及复苏后的细胞形态的研究表明进入VBNC状态后副溶血弧菌HW799形状变为球状,体积比正常状态明显变小,活细胞数也略有减少;采用在培养液中添加营养物质升温培养的方法,使VBNC状态的副溶血弧菌细胞在48h内复苏为可培养状态,复苏后的副溶血弧菌HW799与正常状态的细菌形态相似.     

空肠弯曲菌活而不可培养状态的产毒素能力及毒素基因表达的检测

目的 通过诱导空肠弯曲菌进入活的不可培养(VBNC)状态建立细胞学模型,用VeroE6细胞和HeLa细胞检测空肠弯曲菌产细胞膨胀性肠毒素(cytotoxic distendingtoxin,CDT)能力的改变,并在RNA水平上进一步验证毒素的表达。方法 采用4℃冷藏及-70℃冷冻的方法诱导空肠弯曲菌进入VBNC状态,利用细胞总数计算、活细胞染色计数及可培养细胞计数间接确证VBNC状态细菌的存在,细胞对于VBNC状态下的细菌仍然敏感,并用反转录-聚合酶链反应(RT-PCR)证实空肠弯曲菌毒素基因的表达。结果 3种不同状态的细胞计数结果表明存活细胞进入了VBNC状态,细菌经4℃冷藏及-70℃冷冻后数量减少约1个数量级,在冷冻前,细菌对细胞毒性较强,72h时70%以上细胞凋亡,冷冻后的VBNC状态细菌对VeroE6和HeLa细胞的生长仍有影响,72h时约有40%细胞进入凋亡状态。结论 低温、贫养条件下空肠弯曲菌可进入VBNC状态,用常规传统的羊血平板培养基仅能使不到10%的空肠弯曲菌复苏生长,从而可以检测,在VBNC状态时,空肠弯曲菌产毒素能力改变较大,远远低于正常状态,但仍具备产毒素的能力,也从RNA水平实验得到证实。     

微生物在水环境中的存活和生长

在灭菌自来水模拟水体中,研究了7种细菌的存活和生长规律。Klebsiella pneumo-niae,Enterobacter aerogenes,Agrobacterium tumefatciens,在7天内平板计数降至0,而水体中镜检细菌总数(AODC)和活菌直接计数(DVC)结果无大变化,说明细菌已变成活的非可培养状态。Micrococcus,flavus 和 Streptococcus faecalis 的可培养菌数也可降至0。Pseudomonas sp.在48小时内由10~5降至10~2cfu/ml,随即升至10~6 cfu/ml 并持续到实验终了(41天)。Bacillus subtilis 在48小时平板计数降至10~2cfu/ml 并维持在该水平至实验结束(38天)。研究结果表明仅用涂布平板法检测多种细菌在水环境中的生存和分布是不合适的。     

氧化胁迫诱导苍白杆菌JP1形成VBNC状态及其特征

石油降解菌在各种有害环境因素作用下会进入活的非可培养(viable but non-culturable, VBNC)状态，从而影响其生长及石油降解率。为了研究有害环境因素对石油降解菌生长及石油降解率的影响，采用分光光度法、荧光染色-激光共聚焦显微镜观测H₂O₂胁迫下苍白杆菌(Ochrobactrum sp.)JP1细胞的生长及VBNC状态形成情况。结果表明，不同浓度H₂O₂对其生长有一定抑制作用，当培养液中H₂O₂浓度为75.0 mmol/L时，可有效抑制苍白杆菌JP1生长，处理12 h后苍白杆菌JP1进入VBNC状态。VBNC状态的苍白杆菌JP1细胞缩小变成球体，周质间隙增大；在适宜条件下，VBNC状态苍白杆菌JP1能够复苏为可培养状态，添加丙酮酸钠能够促进VBNC状态细菌细胞的复苏。复苏后的苍白杆菌RJP1具有良好的环境适应性和石油降解能力，为石油污染生物修复的菌种筛选及应用提供了新的策略。     

绿色荧光蛋白标记的嗜水气单胞菌在温暖水体的存活及稳定性

用绿色荧光蛋白基因(GFP)标记嗜水气单胞菌4332株(Ah4332GFP),检测其在温暖水体中的存活及稳定性.在灭菌自然塘水中,Ah4332GFP存活47d之久,该菌浓度随时间而变化,呈现两个峰值;在非灭菌自然塘水中,Ah4332GFP存活18d,该菌浓度均随时间延长呈下降趋势.表明Ah4332GFP和天然细菌之间的相互作用影响其稳定性.用M9培养基检测Ah4332GFP质粒的稳定性,结果显示在传代培养40、70代后,质粒稳定率分别保持在473%和205%.可见pGFP是一种相对稳定的质粒,Ah4332GFP可用于微生物生态的研究.       

"活的非可培养状态"细菌荧光观察方法

目的：建立一套稳定、简便、快捷,可靠的细菌活的非可培养状态（VBNC）荧光显微镜观察方法。利用市售的黑色钢笔墨水着染微孔滤膜,然后采用手动压片方式,将经LIVE/DEAD BacLightTM Bacterial Viability Kit（13152）染色的细菌样本固定在滤膜上,用落射荧光显微镜直接观察。结果：该法所得的荧光图像背景无光亮且黑暗,菌体荧光、形态、排列等均清晰可见,而且呈现绿色荧光的活菌与呈现红色荧光的死菌较易分辨。与现行技术相比,还能缩短染色时间近12～16h。结论：建立的VBNC细菌荧光观察方法能有效地克服现有技术存在的适用范围狭窄、取材困难、观察结果不准确、操作时间长等缺点,可为细菌VBNC研究提供值得借鉴的方法。     

细菌VBNC态检测技术的研究进展

细菌在一些不良压力条件下会进入"活的非可培养状态"(viable but non-culturable,VBNC),其仍具有活力但不能采用常规的平板培养基进行培养,VBNC态细菌一旦培养条件适宜仍能继续生长繁殖,有些致病菌依旧具有毒力。如果检测方法不当,会造成假阳性或假阴性的结果,需要采取适合的检测方法进行检测。文章概述了几种检测VBNC态细菌的方法,如活菌直接计数法、核酸染料检测法、呼吸检测法、分子生物学法、免疫学法、流式细胞仪检测法等,并对检测方法的应用现状进行了评述。     

细菌有活力但不可培养状态及其机制研究进展

有活力但不可培养(viable but non-culturable,VBNC)状态是细菌遭遇逆境时进入的一种特殊状态,该状态下的菌体在条件适宜时可复苏并恢复其致病性,被认为是细菌躲避不良环境的一种生存策略。VBNC状态菌体对人类医学和工农业生产具有巨大的潜在威胁,开展关于VBNC状态的检测及诱导、复苏及其机制研究可为减少或避免该状态细菌的危害提供理论基础。本文简要综述了细菌VBNC状态在诱导、复苏及致病性等方面的研究进展,并结合本实验室及国内外相关团队近年来在植物病原细菌VBNC状态研究中的结果,详细总结了VBNC状态细菌的形成和复苏机制,对植物病原细菌在环境胁迫下的存活机制、病害田间初侵染来源分析及VBNC状态菌体在病害循环中的作用等相关研究具有重要参考意义。     

细菌“活的不可培养状态”的生态意义及研究进展

"活的不可培养(VBNC)"状态是细菌在不良条件下的一种生存方式.VBNC状态作为细菌的一种生理状态,对传统微生物学产生了深远的影响.进入VBNC状态的细胞发生了一系列变化,无法继续用常规培养方法检测,在医学健康,环境科学等领域产生了巨大的影响,改进检测方法具有重要的意义.本文介绍了进入VBNC状态细菌在DNA、蛋白质组成等方面发生的变化,复苏过程.同时还介绍了VBNC状态的最新检测方法,最后对VBNC状态未来的研究方法进行了讨论.     

微生物VBNC状态形成及复苏机制

99%以上的微生物因处于活的但非可培养(viable but non-culturable,VBNC)状态而无法分离培养。复苏促进因子(resuscitation-promoting factors,Rpfs)是培养获取VBNC菌的最重要突破。结合课题组近十余年从环境功能视角利用Rpf复苏培养VBNC菌的研究,本文在阐述微生物VBNC状态的形成及复苏进展的基础上,从VBNC菌形成及复苏过程出发,探究"探索因子"与群体感应的内在关系。并总结了课题组利用Rpf所复苏培养的具有潜在环境功能的VBNC菌种。本论文将为揭示微生物VBNC状态的形成及复苏机制提供新的思路,并为认识和重新评价Rpf法复苏培养VBNC菌在污染环境微生物修复中的作用提供理论依据。     

Tuning of electronic properties of one-dimensional cyano-bridged Cu-Ni, Cu-Pd, and Cu-Pt bimetallic assemblies by stereochemistry of ligands

Preparations, XPS and electronic spectroscopy, and magnetism of seven new one-dimensional cyano-bridged coordination polymers, chiral [Cu(RR-chxn)₂][Pd(CN)₄] · 2H₂O (1), [Cu(trans-chxn)₂][M(CN)₄] · 2H₂O (2, 4, and 6 for M = Pd, Ni, and Pt), and [Cu(cis-chxn)₂][M(CN)₄] · 2H₂O (3, 5, and 7 for M = Pd, Ni, and Pt) (RR-chxn = cyclohexane-(1R,2R)-diamine, trans-chxn = racemic trans-cyclohexane-(1,2)-diamine, and cis-chxn = racemic cis-cyclohexane-(1,2)-diamine) have been reported in view of tuning of their electronic properties by stereochemistry of chxn ligands and metal-substitution. Comparison of Cu 2p_1/2 and 2p_3/2 peaks of XPS and broad d-d bands around 18 000 cm⁻¹ of electronic spectra are described systematically for 1-7. Variable-temperature magnetic measurement shows that complexes 1-7 indicate weak antiferromagnetic interactions via cyano-bridges. Because of semi-coordination coupled with pseudo Jahn-Teller elongation and electrostatic interaction for 1, the axial Cu-N coordination bond distances of 2.330(7) and 3.092(8) Å are considerably longer than those of equatorial ones in the range from 2.016(6) to 2.030(6) Å. The former bond distances of 1 are intermediate values among the related Ni (2.324(6) and 3.120(8) Å) and Pt (2.34(1) and 3.09(1) Å) complexes.     

Land,livestock, and livelihoods: Changing dynamics of gender,caste, and ethnicity in a Nepalese Village

Over the past 10 years, Ghusel VDC, Lalitpur District has moved from primarily subsistence agriculture into the wider cash economy aided by the Small Farmers' Development Program (SFDP), which provides credit to farmers mainly for the purchase of buffalo for milk production, and by the National Dairy Corporation, which supports local dairy cooperatives. Analysis reveals that buffalo-keeping and milk sales are increasing the well-being of many households, while at the same time creating new inequalities in gender roles and responsibilities, greater inequities between Brahmin and Tamang residents in Ghusel, and placing pressures on the ecosystem for increased supplies of fodder and fuelwood. Evidence suggests that there is critical, need for attention to the social, and particularly gender-based, implications of maintaining livestock for milk sales and to the ecological underpinnings of this livelihood system.     

Structure and function of S-adenosylhomocysteine hydrolase

In mammals, S-adenosylhomocysteine hydrolase (AdoHcyase) is the only known enzyme to catalyze the breakdown of S-adenosylhomocysteine (AdoHcy) to homocysteine and adenosine. AdoHcy is the product of all adenosylmethionine (AdoMet)-dependent biological transmethylations. These reactions have a wide range of products, and are common in all facets of biometabolism. As a product inhibitor, elevated levels of AdoHcy suppress AdoMet-dependent transmethylations. Thus, AdoHcyase is a regulator of biological transmethylation in general. The three-dimensional structure of AdoHcyase complexed with reduced nicotinamide adenine dinucleotide phosphate (NADH) and the inhibitor (1′R, 2′S, 3′R)-9-(2′,3′-dihyroxycyclopenten-1-yl)adenine (DHCeA) was solved by a combination of the crystallographic direct methods program, SnB, to determine the selenium atom substructure and by treating the multiwavelength anomalous diffraction data as a special case of multiple isomorphous replacement. The enzyme architecture resembles that observed for NAD-dependent dehydrogenases, with the catalytic domain and the cofactor binding domain each containing a modified Rossmann fold. The two domains form a deep active site cleft containing the cofactor and bound inhibitor molecule. A comparison of the inhibitor complex of the human enzyme and the structure of the rat enzyme, solved without inhibitor, suggests that a 17° rigid body movement of the catalytic domain occurs upon inhibitor/substrate binding.     

Chiral Separation of G‐type Chemical Warfare Nerve Agents via Analytical Supercritical Fluid Chromatography

Chemical warfare nerve agents (CWNAs) are extremely toxic organophosphorus compounds that contain a chiral phosphorus center. Undirected synthesis of G‐type CWNAs produces stereoisomers of tabun, sarin, soman, and cyclosarin (GA, GB, GD, and GF, respectively). Analytical‐scale methods were developed using a supercritical fluid chromatography (SFC) system in tandem with a mass spectrometer for the separation, quantitation, and isolation of individual stereoisomers of GA, GB, GD, and GF. Screening various chiral stationary phases (CSPs) for the capacity to provide full baseline separation of the CWNAs revealed that a Regis WhelkO1 (SS) column was capable of separating the enantiomers of GA, GB, and GF, with elution of the P(+) enantiomer preceding elution of the corresponding P(–) enantiomer; two WhelkO1 (SS) columns had to be connected in series to achieve complete baseline resolution. The four diastereomers of GD were also resolved using two tandem WhelkO1 (SS) columns, with complete baseline separation of the two P(+) epimers. A single WhelkO1 (RR) column with inverse stereochemistry resulted in baseline separation of the GD P(–) epimers. The analytical methods described can be scaled to allow isolation of individual stereoisomers to assist in screening and development of countermeasures to organophosphorus nerve agents. Chirality 26:817–824, 2014. © 2014 The Authors. Chirality published by John Wiley Periodicals, Inc.     

Influence of Fluoride on Rat Kidney Antioxidant System: Effects of Methionine and Vitamin E

The aim of the study has been to determine and compare the influence upon the kidney antioxidative system, exercised by administration of vitamin E, and vitamin E in combination with methionine, under conditions of oxidative stress induced by sodium fluoride. The experiment was carried out on Wistar FL rats (adult males) that, for 35 days, were administered water, NaF, NaF with vitamin E, or vitamin E with methionine (doses: 10 mg NaF/kg of body mass/24 h, 3 mg vitamin E per 10 μl per rat for 24 h, 2 mg methionine per rat for 24 h). The influence of administered sodium fluoride and antioxidants upon the antioxidative system in kidney was examined by analyzing the concentration of malondialdehyde (MDA) and the activity of the most important antioxidative enzymes (SOD, total and both its isoenzymes, GPX, GST, GR, and CAT). The studies carried out confirmed the disadvantageous effect of the administered dose of NaF upon the antixodiative system in rats (increase in the concentration MDA, decrease activity of all antioxidative enzymes). The administration of vitamin E increased the activity of studied enzymes with the exception of glutathione reductase GR; it also reduced the procesess of lipid peroxidation. It has been found that combined doses of vitamin E and methionine were most effective in inhibiting lipid peroxidation processes. The results confirmed the antioxidative properties of methionine.     

Reorientation of Microfibrils and Microtubules at the Outer Epidermal Wall of Maize Coleoptiles During Auxin-Mediated Growth

Auxin-mediated elongation growth of isolated subapical coleoptile segments of maize (Zea mays L.) is controlled by the extensibility of the outer cell wall of the outer epidermis (Kutschera et al., 1987). Here we investigate the hypothesis that auxin controls the extensibility of this wall by changing the orientation of newly deposited microfibrils through a corresponding change in the orientation of cortical microtubules. On the basis of electron micrographs it is shown that cessation of growth after removal of the endogenous source of auxin is correlated with a relative increase of longitudinally orientated microfibrils and microtubules at the inner wall surface. Conversely, reinduction of growth by exogenous auxin is correlated with a relative increase of transversely orientated microfibrils and microtubules at the inner wall surface. These changes can be detected 30–60 min after the removal and addition of auxin, respectively. The functional significance of directional changes of newly desposited wall microfibrils for the control of elongation growth is discussed.     

Gamma-glutamylcysteine protects ergothioneine-deficient Mycobacterium tuberculosis mutants against oxidative and nitrosative stress

Mycobacterium tuberculosis (M.tb.), the causative agent of tuberculosis (TB), cannot synthesize GSH, but synthesizes two major low molecular weight thiols namely mycothiol (MSH) and ergothioneine (ERG). Gamma-glutamylcysteine (GGC), an intermediate in GSH synthesis, has been implicated in the protection of lactic acid bacteria from oxidative stress in the absence of GSH. In mycobacteria, GGC is an intermediate in ERG biosynthesis, and its formation is catalysed by EgtA (GshA). GGC is subsequently used by EgtB in the formation of hercynine-sulphoxide-GGC. In this study, M.tb. mutants harbouring unmarked, in-frame deletions in each of the fives genes involved in ERG biosynthesis (egtA, egtB, egtC, egtD and egtE) or a marked deletion of the mshA gene (required for MSH biosynthesis) were generated. Liquid chromatography tandem mass spectrometry analyses (LC-MS) revealed that the production of GGC was elevated in the MSH-deficient and the ERG-deficient mutants. The ERG-deficient ΔegtB mutant which accumulated GGC was more resistant to oxidative and nitrosative stress than the ERG-deficient, GGC-deficient ΔegtA mutant. This implicates GGC in the detoxification of reactive oxygen and nitrogen species in M.tb.     

In vitro studies of the rabbit immune system. IV. Differential mitogen responses of isolated T and B cells.

The mitogenic responses of separated rabbit lymphocyte populations functionally analogous to mouse T and B cells have been tested in vitro. Purified T cells were prepared by passage over nylon wool (NW) and purified B cells prepared by treatment with antithymocyte serum and complement (ATS + C). ATS + C kills 70% of peripheral blood lymphocytes (PBL's) and 50% of the spleen cells while passage over NW yields 40% of the applied PBL's and 5–23% of the applied spleen cells. NW-purified T cells from the spleen or PBL's respond fully to concanavalin A (Con A) but have a reduced response to phytohemaglutinin (PHA) and little or no response to goat anti-rabbit immunoglobulin (anti-Ig). PBL's that survive ATS + C (B cells) are stimulated by anti-Ig but not by Con A or PHA. B cells purified from spleen do not respond to Con A or PHA but will respond to anti-Ig under appropriate conditions. A full spleen B-cell response to anti-Ig required removal of Ig produced by the cultures that blocked anti-Ig stimulation. It is concluded that, for rabbit lymphocytes, Con A and PHA are primarily T-cell mitogens and that anti-Ig is primarily a B-cell mitogen. However, the mitogen response of unfractionated PBL or spleen cell populations indicates an overlap in reactivity. This could be due to cells sharing T and B properties, alteration of cell populations by the fractionation procedures used, or recruitment of one population in the presence of a mitogenic response of the other population.     

八种脑-肠肽侧脑室内注射对大鼠基础胃酸分泌的影响

用乌拉坦麻醉大鼠作急性实验,采用连续灌流胃并收集流出液的方法,观察向侧脑室内注射微量脑-肠肽对大鼠基础胃酸分泌的影响。实验结果如下:(1)雨蛙肽、八肽胆囊收缩素、促甲状腺素释放激素及四肽胃泌素均使总酸排出量增加;(2)生长抑素、胰多肽、P 物质、胰高血糖素则使总酸排出量减少;(3)上述肽类用侧脑室注射的剂量作肌肉注射,除四肽胃泌素也产生明显的刺激胃酸分泌作用外,对胃酸分泌均无明显影响。以上结果提示,脑内的一些肽类可能以神经递质或调制物的方式,参与中枢对胃酸分泌的调节。     

A chromium-tolerant plant growing in Cr-contaminated land

In this paper, a kind of Typhaceae plant species, Typha angustifolia L, with a high tolerance to Cr is described. Experiments were carried out to examine its ability to tolerant Cr and its physiological response. The results showed that there was no difference in growth, plant height, and biomass response to external Cr (VI) between the plants exposed to 100 microM Cr (VI) and control (0 microM), while increasing Cr levels to 200-800 microM induced a significant decrease in plant height and biomass, but no significant injury was detected, even for the plants exposed to 800 microM Cr. Chromium induced significant increases in superoxide dismutase (SOD) and peroxidase (POD) activities. Meanwhile, a significantly positive correlation was found between Cr and Mn or Cu in leaves and roots, respectively. The Cr tolerance of the plant appeared to be associated with the enhancement of SOD and POD activities and the improvement in uptake and translocation of the essential microelements.