金黄色葡萄球菌AgrA/C双组分信号转导模型的构建与功能评价

【背景】抗生素的无序使用加剧了耐药性金黄色葡萄球菌超级菌株的出现,由其引发的感染已成为最难解决的感染性疾患。在生物体系外构建AgrA/C双组分系统的跨膜信号转导过程,对解决金黄色葡萄球菌的耐药性问题和发现新型抗菌药物具有重要的研究意义。【目的】人工模拟构建金黄色葡萄球菌AgrA/C双组分信号转导模型,为生物体外研究金黄色葡萄球菌双组分信号转导的机制及以其为靶点的药物筛选提供新途径。【方法】在大肠杆菌宿主细胞中大量表达AgrA和Agr C蛋白,利用亲和层析和分子筛凝胶层析对其进行分离纯化,利用非放射性凝胶阻滞实验(EMSA)检测AgrA蛋白活性,并检测Agr C激酶活性;进而利用脂质体介导法在体外组装AgrA/C双组分信号转导模型,应用EMSA方法进行评价。【结果】分离纯化得到AgrA和Agr C蛋白,二者纯度均达到90%以上,均具有活性。在生物体系外构建了金黄色葡萄球菌AgrA/C双组分信号转导模型,该系统可增强AgrA对DNA的延滞作用,具有信号传递功能。【结论】初步构建AgrA/C双组分信号转导模型,该模型具有信号传递能力,有望作为针对金黄色葡萄球菌开发新型抗菌药物的筛选平台。     

金黄色葡萄球菌基因调节系统研究进展

金黄色葡萄球菌是人类的一种重要病原菌,可以引起许多临床表现不同的感染性疾病。它所致感染的多样性和严重度取决于不同毒力因子的协同表达,而这些数量众多的毒力因子的表达会受到不同调节系统的控制,同时这些调节系统之间也存在着复杂的相互作用关系。这些基因调节系统主要有两大类：一类是双组分信号转导系统（如Agr、SaeRS、SrrAB、ArlSR、LytRS、WalKR）;另一类是转录因子f如Sar、Rot、MgrA、SigmaB）。它们的协同作用有助于金黄色葡萄球菌对外界环境信号做出反应,调节致病过程中毒力因子在不同情况下的表达。     

金黄色葡萄球菌arl双组分信号转导系统受体蛋白ArlS_(CA)的表达、纯化及活性研究

金黄色葡萄球菌是一类重要的病原菌,其毒力因子的表达及分泌过程由多种双组分信号转导系统(two component signal transduction system,TCSTS)共同调控,其中ArlRS双组分信号转导系统与细菌的生长和分裂密切相关。ArlRS双组份系统的信号传递通过组氨酸激酶ArlS磷酸化实现,ArlS的胞内域被认为是调控毒力因子表达的重要功能域,以ArlS蛋白的胞内域部分即ArlS_(CA)为目标蛋白进行相关的活性研究。首先,构建pProEX-HTa-arls和pProEX-HTa-arlr重组质粒,对目的蛋白进行诱导表达。其次,利用金属离子螯合层析、离子交换层析以及凝胶过滤层析方法对目的蛋白进行分离纯化,纯化后的ArlR蛋白纯度可达98%,产量约为25mg/L;纯化后的ArlS蛋白纯度可达90%,产量约为15mg/L。圆二色谱检测结果显示纯化后的目的蛋白有完整的二级结构,体外磷酸化结果显示,ArlS蛋白具有激酶活性,自磷酸化后可以将磷酸基团转移给反应调控蛋白ArlR。最后,利用定点突变的方法,构建了418位和420位氨基酸残基突变的表达载体pProEX-HTa-ArlS_(CAG418A)和pProEX-HTa-ArlS_(CAG420A)。ArlS_(CAG418A)和ArlS_(CAG420A)蛋白不具有激酶活性,说明418位和420位氨基酸残基在ArlS蛋白的自磷酸过程中起着关键作用。     

金黄色葡萄球菌杀白细胞素ED的研究进展

杀白细胞素ED(leukocidin ED,LukED)是金黄色葡萄球菌产生的双组分成孔杀白细胞素之一,由共转录于一条mRNA的lukE和lukD两个基因编码。LukED可与趋化因子受体CCR5结合以杀伤巨噬细胞、T细胞和树突细胞,或与中性粒细胞、单核细胞和自然杀伤(natural kiler,NK)细胞上的表面受体CXCR1/2结合以促进金黄色葡萄球菌的致病性及系统性感染宿主的死亡。此外,LukED还可结合Duffy 抗原趋化因子受体,使裂解红细胞释放血红蛋白,促进细菌的铁吸收和生长繁殖。LukED的表达受双组分信号转导系统附属基因调节子--毒素抑制子(Agr-Rot)通路和转录调节子RpiRc、SpoVG的调控。lukED基因在金黄色葡萄球菌中广泛流行,与金黄色葡萄球菌所致血流感染、脓疱病及抗生素相关性腹泻密切相关。这些进展对了解LukED的表达调控机制、临床意义及其在细菌致病机制中的作用,开发新的金黄色葡萄球菌感染抗毒素治疗药物具有重要意义     

ArlR反应调节蛋白在表皮葡萄球菌不同生长期表达的检测

表皮葡萄球菌(Staphylococcus epidermidis,SE)是寄居在人体和黏膜表面的条件致病菌,因可在医疗植入材料表面形成生物膜(biofilm)而具有致病性。细菌双组分信号转导系统可调控生物膜形成,但其调控机制在SE中研究甚少。本课题对arlRS双组分信号转导系统的反应蛋白ArlR在细菌不同生长期的表达情况进行初步研究。首先构建ArlR原核表达质粒,用纯化重组ArlR免疫小鼠,获得多克隆抗-ArlR抗体,免疫Dot方法检测结果显示小鼠抗-ArlR血清效价>1∶100000。进一步采用蛋白免疫印迹法检测ArlR在SE1457野生株不同生长期中的表达水平,结果显示,ArlR在2h表达量较低,到4h达高峰,6～10h表达量较4h降低。利用反转录实时荧光定量聚合酶链反应检测arlR基因在不同生长期的转录水平,结果显示相应时间点ArlR蛋白表达水平与arlR基因转录水平一致。本研究结果为后期研究双组分信号转导系统arlRS对SE生物膜形成的影响奠定基础。     

基于万古霉素建立荧光酶联免疫吸附法检测金黄色葡萄球菌

以猪IgG作为捕获抗体固定金黄色葡萄球菌,修饰有万古霉素的量子点荧光微球作为"检测抗体",建立荧光酶联免疫吸附法检测金黄色葡萄球菌。文中制备了平均粒径为100 nm的量子点荧光微球并与万古霉素偶联;摸索了反应最佳盐离子浓度为0.01 mol/L,反应最佳pH为6.0。在该实验条件下,金黄色葡萄球菌的检测灵敏度为104 CFU/m L,与其他致病菌无交叉反应。以上结果表明,该方法可用于快速检测金黄色葡萄球菌,为金黄色葡萄球菌的临床监控和食品检测提供参考。     

沙门菌PhoP-PhoQ双组分信号转导系统

PhoP-PhoQ是调控沙门菌毒力的重要双组分信号转导系统,由组氨酸蛋白激酶PhoQ和反应调节蛋白PhoP组成。PhoP-PhoQ可调节沙门菌对Mg2+及其他周质环境的适应性,并调控沙门菌感染中毒力基因的转录和表达。PhoP-PhoQ调控的毒力基因参与沙门菌对上皮细胞的侵袭、胞内生存、对抗菌肽的抵抗反应、脂质A的修饰、Ⅲ型分泌系统效应蛋白的分泌等环节。PhoP-PhoQ还可与其他双组分信号转导系统或调节子合作,调控沙门菌的毒力。因此,PhoP-PhoQ双组分信号转导系统在沙门菌的毒力调控中发挥重要作用。     

以J-Lat 11.1为HIV-1潜伏感染模型再激活效果检测方法的初步探讨

目的:比较3种检测方法的优缺点,探索全面评价人免疫缺陷病毒1型(HIV-1)潜伏感染再激活剂的检测方法。方法:以HIV-1潜伏细胞株J-Lat 11.1为潜伏感染模型、豆蔻酰佛波醇乙酯(PMA)为潜伏再激活剂,用流式细胞术检测绿色荧光蛋白(GFP)阳性细胞所占比例,酶标仪检测GFP表达强度,活细胞成像系统检测GFP的动态表达情况。结果:流式细胞术检测显示PMA再激活出GFP阳性细胞的比例随作用浓度的增加(0~10 nmol/L)而增加,但当PMA浓度高于10 nmol/L后变化不再明显;酶标仪检测显示PMA处理后24 h内,GFP荧光强度逐渐增高,之后可在高水平保持至48 h;活细胞成像系统则可以动态反映PMA处理后的再激活过程。结论:可采用流式细胞术检测GFP阳性细胞比例对HIV-1潜伏感染再激活剂进行初步筛选,再结合酶标仪或活细胞成像系统动态监测GFP的表达情况,综合评价再激活剂的作用强度和起效时间。     

乳酸乳球菌表面展示表达系统的构建及其鉴定

目的:旨在构建一个将抗原靶向于乳酸乳球菌细胞表面的表达系统。方法:运用PCR技术从金黄色葡萄球菌基因组中克隆出蛋白A(SPA)C-末端544个碱基对的锚定域序列(Spax)。通过酶切、连接将Spax构建入分泌型质粒pAMJ399形成携有整合外源基因位点BglⅡ的pAMJ400质粒。将报告蛋白—绿色荧光蛋白的基因(Gfp)插入载体pAMJ400的整合位点产生模式质粒pAMJ401并电转化其于乳酸乳球菌MG1363。绘制转化子MG1363(pAMJ401)生长曲线确认诱导期。调节pH值(6.0~6.5)诱导转化子并在荧光显微镜下观察杂合蛋白(GFP:SPAX)的表达情况。结果:在395nm的蓝色激发光下,诱导后的细菌发出较明亮的绿色荧光,而未诱导的细菌几乎不产生荧光。结论:成功地构建了乳酸乳球菌表面展示表达系统,此系统可以作为口服活菌疫菌研究的可行性操作平台。     

氧化葡萄糖酸杆菌中一种潜在的双组分系统蛋白的功能研究

氧化葡萄糖酸杆菌(Gluconobacteroxydans)基因组编码的蛋白质中,有相当数量的传感器激酶和反应调控蛋白组成了细菌的多个双组分信号转导系统(two-componentsignaltransduction systems, TCSs),这些系统能够介导细菌对外界环境变化做出反应。但目前对G. oxydans中潜在的双组分系统成员蛋白质结构和功能缺少必要的研究【。目的】研究菌株G. oxydans 621H中GOX0645基因序列所编码蛋白质的自磷酸化活性,探究其与细菌趋化性运动的关联,揭示其是否作为一种双组分系统成员蛋白在细胞内发挥作用。【方法】以菌株G. oxydans 621H基因组中一段可能编码双组分系统蛋白质的基因GOX0645为基础,通过生物信息学分析其保守结构域;采用体外化学发光实验证明其编码蛋白的自磷酸化活性;利用基因定点突变筛选出与自磷酸化活性相关的氨基酸位点;通过差速离心法寻找双组分蛋白的亚细胞定位;最后运用体内双分子荧光互补和体外生物大分子相互作用实验印证其与下游鞭毛马达蛋白之间的相互作用。【结果】生物信息学分析发现GOX0645编码蛋白同时具有组氨酸激...     

Distribution of N,P, K,Ca, Mg,S, Zn,Fe, Mn and Cu in groundnut

Summary Concentration of N, P, K, Ca, Mg and S in summer groundnut crop was higher than in kharif while Zn, Fe, Mn and Cu contents were higher in summer crop. Kernel's N, P and Zn; Leaflet's Ca and Mn; Stem's K and Fe; Root's S and Cu and Petiole's Mg contents were highest. Shell's N, P, K, Mg, S, Zn and Cu; Kernel's Ca, Fe and Mn contents were the least. N, P, K, S, Zn and Cu concentrations decreased linearly as the crop grew. Ca, Mg, Fe and Mn concentrations did not display any distinct pattern. Ca concentration was positively correlated with pod yield in both the seasons.     

Uniform hypothesis of cell behavior--movement, contact inhibition of movement, adhesion, chemotaxis, phagocytosis, pinocytosis, division, contact inhibition of division, fusion.

The cell has been represented as a charged liquid drop. Contrary to the DLVO-theory, the effect of the surface potential upon the value of the interfacial tension of the cell membrane has also been taken into consideration. The cell membrane has visco-elastic properties and its constituents may move against each other. Cell movement is caused by the appearance of a small number of the electrically charged constituents of the cell membrane on the leading edge of the cell. This produces a local decrease in the surface tension and the cell membrane expansion. At the moment of contact between two cells proton transfers occur between the strongly negatively charged microvilli of one cell and the body of the other, analogous to a condenser breakdown. This, through the effect on the surface tension, causes contact inhibition of movement. The distribution of the proton dissociable groups modifies the interaction between the cells (differentiation) and between the cell and the substratum (adhesion). Adsorption of the charged compounds at the surface of the cell membrane, decreasing the surface potential and increasing the surface tension, causes the phenomena of chemotaxis, phagocytosis and pinocytosis. Cell division, considered in the terms of the surface energy, requires an adequate supply of considerable quantities of energy inversely proportional to the surface potential value. In case of a reduction of the distance between the cells, their surface potential and the energetic barrier of the cell division processes increases, and causes contact inhibition of cell division. Due to their high charge, division of neoplastic cells is inhibited much later than division of normal cells, or is completely ininhibited due to geometric conditions. Fusion of the cell membrane in the intra-cellular and intercellular processes is a reverse process in relation to the cell division.     

The effect of malathion,diazinon, and various concentrations of zinc,copper, nickel,lead, iron,and mercury on fish

Acute and chronic toxicity tests for malathion, diazinon, copper (Cu), mercury (Hg), lead (Pb), zinc (Zn), nickel (Ni), and iron (Fe) were conducted. Mortalities ofBarilius vagra andCyprinus carpio (common carp) were variable but LC50-96 hr were similar for pesticides. AdultB. vagra seem to be more sensitive to malathion than juvenile carp. Both juvenile carp and adultB. vagra were extremely sensitive to diazinon. Long-term exposure to pesticides modified morphology and behavior. The LC50-96 values for Cu, Hg, and Pb were 0.3, 0.16, and 0.44, respectively, for smaller fish and 1.0, 0.77, and 1.33, respectively, for larger fish. Replicate LC50 values for Zn, Ni, and Fe were somewhat variable, and for these metals, the size of the fish seemed to affect response because LC50 values increased as fish size increased. Cooper, Pb, Zn, and Fe residues following exposure to sublethal concentrations of these metals for 15 d were significantly greater in whole juvenile common carp than in controls.     

Further studies of the sieve-element plastids of theCaryophyllales includingBarbeuia,Corrigiola, Lyallia,Microtea, Sarcobatus,andTelephium

The sieve-element plastids of 69 species of theCaryophyllales were investigated by transmission electron microscopy. All contained the specific subtype-P3 plastids characterized by a peripheral ring of protein filaments. The presence or absence of an additional central protein crystal and their shape being either polygonal or globular as well as the average sizes of the sieve-element plastids are useful features in the characterization of some families.—Barbeuia contains sieve-element plastids that confirm its placement within thePhytolaccaceae. Lyallia differs fromHectorella by including small starch grains in their sieve-element plastids, which otherwise by their globular crystals negate a closer connection to theCaryophyllaceae. The lack of a central protein crystal in its form-P3fs plastids placesMicrotea best within theChenopodiaceae. Sarcobatus, a so far uncontested member of theChenopodiaceae, contains form-P3cf plastids, i.e., including a central crystal not found elsewhere in this family.Telephium andCorrigiola, shifted back and forth betweenMolluginaceae andCaryophyllaceae, have form-P3cf(s) plastids with a polygonal crystal which favor their placement within theCaryophyllaceae.     

Simultaneous measurement of the trace elements Al, As, B, Be, Cd, Co, Cu, Fe, Li, Mn, Mo, Ni, Rb, Se, Sr, and Zn in human serum and their reference ranges by ICP-MS

The goal of this article was to establish reference ranges of the concentration of trace elements in human serum and to compare these results with those reported by other authors. We describe the sample preparation and measurement conditions that allow the rapid, precise, and accurate determination of Al, As, B, Be, Cd, Co, Cu, Fe, Li, Mn, Mo, Ni, Rb, Se, Sr, and Zn in human serum samples (n=110) by inductively coupled plasma-mass spectrometry (ICP-MS). Accuracy and precision were determined by analyzing three reconstituted reference serum samples by comparison with other methods and by the standard addition procedure. The advantages of the ICP-MS method include short time of analysis of the elements mentioned, low detection limit, high precision, and high accuracy. Disadventages include a high risk of contamination due to the presence of some of the elements of interest in the environment, the relatively delicate sample handling, and the high cost of the equipment.     

The Effect of Age and Gender on Al, B, Ba, Ca, Cu, Fe, K, Li, Mg, Mn, Na, P, S, Sr, V, and Zn Contents in Rib Bone of Healthy Humans

The effect of age and gender on major, minor, and trace element contents in the intact rib bone of 80 relatively healthy 15–55-year-old women and men was investigated. Contents or upper limit of contents of 16 chemical elements in the rib bone were determined by inductively coupled plasma atomic emission spectrometry (ICP-AES). Mean values (M?±?SΕΜ) for the mass fraction of Ba, Ca, Cu, Fe, K, Li, Mg, Na, P, S, Sr, and Zn (milligram per kilogram of dry bone) were as follows: 2.54?±?0.16, 171,400?±?4,050, 1.35?±?0.22, 140?±?11, 1,874?±?71, 0.049?±?0.011, 2,139?±?38, 5,378?±?88, 75,140?±?1,660, 1,881?±?51, 291?±?20, and 92.8?±?1.5, respectively. The upper limits of contents of Al, B, Mn, and V were <7.20, <0.65, <0.36, and <0.03, respectively. Statistically significant tendency for the Ca, Mg, and P content to decrease with age was found in the human rib bone, regardless of gender. The mass fraction of Fe in the male rib bone increases with age. It was shown that higher Ca, Mg, Na, P, and Sr mass fractions as well as lower Fe content were typical of female ribs as compared to those in male ribs.     

Synthesis,tautomerism, and antimicrobial,anti-HCV,anti-SSPE,antioxidant, and antitumor activities of arylazobenzosuberones

2-Dimethylaminomethylene-1-benzosuberone 1 was coupled with diazotized aniline derivatives to afford a series of the hitherto unreported 2-arylazo-1-benzosuberones 3a–i. The tautomeric structure and the effect of substituents on the tautomeric form (s) of the products 3a–i were discussed. Similar coupling of the enaminone 1 with diazonium salts of heterocyclic amines gave the respective fused azolotriazino-benzosuberones. Some of the newly synthesized compounds showed potent antimicrobial, anti-HCV, antioxidant, antitumor (as topoisomerase I inhibitors), and antimicrobial activities.     

Definitions,measurements, and classifications of stimuli,situations, and environments

A review is presented of issues relevant to the definition, measurement, and classification of stimuli, situations, and environments. Problems such as the lack of adequate definitions of concepts, error and bias in measurement procedures, confusion between measurement of a concept and measurement of its behavioral effects, and the lack of agreement among alternative measures are emphasized. It is suggested that concepts be defined in terms of objective characteristics while allowing for the study of the transactional relationship between organism and environment. The work of the ethologists in defining stimuli while studying their relationship to different organismic states and situational contexts is emphasized in this regard. Following Brunswik, it is also suggested that wherever possible there be a representative sampling of variables in natural settings. Note from the editors: From time to time, Human Ecology will publish a review article. Our first in this series is a review by a psychologist of basic definitional and conceptual problems in environmental studies.This paper was prepared while the author was a Visiting Research Fellow at the Educational Testing Service. The support of ETS and my colleagues in the Division of Psychological Studies is gratefully acknowledged. The review was also supported in part by a grant from the Rutgers University Research Council.