VirAB在单核细胞增生李斯特菌耐药性及生物被膜形成中的作用

【背景】单核细胞增生李斯特菌(Listeria monocytogenes,Lm)对一些临床常用抗生素、乳酸链球菌素(Nisin)等抗菌药物的敏感性下降,然而其背后的机制仍未完全阐明。【目的】调查转运蛋白VirAB在Lm对抗菌药物的耐药性及生物被膜形成中的作用。【方法】利用同源重组技术构建Lm基因缺失突变株,比较野生株和缺失株对抗菌药物的耐药性;利用微孔板法观测突变株生物被膜形成能力的变化;利用平板泳动法研究菌株的泳动能力。【结果】与野生株相比,virAB缺失突变株对头孢类抗生素、Nisin和溴化乙锭的敏感性增加;当培养基中分别添加亚致死浓度的苯扎氯铵、卡那霉素和四环素时,突变株均表现出不同程度的生长缺陷。缺失virAB后菌株形成生物被膜的能力下降。【结论】VirAB在Lm对头孢类等抗菌药物的耐药及生物被膜形成方面具有重要作用。     

单核细胞增生李斯特菌LadR蛋白对外排泵MdrL的调控机制研究

研究单核细胞增生李斯特菌(Listeria monocytogenes,Lm)调控子LadR对外排泵MdrL的调控作用及方式。采用同源重组技术构建ladR基因缺失菌株ΔladR;通过实时荧光定量PCR检测mdrL基因在野生株EGD-e和突变株ΔladR中的转录水平;构建重组蛋白表达菌株BL21(DE3)-ladR并纯化重组蛋白His6-LadR;通过凝胶阻滞试验检测LadR蛋白与mdrL基因启动子区域的结合活性。成功构建ladR基因缺失菌株ΔladR。与野生株EGD-e相比,突变株ΔladR中mdrL基因的转录水平提高约51倍;成功构建重组蛋白表达菌株BL21(DE3)-ladR,纯化得到浓度为1 mg/mL的重组蛋白His6-LadR;凝胶阻滞实验结果显示,重组蛋白His6-LadR可与mdrL基因的启动子区域特异性结合。Lm的调控子LadR通过与mdrL基因启动子区域特异性结合实现对外排泵MdrL的负调控。     

lpxL和lpxM基因对O1、O78血清型禽致病性大肠杆菌毒力的影响

【目的】为探究脂多糖对O1、O78血清型禽致病性大肠杆菌(Avian pathogenic Escherichia coli,APEC)致病作用的影响。【方法】选取负责脂质A生物合成相关基因lpx L和lpx M,利用λ噬菌体的Red同源重组系统分别构建APECE516(O1血清型)和APECE522(O78血清型)缺失株E516Δlpx L、E516Δlpx M、E516Δlpx LΔlpx M、E522Δlpx L、E522Δlpx M和E522Δlpx LΔlpx M,并通过体内外试验对其生物学特性及致病性进行研究。【结果】各菌株生长速度基本一致。E516Δlpx L、E516Δlpx LΔlpx M、E522Δlpx M和E522Δlpx LΔlpx M的抗血清补体杀菌能力和抗鸡巨噬细胞HD-11吞噬能力较野生株显著下降,而缺失株E516Δlpx M、E522Δlpx L与野生株相比无明显差异;半数致死剂量测定结果显示,除E516Δlpx M、E522Δlpx L外,各缺失株毒力降低1000倍左右;SPF鸡体内动态分布试验结果显示,各缺失株在鸡体内定殖能力较野生株显著下降,但回补株的毒力未能恢复至野生株水平。【结论】lpx L和lpx M基因与O1血清型APECE516株和O78血清型APECE522株的毒力有关,但是lpx L和lpx M基因对E516和E522菌株毒力的影响存在差异。     

甲基营养菌MB200中mutS的缺失及高浓度甲醇和甲醛诱变

【背景】甲基营养菌(Methylobacterium)是一类能够以单碳或非C-C键低碳化合物(如甲烷、甲醇、甲醛等)为底物生长,并可生产多种代谢产物如氨基酸、工业酶和辅助因子、多羟基烷酸酯(polyhydroxyalkanoates,PHA)、多糖和类胡萝卜素等的革兰氏阴性细菌。【目的】通过突变甲基营养菌MB200的mutS基因,在胁迫条件下定向诱导,以获得可以耐受高浓度甲醇和甲醛的生产菌株。【方法】利用三亲本结合构建mutS基因缺失的高突变菌株MB200sTB,逐步提升培养液中甲醇、甲醛的浓度进行定向诱导突变,对获得的高耐受性突变株进行回补,分析菌株的生长情况。【结果】构建了mutS基因的缺失突变体MB200sTB,并且得到了高耐受甲醇和甲醛的菌株MB200sHBc和MB200sHBq。MB200sHBc与野生株MB200相比其甲醇耐受性得到了极显著的提高,甲醇耐受浓度从8g/L提升到44g/L,但生长量不受影响。MB200sHBq在以甲醛为0.45g/L的碳源条件下,生长量相较于野生型MB200提高了1.69倍。【结论】通过定向诱导缺失mutS基因的突变体,可获得具有生产应用潜力的...     

单核细胞增生性李斯特菌prfA基因缺失株的构建及其生物学特性

【目的】单核细胞增生性李斯特菌(Lm)是人兽共患李斯特菌病的病原菌,其致病性与调控因子PrfA蛋白作用下毒力基因的表达有着密切关系,本文初步探讨了PrfA蛋白对细菌毒力因子的调控作用。【方法】利用同源重组技术对血清型分别为1/2a和4b的LM4、F4636进行prfA基因的敲除,并构建其回复突变株,对获得的突变株LM4ΔprfA、F4636ΔprfA进行生物学特性研究。【结果】实验结果表明:两株缺失株的溶血活性丧失、回复突变株的溶血活性得到恢复,突变株还丧失磷脂酶活性,黏附和侵袭特性显著下降(P<0.05),对BALB/c小鼠的半数致死剂量提高了105个数量级。【结论】由此表明,PrfA蛋白对hly、plcB、inl家族基因的表达及细菌毒力具有重要的调控作用。prfA基因缺失株的构建为进一步研究PrfA蛋白的调控功能提供了材料,为研究其在Lm致病性中的作用奠定了基础。     

转录调节因子σ~(38)介导铜绿假单胞菌绿脓菌素合成代谢调控

【目的】为了进一步鉴定铜绿假单胞菌转录调控因子σ~(38)对2个拷贝吩嗪合成基因簇(phz A1-G1和phz A2-G2)的具体调控方式并推定介导绿脓菌素合成代谢的可能调控机制。【方法】根据铜绿假单胞菌基因组信息,利用同源重组原理构建rpo S基因缺失突变株Δrpo S以及克隆全长rpo S基因作互补分析;再以单一吩嗪基因簇缺失突变株Δphz1和Δphz2为出发菌株,分别构建rpo S缺失突变株Δrpo Sphz1和rpo S插入突变株Δrpo Sphz2,测定并比较野生株及相关突变株的绿脓菌素合成量,初步推定σ~(38)因子对2个不同吩嗪基因簇表达的调控方式。【结果】在GA培养基中,突变株Δrpo S的绿脓菌素合成量比野生株显著增加;互补分析证实,σ~(38)可使突变株Δrpo S的绿脓菌素降低并接近野生株PAO1水平;与对照株Δphz1相比,突变株Δrpo Sphz1的绿脓菌素合成量因σ~(38)因子缺失而显著减少;而与对照株Δphz2相比,突变株Δrpo Sphz2的绿脓菌素合成量因σ~(38)因子缺失显著增加。【结论】转录调控因子σ~(38)对铜绿假单胞菌绿脓菌素的合成代谢的确具一定的负调控作用;结合已报道的研究结果,初步推定:σ~(38)因子通过负调控吩嗪基因簇phz1,正调控吩嗪基因簇phz2的表达实现对绿脓菌素合成代谢的调控。     

单增李斯特菌二硫键形成蛋白DsbG介导酸耐受及鞭毛运动性调控北大核心

【目的】本研究旨在通过构建单增李斯特菌(Listeria monocytogenes)二硫键形成蛋白编码基因dsbG缺失株和回补株,探究该蛋白在酸耐受和鞭毛介导的运动性中发挥的生物学功能。【方法】利用同源重组方法构建单增李斯特菌dsbG缺失株和回补株,比较野生株和突变株在不同pH梯度培养基条件下的生长速率和存活率;通过实时荧光定量PCR方法,比较致死酸应激条件下野生株和缺失株酸耐受基因转录水平。通过半固体培养基、荧光定量PCR方法和鞭毛负染色透射电镜观察,比较野生株和突变株的运动能力、鞭毛相关基因转录水平变化和鞭毛形态差异。【结果】与野生株相比,dsbG缺失株的生长能力和速率差异不显著;在pH 3.5(盐酸和柠檬酸)培养条件下,存活率显著降低;精氨酸合成途径基因argD和argF转录水平分别下调2.4和3.7倍。同时,dsbG缺失株的运动能力减弱,且鞭毛形成相关的flaA、flgB和flgD等基因转录水平显著下调(分别为29.7、6.7和6.9倍),鞭毛形成能力减弱。【结论】本研究首次证实了单增李斯特菌二硫键形成蛋白DsbG能感应低pH应激,并形成耐受;证实了DsbG通过调控鞭毛相关基因的转录进而影响细菌的鞭毛形成和运动性。本研究有助于深入了解二硫键形成蛋白家族介导单增李斯特菌环境适应的分子机制,为食源性致病菌的防控提供理论基础。     

多杀性巴氏杆菌aroA基因缺失突变株的构建及鉴定

【目的】构建多杀性巴氏杆菌aroA基因缺失突变株,并验证其致病性。【方法】采用正向筛选同源重组技术构建多杀性巴氏杆菌aroA基因缺失突变株,利用PCR对突变株进行鉴定,分析其遗传稳定性、生长特性和致病性。【结果】成功构建多杀性巴氏杆菌aroA基因缺失突变株,连续传代20代,遗传稳定;突变株体外生长曲线表明,在前6h生长速度稍慢于亲本菌,随后两者生长速度一致。对小鼠的致病性试验表明:经腹腔注射aroA基因缺失突变株在1.0×106 CFU对小鼠无致死性,而亲本菌株在1.0×102 CFU对小鼠是致死性的。【结论】本研究获得多杀性巴氏杆菌aroA基因缺失突变株,对小鼠的致病性是减弱的。多杀性巴氏杆菌突变株的构建有助于研究其致病机理。     

鼠伤寒沙门菌baeSR基因缺失株的构建及其对抗生素敏感性分析

【背景】沙门菌的多重耐药现象日渐严重,对其耐药机理的研究尤为迫切,双组分系统与细菌耐药性密切相关。【目的】构建鼠伤寒沙门菌baeSR基因缺失株及回补株,探究双组分系统BaeSR对鼠伤寒沙门菌耐药性的影响。【方法】以鼠伤寒沙门菌体外诱导耐药株CR为研究对象,通过自杀质粒p LP12介导的同源重组方法,以氯霉素抗性标记和阿拉伯糖诱导的致死基因vmt进行正、反向双重筛选,获得基因缺失株CRΔbaeSR,并将重组表达质粒pBAD-baeSR转化于CRΔbaeSR构建回补株CR CΔbaeSR。采用微量肉汤稀释法测定11种常见代表药物对野生株、缺失株及回补株的最小抑菌浓度(Minimum inhibitory concentration,MIC),并测定3株菌的生长曲线、运动性及生物膜形成能力。【结果】与野生株相比,环丙沙星(CIP)、恩诺沙星(ENR)、沙拉沙星(SAR)、头孢噻呋(CEF)、庆大霉素(GEN)、阿米卡星(AMK)、安普霉素(APR)对缺失株的MIC值有所下降;缺失株的生长速率稍显缓慢,且最终浓度也相对较低,但并无统计学上的显著差异(P0.05);缺失株的运动性(P0.05)及生物膜形成能力(P0.01)均显著下降。【结论】鼠伤寒沙门菌baeSR基因缺失后,可通过影响其运动性及生物膜形成能力而对抗生素的敏感性产生影响。     

集胞藻PCC 6803中丝氨酸/苏氨酸激酶SpkC对高温胁迫的响应

丝氨酸/苏氨酸激酶是蓝藻感知和转导外界刺激的重要元件,但至今蓝藻中很多丝氨酸/苏氨酸激酶的功能尚属未知。【目的】研究集胞藻PCC6803中的丝氨酸/苏氨酸激酶Spk C是否参与对高温胁迫的响应。【方法】本研究采用同源重组的方法构建spC基因完全敲除突变株,检测突变株与野生株在高温胁迫下的生长状况、色素组成,并对高温胁迫下叶绿素荧光参数差异进行分析,比较光合系统Ⅱ活性差异。此外,通过测定生长速率来判断高温胁迫后藻株的恢复情况。【结果】经过42℃高温胁迫后,与野生株相比,突变株ΔspkC生长减缓,光合色素(叶绿素、类胡萝卜素和藻胆色素)的含量降低;45℃高温胁迫下突变株ΔspkC的光合系统Ⅱ活性下降幅度更大;经过5 d 42℃高温处理后,突变株生长几乎停滞,存活率较野生株明显降低。【结论】集胞藻PCC 6803中spkC基因的缺失导致突变株对高温胁迫响应出现缺陷,提示丝氨酸/苏氨酸激酶SpkC参与响应高温胁迫。     

Role for dopamine in malonate-induced damage in vivo in striatum and in vitro in mesencephalic cultures

Defects in mitochondrial energy metabolism have been implicated in the pathology of several neurodegenerative disorders. In addition, the reactive metabolites generated from the metabolism and oxidation of the neurotransmitter dopamine (DA) are thought to contribute to the damage to neurons of the basal ganglia. We have previously demonstrated that infusions of the metabolic inhibitor malonate into the striata of mice or rats produce degeneration of DA nerve terminals. In the present studies, we demonstrate that an intrastriatal infusion of malonate induces a substantial increase in DA efflux in awake, behaving mice as measured by in vivo microdialysis. Furthermore, pretreatment of mice with tetrabenazine (TBZ) or the TBZ analogue Ro 4-1284 (Ro-4), compounds that reversibly inhibit the vesicular storage of DA, attenuates the malonate-induced DA efflux as well as the damage to DA nerve terminals. Consistent with these findings, the damage to both DA and GABA neurons in mesencephalic cultures by malonate exposure was attenuated by pretreatment with TBZ or Ro-4. Treatment with these compounds did not affect the formation of free radicals or the inhibition of oxidative phosphorylation resulting from malonate exposure alone. Our data suggest that DA plays an important role in the neurotoxicity produced by malonate. These findings provide direct evidence that inhibition of succinate dehydrogenase causes an increase in extracellular DA levels and indicate that bioenergetic defects may contribute to the pathogenesis of chronic neurodegenerative diseases through a mechanism involving DA.     

SSTR5 ablation in islet results in alterations in glucose homeostasis in mice

Somatostatin (SST) peptide is a potent inhibitor of insulin secretion and its effect is mediated via somatostatin receptor 5 (SSTR5) in the endocrine pancreas. To investigate the consequences of gene ablation of SSTR5 in the mouse pancreas, we have generated a mouse model in which the SSTR5 gene was specifically knocked down in the pancreatic beta cells (betaSSTR5Kd) using the Cre-lox system. Immunohistochemistry analysis showed that SSTR5 gene expression was absent in beta cells at three months of age. At the time of gene ablation, betaSSTR5Kd mice demonstrated glucose intolerance with lack of insulin response and significantly reduced serum insulin levels. Insulin tolerance test demonstrated a significant increase of insulin clearance in vivo at the same age. In vitro studies demonstrated an absence of response to SST-28 stimulation in the betaSSTR5Kd mouse islet, which was associated with a significantly reduced SST expression level in betaSSTR5Kd mice pancreata. In addition, betaSSTR5Kd mice had significantly reduced serum glucose levels and increased serum insulin levels at 12 months of age. Glucose tolerance test at an older age also indicated a persistently higher insulin level in betaSSTR5Kd mice. Further studies of betaSSTR5Kd mice had revealed elevated serum C-peptide levels at both 3 and 12 months of age, suggesting that these mice are capable of producing and releasing insulin to the periphery. These results support the hypothesis that SSTR5 plays a pivotal role in the regulation of insulin secretion in the mouse pancreas.     

2018年中国植物科学若干领域重要研究进展

2018年中国植物科学继续呈现快速发展的态势, 我国科学家在国际植物科学主流学术刊物发表论文数量大幅增加, 取得了多项具有重要影响的成果。调控植物生长-代谢平衡实现可持续农业发展入选2018年度中国科学十大进展; 中国被子植物区系进化历史研究入选2018年度中国生命科学十大进展。以水稻为代表的农作物和果蔬等经济作物研究在国际上已呈现出明显的优势, 若干领域已从“追赶”状态跨越到“领跑”地位。该文对2018年中国科学家在植物科学若干领域取得的重要研究成果进行了概括性评述, 旨在全面追踪和报道当前中国植物科学领域的发展前沿和热点, 展示中国科学家所取得的杰出成就。     

我国葫芦科植物离体培养研究进展

葫芦科植物包括多种瓜类蔬菜,对其进行离体培养研究具有重要的理论和实践意义。综述了国内在葫芦科植物器官培养、体细胞胚胎发生、花药培养、原生质体培养和体细胞杂交及离体遗传转化等方面取得的研究进展,并对葫芦科植物离体培养、遗传转化与育种的前景作了展望。     

2018年中国植物科学若干领域重要研究进展

2018年中国植物科学继续呈现快速发展的态势, 我国科学家在国际植物科学主流学术刊物发表论文数量大幅增加, 取得了多项具有重要影响的成果。调控植物生长-代谢平衡实现可持续农业发展入选2018年度中国科学十大进展; 中国被子植物区系进化历史研究入选2018年度中国生命科学十大进展。以水稻为代表的农作物和果蔬等经济作物研究在国际上已呈现出明显的优势, 若干领域已从“追赶”状态跨越到“领跑”地位。该文对2018年中国科学家在植物科学若干领域取得的重要研究成果进行了概括性评述, 旨在全面追踪和报道当前中国植物科学领域的发展前沿和热点, 展示中国科学家所取得的杰出成就。     

2017年中国植物科学若干领域重要研究进展

2017年中国植物科学继续保持高速发展态势, 重大成果频出, 具体表现在中国植物学家在国际顶级学术期刊发表的文章数量平稳上升。中国植物科学领域的研究工作者成果精彩纷呈, 如新型广谱抗病机制的发现、水稻广谱抗病遗传基础及机制和疫霉菌诱发病害成灾机制研究等。2017年中国生命科学领域十大进展评选中, 有两项植物科学领域的研究成果入选。水稻生物学、进化与基因组学和激素生物学等领域学科发展突出。另外, 值得一提的是, 长期从事高等植物与代谢途径调控分子网络研究和水稻品种设计育种的李家洋院士的研究成果“水稻高产优质性状形成的分子机理及品种设计”荣获2017年国家自然科学一等奖。这一具有重大国际影响的开创性贡献标志着中国植物科学在该领域的国际科学前沿居于引领和卓越地位。该文对2017年中国本土科学家在植物科学若干领域取得的重要研究成果进行了系统梳理, 旨在全面追踪和报道当前中国植物科学领域发展的最新前沿动态, 与广大读者共同分享我国科学家所取得的辉煌成就。     

Apoptosis in experimental myocardial infarction in situ and in the perfused heart in vitro

We report the appearance of apoptotic cells in experimental myocardial infarction (rabbit heart) in in situ and in vitro preparations. Apoptosis was recognized by intravital staining with Hoechst 33342 (Ho342), by nick-end labeling (TUNEL) and by DNA laddering. A steady rise in the relative number of apoptotic cardiomyocytes (apoptotic index) was noted in in situ preparations. Apoptosis was first noted 6 h after the onset of ischemia with its highest value occurring after 72 h. Apoptotic nuclei were absent in remote areas of the left and right ventricles. Apoptotic nuclei within the infarcted area showed diminished intensity of Ho342 fluorescence. Three days after ischemia, a border zone adjacent to the infarcted area consisting of apoptotic macrophages was recognized. A novel finding was the appearance of apoptotic cardiomyocytes in the isolated perfused ischemic heart. Occurring as early as 50 min after the onset of ischemia, a high apoptotic index was present adjacent to the ligature placed around the coronary artery. This observation provides the opportunity to selectively examine factors leading to apoptosis in the ischemic heart under controlled experimental conditions.     

我国葫芦科植物离体培养研究进展

葫芦科植物包括多种瓜类蔬菜,对其进行离体培养研究具有重要的理论和实践意义.综述了国内在葫芦科植物器官培养、体细胞胚胎发生、花药培养、原生质体培养和体细胞杂交及离体遗传转化等方面取得的研究进展,并对葫芦科植物离体培养、遗传转化与育种的前景作了展望.     

Morphine-potentiated platelet aggregation in in vitro and platelet plug formation in in vivo experiments

The detailed mechanisms underlying morphine-signaling pathways in platelets remain obscure. Therefore, we systematically examined the influence of morphine on washed human platelets. In this study, washed human platelet suspensions were used for in vitro studies. Furthermore, platelet thrombus formation induced by irradiation of mesenteric venules with filtered light in mice pretreated with fluorescein sodium was used for an in vivo thrombotic study. Morphine concentration dependently (0.6, 1, and 5 microM) potentiated platelet aggregation and the ATP release reaction stimulated by agonists (i.e., collagen and U46619) in washed human platelets. Yohimbine (0.1 microM), a specific alpha(2)-adrenoceptor antagonist, markedly abolished the potentiation of morphine in platelet aggregation stimulated by agonists. Morphine also potentiated phosphoinositide breakdown and intracellular Ca(2+) mobilization in human platelets stimulated by collagen (1 microg/ml). Moreover, morphine (0.6-5 microM) markedly inhibited prostaglandin E(1) (10 microM)-induced cyclic AMP formation in human platelets, while yohimbine (0.1 microM) significantly reversed the inhibition of cyclic AMP by morphine (0.6 and 1 microM) in this study. The thrombin-evoked increase in pH(i) was markedly potentiated in the presence of morphine (1 and 5 microM). Morphine (2 and 5 mg/g) significantly shortened the time require to induce platelet plug formation in mesenteric venules. We concluded that morphine may exert its potentiation in platelet aggregation by binding to alpha(2)-adrenoceptors in human platelets, with a resulting inhibition of adenylate cyclase, thereby reducing intracellular cyclic AMP formation followed by increased activation of phospholipase C and the Na(+)/H(+) exchanger. This leads to increased intracellular Ca(2+) mobilization, and finally potentiation of platelet aggregation and of the ATP release reaction.     

Changes in aquatic vegetation in Rhine floodplain streams in Alsace in relation to disturbance

Abstract. Changes are described in aquatic vegetation in oligotrophic, groundwater-fed Rhine floodplain streams in Alsace (eastern France), resulting from disturbance. Disturbance factors include changes in nutrients, either permanent ones - effluent from a waste water treatment plant or trout hatcheries - or periodic ones: flooding. Regular inputs of high levels of phosphate and ammonia modified the macrophyte vegetation in these streams. The floristic composition, which was characteristic of oligotrophic waters upstream of the eutrophicated sector, changed to that of a eutrophic situation as originally found downstream. Periodic disturbance by floods which normally occur once a year, irregularly eutrophicates the small streams, causing the development of a mixture of eutrophic and oligotrophic species. Six macrophyte communities are distinguished, indicating different trophic levels. The aquatic vegetation is adapted to the variations of phosphate and ammonia levels. Hence, aquatic macrophytes can be used as bio-indicators of fluctuations in water nutrient levels in relation to the type of disturbance.