细菌抗氧化系统-oxyR调节子研究进展

细菌抗氧化系统是细菌抵抗呼吸作用及环境因素导致的氧化损伤的一套防卫系统.oxyR调节子是最早发现的具有抗氧化作用的系统之一,由OxyR调节蛋白的编码基因oxyR及其调控的基因和操纵子所构成.oxyR调节子参与了细菌的抗氧化作用、抑制自发突变、致病性、铁代谢及外膜蛋白相变等多种生理代谢作用,这些发现促进了该调节子在细菌耐药性以及致突变物质筛查等方面的研究应用.作者主要从细菌oxyR调节子的结构组成、参与的生理代谢作用、OxyR调控转录的分子机制及影响因素等方面结合最新研究成果展开了介绍,以期对开展细菌抗药性研究及致突变物质的筛查等提供参考.     

Invasion of Cryptococcus neoformans into human brain microvascular endothelial cells requires protein kinase C-alpha activation

Pathogenic fungus Cryptococcus neoformans has a predilection for the central nervous system causing devastating meningoencephalitis. Traversal of C. neoformans across the blood–brain barrier (BBB) is a crucial step in the pathogenesis of C. neoformans . Our previous studies have shown that the CPS1 gene is required for C. neoformans adherence to the surface protein CD44 of human brain microvascular endothelial cells (HBMEC), which constitute the BBB. In this report, we demonstrated that C. neoformans invasion of HBMEC was blocked in the presence of G109203X, a protein kinase C (PKC) inhibitor, and by overexpression of a dominant-negative form of PKCα in HBMEC. During C. neoformans infection, phosphorylation of PKCα was induced and the PKC enzymatic activity was detected in the HBMEC membrane fraction. Our results suggested that the PKCα isoform might play a crucial role during C. neoformans invasion. Immunofluorescence microscopic images showed that induced phospho-PKCα colocalized with β-actin on the membrane of HBMEC. In addition, cytochalasin D (an F-filament-disrupting agent) inhibited fungus invasion into HBMEC in a dose-dependent manner. Furthermore, blockage of PKCα function attenuated actin filament activity during C. neoformans invasion. These results suggest a significant role of PKCα and downstream actin filament activity during the fungal invasion into HBMEC.     

Antibodies differentiate desmosome-form and nucleus-form pinin: evidence that pinin is a moonlighting protein with dual location at the desmosome and within the nucleus.

Pinin is a desmosome-associated protein occurring in epithelia, cardiac muscle, and meninges. This molecule was found to be capable of enhancing cell junction formation and thought to play a key role in reorganization and stabilization of the desmosome-intermediate filament complex in epithelial cells (J. Cell Biol. (1996) 135, 1027-1042). Recently a protein, claimed to be localized exclusively in the nucleus, however, with amino acid sequence identical to pinin, was reported (E. J. Cell Biol. (1998) 75, 295-298). Here I present evidence that pinin exists simultaneously at the desmosome and within the nucleus by generating location-specific monoclonal antibodies. Although the desmosome-form (d-form) and the nucleus-form (n-form) pinin share identical amino acid sequences as demonstrated by cDNA library screening and DNA sequencing, they exhibit remarkably different biochemical properties, reflecting the apparent different multiprotein nature of their differential cellular locations. In addition, the d-form pinin is characterized by a dynamic transport process which involves the gradual diminishing of nuclear materials relative to enhanced anchoring of pinin to the desmosome upon mature cells. Finally I demonstrate that pinin exists in two forms of different gene product: pinin1 and pinin2. These data argue strongly against the statement that pinin is an exclusive nuclear protein and support the notion that pinin is a moonlighting protein with more than one function as a consequence of its dual cellular location.     

慢性缺氧对大鼠肺内皮素表达的影响

本文以ＡＢＣ法和原位杂交技术,观察了慢性缺氧时大鼠肺组织内内皮素－１（ＥＴ－１）的表达情况,结果发现：①正常肺血管内皮细胞有少许ＥＴ－１样阳性染色物质呈现。②缺氧ＩＷ后,肺内ＥＴ－１含量增加,主要位于肺血管内皮细胞和支气管粘膜上皮细胞。③缺氧２Ｗ和３Ｗ后,ＥＴ１阳性免疫物质进一步增加,于肺泡细胞内也见到阳性染色。④缺氧１Ｗ后肺内ＥＴ－１ｍＲＮＡ表达增加,缺氧２Ｗ和３Ｗ后,ＥＴ－１ｍＲＮＡ的表达进一步加强。提示缺氧可刺激肺内ＥＴ－１ｍＲＮＡ的表达,慢性缺氧时肺内ＥＴ－１持续分泌增加,这可能是缺氧性肺动脉高压发生的重要因素之一。     

在基于BAC的EB病毒基因组中引入突变

为了在Epstein-Barr病毒(EBV)172kb的基因组中引入突变以研究基因功能,建立了一种简单有效的基因操作方法.在载体pcDNA3.1( )上操作,将两端含有重组蛋白FLP识别位点(FRT)的卡那霉素筛选标记基因(kan)与鼻咽癌(NPC)来源的、包含LMP1基因全长ORF的gDNA"无缝"连接(无外源序列插入).连接后的kan-LMP1线性DNA片段经转化、由λ噬菌体中redαβγ系统介导在E.coli中发生同源重组(ET克隆),用kan-LMP1替代了BAC-EBV(p2089)中相应的LMP1基因区域,然后经过重组蛋白FLP对FRT-kan-FRT特异性的识别,切除了引入的kan基因,留下一个69bp的FRT"疤痕".通过抗性筛选和对菌液进行PCR扩增可以鉴定突变子.这种经改进并程序化的方法.也适应于引入其它突变或在其它BAC-疱疹病毒基因组中引入突变.     

臭氧浓度升高对香樟叶片光合色素及抗过氧化的影响及其氮素响应

为阐明氮(N)沉降条件下我国亚热带典型常绿树种香樟(Cinnamomum camphora)的臭氧(O3)胁迫响应特征,以1年生幼苗为试验对象,采用开顶气室(OTC)进行模拟研究。O3处理设大气O3(AA)、大气O3+60 nmol/mol(AA+60)、大气O3+120nmol/mol(AA+120)3个水平;N素以硝酸铵(NH4NO3)溶液形式施加,施N量设0(N0)、30(N30)、60(N60)(kg.hm-.2a-1)3个水平。主要考察了叶片膜质过氧化、光合色素、胞质抗坏血酸含量及总抗氧化能力的胁迫响应特征。结果表明:一个生长季(2009年5月15日—2009年9月10日)高浓度O3处理导致试验幼苗叶肉细胞膜脂过氧化程度显著加剧,叶绿素a、叶绿素b、类胡萝卜素含量显著降低;N素施加显著提高了叶绿素含量,但未改变细胞膜脂过氧化水平。叶片总抗氧化能力在AA+120处理下显著提高,但未受N施加影响。还原型抗坏血酸含量在N0处理下随O3浓度升高而降低;在N30及N60处理下随O3浓度升高而增高。施N可通过提高叶片抗坏血酸含量来增强香樟对O3升高的适应能力。研究结论可为O3与N复合污染下,我国亚热带地区典型常绿树种的保护提供科学参考。     

不同森林恢复类型对土壤微生物群落的影响

为了评价不同森林恢复类型与方式对南方红壤丘陵区退化生态系统土壤微生物群落的影响,借助氯仿熏蒸法、平板涂抹法和BIOLOG检测法,比较研究了4种森林恢复类型土壤微生物的群落特征．结果表明,4种森林恢复类型土壤微生物生物量碳、细菌数量差异显著,2项指标均以天然次生林土壤最高,人工林次之,荒地最差;碳源平均颜色变化率(AWCD法)和微生物代谢多样性指数(丰富度和多样性)在5种植被类型的土壤中也有明显差异,其趋势与微生物量碳、细菌数量基本相同;天然次生林土壤微生物群落利用碳源的整体能力和功能多样性比人工林和荒地强．相关分析表明,0～20和20～40cm土壤微生物的代谢多样性与根系生物量紧密相关(r=0．933,P<0．05;r=0．925,P<0．05)．自然恢复更有利于改善土壤微生物的结构和功能．     

两株海洋真菌的鉴定及其次级代谢产物抑制烟草花叶病毒及抗肿瘤活性

摘要：【目的】通过对2株活性海洋真菌发酵产物提取物抑制烟草花叶病毒和抗肿瘤活性进行研究,为进一步得到活性纯品化合物作为抗病毒及抗肿瘤的先导化合物奠定基础。【方法】菌株发酵产物的粗提物是通过甲醇浸取并在真空条件下蒸干得到的。粗提物中溶于水的部分为水溶性部分,不溶于水的部分为脂溶性部分。通过间接酶联免疫法检测样品抑制烟草花叶病毒的活性,通过四甲基偶氮唑盐微量酶反应比色法（MTT法）检测样品抗肿瘤活性,通过形态及ITS rDNA序列法进行菌株鉴定。【结果】两株海洋真菌抑制烟草花叶病毒活性和抗肿瘤的活性均较高。分子鉴定结果显示,两株真菌分别与Penicillium oxalicum 和 Neosartorya fischeri 的同源性极高。菌株0312F1发酵液的水溶性部分具有抗病毒及抗肿瘤活性,菌株1008F1发酵液的脂溶性部分具有抑制烟草花叶病毒活性,而水溶性部分具有抗肿瘤活性。【结论】菌株0312F1和菌株1008F1发酵液的提取物抑制烟草花叶病毒的活性部位不同,而抗肿瘤活性部位相同。菌株0312F1发酵液提取物的水溶性活性部位对肝癌细胞BEL-7404的抑制效果比对胃癌细胞SGC-7901的抑制效果明显,而菌株1008F1发酵液提取物的水溶性活性部位对胃癌细胞SGC-7901的抑制效果比对肝癌细胞BEL-7404的抑制效果明显。     

一株产细菌纤维素菌株的筛选鉴定及其产物分析

从红茶菌液中筛选获得一株产细菌纤维素的菌株BC-41,经生理生化分析和分子生物学鉴定,现证实该菌株为中间葡糖酸醋杆菌(Gluconacetobacter intermedius)。对该菌株所产生的细菌纤维素进行了物理特性的表征和分析,获得以下数据:BC-41所产的纤维素纯度达到91.32%,湿纤维素膜含水率达99.16%,每克干纤维素膜能吸水28.59 g;扫描电子显微镜观察,显示该纤维素具有网状结构,且纤维束宽度分布在40-100 nm之间;X射线衍射分析,证实该纤维素的晶型为纤维素I型,结晶指数为48.8%;通过黏度测定法,得出该纤维素的平均聚合度达2 100。