氦氧饱和高气压暴露对铜绿假单胞菌毒力表型的诱导调节

研究氦氧饱和高气压暴露对铜绿假单胞菌与急性侵袭感染相关的主要毒力因子表型的影响。分别检测暴露前后铜绿假单胞菌的生长速度、运动能力,对乙酰胺的分解能力和培养细胞外液中绿脓菌素、弹性蛋白酶的分泌水平和活性。在动物水平验证毒力的变化。结果显示,经4.5 MPa氦氧饱和加压暴露后,与对照组相比铜绿假单胞菌的生长增殖、运动能力和对乙酰胺的分解能力增强,绿脓菌素分泌增加,弹性蛋白酶活性增强;暴露后细菌对实验小鼠的毒性增加。因此氦氧饱和高气压环境对铜绿假单胞菌的毒力表型有正调控作用。     

铜绿假单胞菌Arr基因突变对生物膜和绿脓菌素合成的影响

为了研究铜绿假单胞菌Arr基因对生物膜和绿脓菌素合成的影响,采用抗庆大霉素基因序列(Gentamycin resistance cassette,aacC1)插入失活的策略构建了铜绿假单胞菌Arr基因突变株PA-AG,通过96孔板静止培养、结晶紫染色的方法检测其生物膜的形成量,利用抽提的方法检测绿脓菌素的合成量。结果在KMB或LB培养基中,突变株PA-AG形成生物膜的量均有所减少,野生株约是突变株的2倍,然而突变株合成绿脓菌素的能力却明显加强,约为野生株的2.5倍。由此推测,铜绿假单胞菌Arr基因在一定程度上促进了生物膜的形成,抑制了绿脓菌素的合成。     

植物发酵液提取物对铜绿假单胞菌生物膜的作用

【目的】探讨植物发酵液提取物(plant fermentation extract,PFE)对铜绿假单胞菌生物膜的抑制作用,为临床上铜绿假单胞菌感染相关疾病的治疗提供参考。【方法】通过划线法分离临床标本中的铜绿假单胞菌并进行鉴定,通过报告菌株测定铜绿假单胞菌的毒力因子,采用试管法和激光共聚焦扫描显微镜测定生物膜的形成。【结果】在分离出的16株铜绿假单胞菌中,PFE对PA007菌株的作用效果最好,1%PFE显著降低PA007菌株生物膜、绿脓菌素和N-(3-oxododecanoyl)-HSL(3-oxo-C12-HSL)的产量(P0.05)。同时,也显著降低Las A蛋白酶的活性以及持留菌存活率(P0.05)。荧光定量PCR实验结果表明PFE能显著抑制las I和pqs A基因的表达(P0.05)。【结论】PFE具有抗铜绿假单胞菌感染能力,在临床上铜绿假单胞菌感染疾病的治疗中具有巨大的潜在价值。     

铜绿假单胞菌LasR基因反义核酸原核表达载体的构建及对该菌毒力的影响

PCR扩增LasR基因,用反向克隆法构建LasR基因反义核酸原核表达载体pUCP18/lasRantisense并转化铜绿假单胞菌,经酶切、PCR及测序鉴定重组载体.RT-PCR检测LasB基因和LasI基因mRNA的表达,NAD法测定外毒素A的活性,紫外分光光度计测定绿脓菌素的产生水平.用转化pUCP18/lasRantisense质粒菌株感染大鼠呼吸道并进行病理组织切片检查.PCR扩增出约720bp片段,与GenBank(No:NC_002516)报道的基因片段大小一致,构建了LasR基因反义核酸原核表达载体pUCP18/lasRantisense,并成功转化铜绿假单胞菌,且有效表达,使转化菌的毒力因子弹性蛋白酶、外毒素A和绿脓菌素表达水平均降低.与标准株感染的大鼠比较,转化pUCP18/lasRantisense质粒菌株感染的大鼠支气管炎症明显减轻.上述结果表明,LasR基因反义核酸原核表达载体pUCP18/lasRantisense可以降低铜绿假单胞菌的毒力.     

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。     

铜绿假单胞菌lasI,rhlI基因功能缺陷株的构建和生物学功能验证

构建铜绿假单胞菌lasI,rhlI基因功能缺陷株,为进一步阐明氦氧饱和高气压暴露条件诱导lasI,rhlI基因介导铜绿假单胞菌毒力调节的分子机制研究奠定基础。用双亲株接合转移法删除lasI,rhlI基因ORF编码区,通过RT-PCR方法验证目标基因编码序列mRNA的缺失;通过对细菌生长增殖能力、弹性蛋白酶代谢活性和细菌绿脓菌素分泌能力等表型的测定,验证目标基因编码序列缺失后的基因调节功能的缺陷。结果表明成功构建铜绿假单胞菌lasI,rhlI基因功能缺陷株,可作为进一步研究的基因工程菌。     

谷胱甘肽对铜绿假单胞菌exoS 和exoY基因的影响

摘要: 【目的】研究谷胱甘肽对铜绿假单胞菌exoS和exoY基因表达的影响。【方法】利用丁硫氨酸亚砜胺和马来酸二乙酯同时耗竭细胞内的谷胱甘肽,并构建包含被lacZGm破坏的谷胱甘肽合成酶基因的突变体。通过分别连有exoS 和exoY基因启动子的pMS402质粒上Lux报道子发光值大小检测exoS 和exoY基因表达变化情况。【结果】exoS和exoY基因的表达在用化学药品耗竭的细胞中或是在谷胱甘肽合成酶突变体中都降低。【结论】铜绿假单胞菌细胞内的谷胱甘肽可以促进exoS和exoY的表达。这将为进一步研究铜绿假单胞菌的感染以及致病性机理提供一定的理论基础。     

绿脓菌素分型的研究

早在1925年Gratia发现某些大肠菌具有抑制其它大肠菌生长的物质,命名为大肠菌素。以后Fredericq等人进一步研究又发现了巨形杆菌产生巨杆菌素,鼠疫杆菌产生鼠疫菌素,荧光假单胞菌产生荧光菌素及绿脓假单胞菌产生绿脓菌素。这些物质统称为细菌素。     

铜绿假单胞菌和白假丝酵母的跨界相互作用

铜绿假单胞菌和白假丝酵母(又称白念珠菌)这两种条件致病菌可共存于人体。两者间存在复杂的相互关系,即跨界相互作用。铜绿假单胞菌抑制白念珠菌形态转换,抑制其生物膜形成并毒杀其菌丝;白念珠菌则抑制铜绿假单胞菌绿脓素形成并抑制其丛集运动。跨界相互作用可能存在3种机制:信号转导通路、生物膜和毒力因子。铜绿假单胞菌通过信号分子N-3-氧代十二烷酰-L-同型丝氨酸内酯(3-oxo-C12-HSL)抑制白念珠菌形态转换,而白念珠菌通过信号分子法呢醇抑制铜绿假单胞菌绿脓素生成和丛集运动,即存在信号分子介导的跨界相互作用。跨界相互作用影响铜绿假单胞菌和白念珠菌各自的致病性。如能充分利用跨界相互作用,将有助于优化治疗的选择。     

铜绿假单胞菌感染的非抗生素治疗研究进展

铜绿假单胞菌是引起多种人体感染的常见致病菌,目前主要采用抗生素治疗铜绿假单胞菌感染.由于铜绿假单胞菌能对多种抗生素产生抗性,导致传统的抗生素治疗面临非常大的挑战.因而铜绿假单胞菌感染的非抗生素治疗方法受到了广泛重视,并取得了可喜的研究进展.本文从抗原抗体免疫疗法、噬菌体疗法、抗毒力因子疗法三个方面就铜绿假单胞菌的非抗生...     

Recent advances in the study of Kaposi's sarcoma-associated herpesvirus replication and pathogenesis

It has now been over twenty years since a novel herpesviral genome was identified in Kaposi's sarcoma biopsies. Since then, the cumulative research effort by molecular biologists, virologists, clinicians, and epidemiologists alike has led to the extensive characterization of this tumor virus, Kaposi's sarcoma-associated herpesvirus(KSHV; also known as human herpesvirus 8(HHV-8)), and its associated diseases. Here we review the current knowledge of KSHV biology and pathogenesis, with a particular emphasis on new and exciting advances in the field of epigenetics. We also discuss the development and practicality of various cell culture and animal model systems to study KSHV replication and pathogenesis.     

The structure, reproduction and taxonomy of Vidalia and Osmundaria (Rhodophyta, Rhodomelaceae)

Comprises species occurring mostly in subtidal habitats in tropical, subtropical and warm-temperate areas of the world. An analysis of the type species, V. spiralis (Sonder) Lamouroux ex J. Agardh, a species from Australia, establishes basic characters for distinguishing species in the genus. These characters are (1) branching patterns of thalli, (2) flat blades that may be spiralled on their axis, (3) width of the blade, (4) primary or secondary derivation of sterile and fertile branchlets and (5) position of sterile and fertile branchlets on the thalli. Application of the latter two characters provides an important basic method for separation of species into three major groups. Osmundaria , a genus known only in southern Australia, was studied in relation to Vidalia , and its separation from the Vidalia assemblage is not accepted. Species of Vidalia therefore are transferred to the older genus name, Osmundaria. Two new species, Osmundaria papenfussii and Osmundaria oliveae are described from Natal. Confusion in the usage of the epithet, Vidalia fimbriala Brown ex Turner has been clarified, and Vidalia gregaria Falkenberg, described as an epiphyte on Osmundaria pro/ifera Lamouroux, is revealed to be young branches of the host, Osmundaria prolifera.     

New or rare chromosome counts in Artemisia L. (Asteraceae, Anthemideae) and related genera from Kazakhstan

Fifteen chromosome counts of six Artemisia taxa and one species of each of the genera Brachanthemum, Hippolytia, Kaschgaria, Lepidolopsis and Turaniphytum are reported from Kazakhstan. Three of them are new reports, two are not consistent with previous counts and the remainder are confirmations of very scarce (one to four) earlier records. All the populations studied have the same basic chromosome number, x = 9, with ploidy levels ranging from 2x to 6x. Some correlations between ploidy level, morphological characters and distribution are noted.     

肝癌中HBV和HCV基因和抗原的分布及意义

采用原位分子杂交方法检测HCV RNA及HBV X基因;采用免疫组织化学方法研究HCV核心抗原,非结构区C33c抗原及HBxAg在肝细胞肝癌中的定位及分布.结果表明(1)HCV RNA、HBV X基因在肝细胞肝癌组织检出率分别为40%(55/136)和82%(112/136).HCV RNA定位于癌细胞的胞浆内,阳性细胞呈散在、灶状及弥漫分布三种形式;HBV X基因在肝癌细胞中的分布呈胞浆型、核型及核浆型,阳性细胞也呈上述三种分布形式;(2)HCV C33c抗原、核心抗原在肝细胞肝癌中的阳性率为81%(133/164)及86%(141/164).C33c抗原定位于癌细胞及肝细胞的胞浆内;核心抗原既定位于癌细胞核中,又可定位于胞浆中.C33c抗原阳性细胞以灶状分布为主;而核心抗原阳性细     

Selection with two alleles and complete dominance

For a plant selection model with frequency-independent viabilities, fertilities and selfing rates, it is shown that apart from global fixation, for certain parameter combinations a protected polymorphism and facultative fixation (either allele may become fixed according to initial frequencies) may both occur. Facultative fixation requires different selling rates for the dominant and recessive type. Protection of the polymorphism requires resource allocation for male and female function. In this connection the problem of purely genetically caused population extinction is discussed.
For general frequency dependence and regular segregation, the chances for establishment of a completely recessive gene are compared to those of a completely dominant gene. It is proven that the process of establishment of the recessive gene, despite a fitness advantage, may be considerably endangered by drift effects if random mating prevails. The recessive gene may reach the same effectivity in establishment as a dominant gene, only if the recessive homozygote mates exclusively with its own type during the period of establishment.