木霉属真菌拮抗金钗石斛病原菌的研究

目的考察石斛属药用植物内生真菌拮抗软腐病的真菌。方法将179株内生真菌进行金钗石斛软腐病生物防治盆栽试验,以获得拮抗病原菌终极腐霉的内生真菌。将具有拮抗效果的真菌在PDA培养基中与终极腐霉进行双重培养平皿对峙试验并对其显微形态进行观察,利用3,5-二硝基水杨酸法检测真菌的β-1,3和β-1,4葡聚糖酶活性。结果 T.koningiopsis与T.rogersonii在盆栽试验和双重培养平皿对峙试验中对终极腐霉具有抑制作用。T.koningiopsis与T.rogersonii菌丝将终极腐霉菌丝包围并将其破坏;两者β-1,3和β-1,4葡聚糖酶活性在所测试的5种真菌中最高。结论 T.koningiopsis与T.rogersonii可有效抑制终极腐霉;由两者分泌的β-1,3和β-1,4葡聚糖酶可能在破坏终极腐霉细胞壁方面发挥作用。T.koningiopsis和T.rogersonii可作为潜在的生防菌发挥拮抗终极腐霉作用。     

贵阳腐霉对致倦库蚊幼虫致病的组织学研究?

为了观察灭蚊真菌贵阳腐霉感染致倦库蚊幼虫后的组织形态学变化,探讨贵阳腐霉对致倦库蚊幼虫的致死机理,采用石蜡切片、 H． E．染色和比较组织学方法,以未经贵阳腐霉作用的致倦库蚊幼虫为对照,连续观察用贵阳腐霉感染24～72 h致倦库蚊幼虫的组织学变化。结果发现被贵阳腐霉感染的致倦库蚊幼虫中肠上皮细胞出现细胞间隙增大,随着感染时间的延长,细胞间隙进一步增大并出现空泡化、质膜破损、细胞裂解一系列病理变化。结果表明贵阳腐霉可导致致倦库蚊幼虫中肠上皮细胞的破坏,推测这可能是导致其死亡原因之一。     

巨细胞病毒感染对人胚肺成纤维细胞增殖及凋亡的影响

目的探讨人巨细胞病毒（human cytomegalovirus,HCMV）感染对宿主细胞增殖及凋亡的影响。方法用HCMV AD169感染人胚肺成纤维细胞（human embryonic lung fibroblast cells,HEL）,倒置显微镜观察不同感染时间细胞病变,采用MTT法检测HCMV感染对HEL细胞增殖的抑制作用,同时采用流式细胞术检测HCMV感染细胞对照组细胞凋亡指数。结果在HCMV感染48h内,细胞增殖及凋亡指数与细胞对照组无明显差异。自HCMV感染72h后,细胞增殖明显受抑制,凋亡指数明显增高,细胞病变逐渐加重。结论在HCMV感染早期,对宿主细胞的增殖及凋亡影响不明显,在感染后期,HCMV通过抑制宿主细胞增殖,加重细胞病变,促进感染细胞的凋亡而发挥致病作用。     

肠源性脂多糖的研究进展

成人肠道内约有10^13～10^14个共生菌,脂多糖（lipopolysaccharide,LPS）是革兰阴性细菌细胞壁的组成部分,也是革兰阴性菌感染的主要致病分子。近年来研究发现肠源性LPS是慢性非感染性疾病如代谢综合征等的致病因素之一。因此,LPS与人类健康的关系受到高度重视。近年来一些研究探讨了肠源性LPS的结构功能、吸收代谢及与低度炎症综合征的关系、如何降低肠源性的LPS进入血液循环以及内毒素血症的治疗措施,本文对此作一综述。     

风疹病毒野生株感染ECV304血管内皮样细胞的细胞病变效应

目的通过体外培养和风疹病毒野生株感染ECV304血管内皮样细胞,初步探讨中国大陆流行的风疹病毒野生株的致病变性特征.方法常规方法培养ECV304血管内皮样细胞和病毒接种,RT-PCR和间接免疫荧光法检测风疹病毒包膜糖蛋白E1基因的表达,相差显微镜及电子显微镜下观察细胞受病毒感染后形态变化,脱氧核糖核酸末端转移酶法（TUNEL）检测细胞凋亡.结果感染后2～3 d即可检测到风疹病毒包膜糖蛋白E1基因表达;感染后4 d,相差显微镜观察呈现明显的细胞病变效应,即细胞悬浮脱壁、细胞体变圆缩小、细胞膜皱缩、染色质浓缩等;电镜下观察可见明显病毒颗粒,核染色质浓集、边缘化,线粒体聚集于细胞核周围呈溶酶体化、空泡化和自噬现象.TUNEL法检测细胞凋亡指数（AI）在病毒感染组与对照组间有显著性差异（P＜0.01）.结论风疹病毒野生株在ECV304细胞中有明显的致细胞病变效应,在体外细胞培养下可诱导ECV304血管内皮样细胞凋亡.     

尿道致病性大肠埃希菌感染对人膀胱上皮细胞基因表达谱的影响

目的 研究尿道致病性大肠埃希菌(UPEC)菌株132与人膀胱上皮EJ细胞的相互作用,分析该菌株感染对EJ细胞基因表达谱的改变.方法 UPEC132感染EJ细胞,用倒置显微镜观察细菌与细胞的黏附,计算黏附率,并通过激光共聚焦显微镜观察UPEC132对细胞的侵袭.感染UPEC132的EJ细胞与未经细菌感染的细胞提取总RNA,用人类全基因组寡核苷酸微阵列芯片分析差异表达基因,并采用RT-PCR对基因芯片数据进行验证.结果 UPEC132能够黏附于EJ细胞表面,黏附率为(73.20±5.26)%;激光共聚焦显微镜观察发现部分细菌位于细胞内部,证实该菌对EJ细胞具有侵袭性.UPEC132感染后的EJ细胞与未经感染的细胞相比,共有28个基因上调,1个基因下调,主要涉及细胞增殖、炎症反应、细胞凋亡等相关基因.结论 UPEC与尿路上皮细胞的相互作用激活宿主细胞内部多种应答反应与信号转导途径,本研究为深入探索UPEC致病机制奠定基础.     

病原菌入侵宿主细胞的机制研究进展

传染病病原菌是对人类和动物健康的最大威胁。深入研究病原菌如何入侵宿主细胞的机制是揭示传染病的关键。经过长期的进化革兰氏阴性菌发展形成几种分泌途径破坏宿主细胞,包括普通分泌途径、Ⅰ型、Ⅱ型、Ⅲ型和Ⅳ型分泌途径。这些病原菌主要是通过摧毁宿主细胞的细胞骨架、调节信号转导途径和破坏宿主细胞的免疫机制达到感染宿主细胞的目的。本文首先就病原菌毒力因子的分泌途径进行了初步介绍,然后对目前有关病原菌如何入侵宿主细胞以及入侵宿主细胞后如何破坏细胞的结构和功能进行了概述。     

白念珠菌不同菌态成分诱导小鼠DCs分化成熟的差异性比较

白念珠菌是人类最常见的条件致病菌之一,可引起从皮肤黏膜到内脏的广泛感染.白念珠菌从形态观察为双相真菌,有酵母相和菌丝相,其中菌丝相更易黏附入侵宿主组织,是该菌在体内的主要致病形式.DCs是体内最重要的抗原提呈细胞,正常情况下绝大多数处于非成熟状态,一旦摄取抗原或接受某些因素刺激,DCs就会发育成熟,通过多种因素可诱导T细胞向不同类型Th细胞分化,形成抗感染免疫的起点和调节点.本研究通过将粉碎后白念珠菌酵母相、菌丝相成分分别刺激小鼠骨髓源DCs,观察DCs形态、表达表面分子CD80、CD86及分泌IL-12情况,比较两者间的差异.     

伞枝犁头霉在体外诱导人血管内皮细胞凋亡

目的 研究伞枝犁头霉对人脐静脉血管内皮细胞活性的影响及其机制.方法 采用锥虫蓝染色法分析不同时间段伞枝犁头霉对人血管内皮细胞存活率的影响.细胞凋亡检测试剂盒在荧光显微镜下分析伞枝犁头霉诱导人血管内皮细胞的凋亡情况.流式细胞仪定量检测伞枝犁头霉诱导人血管内皮细胞在不同时间段的凋亡情况,并观察caspase-3抑制剂对凋亡反应的影响.用Western blot法检测伞枝犁头霉引发人血管内皮细胞caspase-3在不同时间段活化情况.结果 锥虫蓝染色法显示伞枝犁头霉以时间依赖的方式抑制人血管内皮细胞的存活率.伞枝犁头霉在共培养第12小时开始抑制人血管内皮细胞存活率(P=0.001).荧光显微镜显示伞枝犁头霉诱导人血管内皮细胞发生凋亡,而并非坏死.流式细胞仪分析显示伞枝犁头霉以时间依赖的方式诱导人血管内皮细胞发生凋亡反应.凋亡细胞在共培养第12小时显著增高(P=0.0036).Caspase-3抑制剂几乎可以完全逆转伞枝犁头霉诱导的凋亡反应.Western blot法显示伞枝犁头霉引起人血管内皮细胞caspase-3活化随时间逐渐增强.结论 伞枝犁头霉在体外试验中可以诱导人血管内皮细胞发生凋亡.该凋亡信号的传导是通过caspase级联反应.     

Rab4蛋白的细胞内定位及其活性改变对DC内源抗原递呈的影响

目的观察Rab4突变体在树突状细胞(DC)内的定位,研究其对DC内源抗原递呈的影响。方法构建Rab4突变体的慢病毒表达质粒,以DNA-磷酸钙共沉淀法制备慢病毒,病毒感染DC-OVA细胞并使用流式细胞术检测感染效率,激光共聚焦显微镜观察DC-OVA细胞中Rab4突变体的表达与定位,最后用识别MHC I类分子-OVA肽复合物的T细胞杂交瘤B3Z细胞检测DC-OVA细胞内源抗原递呈的变化。结果慢病毒可高效感染DC-OVA细胞,感染后Rab4突变体可在DC-OVA中稳定表达,主要分布在细胞膜周围的囊泡结构中。抗原递呈检测结果显示Rab4对DC内源性抗原递呈具有正相关性影响。结论成功观察到不同Rab4突变体在DC内的表达与分布,初步证实Rab4蛋白可通过其活性的改变参与调节DC的内源性抗原递呈。     

Invasion of wood by basidiomycetous fungi VI. Quantitative but not qualitative differences in the pathovirulence of pathogenic and saprophytic decay fungi

Wood dowels sterilely overgrown with test mycelia of pathogenic and saprophytic basidiomycetous wood-decay fungi, respectively, were inserted into drill holes in the stems of 3 hardwood (Fagus sylvatica, Betula verrucosa, Sorbus aucuparia) and 1 softwood tree species (Picea abies). The host trees 40-120 mm in diameter were part of the light-starved understory of a timber forest. Eighteen to 52 months after inoculation the trees were dissected and the colonized stemwood was microbio-logically examined. Extension growth and survival of the test mycelia in trees of known vitality were taken as a basis for fungal pathovirulence rating. Basidiomycetous wood-decay fungi reach at best the status of physiologically facultative pathogens. They only colonized trees of the lowest vitality classes in the understory. For the expected range of pathovirulence, an appropriate host tree to be chosen had a proper range of vitality which expressed itself in a crown volume of 5 to 70% that of the crown of a free-standing tree and a width of the current annual growth ring from 0.5 to 2.5 mm in dependence of the tree species. Increasingly moribund trees with low crown volumes and narrow growth rings favoured an unlimited mycelial expansion and survival, while in vital trees fungal expansion gradually came to a standstill within 18 to 52 months, frequently combined with an active killing of both the pathogenic and saprophytic mycelia. Development of pathogenic test mycelia much more depended on the choice of an appropriate host tree species than did development of saprophytic test mycelia. The stem colonization by several late saprophytes was nearly as extensive and durable as the stem colonization by notorious tree pathogens. Pathosism in wood-decay basidiomycetes appeared thus to be a quantitative rather than a qualitative feature. For the dieback of the basidiomycetous test mycelia in the live stem reasons such as wood substrate depletion or antagonistic microbial activities could be excluded. The dieback rate of the test mycelia however was clearly enhanced in trees of increasing vitality, and it was enhanced with prolonged host-fungus interaction times. In comparison with sound dominant trees, the endophyte incidence in the sapwood of moribund P. abies trees was not increased. At the instant of tree death or crown decapitation however a severe colonization with blue stain and soft rot fungi was observed that exerted considerable antagonism on test mycelia low in kratovirulence. The implication of this antagonistic action for the colonization of dying trees by pathogenic decay fungi are discussed.     

Tobacco mosaic virus replication in resistant and susceptible plants: in some resistant species virus is confined to a small number of initially infected cells

Only small amounts of tobacco mosaic virus (TMV) are recoverable from directly inoculated leaves of some plant species, a phenomenon investigated by P. C. Cheo (1970, Phytopathology 60, 41-46) and termed subliminal infection. To interpret this phenomenon in two varieties of cowpea (Vigna sinensis Emil.), primary leaves were inoculated on their lower surfaces with TMV (common strain), and at various times postinoculation, mesophyll protoplasts were isolated, incubated for 36 hr, and stained with a TMV-specific fluorescent-labeled antibody. It was determined that only 1 in 50,000 to 150,000 protoplasts contained TMV antigen; this number remained essentially unchanged for experimental periods of from immediately after inoculation to up to 11 days postinoculation (the longest period examined). Cytological staining of epidermis from another subliminally infected host, cotton, also revealed infection of only a few cells. These data suggest that leaves of subliminally infected plants support TMV replication in those cells which receive virus during mechanical inoculation, but that the infectious principle is unable to move from those original centers in these hosts. Control experiments with tobacco (Nicotiana tabacum L. cv. Turkish Samsun), in which virus spreads extensively in the inoculated leaves, suggest that a rapid cell-to-cell movement of the infectious entity begins after about 6 hr following inoculation. An unexpected observation was that some cowpea and tobacco mesophyll cells become infected immediately upon mechanical inoculation, suggesting that mesophyll cells can be primary sites of viral ingress into the leaf.     

Plant virus-specific transport function. I. Virus genetic control required for systemic spread

Complementation of tobacco mosaic virus (TMV) mutant LS1 which is temperature-sensitive (ts) in virus transport function was studied in two model systems. The first one was aimed at complementing LS1 cell-to-cell spread through the tissues of the plant systemically preinfected with a temperature-resistant (tr) virus. Two different experiments were performed in this model system. (a) Use was made of a host plant which reacted differently to LS1 (local lesions) and to the helper virus (systemic reaction) at a permissive (24 degrees) temperature. Complementation occurred in a mixedly infected plant at a temperature which is nonpermissive for LS1 (32 degrees), when the necrotic reaction of the host was switched off; the complementation could be revealed upon the temperature shift treatment (TST) (24 --> 32 --> 24 degrees ). (b) Transport of LS1 from cell to cell in the presence of the helper virus was tested directly by immunofluorescent microscopy. In the second model system, the effect of virus transport from the conducting tissues of the stem into the cells of the leaf mesophyll, preinfected with the helper virus, was studied. It was shown that ts mutant LS1, imbibed through the stem, can move into and replicate in the mesophyll cells of the leaf which was preinfected with a tr-helper virus. This was true both at 24 and 32 degrees . On the other hand, LS1 itself could serve as a helper virus only at 24, but not at 32 degrees . In both model systems, it was demonstrated that ts transport can be complemented not only by a related tr TMV strain, but also by an unrelated virus-potato virus X. It has been suggested that a virus-specific transport function performed by the helper virus "opens the gates" for the dependent virus by an unknown modification of host cells.     

Genetic analysis of bipartite geminivirus tissue tropism

The bipartite geminiviruses bean golden mosaic virus (BGMV), cabbage leaf curl virus (CabLCV), and tomato golden mosaic virus (TGMV) exhibit differential tissue tropism in Nicotiana benthamiana. In systemically infected leaves, BGMV remains largely confined to vascular-associated cells (phloem-limited), whereas CabLCV and TGMV can escape into the surrounding mesophyll. Previous work established that TGMV BRi, the noncoding region upstream from the BR1 open reading frame (ORF), is required for mesophyll invasion, but the virus must also contain the TGMV AL23 or BL1/BR1 ORFs. Here we show that, in a BGMV-based hybrid virus, CabLCV AL23 also directed efficient mesophyll invasion in conjunction with TGMV BRi, which suggests that host-adaptation of AL23 is important for the phenotype. Cis-acting elements required for mesophyll invasion were delineated by analyzing BGMV-based hybrid viruses in which various parts of BRi were exchanged with those of TGMV. Interestingly, mesophyll invasion efficiency of hybrid viruses was not correlated with the extent of viral DNA accumulation. In conjunction with TGMV AL23, a 52-bp region of TGMV BRi with sequence homology to DNA A was sufficient for mesophyll invasion. This 52-bp sequence also directed mesophyll invasion in combination with the TGMV BL1/BR1 ORFs. Overall, these results are consistent with a model for mesophyll invasion in which AL2 protein, in association with host factors, acts through the 52-bp region in TGMV BRi to affect expression of the BR1 gene.     

The biological effect of dentin noncollagenous proteins (DNCPs) on the human periodontal ligament stem cells (HPDLSCs) in vitro and in vivo

It was recognized that periodontal progenitor cells penetrate disintegrated Hertwig's epithelial root sheath, and contact with root dentin give rise to periodontium formation. Clinically, direct contact of the conditioned or denuded root surfaces with periodontal cells seems to be a prerequisite for periodontal regeneration. In this study, we investigated the biological effect of dentin noncollagenous proteins (DNCPs) on the human periodontal ligament stem cells (HPDLSCs) in vitro and in vivo. Chemical-conditioned root dentin (CCRD) was prepared by process of partly demineralization and deproteinization. Treated HPDLSCs with DNCPs showed increased proliferation and adhesion ability. Induced HPDLSCs presented several features of cementoblast differentiation, as indicated by morphologic changes, enhanced alkaline phosphatase (ALP) activity, increased matrix mineralization, and upregulated expression of mineralization-associated genes. Incubation of treated HPDLSC aggregate in vivo revealed that cementum-like tissues formed along the CCRD surface with fibrous tissue adjacent to or inserted into it, but untreated HPDLSCs cannot form similar structure. To our knowledge, this is the first study to apply active proteins derived from dentin with periodontal stem cells to construct periodontal structure, which may shed light on human periodontal tissue regeneration.     

Cauliflower mosaic virus gene II product forms distinct inclusion bodies in infected plant cells.

Turnip leaves infected with the aphid transmissible isolate of cauliflower mosaic virus (CaMV Cabb B-JI) showed two types of virus-containing inclusion bodies (IBs), which differed morphologically and in their protein composition when analyzed by immunogold labeling of ultrathin sections. Vacuolated IBs, typical of CaMV infections, contained P62 (the generally accepted IB protein) but lacked P18 (the aphid transmission factor), while electron-lucent IBs did not contain P62 but were the only detectable sites of P18 accumulation within the infected leaf cells. Both types of inclusions were detected in cells of the epidermis, vascular bundles, mesophyll, and spongy parenchyma. Electron-lucent IBs were not found in the aphid nontransmissible isolates of CaMV, Campbell and CM4-184.     

Viral and host RNA synthesis in BSMV-infected barley

BSMV (barley stripe mosaic virus) nucleoprotein, BSMV-RNA (ribonucleic acid), and host RNA synthesis were characterized in the mature, inoculated leaf one and the systemically infected leaf two at 24-hr intervals from 1–8 days after inoculation. BSMV-RNA synthesis preceded virion appearance by approximately 24 hr in either leaf. In leaf one, viral RNA synthesis was occurring by 24 hr after inoculation. An excess of up to 150-fold of viral RNA was apparent during early stages of virion accumulation. During periods of rapid viral RNA accumulation, the in vivo rate of ³²P incorporation into viral RNA was much higher than into host RNA in infected tissue or in comparable healthy tissue. Host RNA synthesis was reduced during or after periods of rapid viral RNA accumulation, as determined by ³²P-labeling. The quantity of sedimenting host RNA was also reduced. The maximum level of viral RNA was comparable in either leaf, but the systemically infected leaf two produced about three times the amount of viral nucleoprotein as did leaf one. The severe inhibition of host RNA synthesis in leaf one may be one of the factors which results in the low “efficiency of encapsidation” in leaf one.     

Do T lymphocytes correlate danger signals to antigen?

When a cell is infected by a virus, or becomes transformed into a malignant state, it presents clues to its disease on the outer surface of its membrane. Such clues include peptide fragments of proteins produced inside the cell; when the cell is infected by a virus, viral peptides, as well as the cell's normal peptides, are displayed on the cell's membrane as potential antigens. Infected and malignant cells also externally present special molecules that ligate NKG2D receptors on immune cells. When patrolling T lymphocytes detect the presence of both their cognate peptide antigen and NKG2D ligands on one target, they proliferate and increasingly kill other cognate target cells. The danger model of immunity recognizes NKG2D ligands as stimulators of T cell cytotoxicity, but heretofore could not explain how T cells specific to normal peptides typical of healthy host cells outside the thymus, could avoid activation by danger signals on diseased cells. The problem is that T cells specific to host-peptides are also stimulated when those peptides are by chance also displayed on diseased cells displaying NKG2D ligands. However, if T cells predicated their cytotoxicity not only on the presence of their cognate antigen found in conjunction with danger signals, but also on the absence of their cognate antigen on cells not presenting danger signals, then only T cells specific for disease antigens would become activated. Since Fas display is correlated with viral or malignant transformation, it may be a danger-signal like NKG2D ligands. T cells which encounter Fas on malignant, cognate cells, increasingly bind Fas on healthy bystander cells not displaying cognate antigens. Perhaps such healthy bystander cells provide T cells with reference-levels of danger-signals for local tissue cells, allowing T cells to select between tolerance and cytotoxic reaction to their cognate antigen, as they circulate in the periphery. This paper will analyze cytotoxicity assays that show that T cells challenge syngeneic, non-cognate bystanders with Fas ligand (FasL), and why syngeny is a requirement for danger-reference cells. Some heretofore unexplained effects of superantigens will be suggested to be due to their obstruction of reference-target detection. This paper will also suggest that established tumors often evolve a subpopulation of high-danger-signal, low tumor-antigen cells that protect the tumor against T cells; that characteristics of dendritic cells (DC) complement the danger sensing of T cells; and that DC may also use quantitatively comparative, self-referential, danger-correlation measurements to recognize transformed cells interspersed among healthy host tissue cells.     

Adhesins and invasins of pathogenic Escherichia coli

Pathogenic E. coli cause both intestinal and extra-intestinal infections in humans and animals. Bacteria must be able to adhere to host cells if they are to colonize and to invade their hosts. Numerous E. coli adhesins with different morphological features and receptor specificities have been identified. Many bacteria produce several adhesins with different receptor specificities. Although not all adhesin receptors have been identified yet, it appears that adhesins generally behave as lectins, recognizing oligosaccharide residues of glycoproteins or glycolipids. This review summarizes recent advances concerning host tissue colonization properties, providing new insights into adhesive organelle biogenesis in pathogenic E. coli and into the development of reservoirs of pathogenic bacteria in the host. To limit the length of this review, I will use examples of structural characteristics and invasive properties of a few bacterial adherence factors: type 1 pili, Afa adhesive sheath and some outer membrane adhesins.     

CD117-positive cells and mast cells in adult human cardiac valves--observations and implications for the creation of bioengineered grafts.

BACKGROUND: There is no report to date of stem cells in human cardiac valves. We examined their possible presence, number, and distribution in valves removed at cardiac surgery from patients with a variety of underlying valve pathologies. METHODS: Grossly normal aortic and mitral valves were obtained from live heart transplant patients. Surgically excised valves with rheumatic mitral stenosis, aortic valve age-related degeneration, aortic valve changes of aortoannular ectasia, and mitral valves with myxomatous degeneration were studied. Immunohistochemical and histochemical studies were performed on sequential valve sections, including hematoxylin and eosin, hematoxylin phloxine saffron, Movat pentachrome, toluidine blue, CD31, CD34, and CD117. RESULTS: There were small clusters of CD117-positive cells in the fibrosa and spongiosa of mitral and aortic valves from all groups of valves. Sequential sectioning and staining showed that almost all of these cells were mast cells. However, in the mitral myxomatous valves and the mitral rheumatic valves, there were rare CD117-positive cells that did not have corresponding toluidine blue staining and thus could be valve mesenchymal stem cells. CONCLUSIONS: Most of the CD117-positive cells in normal and diseased adult heart valves are mast cells. These valve cells could play a role in valve pathology and injury. A very small number of possible valve stem cells were also identified. It is unlikely that these valve stem cells are sufficient in number to allow isolation and expansion for tissue engineering purposes.