肝螺杆菌与幽门螺杆菌的功能基因组比较分析

肝螺杆菌(Hh)是迄今为止发现与肝脏疾病相关的5种螺杆菌之一.Hh株ATCC51449的全基因组测序已经完成.研究发现Hh有和幽门螺杆菌(Hp)一样的尿素酶、鞭毛蛋白和黏附素同源基因,而编码趋化蛋白及细胞致死性肿胀毒素(CDT)的特性则与空肠弯曲菌类似.Hh缺乏大多数与Hp同源的定植和毒力因子如cag致病岛.此外Hh还有一个与霍乱弧菌相似的致病岛HHGI1.上述基因特点导致该菌肠道定植的适应性与致病潜能.     

格林巴利综合征相关及单纯腹泻相关空肠弯曲菌细胞毒作用分析

目的分析格林-巴利综合征(GBS)相关空肠弯曲菌细胞毒素(CDT)作用。方法9株GBS相关空肠弯曲菌以及4株腹泻患者分离菌株的全菌蛋白分别以0.001μg～5μg/mL的浓度作用于HeLa细胞,通过观察细胞形态的改变,获得不同菌株对细胞作用的差异。结果不同菌株间产生细胞毒作用的浓度范围从0.01μg/mL到5μg/mL。结论GBS相关菌株对HeLa细胞的细胞毒素作用与腹泻患者分离株相比没有显著差异。不同空肠弯曲菌菌株对HeLa细胞的细胞毒素作用具有菌株特异性。     

10种中草药对空肠弯曲菌粘附抑制作用的观察

空肠弯曲菌是引起人类急性腹泻的病原菌之一,其致病作用与其粘附作用、侵袭力及毒素有关。目前,临床治疗空肠弯曲菌引起的腹泻多采用抗生素药物,但由于耐药株的不断产生,效果常不理想,因此,有必要开辟新的药物治疗途径,故我们采用十种中草药对空肠弯曲菌的粘附抑制作用进行了初步研究,并用D~+……甘露糖、抗弯曲菌抗血清进行对照研究,现将结果报道如下。     

空肠弯曲菌主要外膜蛋白OMP18的B细胞抗原表位分析及功能鉴定

目的 从生物信息学角度了解空肠弯曲菌主要外膜蛋白OMP18的跨膜结构、B细胞抗原表位及其抗原性、基因序列保守性等特征,并鉴定空肠弯曲菌主要外膜蛋白优势B细胞抗原表位,为后续抗体检测和疫苗研究提供实验依据。方法 使用NCBI/Blast,TMHMM Server V2.0,DNA Star等生物信息学分析软件对空肠弯曲菌主要外膜蛋白OMP18进行蛋白跨膜结构预测、线性B细胞抗原表位及其抗原性分析、并对不同空肠弯曲菌OMP18蛋白的氨基酸序列同源性进行比较。采用空肠弯曲菌全菌抗体IgG为一抗,基于ELISA技术对优势线性B细胞抗原表位进行筛选鉴定。结果 生物信息学预测结果表明外膜蛋白OMP18定位于空肠弯曲菌外膜且不存在跨膜结构。蛋白序列保守存在于各空肠弯曲菌株中,序列同源性达95%以上。同时,我们发现该外膜蛋白中存在3个明显的B细胞线性表位,且具有很强的抗原性。ELISA检测鉴定结果表明3个B细胞抗原表位都能有效识别空肠弯曲菌全菌抗体。结论 空肠弯曲菌主要外膜蛋白OMP18保守存在于细菌表面,存在3个重要的B细胞线性抗原表位,可用于后续的抗体检测和疫苗开发研究。     

空肠弯曲菌AhpC蛋白B细胞抗原表位预测及抗原性分析

目的利用生物信息学预测分析空肠弯曲菌AhpC的跨膜结构、信号肽及B细胞抗原表位,并分析空肠弯曲菌AhpC优势B细胞抗原表位的抗原性,为后续疫苗研究提供依据。方法使用TMHMM Server V2.0、SignalP 4.1、IEDB等生物信息学分析软件对空肠弯曲菌AhpC进行蛋白跨膜结构预测、信号肽及线性B细胞抗原表位进行预测分析。分别以原核表达的空肠弯曲菌AhpC蛋白和化学合成的表位肽为抗原,空肠弯曲菌AhpC抗体为一抗,通过ELISA及Dot blot对优势线性B细胞抗原表位的抗原性进行分析及筛选。结果生物信息学预测结果表明AhpC定位于空肠弯曲菌外膜,无跨膜结构,无信号肽,同时该蛋白中存在7个的B细胞线性表位。经Dot blot和ELISA检测发现其中AhpC4-16表位具有较强的抗原性,为优势表位。结论空肠弯曲菌AhpC保守存在于细菌表面,存在1个优势的B细胞线性抗原表位(AhpC4-16),可用于后续的疫苗开发研究。     

贵州省首例空肠弯曲菌菌血症病例病原鉴定和亚种分型

目的 对贵州省1例菌血症患儿血液中分离的疑似空肠弯曲菌进行鉴定。方法 运用传统细菌学方法和分子生物学方法,对从菌血症患者血液分离的可疑空肠弯曲菌进行鉴定和亚种分型。结果 来自菌血症患儿血液的可疑菌株,经传统生化鉴定为空肠弯曲菌空肠亚种,特异性多重PCR方法鉴定为弯曲菌属空肠弯曲菌,NAP-mPCR方法鉴定为空肠弯曲菌空肠亚种。结论 分离自贵州省菌血症患儿血液的菌株确认为空肠弯曲菌空肠亚种,NAP-mPCR方法可将空肠弯曲菌鉴定到亚种水平。     

空肠弯曲菌鞭毛蛋白单克隆抗体的制备

目的为获得空肠弯曲菌鞭毛蛋白单克隆抗体,用于空肠弯曲菌的检测和研究。方法用空肠弯曲菌ATCC29428株灭活全菌作为抗原,免疫BALB/c小鼠,待抗体水平达到1∶51 200时,取脾细胞与生长良好的对数期Sp2/0进行融合。同时以粗提天然鞭毛蛋白和人工表达重组鞭毛蛋白包板,用间接ELISA方法筛选,多次克隆化培养后获得的单抗用Western blot进行生物学特性鉴定。结果获得3株持续、稳定分泌抗空肠弯曲菌鞭毛蛋白单克隆抗体的杂交瘤细胞株,分别命名为1G8、1G9和3F2。结论获得的空肠弯曲菌鞭毛蛋白单克隆抗体为建立空肠弯曲菌的蛋白检测及后续研究奠定基础。     

空肠弯曲菌实时荧光定量PCR检测方法的研究

目的建立一种快速廉价的检测空肠弯曲菌的实时荧光定量PCR方法。方法根据空肠弯曲菌flaA、gyrA基因设计引物,建立空肠弯曲菌实时荧光定量PCR检测方法。结果以flaA基因引物对空肠弯曲菌DNA进行实时荧光定量PCR,扩增曲线呈S形指数增长,大肠埃希菌等对照菌扩增阴性。方法的灵敏度为10CFU/ml菌悬液。结论建立的空肠弯曲菌荧光定量PCR检测方法具有快速、简便、准确等特点,具有一定的使用价值。     

江苏奶牛空肠弯曲菌和结肠弯曲菌流行状况及耐药性分析

目的了解江苏省奶牛空肠、结肠弯曲菌流行及耐药状况。方法采用多重PCR方法对10个奶牛场的产奶牛、育成牛和饲养环境进行空肠弯曲菌和结肠弯曲菌流行状况调查,采用琼脂扩散法测定分离株的耐药性。结果1531份样品中,119份空肠弯曲菌阳性,平均阳性率7.77%;3份结肠弯曲菌阳性,平均阳性率0.20%;空肠弯曲菌、结肠弯曲菌阳性率最高分别为30.91%、3.57%。在三类样品中,产奶牛空肠弯曲菌、结肠弯曲菌阳性率分别为5.02%、0.32%,育成牛空肠弯曲菌阳性率为8.70%、未检出结肠弯曲菌,环境样品空肠弯曲菌、结肠弯曲菌阳性率为10.28%、0.18%。35株奶牛空肠弯曲菌分离株对8大类21种抗生素高度敏感率的是:阿莫西林100%、阿齐红霉100%、链霉素97.14%、庆大霉素94.29%、红霉素91.43%、头孢噻肟82.86%、克林霉素82.86%;高度耐药率的是:头孢哌酮100%、恩诺沙星100%、环丙沙星97.14%、复方新诺明97.14%、左旋氧氟沙星94.29%、萘啶酸94.29%、头孢拉定94.29%、诺氟沙星91.43%、头孢克罗88.57%。菌株耐药谱显示,35株分离株的耐药主要集中在9耐到12耐,占88.57%,产奶牛分离株的多重耐药性较其它分离株更严重。结论我国奶牛群中空肠弯曲菌、结肠弯曲菌的流行和耐药状况呈现多样化和复杂化,研究结果为正确评价我国奶牛群弯曲菌的流行状况和制定切实可行的防控措施提供科学依据。     

幽门螺杆菌对胃上皮细胞粘附作用的研究

通过细胞酶免疫测定法测定了胃上皮细胞与HP、E·coli、空肠弯曲菌的结合情况,结果表明胃上皮细胞与HP有较高的结合力,显著高于E·coli、空肠弯曲菌组(P<0.05).在HP低浓度时,反应OD值随加入菌量的增加而递增,当菌量达10~6～10~7/ml时,OD值不再递增,提示HP与胃上皮细胞的结合呈饱和性、特异性.HP与肝、肠和粘膜细胞结合力低下(P<0.05),提示HP感染具有组织定向世.     

HT-2毒素致DNA损伤的单细胞电泳观察

目的　测定HT-2毒素对Hela细胞DNA的损伤作用,检验单细胞电泳法（SCGE）在人群试验中的可行性。方法　应用SCGE和细胞生长抑制实验（MTT）。结果　HT-2毒素具有致DNA损伤的作用,并随着毒素剂量的增加,DNA损伤程度加重。800ng/ml的尾长为（28.4±5.67）μm,1600ng/ml时为（55.8±10.47）μm,呈剂量反应关系。结论　HT-2毒素具有致细胞DNA损伤的作用。     

Role of platelet activating factor in the intestinal epithelial secretory and Chinese hamster ovary cell cytoskeletal responses to cholera toxin.

With the recent heightened concern about cholera around the world come new questions about the mechanism by which cholera toxin causes diarrhea. Peterson and Ochoa have suggested that prostaglandin synthesis is key to both the intestinal epithelial secretory and the CHO cell responses to cholera toxin [Peterson, J. W. and Ochoa, G. (1989) Science 245, 857-859]. Because platelet activating factor (PAF) can be a potent stimulus for prostaglandin synthesis, we examined its role in the intestinal and tissue culture effects of cholera toxin. We report that the specific PAF receptor antagonists BN 52021 and SR 27417 inhibit the effects of cholera toxin on intestinal secretion in rabbit ileal loops in vivo and on the cytoskeleton of Chinese hamster ovary cells in vitro. We also show that PAF itself can cause net fluid secretion in the rabbit model and that PAF potentiates the effects of cholera toxin on intestinal secretion. Finally, we demonstrate that cholera toxin stimulates significant PAF production (2.6-fold) in isolated T-84 intestinal epithelial cells. We conclude that cholera toxin stimulates PAF production and that PAF is involved in both the secretory and cytoskeletal responses to cholera toxin. These findings further support the involvement of additional mediators of cholera toxin effects other than mucosal cell cyclic AMP and help explain the effects of cholera toxin on prostaglandin synthesis.     

Thapsigargin-induced transport of cholera toxin to the endoplasmic reticulum.

Cholera toxin is normally observed only in the Golgi apparatus and not in the endoplasmic reticulum (ER) although the enzymatically active A subunit of cholera toxin has a KDEL sequence. Here we demonstrate transport of horseradish peroxidase-labeled cholera toxin to the ER by electron microscopy in thapsigargin-treated A431 cells. Thapsigargin treatment strongly increased cholera toxin-induced cAMP production, and the formation of the catalytically active A1 fragment was somewhat increased. Binding of cholera toxin to the cell surface and transport of toxin to the Golgi apparatus were not changed in thapsigargin-treated cells, suggesting increased retrograde transport of cholera toxin from the Golgi apparatus to the ER. The data demonstrate that retrograde transport of cholera toxin can take place and that the transport is under regulation. The results are consistent with the idea that retrograde transport can be important for the action of cholera toxin.     

Autoradiographic assay of mutants resistant to diphtheria toxin in mammalian cells in vitro.

Diphtheria toxin kills mammalian cells by ribosylating elongation factor 2, a protein factor necessary for protein synthesis. The frequency of cells able to form colonies in the presence of the toxin can be used as an assay for mutation to diphtheria toxin resistance. We report here that resistance to diphtheria toxin can also be detected autoradiographically in cells exposed to [3H]leucine after treatment with the toxin. In cultures of Chinese hamster ovary cells, the frequency of such resistant cells is increased by exposure of the cells to gamma-rays, ultraviolet light, ethylnitrosourea, mitomycin c, ethidium bromide, and 5-bromo-2'-deoxyuridine in a dose- and time-dependent manner. The resistant cells form discrete microcolonies if they are allowed to divide several times before intoxication, which indicates that they are genuine mutants. The assay is potentially adaptable to any cell population that can be intoxicated with diphtheria toxin and labeled with [3H]leucine, whether or not the cells can form colonies. It may be useful, therefore, for measuring mutation rates in slowly growing or nondividing cell populations such as breast, brain, and liver, as well as in cells that do divide but cannot be readily cloned, such as the colonic epithelium.     

Receptor-mediated endocytosis of diphtheria toxin by cells in culture.

The binding and uptake of fluorescently labeled diphtheria toxin by cells in culture has been examined by using epifluorescence video intensification microscopy. Rhodamine-labeled diphtheria toxin retained significant toxicity on bioassay and in cell culture and was tested for uptake by human WI-38 and mouse 3T3 fibroblasts grown in culture. When added to cells at 37 degrees C, toxin was observed to become concentrated and internalized in discrete vesicles in both cell lines. The appearance of fluorescent clusters could be prevented by addition of excess unlabeled diphtheria toxin to the medium or by addition of ATP (which has been shown to block toxin binding to cells), indicating that the rhodamine-labeled toxin was binding to diphtheria toxin-specific cell surface binding sites. When the simultaneous uptake of rhodamine-labeled diphtheria toxin and fluorescein-labeled alpha 2-macroglobulin was monitored, the two proteins appeared in the same clusters indicating that the toxin undergoes receptor-mediated endocytosis. Despite the difference in susceptibility to diphtheria toxin of cells derived from sensitive (human) and resistant (mouse) tissues, the behavior of the rhodamine-labeled derivative in both cell lines was indistinguishable in terms of toxin required for formation of clusters or inhibition by unlabeled toxin or by ATP. These results demonstrate that diphtheria toxin-specific cell surface binding sites occur on both insensitive and sensitive cells and suggest that toxin is processed similarly by both cell types during its initial cell surface binding and internalization by this pathway. The possible involvement of this uptake system in the mechanism of action of diphtheria toxin in cells is discussed.     

Diphtheria toxin mutant selectively kills cerebellar Purkinje neurons.

CRM107 (crossreacting material 107), a double point mutant of diphtheria toxin that lacks receptor-binding activity, specifically kills cerebellar Purkinje cells in vivo. After injection into guinea pig cerebrospinal fluid, CRM107 (0.9 micrograms) and CRM107-monoclonal antibody conjugates (10 micrograms) kill up to 90% of the total Purkinje cell population with no detectable toxicity to other neurons. Animals exhibit ataxia, tremor, and abnormalities of posture and tone. Native diphtheria toxin, ricin, and ricin A chain do not cause ataxia and do not reduce the Purkinje cell population after intrathecal injection into guinea pigs at toxic or maximally tolerated doses. However, in rats, which will tolerate higher doses of diphtheria toxin than guinea pigs, Purkinje cells can be killed by both CRM107 and diphtheria toxin. A truncated mutant of diphtheria toxin, called CRM45, can also cause Purkinje cell killing but has additional toxicity not seen with CRM107. Animals treated with intrathecal CRM107 or CRM107 linked to antibodies may serve as models for Purkinje cell loss in a broad spectrum of human diseases and may be used to further study cerebellar physiology. Understanding the basis for the Purkinje cell sensitivity to CRM107 may illuminate other causes of Purkinje cell loss.     

Evidence for the complex nature of the ganglioside receptor for cholera toxin.

Choleragenoid binds more slowly and less strongly than cholera toxin to intestinal mucosal cells, and even less strongly to free ganglioside in solution. However, binding to ganglioside is greatly enhanced when the ganglioside is in the form of an insoluble complex with cerebroside. These findings suggest that both the binding and the active components of the toxin molecule may be necessary for optimal binding of the toxin to the intact cell, and that the ganglioside in the cell receptor is in a complex form. Choleragenoid only partially blocks the action of the toxin on ruptured cells. This observation indicates that, while binding to a membrane receptor is necessary for the action of the toxin on the whole cell, it is possible to activate adenyl cyclase in a perforated cell by a process apparently independent of membrane binding; however, this activation may be possible only if the toxin preparation contains the active component dissociated from choleragenoid.     

Tetanus toxin as a marker for small-cell lung cancer cell lines

Summary Tetanus toxin labeling of human lung cancer cell lines was investigated using direct and indirect immunofluorescence and immunohistochemical staining. Cells of characterized permanent cell lines, eight small-cell lung cancer (SCLC) cell lines of classic subtype, six SCLC cell lines of variant subtype and seven non-small-cell lung cancer (NSCLC) cell lines, were incubated with a saturating concentration of tetanus toxin. For staining, fluorescein-isothiocyanate-(FITC)-conjugated anti-(tetanus toxin) antibodies were used or a mouse monoclonal anti-(fragment C) antibody with subsequent binding of FITC-conjugated anti-(mouse Ig) antibody or peroxidase-antiperoxidase complex. Only SCLC showed an intense fluorescence/immunoreactivity restricted to the cell membrane. Quantitative analysis of tetanus toxin labeling by flow cytometry revealed the percentage of positive cells to be between 35% and 95% in SCLC without obvious differences between the classic and variant subtypes of SCLC. In NSCLC the percentage of positive cells was lower than 10%. These results demonstrate that SCLC in contrast to NSCLC can be labeled with tetanus toxin, emphasizing the neuroendocrine properties of this tumor, and that tetanus toxin labeling may become a useful diagnostic marker for SCLC cells in cytology.Abbreviations used SCLC small-cell lung cancer - NSCLC non-small-cell lung cancer - FITC fluorescein-isothiocyanate - PBS phosphate-buffered saline - FACS fluorescence-activated cell sorter - NB neuroblastoma - APUD amine precursor uptake and decarboxylation - Fuc-GM1 fucosylated GM1 - Fuc-GD1b fucosylated GD1b (ganglioside nomenclature according to Svennerholm 1977)     

Cholera toxin enhances factor-dependent colony growth of murine mast cell progenitors

Mast cell colonies were observed when mouse spleen or bone marrow cells were cultured in the presence of medium conditioned by concanavalin-A-stimulated spleen cells, indicating that the medium contains the factor(s) necessary for the formation of these colonies. This factor-dependent colony growth of mast cell progenitors was enhanced by cholera toxin and prostaglandin E, which act on cellular growth mainly by elevating the intracellular cyclic-AMP level. The effect of the toxin was neutralized by preincubation of the toxin with GM1 ganglioside, the receptor substance for cholera toxin, suggesting that cholera toxin exerts its action through GM1 gangliosides present on mast cell progenitors. The toxin B subunit, which binds to GM1 ganglioside but does not elevate intracellular cyclic AMP level, did not affect the colony growth of mast cell progenitors. From these results, it is suggested that intracellular cyclic AMP levels may be involved in colony growth of mast cell progenitors.     

Clostridium difficile toxin A excites enteric neurones and suppresses sympathetic neurotransmission in the guinea pig

BACKGROUND AND AIMS: Evidence suggests that the intestinal actions of Clostridium difficile toxin A-stimulation of secretion and motility, and an acute inflammatory response-have a neurally mediated component. METHODS: Direct intracellular electrophysiological recording of electrical and synaptic behaviour in enteric neurones was performed in the submucous plexus of guinea pig small intestine during exposure to the toxin. RESULTS: Application of toxin A affected both the electrical behaviour of the neuronal cell bodies and inhibitory noradrenergic neurotransmission to the cell bodies. Altered electrical behaviour included depolarisation and increased excitability. Tetrodotoxin or a histamine H(2) receptor antagonist did not affect the depolarisation evoked by toxin A. Failure of the histamine antagonist to suppress the actions of toxin A is evidence that its actions were not mediated by degranulation of intramural mast cells. The action of toxin A on neurotransmission was suppression of inhibitory postsynaptic potentials evoked in the neuronal cell bodies by stimulation of sympathetic nerve fibres that synapsed with the cell bodies. The inhibitory postsynaptic potentials were mediated by norepinephrine (noradrenaline) acting at postsynaptic alpha adrenoceptors on the cell bodies. Hyperpolarising responses evoked in the cell bodies by micropressure application of norepinephrine were unaffected by toxin A. This fulfils criteria for a presynaptic inhibitory action of toxin A to suppress release of norepinephrine from sympathetic postganglionic axons. CONCLUSIONS: Results suggest that the neural component of the action of toxin A involves both direct excitation of enteric neurones and suppression of norepinephrine release from postganglionic sympathetic nerve fibres in the enteric nervous system.