双歧杆菌及肠致病性大肠杆菌粘附的细胞膜通透性研究

采用乳酸脱氢酶释放法比较研究双歧杆菌１０２７株及肠致生大肠杆菌（ＥＰＥＣ）对体外肠上皮细胞Ｌｏｖｏ细胞株粘附的细胞膜通透性,探讨它们对肠上皮细胞的不同生物学交谈２。结果表明,双歧杆菌粘附Ｌｏｖｏ细胞后,宿主细胞释放ＬＤＨ远较ＥＰＥＣ粘附的效果低,提示双歧杆菌粘附对主细胞膜通生影响不大,而ＥＥＣ则可损伤宿主细胞膜而增加其通性。因此,双歧杆菌作为生理性细菌可与肠上皮细胞和谐共生,这与ＥＰＥＣ的粘附损伤     

UVB诱导NIH3T3细胞凋亡时的信号转导机制:Ⅱ.神经酰胺所致细胞内Ca~(2+)和pH的变化

利用粘附式细胞仪（ＡＣＡＳ－５７０）结合相应的荧光探针分别测定了外源性神经酰胺诱导ＮＩＨ３Ｔ３细胞凋亡时胞浆游离Ｃａ＾２＋水平和ＵＶＢ照射ＮＩＨ ３Ｔ３细胞所致细胞内ＰＨ的变化以及神经酰胺的生民对这一变化的影响。结果表明,１．神经酰胺能够导致ＮＩＨ ３Ｔ３细胞胞浆游离Ｃａ＾２＋升高既来源于胞外叠为源于胞内钙池,但外钙内流是引起和维持胞内Ｃａ＾２＋处于高水平所必要条件,ＮＩＨ ３Ｔ３细胞也上存在着两     

凝血酶对荧光标记的人单个血小板游离[Ca^2＋]和膜电位的动态影响

本文将ｆｌｕｏ－３和ｄ_ｉ－ＢＡ－Ｃ４（３）荧光标记的血小板固定于纤维蛋白原表面,以５７０型粘附式细胞仪（ＡＣＡＳ５７０）动态观察了凝血酶激活的人单个血小板细胞内游离［Ｃａ~（２＋）］（钙离子浓度）和膜电位的变化。静息状态时细胞游离［Ｃａ~（２＋）］和膜电位荧光较低,波动不明显。当加入０．１Ｕ／ｍｌ凝血酶激活时,［Ｃａ~（２＋）］（细胞内游离钙离子浓度）与膜电位迅速升高,随后［Ｃａ~（２＋）］出现反复振荡,幅度达约５００荧光单位,而膜电位基本上保持峰值水平。［Ｃａ~（２＋）］_ｉ升高与膜电位变化在时间和程度上不一致。本文结果提示,凝血酶引起血小板［Ｃａ~（２＋）］振荡和膜去极化,后者不是Ｃａ~（２＋）内流引起的。     

α1-肾上腺素受体亚型介导细胞内游离Ca2+增高的信号转导途径

本文探讨了α１ａ,α１ｂ,α１ｄ三种亚型肾上腺素受体激动时细胞内Ｃａ６２＋浓度升高的信号转导途径。在稳定表达三亚型α１－ＡＲ的ＨＥＫ２９３细胞２系中,用ｆｕｒａ－２方法细胞内Ｃａ＾２＋信号强弱的变化。结果显示,百日咳毒素对去甲肾上腺素激动三亚型α１－ＡＲ而引起的「Ｃａ＾２＋」ｉ升高无影响,Ｕ－７３１２２和ＰＭＡ明显抑制「Ｃａ＾２＋」ｉ升高．     

双歧杆菌粘附素受体的初步观察

本文研究了Ｌｏｖｏ细胞上双歧杆菌粘附素的受体。结果表明,过碘酸钠成或蛋白酶处理Ｌｏｖｏ细胞后,双歧杆菌对Ｌｏｖｏ细胞的粘附力明显降低,呈剂量效应。Ｄ一甘露糖能抑制两者的粘附;葡萄糖,乳糖,山梨糖,蔗糖及Ｄ一果糖不能抑制粘附。提示双歧杆菌粘附素受体是一种糖蛋白,可能与甘露糖有关。     

由蛋白质介导的双歧杆菌对体外培养肠上皮细胞的粘附

本文对双歧杆菌和体外肠上皮细胞系Ｌｏｖｏ细胞间的粘附进行了研究。结果表明,双歧杆菌能特异性地粘附于肠上皮细胞周围,并且具有浓度和时间效应;各株双歧杆菌的粘附力存在着差异,新分离株高于标准株,胰蛋白酶处理耗尽培养液上清可完全抑制其粘附;高温也能降低粘附力;而白蛋白对粘附无影响。提示,双歧杆菌粘附素可能是一种不耐热的蛋白质,主要存在于耗尽培养液上清中     

药物诱导后的人大肠癌细胞的[Ca^2＋]i的变化

采用荧光分光光度计法检测维甲酸（ＲＡ）、１,２５（ＯＨ）２ＶＤ３及佛波酯（ＰＭＡ）诱导ＣＣＬ２２９细胞分化后［Ｃａ２＋］ｉ变化,并观察内质网（ＥＲ）特异的Ｃａ２＋－ＡＴＰａｓｅ抑制剂Ｔｈａｐｓｉｇａｒｇｉｎ（ＴＧ）、ＩＰ３受体抑制剂Ｈｅｐａｒｉｎ对ＲＡ诱导［Ｃａ２＋］ｉ变化的影响,从而探讨ＲＡ诱导［Ｃａ２＋］ｉ变化与ＥＲ的关系。结果显示：ＲＡ和１,２５（ＯＨ）２ＶＤ３在数秒内引起［Ｃａ２＋］ｉ显著升高。在ＥＧＴＡ和Ｖｅｒａｐａｍｉｌ预处理细胞条件下,ＴＧ不能抑制ＲＡ引起Ｃａ２＋从细胞内钙池中外流,ＲＡ作用后ＴＧ仍能升高［Ｃａ２＋］ｉ。另外,Ｈｅｐａｒｉｎ也不能完全抑制ＲＡ升高［Ｃａ２＋］ｉ。提示ＲＡ诱导大肠癌细胞升高［Ｃａ２＋］ｉ可能通过ＥＲ上ＩＰ３敏感性和非敏感性钙池,亦可能细胞内存在除ＥＲ外对ＲＡ敏感的钙池。     

钙离子在颈动脉体化学感受性传递过程中的作用

在颈动脉体（ＣＢ）化学感受性传递过程中,球细胞释放递质的机制至今尚未阐明。有文献报道无Ｃａ２＋时化学感受器释放多巴胺和颈动脉窦神经电活动均减少。另有实验研究显示,在细胞外液无Ｃａ２＋时基础状态下和低氧诱导的球细胞内ｃＡＭＰ明显升高,腺苷酸环化酶激活剂...     

肠道菌感染靶细胞过程中的跨膜信号传递

伤寒沙门菌和肠致病性大肠杆菌（ＥＰＥＣ）都是重要的人类肠道致病原,对它们的控制一直相当困难,尤其在大量广谱、新型抗生素的医院中,它们均可成为医院获得性感染的重要病原菌,本文从１,４,５－三磷酸肌醇（ＩＰ３）、Ｃａ＾２＋及甘油二酯（ＤＧ）、蛋白激酶（ＰＫＣ）等信号传导系统在肠道菌粘附,感染靶细胞时所起的作用入手,对细菌粘附宿主细胞后的细胞形态变化,感染菌的入侵及损伤过程提出新的分子机理。     

平滑肌收缩中Ca^2＋敏感性调节的机理

多种激动剂增加细胞器对Ｃａ＾２＋的敏感性,即Ｃａ＾２＋的敏感性,即Ｃａ＾２＋敏感化作用。激动剂的这种作用可能是通过Ｇ蛋白,经信号分子花生四烯酸和二酰基甘油,反暹号传递到肌球蛋白轻链磷酸酶（ＭＬＣＰ）,增加肌球蛋白磷酸化。细胞内游离Ｃａ＾２＋升高到一定程度,ｃａｌｍｏｄｕｌｉｎ激酶Ⅱ活化,导致ＭＬＣＫ磷酸化,降低了其对Ｃａ＾２＋－ｃａｌｍｏｄｕｌｉｎ亲和力,ＭＬＣＫ对Ｃａ＾２＋敏感性降低,即Ｃａ＾２     

Streptococcus pneumoniae infection of host epithelial cells via polymeric immunoglobulin receptor transiently induces calcium release from intracellular stores

The pneumococcal surface protein C (PspC) is a major adhesin of Streptococcus pneumoniae (pneumococci) that interacts in a human-specific manner with the ectodomain of the human polymeric immunoglobulin receptor (pIgR) produced by respiratory epithelial cells. This interaction promotes bacterial colonization and bacterial internalization by initiating host signal transduction cascades. Here, we examined alterations of intracellular calcium ([Ca(2+)](i)) levels in epithelial cells during host cell infections with pneumococci via the PspC-hpIgR mechanism. The release of [Ca(2+)](i) from intracellular stores in host cells was significantly increased by wild-type pneumococci but not by PspC-deficient pneumococci. The increase in [Ca(2+)](i) was dependent on phospholipase C as pretreatment of cells with a phospholipase C-specific inhibitor U73122 abolished the increase in [Ca(2+)](i). In addition, we demonstrated the effect of [Ca(2+)](i) on pneumococcal internalization by epithelial cells. Uptake of pneumococci was significantly increased after pretreatment of epithelial cells with the cell-permeable calcium chelator 1,2-bis-(o-aminophenoxy)-ethane-N,N,N',N'-tetraacetic acid-tetraacetoxymethyl ester or use of EGTA as an extracellular Ca(2+)-chelating agent. In contrast, thapsigargin, an inhibitor of endoplasmic reticulum Ca(2+)ATPase, which increases [Ca(2+)](i) in a sustained fashion, significantly reduced pIgR-mediated pneumococcal invasion. Importantly, pneumococcal adherence to pIgR-expressing cells was not altered in the presence of inhibitors as demonstrated by immunofluorescence microscopy. In conclusion, these results demonstrate that pneumococcal infections induce mobilization of [Ca(2+)](i) from intracellular stores. This may constitute a defense response of host cells as the experimental reduction of intracellular calcium levels facilitates pneumococcal internalization by pIgR-expressing cells, whereas elevated calcium levels diminished bacterial internalization by host epithelial cells.     

Intracellular elevations of free calcium induced by activation of histamine H1 receptors in interphase and mitotic HeLa cells: hormone signal transduction is altered during mitosis

A broad range of membrane functions, including endocytosis and exocytosis, are strongly inhibited during mitosis. The underlying mechanisms are unclear, however, but will probably be important in relation to the mitotic cycle and the regulation of surface phenomena generally. A major unanswered question is whether membrane signal transduction is altered during mitosis; suppression of an intracellular calcium [( Ca2+]i) transient could inhibit exocytosis; [Ca2+]i elevation could disassemble the mitotic spindle. Activation of the histamine H1 receptor interphase in HeLa cells is shown here by Indo-1 fluorescence to produce a transient elevation of [Ca2+]i. The [Ca2+]i transient consists of an initial sharp rise that is at least partially dependent on intracellular calcium followed by an elevated plateau that is absolutely dependent on extracellular calcium. The [Ca2+]i transient is completely suppressed by preincubation with the tumor promoter, phorbol myristate acetate, but is unaffected by preincubation with pertussis toxin (islet-activating protein). In mitotic (metaphase- arrested) HeLa cells, the [Ca2+]i transient is largely limited to the initial peak. Measurement of 45Ca2+ uptake shows that it is stimulated by histamine in interphase cells, but not in mitotics. We conclude that the histamine-stimulated generation of the second messenger, [Ca2+]i, in mitotic cells is limited by failure to activate a sustained calcium influx. The initial phase of calcium mobilization from intracellular stores is comparable to that in interphase cells. Hormone signal transduction thus appears to be altered during mitosis.     

Species-specific cell adhesion of enteropathogenic Escherichia coli is mediated by type IV bundle-forming pili

Enteropathogenic Escherichia coli (EPEC) is a causative agent of diarrhoea in humans. Localized adherence of EPEC onto intestinal mucosa was reproduced in an in vitro adherence assay with cultured human epithelial cells. We found that the efficiency of EPEC adherence to a mouse-derived colonic epithelial cell line, CMT-93, was remarkably lower than its adherence to human-derived intestinal cell lines, such as Intestine-407 or Caco-2. Although EPEC did adhere to some cell lines derived from non-human species, fixing the cells with formalin to inactivate one or more formalin-sensitive factors allowed us to observe species-specific differences in EPEC adherence. In contrast to these results, an EPEC mutant that is defective in bundle-forming pili (BFP) production adhered as efficiently to CMT-93 cells as to Caco-2 cells. Furthermore, Citrobacter rodentium expressing BFP adhered to Caco-2 cells much more efficiently than to CMT-93 cells. Finally, a purified BfpA-His6 fusion protein showed higher affinity for Caco-2 cells than for CMT-93 cells, and inhibited EPEC adherence. Following BFP-mediated adherence, secretion of EspB from adherent bacteria and reorganization of F-actin in the host cells was observed. EPEC adhering to CMT-93 cells induced far less secretion of EspB, or reorganization of F-actin in the host CMT-93 cells, than did EPEC adhering to Caco-2 cells. These results indicated that BFP plays an important role in the cell-type-dependent adherence of EPEC and in the progression to the later steps in EPEC adherence.     

Inhibition of adhesion of enteroinvasive pathogens to human intestinal Caco-2 cells by Lactobacillus acidophilus strain LB decreases bacterial invasion

Abstract Salmonella typhimurium and enteropathogenic Escherichia coli (EPEC) were found to adhere to the brush border of differentiated human intestinal epithelial Caco-2 cells in culture, whereas Yersinia pseudotuberculosis and Listeria monocytogenes adhered to the periphery of undifferentiated Caco-2 cells. All these enterovirulent strains invaded the Caco-2 cells. Using a heat-killed human Lactobacillus acidophilus (strain LB) which strongly adheres both to undifferentiated and differentiated Caco-2 cells, we have studied inhibition of cell association with and invasion within Caco-2 cells by enterovirulent bacteria. Living and heat-killed Lactobacillus acidophilus strain LB inhibited both cell association and invasion of Caco-2 cells by enterovirulent bacteria in a concentration-dependent manner. The mechanism of inhibition of both adhesion and invasion appears to be due to steric hindrance of human enterocytic pathogen receptors by whole-cell lactobacilli rather than to a specific blockade of receptors.     

Staphylococcal enterotoxin A modulates intracellular Ca2+ signal pathway in human intestinal epithelial cells

We demonstrate here that staphylococcal enterotoxin A (SEA) induces an increase in intracellular calcium ([Ca2+]i) in human intestinal epithelial cells and the [Ca2+]i is released from intracellular stores. SEA-induced increase of [Ca2+]i was clearly inhibited by treatment with a nitric oxide synthase (NOS) inhibitors, N(G)-monomethyl-L-arginine and guanidine. Intestinal epithelial cells express endothelial NOS in resting cell condition, and express inducible NOS after stimulating with tumor necrosis factor (TNF)-alpha. TNF-alpha-pretreated cells showed a significant increase in [Ca2+]i that was also inhibited by the NOS inhibitor. These results suggest that SEA modulated [Ca2+]i signal is dependent on NOS expression in human intestinal epithelial cells.     

Alterations in calcium-mediated signal transduction after traumatic injury of cortical neurons

Calcium influx and elevation of intracellular free calcium ([Ca2+]i), with subsequent activation of degradative enzymes, is hypothesized to cause cell injury and death after traumatic brain injury. We examined the effects of mild-to-severe stretch-induced traumatic injury on [Ca2+]i dynamics in cortical neurons cultured on silastic membranes. [Ca2+]i was rapidly elevated after injury, however, the increase was transient with neuronal [Ca2+]i returning to basal levels by 3 h after injury, except in the most severely injured cells. Despite a return of [Ca2+]i to basal levels, there were persistent alterations in calcium-mediated signal transduction through 24 h after injury. [Ca2+]i elevation in response to glutamate or NMDA was enhanced after injury. We also found novel alterations in intracellular calcium store-mediated signaling. Neuronal calcium stores failed to respond to a stimulus 15 min after injury and exhibited potentiated responses to stimuli at 3 and 24 h post-injury. Thus, changes in calcium-mediated cellular signaling may contribute to the pathology that is observed after traumatic brain injury.     

Detection and localization of a Ca2+-ATPase activity in Toxoplasma gondii

Toxoplasma gondii, the agent causing toxoplasmosis, is an obligate intracellular protozoan parasite. A calcium signal appears to be essential for intracellular transduction during the active process of host cell invasion. We have looked for a Ca2+-transport ATPase in tachyzoites and found Ca2+-ATPase activity (11-22 nmol Pi liberated/mg protein/min) in the tachyzoite membrane fraction. This ATP-dependent activity was stimulated by Ca2+ and Mg2+ ions and by calmodulin, and was inhibited by pump inhibitors (sodium orthovanadate or thapsigargin). We used cytochemistry and X-ray microanalysis of cerium phosphate precipitates and immunolabelling to find the Ca2+, Mg2+-ATPase. It was located mainly in the membrane complex, the conoid, nucleus, secretory organelles (rhoptries, dense granules) and in vesicles with a high calcium concentration. Thus, Toxoplasma gondii possesses Ca2+-pump ATPase (Ca2+, Mg2+-ATPase) as do eukaryotic cells.     

Rapid activation of Na+/H+ exchange by EPEC is PKC mediated

Enteropathogenic Escherichia coli (EPEC) increases sodium/hydrogen exchanger 2 (NHE2)-mediated sodium uptake by intestinal epithelial cells in a type III secretion-dependent manner. However, the mechanism(s) underlying these changes are not known. This study examines the role of a number of known secreted effector molecules and bacterial adhesins as well as the signaling pathways involved in this process. Deletion of the bacterial adhesins Tir and intimin had no effect on the increase in sodium/hydrogen exchanger (NHE) activity promoted by EPEC infection; however, there was a significant decrease upon deletion of the bundle-forming pili. Bacterial supernatant also failed to alter NHE activity, suggesting that direct interaction with bacteria is necessary. Analysis of the signal transduction cascades responsible for the increased NHE2 activity during EPEC infection showed that PLC increased Ca2+, as well as PKCalpha and PKCepsilon were involved in increasing NHE activity. The activation of PKCepsilon by EPEC has not been previously described nor has its role in regulating NHE2 activity. Because EPEC markedly increases NHE2 activity, this pathogen provides an exceptional opportunity to improve our understanding of this less-characterized NHE isoform.