干酪乳杆菌LC2W表面黏附相关蛋白的提取与鉴定

目的提取和鉴定干酪乳杆菌LC2W表面黏附相关蛋白,初步探索LC2W对胃癌细胞MKN-45细胞的黏附机制。方法LiCl处理、Sephadex G-75柱层析分离提取LC2W的表面蛋白,用黏附试验、电镜观察和SDS-PAGE电泳进行黏附相关蛋白的鉴定。结果LC2W经LiCl处理后,扫描电镜结果发现菌体表面粗糙但仍完整,黏附试验表明其对MKN-45细胞的黏附能力显著降低。提取到的表面蛋白的分子量分别为41.6、63.5、66.2 kDa。粗提物经柱层析后发现分子量为41.6 kDa的组分可以明显增强经LiCl处理过的菌体的黏附,而与未经处理的菌体黏附情况类似。结论表面蛋白参与了LC2W对MKN-45细胞的黏附,其主要活性成分的分子量为41.6 kDa。     

混合乳酸菌对小鼠 肠上皮细胞的粘附及抑菌机制

目的研究混合乳酸菌粘附及抑菌机制。方法测定混合菌株粘附相关能力及抑菌方式,初步研究菌株粘附相关的表面活性成分。结果混合菌株的表面疏水性、自聚合能力较高,对ICE-6细胞的粘附性亦较高。可抑制G+菌(金黄色葡萄球菌、枯草杆菌、艰难梭菌)和G-菌(大肠埃希菌、沙门菌、假单胞菌)粘附ICE-6细胞,抑制方式效果为排除竞争替代;热处理对菌株粘附性影响明显高于胃蛋白酶、胰蛋白酶、LiCl和NaIO_4等处理,表面蛋白(80～140kDa)可使LiCl处理菌株的粘附率提升45%。结论混合乳酸菌主要通过排除方式抑制外源菌的粘附,表面蛋白影响混合乳酸菌的粘附能力。     

三株益生菌粘附性质及机制的初步研究

目的研究3株益生菌株的粘附性质,初步探讨细菌与肠细胞的粘附机制。方法选取已被深入研究的LGG作为阳性对照,应用显微观察和平板计数的方法测定3株乳杆菌和肠上皮细胞Caco-2的粘附,选择疏水性、表面电荷和自聚合能力3项指标来描述细菌的表面性质,应用粘附抑制试验和酵母凝集试验来测定糖类专一性粘附。结果无论是用显微观察还是平板计数,ST-Ⅲ均是所测试的4株菌中粘附能力最强的,当加入细菌和细胞比约为60∶1时,ST-Ⅲ在Caco-2上的粘附数为(7.43±0.65)CFU/细胞,强于对照菌LGG[(3.99±0.57)CFU/细胞]。在所测试的4株菌中,粘附能力和疏水性具有很好的相关性,同时自聚合能力对粘附也表现出一定的促进作用。除LGG外,D-甘露糖和甲基-α-D-甘露糖苷均能抑制另外3株乳杆菌的粘附,同时这3株乳杆菌也能不同程度地凝集酵母。结论3株益生菌的粘附均涉及到非特异性的疏水结合和甘露糖特异性粘附两个过程;ST-Ⅲ是1株具有良好粘附能力的益生菌,有待进一步的研究。     

荧光标记法初探植物乳杆菌ST-Ⅲ对Caco-2细胞的粘附机理

利用荧光探针CFDA-SE标记植物乳杆菌ST-Ⅲ,测定其对Caco-2细胞粘附能力的变化,提取相关物质,探讨其粘附机理。化学和酶处理ST-Ⅲ菌悬液发现,经胃蛋白酶、胰蛋白酶、氯化锂、苯酚、盐酸胍和热处理能显著降低ST-Ⅲ的粘附性,表明表面蛋白或脂磷壁酸(Lipoteichoic acid)可能参与了ST-Ⅲ对Caco-2细胞的粘附。粘附抑制试验和可逆性结合实验证实表面蛋白而非脂磷壁酸参与了ST-Ⅲ对Caco-2细胞的粘附。结果表明ST-Ⅲ的表面蛋白粗提物可能含有粘附素类的S-层蛋白(S-layer protein),经SDS-PAGE电泳分析,此粗提物主要成分的分子量分别为72.7、34.1和24.3kD。     

双歧杆菌体外对Caco-2的黏附及其表面性质分析

【目的】体外测定双歧杆菌的黏附能力并对其表面性质进行分析。【方法】利用Caco-2细胞作为黏附模型体外测定七株菌的黏附能力,同时分析其自动聚集能力和表面疏水性,通过采用不同酶及化学物质处理双歧杆菌菌体细胞表面初步确定双歧杆菌细胞表面黏附相关化合物的类型,并对双歧杆菌表面蛋白进行电泳分析。【结果】自动聚集能力和表面疏水性均高的双歧杆菌菌株,其黏附能力高于自动聚集能力和表面疏水性均低的菌株,表现出明显的正相关。此外,受试菌株的黏附能力对蛋白酶和高碘酸钠敏感,利用LiCl对菌体表面蛋白进行提取后,其黏附能力明显下降,SDS-PAGE结果表明LiCl提取物中含有分子量大小不等的多个蛋白。【结论】双歧杆菌体外对Caco-2细胞的黏附具有菌株特异性,其黏附能力与表面疏水性质和自动聚集能力相关,此外,推测双歧杆菌表面可能含有能调节其黏附的糖蛋白类物质。     

数学模型法研究L．casei LC2W对H.pylori SS1黏附MKN-45细胞的抑制作用

目的研究干酪乳杆菌LC2W对幽门螺杆菌（H.pylori）SS1黏附MKN-45的抑制作用,探讨益生菌对致病菌拈抗的机制。方法体外培养人胃癌细胞MKN-45,采用平板计数的方法研究2株细菌的黏附性质;引入数学模型,比较LC2W与H.pylori SS1的竞争、排除和替代作用。结果运用模型可以估算出LC2W和H.pylori SS1对MKN-45最大黏附数和亲和力的大小,并可以预测在混合体系中2种菌黏附的比例;实验发现LC2W对H.pylori SS1的黏附具有很强的竞争作用和排除作用,且这2种作用存在明显的量效关系。LC2W对H.pylori SS1的黏附的替代作用不明显或过程非常缓慢。结论所采用的数学模型能较好的模拟LC2W和H.pylori SS1黏附及LC2W对H.pylori SS1黏附抑制作用,这种抑制作用主要是通过竞争性占位形成的。     

荧光标记法测定植物乳杆菌ST-Ⅲ对肠道致病菌的粘附抑制及其相关机制的研究

目的研究植物乳杆菌ST-Ⅲ对大肠埃希菌和沙门菌与Caco-2细胞粘附的抑制作用,并初探其机制。方法采用CFDA-SE荧光标记的方法测定加入ST-Ⅲ前后对致病菌对Caco-2细胞粘附能力的变化,通过化学和酶处理ST-Ⅲ细胞壁表面成分、提取相关物质,研究ST-Ⅲ对致病菌粘附的抑制机制。结果 ST-Ⅲ对2种致病菌都具有明显的抑制粘附的能力,其在相同情况下对大肠埃希菌粘附的抑制效果好于对沙门菌的效果(P0.05)。化学和酶处理表明ST-Ⅲ的表面蛋白或者磷壁酸可能参与了对致病菌粘附的抑制过程,提取后发现表面蛋白对大肠埃希菌的粘附表现出极强的抑制效果(P0.01),而对沙门菌的粘附没有抑制作用(P0.05);磷壁酸对2种致病菌均不具备抑制粘附的作用。结论 ST-Ⅲ对大肠埃希菌与Caco-2细胞粘附的抑制作用主要是通过竞争性抑制,而对沙门菌粘附的抑制作用主要是通过空间位阻形成的。     

蛋白质表面疏水性的研究

用Ｐｈｅｎｙｌ－ＳｕｐｅｒｏｓｅＨＲ５／５疏水柱在ＦＰＬＣ仪上测定了一些蛋白的表面疏水性。在被测量的蛋白样品中,细胞色素Ｃ的亲水性最强,胰凝乳蛋白酶的疏水性最强。说明蛋白质的疏水性与其表面性质密切相关,而与蛋白质的分子量、疏水残基总数并不直接相关;去辅基细胞色素Ｃ和Ｃ端缩短的金黄色葡萄球菌核酸酶与天然态比较疏水性变化很大。疏水柱层析还用于监测在低浓度胍的作用下蛋白质的构象变化。以Ｎ－乙酰酪氨酸为模型化合物探测盐酸胍对疏水柱结合能力的影响,在０４Ｍ胍存在时,Ｎ－乙酰酪氨酸在疏水柱上的结合能力略有减弱,但核糖核酸酶Ａ的变化较大,表明胍引起的蛋白质的微小构象变化有效地引起其表面性质的变化;在０．１—０．３Ｍ盐酸胍存在时,甘油醛－３－磷酸脱氢酶表面疏水性明显增大,并伴随聚合态的出现。说明在低胍作用下,酶分子发生的构象变化,导致天然态内埋疏水面的暴露,暴露的疏水面间的相互作用是形成聚合的主要原因。     

植物乳杆菌粘附性与其表面特征关系的探究

本文通过16s rDNA鉴定获得4株植物乳杆菌,并以HT29细胞为体外黏附筛选模型,进一步探讨了这些菌株粘附能力与表面疏水性、自聚共聚能力等表型特征的相关性。结果表明,植物乳杆菌AR326菌株对HT29细胞的粘附性最强,并显示高度的自聚性(25%)和共聚性(25%),但其表面疏水性偏低(15%);通过相关性分析发现,植物乳杆菌的自聚性和共聚性与HT29细胞粘附性呈显著相关性(r=1.0和0.8,p0.05),但表面疏水性、自凝聚性和共聚性两两之间并无显著相关性(p0.05)。本研究结果为建立快速筛选高粘附性植物乳杆菌的方法及其菌株在体内定植和分布研究提供一定参考依据。     

高黏附罗伊氏乳杆菌的筛选及其益生特性评价

通过对28株不同罗伊氏乳杆菌(Lactobacillus reuteri)菌株的粘附力、细胞表面疏水性、与大肠杆菌的共聚合等指标的测定及相关性分析,成功筛选出粘附力最高的菌株R28。研究了该菌株的耐酸性和耐胆盐能力。结果表明,R28与其它菌株相比具有良好的粘附能力。该菌株的自聚集能力和疏水性分别为26.25%和75.53%。与大肠杆菌的共聚合能力可高达36.84%。细胞粘附数可达约43个细菌/每个杆菌细胞。菌株R28具有良好的耐酸性(pH=3)和耐胆盐性(0.1%),其能够很好的粘附于宿主肠道细胞发挥益生作用。罗伊氏乳杆菌R28作为一株优良的益生菌在食品和医药领域具有广阔的应用前景。     

Debaryomyces hansenii strains with different cell sizes and surface physicochemical properties adhere differently to a solid agarose surface

The initial adhesion of four Debaryomyces hansenii strains to a solid agarose surface was investigated and correlated with their cell size and some cell surface physicochemical properties, i.e. (i) hydrophobicity and (ii) electron donor/acceptor ability. One strain adhered very poorly, whereas the three other strains were more adhesive. The former strain had a very hydrophilic cell surface, whereas the latter strains had more hydrophobic cell surfaces. In addition, the strain with the lowest adhesion among the adhesive strains had a more hydrophobic cell surface than the two most adhesive strains. Finally, the more adhesive the strain was, the larger it was, and the better it was to donate electrons from its cell surface. These results show a clear relationship between the cell size, the cell surface physicochemical properties, and the initial adhesion of D. hansenii. A possible explanation of this relationship is discussed.     

乳酸杆菌表面结构和粘附特性的研究进展

乳酸杆菌是人和动物肠道中重要的益生菌.乳酸杆菌的表面特性决定其对肠道的粘附和定植及生理功能的发挥.研究乳酸杆菌表面结构对了解乳酸杆菌的粘附过程具有重要意义.对近年来国外关于乳酸杆菌表面结构特征和理化性质的研究进行了概述,并探讨分析了乳酸杆菌表面结构与乳酸杆菌粘附特性之间潜在的关系.     

The state diagram for cell adhesion mediated by two receptors

Leukocyte recruitment from the bloodstream to surrounding tissues is an essential component of the immune response. Capture of blood-borne leukocytes onto vascular endothelium proceeds via a two-step mechanism, with each step mediated by a distinct receptor-ligand pair. Cells first transiently adhere, or "roll" (via interactions between selectins and sialyl-Lewis-x), and then firmly adhere to the vascular wall (via interactions between integrins and ICAM-1). We have reported that a computational method called adhesive dynamics (AD) accurately reproduces the fine-scale dynamics of selectin-mediated rolling. This paper extends the use of AD simulations to model the dynamics of cell adhesion when two classes of receptors are simultaneously active: one class (selectins or selectin ligands) with weakly adhesive properties, and the other (integrins) with strongly adhesive properties. AD simulations predict synergistic functions of the two receptors in mediating adhesion. At a fixed density of surface ICAM-1, increasing selectin densities lead to greater pause times and an increased tendency toward firm adhesion; thus, selectins mechanistically facilitate firm adhesion mediated by integrins. Conversely, at a fixed density of surface selectin, increasing ICAM-1 densities lead to greater pause times and an increased tendency to firm adhesion. We present this relationship in a two-receptor state diagram, a map that relates the densities and properties of adhesion molecules to various adhesive behaviors that they code, such as rolling or firm adhesion. We also present a state diagram for neutrophil activation, which relates beta(2)-integrin density and integrin-ICAM-1 kinetic on rate to neutrophil adhesive behavior. The predictions of two-receptor adhesive dynamics are validated by the ability of the model to reproduce in vivo neutrophil rolling velocities from the literature.     

Thrombin adhesive properties: induction by plasmin and heparan sulfate

We have previously demonstrated that chemically modified thrombin preparations induce endothelial cell (EC) adhesion, spreading and cytoskeletal reorganization via an Arg-Gly-Asp (RGD) sequence and the alpha v beta 3 integrin. Native thrombin, however, did not exhibit adhesive properties, consistent with crystal structure analysis, showing that Gly-Asp residues of the RGD epitope are buried within the molecule. We have now identified a possible physiological mean of converting thrombin to an adhesive protein. Plasmin, the major end product of the fibrinolytic system, converted thrombin to an adhesive protein for EC in a time and dose-dependent manner. EC adhesion and spreading was also induced by a low molecular weight (approximately 3,000 D) cleavage fragment generated upon incubation of thrombin with plasmin. Cell adhesion mediated by this fragment was completely inhibited by the synthetic peptide GRGDSP. Conversion of thrombin to an adhesive molecule was significantly enhanced in the presence of heparin or heparan sulfate, while other glycosaminoglycans (GAGs) (e.g., dermatan sulfate, keratan sulfate, chondroitin sulfate) had no effect. The role of cell surface heparan sulfate in thrombin conversion to EC adhesive protein was investigated using CHO cell mutants defective in various aspects of GAG synthesis. Incubation of both thrombin and a suboptimal amount of plasmin on the surface of formaldehyde fixed wild- type CHO-KI cells resulted in an efficient conversion of thrombin to an adhesive molecule, as indicated by subsequent induction of EC attachment. In contrast, there was no effect to incubation of thrombin and plasmin with fixed CHO mutant cells lacking both heparan sulfate and chondroitin sulfate, or with cells expressing no heparan sulfate and a three-fold increase in chondroitin sulfate. A similar gain of adhesive properties was obtained upon incubation of thrombin and plasmin in contact with native, but not heparinase-treated extracellular matrix (ECM) produced by cultured ECs. It appears that cell surface and ECM-associated heparan sulfate modulate thrombin adhesive properties through its heparin binding site in a manner that enables suboptimal amounts of plasmin to expose the RGD domain. Our results demonstrate, for the first time, a significant modulation of thrombin molecule by heparin, resulting in its conversion to a potent adhesive protein for ECs. This conversion is most effective in contact with cell surfaces, basement membranes and ECM.     

A study of the adhesive, locomotory and invasive behaviour of Walker 256 carcinosarcoma cells

We have succeeded in selecting two variant strains of the Walker 256 carcinosarcoma which display markedly different adhesive properties. Both the high (W256A) and the low (W256S) adhesive variants respond chemotactically towards 10(-8) M f-met-leu-phe (FMLP) although there is a significant difference in their locomotory ability. Nevertheless, the fact that the essentially non-adherent W256S cells can migrate in vitro argues against any simple relationship between adhesion and locomotion. We suggest that traction is important in locomotion but that it need not arise only from direct adhesive interaction. We have also tested the invasive behaviour of the W256 variants using an in vitro model system in which disruption of a cellular barrier by the invasive cells can be recorded electrophysiologically. Although leucocytes can penetrate such a barrier they do so only under chemotactic stimulation, whereas W256 tumour cells of either variant strain will do so spontaneously. The tumour variants induce cell retraction within the barrier and this may lead ultimately to cell detachment and death. The holes which arise may then be colonized by tumour cells, and in this way the invasive process could be promoted. The molecular mechanisms by which tumour cells achieve destruction of the cellular barrier are not clear, but it is likely that a number of enzymes are involved.     

Functional correlation between cell adhesive properties and some cell surface proteins

The adhesive properties of Chinese hamster V79 cells were analyzed and characterized by various cell dissociation treatments. The comparisons of aggregability among cells dissociated with EDTA, trypsin + Ca2+, and trypsin + EDTA, revealed that these cells have two adhesion mechanisms, a Ca2+-independent and a Ca2+-dependent one. The former did not depend on temperature, whereas the latter occurred only at physiological temperatures. Both mechanisms were trypsin sensitive, but the Ca2+- dependent one was protected by Ca2+ against trypsinization. In morphological studies, the Ca2+-independent adhesion appeared to be a simple agglutination or flocculation of cells, whereas the Ca2+- dependent adhesion seemed to be more physiological, being accompanied by cell deformation resulting in the increase of contact area between adjacent cells. Lactoperoxidase-catalyzed iodination of cell surface proteins revealed that several proteins are more intensely labeled in cells with Ca2+-independent adhesiveness than in cells without that property. It was also found that a cell surface protein with a molecular weight of approximately 150,000 is present only in cells with Ca2+-dependent adhesiveness. The iodination and trypsinization of this protein were protected by Ca2+, suggesting its reactivity to Ca2+. Possible mechanisms for each adhesion property are discussed, taking into account the correlation of these proteins with cell adhesiveness.     

Mechanisms of temporary adhesion in benthic animals

Adhesive systems are ubiquitous in benthic animals and play a key role in diverse functions such as locomotion, food capture, mating, burrow building, and defence. For benthic animals that release adhesives, surface and material properties and external morphology have received little attention compared to the biochemical content of the adhesives. We address temporary adhesion of benthic animals from the following three structural levels: (a) the biochemical content of the adhesive secretions, (b) the micro‐ and mesoscopic surface geometry and material properties of the adhesive organs, and (c) the macroscopic external morphology of the adhesive organs. We show that temporary adhesion of benthic animals is affected by three structural levels: the adhesive secretions provide binding to the substratum at a molecular scale, whereas surface geometry and external morphology increase the contact area with the irregular and unpredictable profile of the substratum from micro‐ to macroscales. The biochemical content of the adhesive secretions differs between abiotic and biotic substrata. The biochemistry of the adhesives suitable for biotic substrata differentiates further according to whether adhesion must be activated quickly (e.g. as a defensive mechanism) or more slowly (e.g. during adhesion of parasites). De‐adhesion is controlled by additional secretions, enzymes, or mechanically. Due to deformability, the adhesive organs achieve intimate contact by adapting their surface profile to the roughness of the substratum. Surface projections, namely cilia, cuticular villi, papillae, and papulae increase the contact area or penetrate through the secreted adhesive to provide direct contact with the substratum. We expect that the same three structural levels investigated here will also affect the performance of artificial adhesive systems.     

Quantitative characterization of single‐cell adhesion properties by atomic force microscopy using protein‐functionalized microbeads

A method was developed to characterize the adhesion properties of single cells by using protein‐functionalized atomic force microscopy (AFM) probes. The quantification by force spectroscopy of the mean detachment force between cells and a gelatin‐functionalized colloidal tip reveals differences in cell adhesion properties that are not within reach of a traditional bulk technique, the washing assay. In this latter method, experiments yield semiquantitative and average adhesion properties of a large population of cells. They are also limited to stringent conditions and cannot highlight disparities in adhesion in the subset of adherent cells. In contrast, this AFM‐based method allows for a reproducible and quantitative investigation of the adhesive properties of individual cells in common cell culture conditions and allows for the detection of adhesive subpopulations of cells. These characteristics meet the critical requirements of many fields, such as the study of cancer cell migratory abilities.