Binding of Extracellular Matrix Proteins by Enterococci

Forty-four enterococcal strains isolated from human clinical specimens were investigated for binding of ¹²⁵I-labeled fibronectin, vitronectin, thrombospondin, lactoferrin, and collagen type I and IV, and for cell surface hydrophobicity. Most strains expressed low binding of iodine-labeled human fibronectin, collagen I and IV, and higher binding of human vitronectin, human lactoferrin, and human thrombospondin. Bacteria grown in Todd-Hewitt broth exhibited increased binding to vitronectin and thrombospondin. In particle agglutination assays (PAA), Enterococcus faecalis strains reacted strongly with coated latex beads in contrast to E. faecium strains, which generally did not react. The ability of enterococci to bind ECM proteins was affected by heating and proteolytic digestion, suggesting that some protein-binding components become surface exposed after treatment with proteases. The binding of ¹²⁵I-labeled proteins to E. faecalis strain E70 was inhibited when cells were preincubated with unlabeled proteins. Preincubating cells with sulfated polymers such as dextran sulfate (M _r 5000 and 8000), pentosan sulfate and heparin decreased binding of vitronectin, lactoferrin, and thrombospondin. The binding of lactoferrin and thrombospondin was also decreased when bacteria were preincubated with galactose, fucose, and mannosamine, but not with mannose. All of 30 E. faecalis strains expressed pronounced surface hydrophobicity, but 10 of 14 E. faecium strains showed hydrophilic cell surface. Received: 22 April 1996 / Accepted: 29 June 1996     

Role of Platelets in Hemostasis and Thrombosis

Platelets interact with the coagulation factors in a complex way to arrest bleeding or generate thrombi. Recently, the platelet''s relationship to endothelial alteration and atheroma production has received renewed attention. At present, tests of platelet function better define “hypocoagulable” rather than “hypercoagulable” states.     

The Role of Cellular and Extracellular Matrix Adhesion Proteins in Organ Transplantation

The specific adhesion of cells to other cells or to particular tissue microenvirorvments is a basic function of cell migration and recognition, and underlines many biologic processes including embryogenesis, repair and immunity. Leukocytes express an array of surface receptors broadly known as “accessory adhesion molecules.” which mediate most cell -cell interactions, direct lymphocyte traffic between anatomical compartments, and facilitate cellular adhesion to the inflammation or alloantigenic sites (Springer 1990). In addition, adhesion molecules are involved in the process of antigen recognition, and may costimulate cell activation and transformation. These proteins are thought to affect the very early antigen independent events between host leukocytes and vascular endothelium. Because of these activities, the subject of adhesion molecules is gaining interest in the field of organ transplantation, in both conceptualization and development of novel therapeutic strategies (de Sousa et al. 1991, Kupiec-Weglinski et al. 1993a, Heemann et al. 1993).     

The Role of Cellular and Extracellular Matrix Adhesion Proteins in Organ Transplantation

The specific adhesion of cells to other cells or to particular tissue microenvirorvments is a basic function of cell migration and recognition, and underlines many biologic processes including embryogenesis, repair and immunity. Leukocytes express an array of surface receptors broadly known as “accessory adhesion molecules.” which mediate most cell -cell interactions, direct lymphocyte traffic between anatomical compartments, and facilitate cellular adhesion to the inflammation or alloantigenic sites (Springer 1990). In addition, adhesion molecules are involved in the process of antigen recognition, and may costimulate cell activation and transformation. These proteins are thought to affect the very early antigen independent events between host leukocytes and vascular endothelium. Because of these activities, the subject of adhesion molecules is gaining interest in the field of organ transplantation, in both conceptualization and development of novel therapeutic strategies (de Sousa et al. 1991, Kupiec-Weglinski et al. 1993a, Heemann et al. 1993).     

Evidence for Cell Surface Extracellular Matrix Binding Proteins in Hydra vulgaris

The present study was designed to identify and functionally characterize potential cell surface extracellular matrix binding proteins in Hydra vulgaris. Using [³H]-laminin as a probe, radioreceptor analysis of a dissociated mixed hydra cell preparation indicated that the average number of laminin binding sites per cell was about 10,000 with a dissociation constant of 1.49 nM. These binding sites could be displaced with unlabelled laminin in a dose-dependent manner and with high concentrations (500 nM) of unlabelled fibronectin. No displacement with type-IV collagen and type-I collagen was observed. Immunoscreerting studies with a battery of antibodies raised to mammalian extracellular matrix (ECM) binding proteins indicated potential cell surface binding sites for the anti-β₁ integrin monoclonal antibody, mAb JG22. Cell adhesion studies indicated that mAb JG22 blocked binding of hydra cells to laminin, but did not affect their binding to fibronectin, type-IV collagen, or type-I collagen. Light and electron microscopic immunocytochemical studies indicated that mAb JG22 localized to the basal plasma membrane of ectodermal and endodermal epithelial cells. Immunoprecipitation studies identified two major bands with masses of about 196 kDa and 150 kDa under reducing conditions, and two bands with masses of >200 kDa under non-reducing conditions. Functional studies indicated that mAb JG22 could reversibly block morphogenesis of hydra cell aggregates, and could block in vivo interstitial cell migration in hydra grafts. These observations indicate that hydra has cell surface binding sites for ECM components which are functionally important during development of this simple Cnidarian     

Effect of Extracellular Matrix Proteins on Vascular Smooth Muscle Cell Phenotype

The effect on phenotypic expression of rabbit vascular smooth muscle cells (SMC) of the interstitial matrix proteins collagen I and fibronectin, the basal lamina proteins collagen IV and laminin, and the serum adhesion protein vitronectin was examined in culture. Experiments were performed in foetal calf serum stripped of fibronectin and vitronectin to eliminate their confounding effects. All the proteins promoted adhesion to the plastic culture dish (in a concentration dependent manner) of SMC freshly isolated from the artery wall. These cells had a high volume density of myofilaments (V_vmyo) in their cytoplasm. Laminin was best at maintaining SMC with a high V_vmyo (V_vmyo = 49.8%) followed by collagen IV (41.7%). Cells plated on vitronectin showed the lowest V_vmyo (31.3%). The results support the concept that the SMC basal lamina has a role in maintaining cells in the high V_vmyo phenotype.     

An Ensemble Method with Hybrid Features to Identify Extracellular Matrix Proteins

The extracellular matrix (ECM) is a dynamic composite of secreted proteins that play important roles in numerous biological processes such as tissue morphogenesis, differentiation and homeostasis. Furthermore, various diseases are caused by the dysfunction of ECM proteins. Therefore, identifying these important ECM proteins may assist in understanding related biological processes and drug development. In view of the serious imbalance in the training dataset, a Random Forest-based ensemble method with hybrid features is developed in this paper to identify ECM proteins. Hybrid features are employed by incorporating sequence composition, physicochemical properties, evolutionary and structural information. The Information Gain Ratio and Incremental Feature Selection (IGR-IFS) methods are adopted to select the optimal features. Finally, the resulting predictor termed IECMP (Identify ECM Proteins) achieves an balanced accuracy of 86.4% using the 10-fold cross-validation on the training dataset, which is much higher than results obtained by other methods (ECMPRED: 71.0%, ECMPP: 77.8%). Moreover, when tested on a common independent dataset, our method also achieves significantly improved performance over ECMPP and ECMPRED. These results indicate that IECMP is an effective method for ECM protein prediction, which has a more balanced prediction capability for positive and negative samples. It is anticipated that the proposed method will provide significant information to fully decipher the molecular mechanisms of ECM-related biological processes and discover candidate drug targets. For public access, we develop a user-friendly web server for ECM protein identification that is freely accessible at http://iecmp.weka.cc.     

细胞外间质

细胞外间质由四大家族组成,胶原蛋白,蛋白多糖。弹性蛋白和细胞外间质糖蛋白。细胞外间质成分不仅仅是细胞的惰性支持物,它具有活性的生物功能,例如细胞粘附及迁移,甚至涉及基因表达。细胞外间质研究是一个十分活跃的生物学领域。     

Binding of Selected Extracellular Matrix Proteins to Enterococci and Streptococcus bovis of Animal Origin

Thirty-three enterococcal strains and 10 Streptococcus bovis strains were investigated for their protein-binding cell surface components. Seven extracellular matrix (ECM) proteins were immobilized on Difco latex beads to detect these components on the surface of all enterococcal strains and eight non-autoaggregating S. bovis strains by a particle agglutination assay (PAA). Twenty-three selected strains were also examined in microtiter plate assays. According to the absorbance readings (A_570nm), 11 strains were classified as nonadherent (A_570nm < 0.1), 10 strains as weakly adherent (0.1 < A_570nm > 0.3), and 2 strains as strongly adherent (A_570nm > 0.3) in these assays. A direct correlation was found between the values obtained in PAA and A_570nm readings of microtiter plate assays. Binding of ¹²⁵I-labeled bovine lactoferrin to enterococci and streptococci was in the range of 6%–30% and of ¹²⁵I-labeled human vitronectin in the range of 9%–33% to streptococci. The binding of ¹²⁵I-labeled ECM proteins to selected strains was much more effectively inhibited by sulfated carbohydrates than by non-sulfated hyaluronic acid, indicating the importance of the sulfate groups of these inhibitors. An inhibition effect of heparin on bLf binding to four selected strains was higher in comparison with fucoidan in the microtiter plates. Thirty-five out of 44 strains had agglutinated rabbit erythrocytes. However, these strains showed no ability to agglutinate bovine or sheep erythrocytes. Received: 28 April 1999 / Accepted: 26 July 1999     

基质金属蛋白酶与血管壁细胞外基质重建

细胞外基质（ECM）不仅维持血管壁的完整性,而且还为血管细胞传递增殖,迁移,分化和凋亡的调控信号,基质金属蛋白酶（MMP）及其内源性抑制剂（TIMP）通过调节ECM合成与降解之间的动态平衡,使ECM维持正常的结构与功能。在高血压,动脉粥样硬化与血管再狭窄的发生与发展过程中,MMP和TIMP的合成与分泌出现异常,由此所引起的ECM合成与降解失调使ECM迅速发生重建。     

Enrichment of Extracellular Matrix Proteins from Tissues and Digestion into Peptides for Mass Spectrometry Analysis

The extracellular matrix (ECM) is a complex meshwork of cross-linked proteins that provides biophysical and biochemical cues that are major regulators of cell proliferation, survival, migration, etc. The ECM plays important roles in development and in diverse pathologies including cardio-vascular and musculo-skeletal diseases, fibrosis, and cancer. Thus, characterizing the composition of ECMs of normal and diseased tissues could lead to the identification of novel prognostic and diagnostic biomarkers and potential novel therapeutic targets. However, the very nature of ECM proteins (large in size, cross-linked and covalently bound, heavily glycosylated) has rendered biochemical analyses of ECMs challenging. To overcome this challenge, we developed a method to enrich ECMs from fresh or frozen tissues and tumors that takes advantage of the insolubility of ECM proteins. We describe here in detail the decellularization procedure that consists of sequential incubations in buffers of different pH and salt and detergent concentrations and that results in 1) the extraction of intracellular (cytosolic, nuclear, membrane and cytoskeletal) proteins and 2) the enrichment of ECM proteins. We then describe how to deglycosylate and digest ECM-enriched protein preparations into peptides for subsequent analysis by mass spectrometry.     

Action of the Major Protease from Entamoeba histolytica on Proteins of the Extracellular Matrix

The action of the major protease from the parasitic protozoon Entamoeba histolytica , a cysteine protease of M, 27,000–29,000, on some important proteins of the extracellular matrix has been studied. The isolated protease degraded the extracellular matrix proteins from human tissue collagen type IV and V as well as laminin and fibronectin with different velocities and specificities under native conditions. Whereas the degradation of fibronectin and laminin proceeded rapidly, yielding distinct fragment patterns, the breakdown of the collagen types happened more slowly and incompletely. The digestion of the denatured isolated α2-chain of bovine collagen type I was very fast and unspecific requiring only 1/10 of the enzyme activities as compared with the other substrates mentioned above. Nearly 85% of the overall proteolytic activity of a soluble fraction of E. histolytica was strongly inhibited by antibodies against the purified histolytic protease as well as by cystatin from chicken egg white, a specific protein inhibitor of cysteine proteases. We conclude that the histolytic protease represents by far the highest portion of soluble proteolytic activity in E. histolytica which is sufficient to destroy the extracellular matrix of the host.     

Helicobacter pylori Interactions with Host Serum and Extracellular Matrix Proteins: Potential Role in the Infectious Process

Helicobacter pylori, a gram-negative spiral-shaped bacterium, specifically colonizes the stomachs of humans. Once established in this harsh ecological niche, it remains there virtually for the entire life of the host. To date, numerous virulence factors responsible for gastric colonization, survival, and tissue damage have been described for this bacterium. Nevertheless, a critical feature of H. pylori is its ability to establish a long-lasting infection. In fact, although good humoral (against many bacterial proteins) and cellular responses are observed, most infected persons are unable to eradicate the infection. A large body of evidence has shown that the interaction between H. pylori and the host is very complex. In addition to the effect of virulence factors on colonization and persistence, binding of specialized bacterial proteins, known as receptins, to certain host molecules (ligands) could explain the success of H. pylori as a chronically persisting pathogen. Some of the reported interactions are of high affinity, as revealed by their calculated dissociation constant. This review examines the binding of host proteins (serum and extracellular matrix proteins) to H. pylori and considers the significance of these interactions in the infectious process. A more thorough understanding of the kinetics of these receptin interactions could provide a new approach to preventing deeper tissue invasion in H. pylori infections and could represent an alternative to antibiotic treatment.     

Diabetic Nephropathy and Extracellular Matrix

Diabetic nephropathy (DN) is a serious complication in diabetes. Major typicalmorphological changes are the result of changes in the extracellular matrix (ECM). Thus,basement membranes are thickened and the glomerular mesangial matrix and thetubulointerstitial space are expanded, due to increased amounts of ECM. One important ECMcomponent, the proteoglycans (PGs), shows a more complex pattern of changes in DN. PGs inbasement membranes are decreased but increased in the mesangium and the tubulointerstitialspace. The amounts and structures of heparan sulfate chains are changed, and such changesaffect levels of growth factors regulating cell proliferation and ECM synthesis, with cellattachment affecting endothelial cells and podocytes. Enzymes modulating heparan sulfatestructures, such as heparanase and sulfatases, are implicated in DN. Other enzyme classesalso modulate ECM proteins and PGs, such as matrix metalloproteinases (MMPs) and serineproteases, such as plasminogen activator, as well as their corresponding inhibitors. Thelevels of these enzymes and inhibitors are changed in plasma and in the kidneys in DN.Several growth factors, signaling pathways, and hyperglycemia per se affect ECM synthesisand turnover in DN. Whether ECM components can be used as markers for early kidney changesis an important research topic, whereas at present, the clinical use remains to beestablished.     

Extracellular Matrix in Neural Plasticity and Regeneration

The extracellular matrix (ECM) is a fundamental component of biological tissues. The ECM in the central nervous system (CNS) is unique in both composition and function. Functions such as learning, memory, synaptogenesis, and plasticity are regulated by numerous ECM molecules. The neural ECM acts as a non-specific physical barrier that modulates neuronal plasticity and axon regeneration. There are two specialized types of ECM in the CNS, diffuse perisynaptic ECM and condensed ECM, which selectively surround the perikaryon and initial part of dendritic trees in subtypes of neurons, forming perineuronal nets. This review presents the current knowledge about the role of important neuronal ECM molecules in maintaining the basic functions of a neuron, including electrogenesis and the ability to form neural circuits. The review mainly focuses on the role of ECM components that participate in the control of key events such as cell survival, axonal growth, and synaptic remodeling. Particular attention is drawn to the numerous molecular partners of the main ECM components. These regulatory molecules are integrated into the cell membrane or disposed into the matrix itself in solid or soluble form. The interaction of the main matrix components with molecular partners seems essential in molecular mechanisms controlling neuronal functions. Special attention is paid to the chondroitin sulfate proteoglycan 4, type 1 transmembrane protein, neural-glial antigen 2 (NG2/CSPG4), whose cleaved extracellular domain is such a molecular partner that it not only acts directly on neural and vascular cells, but also exerts its influence indirectly by binding to resident ECM molecules.

     

Regulation of Human (Caco-2) Intestinal Epithelial Cell Differentiation by Extracellular Matrix Proteins

Extracellular matrix regulation of intestinal epithelial differentiation may affect development, differentiation during migration to villus tips, healing, inflammatory bowel disease, and malignant transformation. Cell culture studies of intestinal epithelial biology may also depend on the matrix substrate used. We evaluated matrix effects on differentiation and proliferation in human intestinal Caco-2 epithelial cells, a model for intestinal epithelial differentiation. Proliferation, brush border enzyme specific activity, and spreading were compared in cells cultured on tissue culture plastic with interstitial collagen I and the basement membrane constituents collagen IV and laminin. Each matrix significantly increased alkaline phosphatase, dipeptidyl peptidase, lactase, sucrase-isomaltase, and cell spreading in comparison to plastic. However, the basement membrane proteins collagen IV and laminin further promoted all four brush border enzymes but inhibited spreading compared to collagen I. Proliferation was most rapid on type I collagen and slowest on laminin and tissue culture plastic. Basement membrane matrix proteins may promote intestinal epithelial differentiation and inhibit proliferation compared with interstitial collagen I.     

Effect of Influenza Virus Proteins Modulate Hemostasis in vitro and in vivo

We studied the effect of influenza virus proteins—hemagglutinin, neuraminidase, nucleoprotein, and membrane protein—on hemostasis in vitro and in vivo. The results demonstrated that envelope proteins hemagglutinin and neuraminidase enhanced the fibrinolytic and anticoagulant activities of blood plasma and the activity of human tissue plasminogen activator. The membrane protein proved to have the highest activity among the core proteins of influenza virus; in contrast to hemagglutinin and neuraminidase, it inhibited fibrinolysis, increased the coagulant activity of blood plasma, and decreased the activity of human tissue plasminogen activator. The combined action of hemagglutinin and neuraminidase increased the plasma fibrinolytic and anticoagulant activities to a greater extent than the individual action of each agent. The combined action of hemagglutinin and membrane protein also increased the plasma fibrinolytic and anticoagulant activities, although to a lesser extent than the action of hemagglutinin alone. These data indicate that viral proteins are physiologically active and can cause influenza-specific disturbances of hemostasis.