Antivinculin

Earlier observations suggested that opioids modify cell-substrate adhesion on agar. In this study, we wanted to investigate whether opioids also interfere with cell adhesion to biologically relevant substrates, including interstitial matrix and basement membrane components. Human embryonic kidney 293 cells stably transfected with FLAG-tagged micro-opioid receptor were used as an experimental model. The cells were cultured on tissue culture plastic, collagen types I and IV, fibronectin, laminin and human amnion fragments in the absence or presence of morphine...

Endothelial cell (EC) seeding of small caliber vascular grafts prior to their implantation has proved to significantly improve long-term patency in humans. We have previously demonstrated that a monolayer of EC could be obtained on type I collagen-coated knitted ultrathin polyster grafts (InterVascular, La Ciotat, France). Thus, the aim of the present work was to understand the nature of cell adhesion mechanisms involved in the cell /biomaterial interface, using HemaCarotid (InterVascular) patches made of type I collagen-coated knitted ultrathin polyster (type I collagen is used to coat patches to attain low permeability)...

The FRTL-5 rat thyroid cell line is widely used for studies of thyrocyte function and growth. The objective of the present study was to investigate the effects of hepatocyte growth factor (HGF) on FRTL-5 cells. HGF has previously been known to be a potent regulator of thyrocyte growth and differentiation. Met, the receptor for HGF was expressed in FRTL-5 cells, as well as in primary cultures of porcine thyrocytes included in the study as control. On HGF stimulation Met was tyrosine phosphorylated in both porcine and FRTL-5 cells, indicating an activation of the receptor...

In skeletal muscle fibers the costameres have been defined by Pardo et al. (1983) as transverse circumferential elements of the cytoskeleton associated to the sarcolemma. Since the first immunolocalization, carried out with purified antivinculin antibodies to the present day, about 10 proteins have been located in costameres, as well as some transmembrane proteins of the integrin superfamily. In particular the colocalization of vinculin and talin and the presence of the integrins confers to this system the description of the adherens junctions type cell-ECM, while the presence of dystrophin in correspondence to both A and I bands with Z line negative is important for the stabilization of the membrane of the skeletal muscle fiber...

In several cell types, short-term increases in the concentration of the G-actin-sequestering peptide thymosin-beta4 (Tbeta4) cause the disassembly of F-actin bundles. To determine the extent of cell adaptability to these reductions in F-actin, we overexpressed Tbeta4 in NIH 3T3 cells. In cell lines with Tbeta4 levels twice those of vector controls, G-actin increased approximately twofold as expected. However, F-actin did not decrease as in short-term experiments but rather also increased approximately twofold so that the G-F ratio remained constant...

BACKGROUND: The costameres in skeletal muscle fibers were first described by Pardo et al. (1983a) and have been defined as transverse circumferential elements of the cytoskeleton associated to the sarcolemma. Specific immunostaining for vinculin shows that the costameres overlie I bands. However, an exact correlation between the costameres and the Z line is uncertain, although approximately 10 proteins so far have been localized in the costameres. To define the exact localization of costameres in human skeletal muscle fibers, we carried out an immunofluorescence study using confocal scanning laser microscopy on the fascia lata muscle of adult males...

A subtractive analysis of peptides eluted from major histocompatibility complex (MHC) class I human histocompatibility leukocyte antigen (HLA)-A2.1 molecules purified from either human immunodeficiency virus type-1 (HIV-1)-infected or uninfected cells was performed using micro high-performance liquid chromatography and mass spectrometry. Three peptides unique to infected cells were identified and found to derive from a single protein, human vinculin, a structural protein not known to be involved in viral pathogenesis...

Tumor growth and metastasis are affected by changes in membrane lipid composition, however, little is known regarding the role of specific fatty acids in these pathological events. We investigated the effects of the long-chain saturated fatty acids (LCSFA), myristate (C14:0), palmitate (C16:0) and stearate (C18:0) on two key steps of metastasis: cell adhesion and invasion into extracellular matrix (ECM). Using a new 72-hour ECM (Amgel) invasion assay, we demonstrated that the exposure of highly invasive human fibrosarcoma HT-1080 cells to 0...

Our previous finding that normal serum immunoglobulins bind to internal platelet proteins on Western blots led us to further identify these proteins and determine the possible significance of autoantibodies against them. A 95-Kd protein reactive with immunoglobulins in most normal sera and easily confused with gpIIIa was shown to be a fragment of vinculin generated by calpain proteolysis. Identity was established by peptide sequencing of the protein purified from platelets stored without specific protease inhibitors...

The receptor for plasmin and the receptor for urokinase-type plasminogen activator were characterized on MCF 7 cells either independently or simultaneously, using fluorescence microscopy and confocal microscopy. The plasmin receptor was visualized, as previously described by Correc et al. (Int. J. Cancer 50, 767, 1992) using biotinylated plasminogen and fluoresceinated streptavidin. The urokinase receptor was revealed by both polyclonal and monoclonal antibodies reacting specifically with this receptor and by binding of urokinase aminoterminal fragment...

Immunofluorescent staining of bovine and avian cardiac tissue with affinity-purified antibody to chicken gizzard vinculin reveals two new sites of vinculin reactivity. First, vinculin is organized at the sarcolemma in a striking array of rib-like bands, or costameres. The costameres encircle the cardiac muscle cell perpendicular to the long axis of the fiber and overlie the I bands of the immediately subjacent sarcomeres. The second new site of vinculin reactivity is found in bovine cardiocytes at tubular invaginations of the plasma membrane...

Using immunocytochemical methods we have studied the distribution of vinculin in the anterior and posterior latissimus dorsi skeletal (ALD and PLD, respectively) muscles of the adult chicken. The ALD muscle is made up of both tonic (85%) and twitch (15%) myofibers, and the PLD muscle is made up entirely of twitch myofibers. In indirect immunofluorescence, antivinculin antibodies stained specific regions adjacent to the sarcolemma of the ALD and PLD muscles. In the central and myotendinous regions of the ALD, staining of the tonic fibers was intense all around the fiber periphery...

The localization of actin, myosin, tropomyosin, alpha-actinin, vinculin, and desmoplakin I/II was visualized by immunofluorescence microscopy. Antibodies against myosin, tropomyosin, and alpha-actinin and rhodamine-phalloidin labeled strongly the proximal and distal terminal webs which ultrastructurally consist of dense microfilament bundles. In the distal terminal web, the staining by these reagents occurred mostly perpendicular to the long axis of the incisor. Antivinculin stained the general area where the distal terminal web is located in the ameloblast...

Antifilamin, antivinculin, and antitropomyosin antibody activities were investigated in sera from 43 patients with myasthenia gravis (MG). Antifilamin and antivinculin antibody activities are significantly higher in MG patients compared with normal controls and patients with Duchenne muscular dystrophy (DMD), Parkinson's disease, and spinocerebellar degeneration. The antifilamin antibody was highly positive (100%) in ocular myasthenia. Antitropomyosin antibody activity was similar to that in DMD patients. No correlation was observed between these antibody activities and the antiacetylcholine receptor antibody titers, the duration of the disease, and thymic pathology...

Rat myotubes in tissue culture form broad areas of close contact with the substrate. These areas often display two distinct, interdigitating sets of membrane domains. One, the "contact domain", is close to the substrate; the other, termed the "AChR domain", is further from the substrate and is rich in acetylcholine receptors (AChR). We have used fluorescence techniques to study the organization of the cytoskeleton in these areas. Substrate-apposed membrane of the myotubes was exposed either by shearing or by permeabilizing the cells with a neutral detergent...

The release of intercellular contacts in MDBK cells, initiated by the depletion of Ca2+ ions from the culture medium, results in the endocytotic uptake of membrane vesicles containing specific membrane constituents of the zonula adhaerens (ZA). During this process the junction-derived, endocytosed vesicles remain associated with the ZA plaque components, while the plaque and its attached actin filaments retract as a whole in a ring-like fashion from the plasma membrane, often accumulating, usually in fragments, in the juxtanuclear cytoplasm...

We have characterized a 120-Kd antigen that frequently reacts with serum antibodies from patients with immune thrombocytopenia or normal subjects. Immunoblots made after two-dimensional nonreduced/reduced sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) or two-dimensional isoelectric focusing/SDS-PAGE demonstrated that this 120-Kd protein has the same molecular weight under nonreduced or reduced conditions, is not a surface protein, and has an isoelectric point (pl) of 6.4 to 6.5. From these data, one likely candidate is the intracellular platelet protein, vinculin...

Vinculin is a 1066-amino acid protein found at several types of actin-membrane junction. To locate sites of interest in the primary structure, a map was derived using partial cleavage reactions. Of several different types of cleavage tested, the most useful was the 5-5'-dithio-bis-(2-nitrobenzoic acid) (DTNB) reaction which cuts at cysteine residues. About 30 well defined fragments were obtained from vinculin, and several methods were used to locate these products in the sequence. Comparison of the peptides generated from whole vinculin with those from the 90-kDa amino-terminal proteolytic fragment revealed which originated there...