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Biomedical subjects

J V Small

Publications and source records attributed to J V Small.

At least 91 records · Page 5Linked to original sources
PubMed1979

Antibody staining of 10-nm (100-A) filaments in cultured smooth, cardiac and skeletal muscle cells.

Antibodies were prepared against the SDS-denatured 10-nm filament protein 'skeletin' extracted from chicken gizzard. The specificity of the antibody to the 10-nm filament protein was shown by immunodiffusion before and after purification of the protein on SDS gels by the enzyme-linked immunoabsorbent assay (ELISA) and by its specific absorption with purified skeletin. In immunofluorescence (where preimmune sera and antigen-absorbed antisera gave negative results), cultured cardiac, skeletal and smooth muscle cells and endothelial cells stained intensely. No staining was observed in fibroblasts present in these cultures, nor was there staining in glial cells or nerve cell bodies and fibres from sympathetic ganglion and Auerbach's plexus cultures. Smooth muscle cells (regardless of their source and phenotypic state) and endothelial cells stained intensely in the perinuclear region and in a fine filamentous network that existed throughout the cytoplasm. In both chick and rat skeletal and cardiac muscle (cultures and frozen sections) filamentous network staining was observed, while in rat muscle the antibody was additionally localized in a regular pattern in the region of the Z-disk, and in the case of cardiac muscle associated with the intercalated disk. The addition of 10(-6) M colchicine to the culture medium of smooth and striated muscle and endothelial cells resulted in an aggregation of the filaments in the nuclear region. Cultured smooth and striated muscle and endothelial cells and freshly isolated smooth muscle cells extracted of actomyosin and tubulin by high and low ionic strength solutions gave a staining pattern similar to non-extracted cells and in the electron microscope, exhibited filaments of predominantly 10 nm diameter.

Animals↗
PubMed1979

Viral infections and IgM autoantibodies to cytoplasmic intermediate filaments.

Seventy-four out of 113 sera from patients with infectious hepatitis, chickenpox, measles and mumps reacted with both smooth muscle and cytoplasmic filaments in cultured fibroblasts and neuroblastoma. Five out of eighty-five control sera also reacted in this way. That the cytoplasmic structures are intermediate filaments was suggested by their rearrangement into coils of perinuclear filaments in colchicine- or vinblastine-treated fibroblasts, but not in cytochalasin B-treated cells. The idenity of these structures was confirmed by the demonstration that the same structures reacted with the post-viral sera and a rabbit and human anti-intermediate filament antibody. Immunoabsorption studies showed that twenty-seven out of thirty-two positive sera were neutralised by skeletin, the intermediate filament protein from smooth muscle. In all but one of the sera, the antibody was IgM. Antibody titres fell in the second specimen in eleven out of fourteen pairs of acute and convalescent sera. The association between viral infections and autoantibodies suggest that production of antibodies suggests that production of antibody to intermediate filaments may be initiated by viruses.

Adolescent↗
PubMed1979

Reactivity of smooth-muscle antibodies with F- and G-actin.

The reactivity of human smooth-muscle antibodies (SMA) with F- and G-actin was investigated by means of an enzyme-linked immunosorbent assay (ELISA). Polystyrene tubes were coated with F-actin, G-actin and tropomyosin. The optimal reactivity of SMA with F-actin was achieved with tubes coated with actin concentrations in the range of 10-100 mug/ml, while both lower and higher amounts yielded lower reactivity. The reactivity with G-actin was low and only a little higher than the reactivity with tropomyosin or uncoated tubes, but much lower than the reactivity with F-actin. The differences in reactivity of SMA with F- and G-actin could not be explained by differences in binding of the two forms of actin to the tubes, although 1.7-2.5 times more F-actin than G-actin bound to the polystyrene surface after coating with equivalent amounts of protein. SMA-negative sera did not react with F-actin coated tubes indicating that the binding of antibody was not due to non-immunological binding of immunoglobulins. Human SMA thus react better with F- than with G-actin.

Actins↗
PubMed1978

Correlation between actin polymerization and surface receptor segregation in neuroblastoma cells treated with concanavalin A.

In response to concanavalin A (Con A), neuroblastoma cells undergo marked morphological changes which involve the retraction of neurites and the induction of broad and extensive lamellar regions around the cell periphery. From the use of FITC-Con A it was shown that the membrane formed on the induced lamellar regions lacked receptors to Con A from the onset of lamella formation. These receptors were confined to the cell body; they initially showed a uniform distribution and were subsequently collected into patches and finally into aggregates or caps. When the aggregates occurred on the cell periphery their position coincided with areas free of lamellae. Investigations of the lamellar regions in Triton-extracted cell monolayers showed them to consist of a meshwork of actin filaments containing radiating thin filament bundles or microspikes. With increasing time in the presence of Con A there was a progressive increase in the number of radiating microspikes. Previous studies have shown the actin in these lamellar regions to be singly polarized with respect to the cell body. From the segregation of Con A receptors away from areas of actin polymerization in the lamellae it is concluded that actin is involved in some indirect way in surface receptor movement.

Actins↗
PubMed1978

Direct visualization of the 10-nm (100-A)-filament network in whole and enucleated cultured cells.

Following extraction of actomyosin and tubulin from cultured cells treated with Triton X-100, a cytoskeleton remains which is composed predominantly of the cell nucleus encompassed by a network of 10-nm filaments. After negative staining the dense perinuclear region appears as a densely woven filament net punctuated by patches of high electron density. Enucleation of 3T3 cells with cytochalasin B gives rise to karyoplasts surronunded by 10-nm filaments and cytoplasts in which 10-nm filaments remain situated in the central region of the cytoplasm. While the 10-nm filaments occurred mainly as single filaments in human skin fibroblasts and 3T3 cells, in epithelioid PtK1 and PtK2 cells they were commonly associated in prominent meandering bundles. In addition, in these latter cells after Triton extraction the remaining ribosomes were bound specifically to the 10-nm-filament net. After exposure of 3T3 cells to cytochalasin B the 10-nm filaments formed branches that radiated from the perinuclear region into the immobile cell extensions. Concavalin A had no marked effect on the distribution of the 10-nm-filament net. The results suggest that the 10-nm filaments act primarily as structural elements, serving, in particular, to support and constrain the nucleus in its position in the cell.

Cell Nucleus↗
PubMed1977

Studies on the function and composition of the 10-NM(100-A) filaments of vertebrate smooth muscle.

The extraction of isolated vertebrate smooth muscle cells at high and low ionic strength yields cell ghosts which are seen in the electron microscope to be composed of a complex network of 10-nm filaments, together with residual actin. After SDS-gel electrophoresis of the cell ghosts only 2 bands may be recognized, one corresponding to actin and the other migrating at about 55 000 mol. wt that arises from the 10-nm filaments. The 10-nm filaments are extremely sensitive to proteolysis and are absent from cells exposed to crude collagenase in the presence of Triton X-100. Such cells, lacking 10-nm filaments, still contract in response to ATP. The data indicate that the 10-nm filaments are not essential for contraction, but rather form a specialized intracellular cytoskeleton. While completely insoluble in concentrated salt solutions the 55 000 mol. wt protein is readily extracted with acetic acid from homogenized and salt-extracted smooth muscle residue. The extracted protein reassembles, on dialysis, into filaments of about 10-nm diameter and has an amino acid composition almost identical to that deduced for vertebrate neurofilaments. From the cytoskeletal role that the 10-nm filaments play in smooth muscle and, as appears likely, in other cell types the filament protein has been tentatively termed 'skeletin'. Results relating to the proportion of skeletin in smooth muscle and the structure of the 10-nm filaments are described and discussed.

Amino Acids↗
PubMed1977

Studies on isolated smooth muscle cells: The contractile apparatus.

Smooth muscle cells may be isolated from the taenia coli muscle of the guinea pig which, when made permeable by treatment with Triton X-100 (0-05%) show a sensitivity to Ca for contraction with MgATP. The rate of contraction, about 10 micron s-1, corresponds closely to the maximum velocity of shortening of the intact muscle. Electron microscopy of such partially demembranated muscle cells shows that myosin filaments of about 16-nm diameter are present in both the rigor and the relaxes states. In addition, the actin and myosin filaments are commonly seen to be associated in groups corresponding approximately in size to the fibrils recognizable in cells in rigor in the light microscope. The dense bodies and the 10-nm filaments are found located between the actin-myosin filament groups. The thick myosin filaments may be isolated by fragmentation of the cells under relaxing conditions. These native filaments range up to about 8 micron in length and show the same structural organization as filaments aseembled from purified smooth muscle myosin: there is no central bare zone and bare edges, about 0-2 micrin long, occur at the filament ends. The lack of bipolarity of the native smooth muscle muosin filaments and the absence, in the contractile apparatus, of actin-associated structures equivalent to Z-lines suggests that the amount of shearing that can occur between the actin and myosin filaments is considerably greater than in skeletal muscle.

Animals↗