Search PubMed⌕ Search

Biomedical subjects

M J Karnovsky

Publications and source records attributed to M J Karnovsky.

At least 181 records · Page 10Linked to original sources
PubMed1979

Lipid domains in biological membranes: their structural and functional perturbation by free fatty acids and the regulation of receptor mobility. Co-presidential address.

We have studied the interaction of free fatty acids (FFAs) with cell membranes and lipid bilayers by monitoring changes in the emission polarization of the fluorescent probes diphenylhexatriene (DPH) and anilino-naphthalene sulfonate (ANS). We found that the FFAs readily intercalate into membranes and produce significant changes in the packing of the lipid molecules. The membrane alterations could be divided into two patterns: the cis-unsaturated FFAs (designated Group A) disorder the membranes' interior (as reported by DPH) and order the head group region (as reported by ANS); the trans-unsaturated or saturated FFAs (Group B) do not alter the bilayer interior but also order the head group region. Using solution theory, the shift in transition midpoint temperatures as a function of fatty acid type was used to infer that the Group A FFAs partition into fluid domains, while Group B FFAs partition preferentially into gel-like domains. These results are explained in terms of a domain model of membrane lipid structure. Low concentrations of Group A FFAs inhibit the capping of surface immunoglobulin (Ig), whereas no effect was seen with Group B FFAs. The capping inhibition caused by Group A FFAs was reversible with increasing doses of extracellular calcium. Fluorescence photobleaching recovery showed that the Group A FFAs do not inhibit receptor immobilization associated with patch formation but rather inhibit the final energy-dependent movement of the patched receptors into a cap. We have also shown that the Group A FFAs cause a shift in membrane-bound calcium to the lipid phase from probably protein calcium-binding sites. The data have generated a model of receptor mobility invoking a trans-membrane, calcium-binding, receptor-anchoring protein, linked to the cytoskeleton. Inhibition of capping by Group A FFAs is postulated to be due to perturbation of specific lipid domains associated with this protein, such perturbation leading to conformational changes in the protein, and consequent intramembraneous calcium sequestration in the lipid phase, rendering the calcium unavailable for activation of the cytoskeleton.

Calcium-Binding Proteins↗
PubMed1978

Surface morphology of mouse and rat thymic lymphocytes: an in situ scanning electron microscopic study.

The present paper deals with a scanning electron microscopic investigation which was undertaken in order to make a direct study of geometrical conformations of thymocytes, to determine the effect of external mechanical forces and finally to analyse the relation of the cell surface morphology to the differentiation and release of thymocytes into circulation. Thymocytes in situ revealed a striking polyhedral configuration with distinct edges and angles that permit a close orientation of cells in a minimum space. This conformation is probably acquired under the influence of forces in the microenvironment of the cells. The immature thymocytes in the cortex were smooth surfaced and constituted a homogenous population with regards to surface morphology except for slight variations in the size and angles of varoius facets of the polyhedra. A minority of the cell population occupying the medulla, however, exhibited a departure in possessing surface undulations and stubby protuberances. Thymocytes isolated in suspension and those in postcapillary venules of thymus did not show the polyhedral shape characteristic of the cells in thymic tissue. They were always rounded, with their surfaces often exhibiting undulations or microvilli. The variations observed in situ are discussed in light of external mechanical forces, cell surface characteristics and the inherent properties of differentiating thymocytes.

Animals↗
PubMed1978

The selection of a stable rat hepatoma variant with concomitant increase in ploidy and permeability to glycerol.

By repeated selection for longer survival in an isotonic solution of glycerol, a stable subline of Novikoff rat hepatoma cells has been isolated. The cells exhibit markedly increased resistances to osmotic lysis in isotonic solutions of glycerol. They are twice as large and have twice as many chromosomes as cells of the parental line. It is suggested that the osmotic stress procedure can be extended for the selection of numerous kinds of mutants and can be used as a method of analysis of membrane properties.

Animals↗
PubMed1978

The adhesive interaction between polymorphonuclear leukocytes and endothelial cells in vitro.

The results of this investigation indicate an adhesive specificity between PMN and cultured endothelial cells. This was monitored by the mono-layer collection assay and by direct cell counts using the scanning electron microscope. Both techniques showed that significantly more PMN attached to endothelial cells than to a variety of other cell types. The interaction can be modulated by divalent cations and neuraminidase, implying a role for surface charge. In the presence of chemotactic agents, the number of PMN adhering to endothelial cells increases. This system presents a good model for studying the process of PMN margination which occurs in vivo during the acute inflammatory response.

Blood↗
PubMed1978

Isolation and characterization of rat glomerular epithelial cells in vitro.

Rat glomeruli were isolated by a graded sieving technique, and the nature and purity of the preparation were examined by scanning electron microscopy (SEM). A great majority of the glomeruli (86.0 +/- 6.0%) were not encapsulated, and there was very little contamination of the preparations with tubular fragments. By supplementing tissue culture medium with conditioned medium (CM) and insulin, we were able to grow single cells from dissociated rat glomeruli. From these single cells, we were able to clone and maintain in culture three distinct cell types. Also, with this specialized medium, we were able to clone these three cell types from outgrowths of whole golmeruli. One of these cell types was characterized as glomerular epithelial cells (GEC). GEC, as opposed to the other two cloned cell types obtained in this study, had cilia on their surfaces and also possessed receptors for complement (C3) in vitro. Low concentrations of the aminonucleoside of puromycin (AMNS), which have been shown to be specifically cytotoxic to GEC in vivo, were found to be cytotoxic to GEC but not to the other two glomerular cells in vitro. In addition, GEC did not contain antihemophilic factor (factor VIII), a marker for endothelium, nor were they able to phagocytose polystyrene spherules in vitro, as can mesangial cells in short-term culture.

Animals↗
PubMed1978

Clusters of intramembranous particles on cultured myotubes at sites that are highly sensitive to acetylcholine.

Electrophysiological and autoradiographic studies have shown that the distribution of acetylcholine (AcCho) receptors on uninnervated cultured chicken muscle cells is not uniform. Regions of high receptor density (hot spots 10--40 times more sensitive than surrounding areas are localized as discrete patches or clusters about 10 micrometer in diameter o myotube muscle membranes. Hot spots were also found on fusion-arrested mononucleated myoblasts. We have developed a method for freeze-fracturing monolayer cultures that allows the unambiguous reidentification of membrane patches previously assayed for ACCho sensitivity. The freeze-fractured membranes at physiologically defined hot spots contain aggregates of many (10--20) small clusters of large (10--19 nm in diameter) intramembranous particles. Clusters are found on both fracture faces, but the particle density is much greater on the protoplasmic (P) face than on the extracellular (E) face (about 2000/micrometer2 vs. 700/micrometer2). Some of the particles appear to be composed of five or six "subunits" arranged cylindrically around a central dark dot. Because the aggregates are present at sites of high AcCho sensitivity, it is likely that the intramembranous particles are in some way related to the AcCho receptor molecule.

Acetylcholine↗
PubMed1978

Localization of D-amino acid oxidase on the cell surface of human polymorphonuclear leukocytes.

The ultrastructural localization of D-amino acid oxidase (DAO) was studied cytochemically by detecting sites of hydrogen peroxide production in human polymorphonuclear leukocytes (PMNs). Reaction product, which forms when cerous ions react with H2O2 to form an electron-dense precipitate, was demonstrated on the cell surface and within the phagosomes of phagocytically stimulated cells when D-amino acids were provided as substrate. Resting cells showed only slight activity. The competitive inhibitor D,L-2-hydroxybutyrate greatly reduced the D-amino acid-stimulated reaction while KCN did not. The cell surface reaction was abolished by nonpenetrating inhibitors of enzyme activity while that within the phagosome was not eliminated. Dense accumulations of reaction product were formed in cells which phagocytosed Staphylococcus aureus in the absence of exogenous substrate. No reaction product formed with Proteus vulgaris while an intermediate amount formed when Escherichia coli were phagocytosed. Variation in the amount of reaction product with the different bacteria correlated with the levels of D-amino acids in the bacterial cell walls which are available for the DAO of PMNs. An alternative approach utilizing ferricyanide as an electron acceptor was also used. This technique verified the results obtained with the cerium reaction, i.e., the DAO is located in the cell surface and is internalized during phagocytosis and is capable of H2O2 production within the phagosome. The present finding that DAO is localized on the cell surface further supports the concept that the plasma membrane is involved in peroxide formation in PMNs.

Cell Membrane↗
PubMed1978

The distribution of albumin and immunoglobulin G in the glomerular capillary wall in aminonucleoside nephrosis.

Using an ultrastructural immunoperoxidase technique, the distribution of endogenous albumin and immunoglobulin G was examined in superficial glomeruli of Munich-Wistar rats with aminonucleoside nephrosis. In glomerular capillaries in which the external surface of the glomerular basement membrane (GBM) was completely covered with spread expanses of epithelial cytoplasm, albumin and IgG were distributed normally, with no detectable penetration beyond the endothelial fenestrae. However, at sites of focal loss of the epithelial covering of the GBM, both albumin and IgG were found to penetrate the GBM. These results suggest that, in this experimental model, plasma proteins leak into the urine at sites of glomerular epithelial denudation.

Albumins↗
PubMed1978

Cell surface changes in capping studied by correlated fluorescence and scanning electron microscopy.

A simple method has been devised for correlating fluorescence microscopy and scanning electron microscopy, whereby the identical cell observed by the former can be observed by the latter. With this methodology, we have studied the sequence of cell surface changes which occur when mouse B lymphocytes, bearing immunoglobulin (Ig) on their surfaces, interact with fluoresceinated anti-Ig antibodies. Initially, the pattern of staining is diffuse; then, rapidly, patching occurs, followed by the sweeping of the patches into a cap. Accompanying these events are: the disappearance of microvilli, the formation of ruffles and lamellipodia at the pole of the cell opposite the capped pole, the presence of a constriction ring beneath the cap, ameboid shapes, and the translocation of the cell in a direction opposite the cap. Whereas the cell body is smooth, the capped pole frequently shows the presence of numerous microvilli. However, not all capping cells show the changes, and cells capped in the presence of cytochalasin B usually show none of them, except that their surfaces are smooth. Furthermore, T-cells undergoing translocation show the same cell surface changes as do B-cells. Whereas the contractile apparatus of the cell is considered to underlie all these phenomena, it is concluded that the cell surface changes do not result from patching and capping per se, but rather are an expression of the cell translocation also induced by the anti-Ig antibody.

Animals↗
PubMed1978

A morphologic and permeability study of luminal smooth muscle cells after arterial injury in the rat.

In the carotid air drying model of arterial endothelial injury in the stressed rat, endothelium does not always regenerate over the zone of intimal thickening; instead, a layer of modified smooth muscle cells appears to form a temporary luminal surface. We examined the properties of these luminal smooth muscle cells in injured right carotid arteries from stressed rats at intervals up to 2 months by light, scanning electron, and transmission electron microscopy. Before perfusion fixation, selected animals were given injections of Evans blue dye, ferritin, or horseradish peroxidase. Unlike adjacent endothelium, the luminal smooth muscle cells most closely resembled neighboring intimal smooth muscle cells, lacked morphologic characteristics of normal endothelium, and did not stain with rabbit antibody to rat factor viii. Unlike normal mature endothelium, this layer did not exclude horseradish peroxidase, Evans blue, or ferritin. These data demonstrate that a nonthrombogenic layer composed of modified smooth muscle cells can appear at the luminal surface of a zone of injury-induced myointimal thickening; however, this layer does not form a permeability barrier to large molecules.

Animals↗
PubMed1978

Focal glomerular sclerosis in the fawn-hooded rat.

We have examined the nature of focal glomerular sclerosis (FGS) in fawn-hooded (FH) rats. The fawn-hooded rat develops pathologic features similar to those observed in steroid-resistant focal glomerular sclerosis, ie, by light microscopy some of the glomeruli appear normal but others show areas of solidification confined to one or two lobules of the tuft. The pathogenesis of this disease is not well known and there is a great need for an animal model. In the FH animal, a marked difference in the development of the lesion was noted between male and female rats. Fifty percent of 4-month-old males had proteinuria in excess of 10 mg/day (none of the females had significant proteinuria), while all 12-month-old males had proteinuria in excess of 45 mg/day (female 12-month-old FH rats had mean proteinuria of 7 mg/day). At 6 months of age continuing through 12 months of age, male FH rats had mesangial deposits of IgG, IgM, and, occasionally, C3, demonstrable by immunofluorescence, whether or not FGS was present. Subepithelial electron-dense deposits were never seen by electron microscopy either at 6 of 12 months. Six-month-old animals frequently did not exhibit FGS. Instead, the glomerular epithelial cells, exhibited fusion of foot processes, vacuolization, and, in some areas, focal loss of the epithelial covering on the glomerular basement membrane (GBM). Six-month-old males with proteinuria exhibited focal loss of negative charge from all layers of the filtration barrier. The GBM from sclerotic glomeruli of 12-month-old rats was commonly denuded of epithelium. None of the animals in this study was uremic. FH rats demonstrated FGS associated with progressive glomerular epithelial cell injury.

Age Factors↗