Biomedical subjects

M J Karnovsky

Publications and source records attributed to M J Karnovsky.

At least 199 records · Page 11Linked to original sources

PubMed1977

Decrease in adhesion of cells cultured in polyunsaturated fatty acids.

The addition of long chain unsaturated fatty acids (linoleic, linolenic and arachidonic acids) to BHK cells reduces the cell to substrate adhesion, causes morphological changes and alters the cellular growth properties. The new characteristics are similar to those of transformed cells. The data indicate that the effects are probably due to actual changes in the surface membrane lipids and not due to prostaglandin synthesis.

Arachidonic Acids↗

PubMed1977

Rates of aggregation, loss of anchorage dependence, and tumorigenicity of cultured cells.

The net rate of spontaneous aggregation of cells suspended with EDTA was measured for various cell types including spontaneous transformants and cells transformed with DNA and RNA viruses. The anchorage dependence as determined by growth in methyl cellulose and the tumorigenicity in vivo were also determined. All cells that had lost their anchorage dependency and were tumorigenic showed a high net rate of spontaneous adhesion. A31 was the only nontransformed cell line to have a high net rate of adhesion. The net rate of spontaneous aggregation of cells is a quick and reliable index of tumorigenicity and offers a new approach to understanding the mechanisms of cell surface changes associated with transformation.

Cell Adhesion↗

PubMed1977

Hydrogen peroxide production in chronic granulomatous disease. A cytochemical study of reduced pyridine nucleotide oxidases.

The ability of polymorphonuclear leukocytes (PMN) to produce H(2)O(2) in response to phagocytic stimulation was examined cytochemically using leukocytes from normal individuals and patients with chronic granulomatous disease (CGD). Normal PMN oxidized diaminobenzidine within the phagocytic vacuole by a reaction dependent upon endogenous H(2)O(2) and myeloperoxidase. CGD PMN failed to oxidize diaminobenzidine, which is consistent with the biochemical data showing a lack of H(2)O(2)-generating capacity. A plasma membrane enzyme (oxidase) activated by phagocytosis is capable of H(2)O(2) production in PMN. The localization of this oxidase activity was explored in CGD PMN using a cytochemical technique specific for H(2)O(2). The enzyme activity is stimulated by exogenous NADH, but not NADPH. Reaction product formation, indicative of activity of the oxidase, is dependent upon precipitation of cerium ions by the enzymatically generated H(2)O(2). The advantage of this approach is that enzyme activity of individual cells can be assessed, allowing determination of numbers of reactive cells in the population and their relative degrees of reactivity. NADH oxidase was found to be active both on the plasma membrane and within the phagocytic vacuoles of control PMN, whereas those cells from three CGD patients showed greatly reduced activity in both these sites. Assessment of the reactivity of individual cells showed the number of cells with oxidase activity in CGD to be significantly reduced when compared to control values. Additionally, of those cells that do react, a higher percentage of them are only weakly reactive. Omission of NADH from the incubation medium reduced the percentage of control cells showing enzyme activity but had no effect on CGD PMN, implying that the enzyme is not saturated with substrate in control cells, but in CGD the diminished enzyme is fully saturated. The defect may lie in the fact that in CGD patients there are fewer cells capable of peroxide generation, and a majority of these reactive cells produce only reduced amounts of this bactericidal agent.

3,3'-Diaminobenzidine↗

PubMed1977

Surface localization of 5'-nucleotidase on the mouse lymphocyte.

The optimal conditions for the cytochemical localization of 5'-nucleotidase (AMPase) in the mouse lymphocyte have been established. Quantitative monitoring of the effects of fixation and the components of the cytochemical medium showed that the cytochemistry can be performed under conditions that do not lead to loss of AMPase activity, and also under conditions where penetration of the substrate into the cell has occurred. The cytochemical reaction product was seen only on the surface of a proportion of splenic lymphocytes, regardless of the fixative used. Biochemical data confirmed that AMPase is an ectoenzyme and is the only protein in splenic lymphocytes capable of catalysing the hydrolysis of AMP. The activity of 5'-nucleotidase was studied also by harvesting cells either from thymus or spleen of A/ST or Cd-1 mouse strains. The enzymatic activity in splenic lymphocytes was more than six time higher than the activity of intact thymus cells. Cytochemically it was evident that within splenic lymphocytes there was a distinct population of lymphocytes with readily demonstrable AMPase activity, and another with no cytochemically demonstrable AMPase activity. It was concluded that murine lymphocytes vary in their activity of AMPase, and that the enzyme is exclusively confined to the cell surface.

Adenosine Monophosphate↗

PubMed1977

5'-Nucleotidase in different populations of mouse lymphocytes.

The activity of 5'-nucleotidase in different populations of intact lymphocytes was studied using biochemical, cytochemical and radioautographic methods. In some strains of mice the results showed a consistent difference in 5'-nucleotidase (AMPase) content between intact thymic and splenic lymphocytes. In the R III, C 57, BALB/c, CBA and Cd-1 strains AMPase activity in the isolated splenic cells was foru to 10 times the activity of intact thymocytes. In highly enriched populations of splenic T and B cells the average AMPase activity was about the same. From separate assays it was seen that the AMPase activity in highly enriched populations of lymphoctes was variable so that within one experiment the T cells seemed to have the higher AMPase activity while in other experiments B cells shown to be more active than T cells. Ultrastructural radioautography was done to count AMPase positive cells within T and B cell populations, the latter identified b binding of I125-labelled anti-immunoglobulin. It was seen that about 50% of B cells, but only about 10% of T cells, were positive for AMPase. It is suggested that there is a subpopulation within B and T cell populations with a high membrane AMPase activity and another subpopulation with less or no enzyme activity. It is also suggested that the activity and/or the proportion of these positive cells is changing within the splenic cell population. By using cortisone to deplete the immature cells from the thymus it was seen that the remaining mature cells have about the same AMPase activity as did the immaturecells, and thus mature T cells must gain their high acitivity after leaving the thymus. By incubating splenic lymphocytes with Concanavalin A it was also seen that the immature transformed cells had the same amount of enzyme as did untransformed cells.

Animals↗

PubMed1977

Cytochalasin B-induced pseudo-cleavage of mouse oocytes in vitro. II. Studies of the mechanism and morphological consequences of pseudocleavage.

Mouse oocytes are induced by cytochalasin B to undergo 'pseudocleavage' in vitro into 2 compartments, only one of which possesses microvilli. It has been found that this particular response to cytochalasin B is related to oocyte size and, possibly, to the acquisition of meiotic competence by the oocyte during its growth phase. Certain of the morphological events which characterize pseudocleavage have been determined using transmission and scanning electron microscopy. These events include: (i) an initial withdrawal of microvilli from the surface of the oocyte, together with the concomitant disappearance of microfilaments normally associated with the microvilli; (ii) the subsequent formation of a pseudocleavage furrow and contractile ring; and (iii) the reappearance of microvilli and associated microfilaments in one of the two resulting oocyte compartments. These changes in surface architecture are reflected in the distribution of fluorescein-conjugated lectins bound to the oocyte surface during pseudocleavage.

Animals↗

PubMed1977

An ultrastructural study of the mechanisms of proteinuria in rat nephrotoxic nephritis.

Nephrotoxic nephritis was induced in Sprague-Dawley and Munich-Wistar rats by the injection of rabbit antirat kidney serum. A biphasic pattern of proteinuria was induced: the heterologous phase with a peak of proteinuria occurring at 10 to 16 hours, and the autologous phase with a peak at 10 to 15 days. For morphologic studies, glomeruli were fixed by perfusion, or by drip-fixation during good blood flow. In the heterologous phase, glomerular endothelial detachment or loss and leukocytic infiltration were prominent. In the autologous phase, focal detachment of glomerular endothelium and epithelium was commonly found. At sites of endothelial loss, in both phases, endogenous albumin (demonstrated by an ultrastructural immunoperoxidase technique), but not intravenously injected ferritin, showed abnormally deep penetration into the glomerular basement membrane. At sites of epithelial loss, found in the autologous phase, both albumin and ferritin were detected throughout the glomerular basement membrane. It is proposed that, in glomerular disease, leakage of plasma proteins may occur across the glomerular basement membrane at sites of endothelial or epithelial detachment.

Albumins↗

PubMed1977

Regression of myointimal thickening following carotid endothelial injury and development of aortic foam cell lesions in long term hypercholesterolemic rats.

In an earlier report (Clowes AW, Ryan GB, Breslow JL, Karnovsky MJ: Lab Invest 35:6, 1976) we demonstrated that cholesterol feeding of rats led to hypercholesterolemia but no increase in smooth muscle cell (SMC) proliferation in right carotid arteries subjected to a standard endothelial injury when compared with normolipemic control animals. We have now examined these plaques at 6 months and 1 year after injury. In control animals, the carotid initimal thickening regressed to a relatively small, acellular, fibrous scar; there was no evidence of renewed endothelial injury and secondary SMC proliferation. Regression of the intimal thickening in the injured carotids of cholesterol-fed animals proceeded exactly as in control animals except for the accumulation of lipid. Unlike control animals, cholesterol-fed rats developed aortic intimal lesions containing extracellular lipid crystals and lipid-laden macrophages derived from mononuclear phagocytes in the blood. In addition to the lack of continued intimal SMC proliferation in the injured carotid, in the face of severe hypercholesterolemia the intima of the aorta did not contain mature SMC, or SMC-derived collagen and elastin. There was also no evidence of increased permeability to Evans blue, injected intravascularly. These findings suggest that hypercholesterolemia in the rat does not produce chronic endothelial injury, development of proliferative fibrous plaques, or enhancement of established SMC lesions.

Animals↗

PubMed1977

Failure of certain antiplatelet drugs to affect myointimal thickening following arterial endothelial injury in the rat.

The effect of aspirin, reserpine, and flurbiprofen on in vivo platelet function and intimal smooth muscle cell hyperplasia in rat carotid arteries subjected to endothelial injury was investigated and related to the effect of these drugs on in vitro platelet aggregation. Endothelial injury was achieved by infusing air briefly into a segment of right common carotid artery. Beginning before or after surgery, experimental animals were given sufficient drug to suppress platelet aggregation in vitro in response to collagen, adenosine diphosphate, or thrombin. The carotid arteries were fixed by perfusion at 5 and 14 days after injury and examined by light, scanning electron, and transmission electron microscopy for platelet activity and intimal smooth muscle cell proliferation in the denuded segment. Platelets in platelet-rich plasma from control animals aggregated in response to collagen, adenosine diphosphate, and thrombin; platelets from aspirin-, flurbiprofen- and reserpine-treated rats showed markedly diminished aggregation in response to collagen and normal or slightly diminished aggregation in response to ADP and thrombin. At 5 days, platelets from control animals formed a dense layer in the denuded segment: at 14 days, marked intimal thickening due to smooth muscle cell hyperplasia was observed. In experimental animals, the platelets were morphologically identical with controls and covered the denuded segment; serotonin granules were missing in platelets of reserpine-treated rats. Intimal thickening at 14 days was the same as controls. We conclude that in the rat no correlation may be made between the effect of aspirin, reserpine, and flurbiprofen on in vitro platelet aggregation and the effect of these drugs on the function of platelets on an arterial wall denuded of endothelium, as judged by morphology; furthermore, even when these drugs are used in sufficient dose to inhibit in vitro aggregation of platelets, myointimal thickening is not inhibited.

Adenosine Diphosphate↗

PubMed1976

Lack of correlation between agglutinability, the surface distribution of con A and post-confluence inhibition of cell division in ten cell lines.

Agglutinability by concanavalin A, distribution of surface-bound concanavalin A, and maximal cell density in monolayer culture were examined under similar conditions in parallel cultures of ten established cell lines. The degree of agglutinability of the cell lines did not correlate with the presence or absence of patching of concanavalin A bound to the cell surface, as determined with a hemocyanin marker. Agglutinability was also not always correlated with the loss of post-confluence inhibition of cell division. Two clones of mouse 3T3 fibroblasts that maintained post-confluence inhibition of cell division and low agglutinability differed substantially with respect to the surface distribution of concanavalin A. Patching of concanavalin A binding sites is neither necessary nor sufficient to explain differences in agglutinability between cell lines.

Agglutination↗

PubMed1976

Modifications of mammalian cell surfaces induced by sugars: scanning electron microscopy.

Substitution of galactose, xylose, or mannose for glucose in the growth medium of L cells or the addition of equal concentrations of the alternate sugars to glucose-containing medium results in marked morphologic changes. The changes are revealed by conventional staining for light microscopy and by scanning electron microscopy. L cells grow indefinitely on combinations of equal concentrations of glucose and galactose, xylose, or mannose. There appear to be no significant differences in growth rate on glucose compared to the combinations of sugars cited. Cells subcultured from glucose to the combinations while undergoing rapid multiplication show marked morphologic changes by light and scanning electron microscopy within 36 hr. Of particular note are the loss of microvilli; flattening of the cells; assumption of polygonal shape; prominence of nuclei and nucleoli; and changes in the structure and distribution of filopodia. Virtually all cells in the population exhibit the changes noted.

Cell Division↗

PubMed1976

Distribution of endogenous albumin in the rat glomerulus: role of hemodynamic factors in glomerular barrier function.

Using an ultrastructural immunoperoxidase technique, the distribution of endogenous albumin in the rat glomerulus was delineated under normal and abnormal hemodynamic conditions. Superficial glomeruli in anesthetized Munich-Wistar rats were rapidly fixed in situ by applying glutaraldehyde to the renal surface. Fixed tissue slices were treated with anti-rat albumin Fab fragments conjugated to horseradish peroxidase (HRP), and were then subjected to the Graham-Karnovsky ultrastructural peroxidase localization procedure. During normal blood flow, dense reaction product specific for albumin was largely confined to the glomerular capillary lumen and endothelial fenestrae, with only small amounts detectable in the lamina rara interna, and none deeper in the basement membrane (GBM) or in the urinary space. If cortical tissue was subjected to routine immersion fixation, or if fixation was performed in situ after ligation of the renal artery, reaction product was detected throughout the GBM and in the urinary space. If fixation was performed in situ after ligation of the renal artery and vein (or artery, vein and ureter), reaction product was found in the GBM and, in very large amounts, in the urinary space. If blood flow was restored for ten minutes after five minutes of renal pedicle (artery and vein) occlusion, the distribution of albumin returned to normal. Thus, glomerular barrier function depends upon the maintenance of normal blood flow conditions.

Animals↗

PubMed1976

Contraction of isolated brush borders from the intestinal epithelium.

Brush borders isolated from epithelial cells from the small intestine of neonatal rats are able to contract in the presence of ATP and Mg2+; Ca2+ is not required. Contraction is characterized by a pinching-in of the plasma membrane in the region of the zonula adherens and a subsequent rounding of the brush borders. No movement or consistent shortening of the microvilli is observed. The contraction appears to involve the 5- to 7-nm diameter microfilaments in the terminal web which associate with the zonula adherens. These filaments bind heavy meromyosin as do the actin core filaments of the microvilli. A model for contraction is presented in which, in the intact cell, terminal web filaments and core filaments interact to produce shortening of the microvilli.

Animals↗

PubMed1976

Substructure of intercellular junctions in freeze-fractured alveolar-capillary membranes of mouse lung.

The purpose of this study was to examine by freeze-fracture the ultrastructure of intercellular junctions between mouse pulmonary endothelial and epithelial cells, and to relate this fine structure to that deduced from previous physiological and ultrastructural studies using tracer techniques. Junctions between capillary endothelial cells consist of one to three interconnected rows of particles which show occasional discontinuities. Small gap junctions are associated with these rows of particles in the arteriolar end of the capillary bed. At the venular end, the junctions consist of low profile ridges on the protoplasmic fracture (PF) face or complimentary grooves on the exoplasmic fracture (EF) face some of which have a sparse number of associated particles. The vascular junctions are similar to those of vessels in rat omentum and mesentery, and resemble "leaky" junctions described in renal proximal convoluted tubular cells. Tight junctions (zonulae occludentes) between type I pneumocytes or between types I and II pneumocytes consist of a band of interconnecting ridges on the PF face and complimentary interconnecting grooves on the EF face. These continuous epithelial junctions have a structure that is typical of tight occluding junctions. Occasionally zonulae occludentes between type I and type II pneumocytes are discontinuous; this may be the result of cell translocation as pneumocytes are shed into the alveolus. Intravascular perfusion fixation at high pressure (140 cm H2O) had no discernible effect on the structure of endothelial or epithelial junctions. The appearance in our study of freeze-fractured pulmonary endothelial and epithelial junctions reveals clearly the physical basis for the results of ultrastructural tracer and physiological studies which have suggested that it is the alveolar epithelium rather than the endothelium that is the chief permeability barrier to small, water-soluble molecules.

Animals↗

PubMed1976

Irreversible deformation of the spectrin-actin lattice in irreversibly sickled cells.

Irreversibly sickled cells (ISC's) are circulating erythrocytes in patients with sickle cell disease that retain a sickled shape even when oxygenated. Evidence points to a membrane defect that prevents the return of these cells to the normal biconcave shape. The erythrocyte membrane protein spectrin is believed to help control erythrocyte shape and deformability. Recent studies suggest that normally spectrin and an erythrocyte actin form a self-supporting, fibrillar, lattice-like network on the cytoplasmic membrane surface. When normal erythrocyte ghosts are extracted with Triton X-100 all the integral membrane proteins and most of the membrane lipids are removed, leaving a ghost-shaped residue composed principally of spectrin and actin. We concentrated ISC's from patients with sickle cell anemia and compared the morphology and protein composition of ghosts and Triton-extracted ghost residues prepared from these ISC's with similar preparations of reversibly sickable cells and normal cells. (a) Many ISC's formed ISC-shaped ghosts. (b) All ISC-shaped ghosts formed ISC-shaped Triton residues. (c) Spectrin, erythrocyte actin (Band 5), an unidentified Band 3 component, and Band 4.1 were the major protein components of the Triton residues. All membrane-associated sickle hemoglobin was removed by the Triton treatment. (d) No ISC-shaped ghosts or ISC-shaped Triton residues were formed when deoxygenated, sickled RSC's were lysed or Triton-extracted. ISC-shaped ghosts and Triton residues were never formed from normal cells. These observations suggest that a defect of the "spectrin-actin lattice" may be the primary abnormality of the ISC membrane. Since ISC's are rigid cells, the data support the postulate that spectrin is a major determinant of membrane deformability. Finally, they provide direct evidence that spectrin is important in determining erythrocyte shape.

Actins↗

PubMed1976

Patching, microvilli, and the agglutination of normal and transformed cells.

Transmission and scanning electron microscopy were used to study possible structural correlates in the process of agglutination of several types of normal and transformed cells by Concanavalin A. In parallel studies we found that post-confluence inhibition of cell division and agglutiniability of cells by Concanavalin A were not correlated with patching of surface bound lectin molecules as determined with a hemocyanin marker. Transformed cells growing in monolayer cultures were found to have many more microvilli than the corresponding normal cells. However, when cells were brought into suspension with EDTA, all cells developed numerous microvilli and we were not able to distinguish between agglutinable and nonagglutinable cells on the basis of morphological appearance. Cells agglutinated by Concanavalin A had numerous interdigitated microvilli at points of cell-cell contact. The appearance of spontaneously agglutinated cells and lectin agglutinated cells was very similar with respect to the involvement of microvilli in cell-cell attachments, and labeling studies with hemocyanin indicated that Concanavalin A bound to microvilli is rapidly cleared from these surface specializations in a manner analogous to that observed with patching of surface bound lectin. Several lines of SV-40 transformed fibroblasts were shown to be considerably more spontaneously agglutinable than untransformed cells. These results indicate that Concanavalin A may amplify an intrinsic membrane property common to many transformed cells that is expressed as an increase in the rate of adhesion of suspended cells. It is proposed that the membrane change detected by the agglutination reaction may also be involved in the loss of post-confluence inhibition of cell division and growth of transformed cells in semisolid media, due to a surface interaction that allows transformed cells to use each other as growth substrata.

Agglutination↗

PubMed1976

The distribution of Ia antigens on the surfaces of lymphocytes.

The distribution of Ia antigens was studied on murine spleen lymphocytes by an ultrastructural technique employing deep freeze-etched replicas. Ia antigens were labeled on cells from appropriate congenic and recombinant strains of mice by incubating the cells with FITC-conjugated anti-Iak antibody, followed by ferritin-coupled Fab anti-FITC. Ia antigens were detected predominantly on immunoglobulin (Ig)-bearing B lymphocytes. Antigens coded for by the entire Ik region were present on the surfaces of 95% of the positive cells (from B10.BR mice) in densely packed microclusters. Ia specificities coded for by the I-A and I-C subregions (on 4R and B10.HTT mice) exhibited a more variable pattern, with 30 to 35% of the labeled cells having sparsely distributed Ia antigens in relatively discrete microclusters. Binding of anti-Iak antibody at 37 degrees C led to patch formation but not to capping. Modulation of surface Ig left Ia antigens diffusely distributed on the cell surface, indicating that these two membrane proteins are independent molecules.

Animals↗

PubMed1976

Glomerular permeability to proteins. Effects of hemodynamic factors on the distribution of endogenous immunoglobulin G and exogenous catalase in the rat glomerulus.

The distribution of endogenous immunoglobulin G (IgG) and exogenous catalase was delineated in the rat glomerulus under normal and abnormal hemodynamic conditions. IgG was identified by an ultrastructural immunoperoxidase technique using antirat IgG Fab fragments conjugated to horseradish peroxidase; catalase was identified by a cytochemical reaction. When superficial glomeruli in anesthetized Munich-Wistar rats were rapidly fixed in situ by dripping glutaraldehyde onto the renal surface, IgG and catalase were largely confined to the glomerular capillary lumen, with only small amounts in the lamina rara interna immediately beneath the endothelial fenestrae, and none deeper in the basement membrane (GBM) or in the urinary space. If cortical tissue was subjected to routine immersion fixation, or if fixation was performed in situ after ligation of the renal artery, IgG and catalase were found throughout the GBM but not in the urinary space. If fixation was performed in situ after ligation of the renal artery and vein (or artery, vein, and ureter), IgG and catalase were found in the GBM and in the urinary space. If blood flow was restored for 10 minutes after 5 minutes of occlusion of the renal artery and vein, the distribution of IgG and catalase returned to that seen during good blood flow, i.e. neither showed significant penetration beyond the endothelial layer. Thus, as was found previously for albumin, glomerular barrier function for IgG and catalase depends upon the maintenance of normal blood flow conditions. We propose that such conditions impose functional restrictions may be mediated by molecular sieving phenomena during normal ultrafiltration across the GBM, perhaps in association with concentration-polarization or charge effects or both. The epithelial slit pores may significantly modulate solute flux across the GBM by controlling the over-all rate of hydrodynamic flow during ultrafiltration.

Animals↗