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M J Karnovsky

Publications and source records attributed to M J Karnovsky.

At least 217 records · Page 12Linked to original sources
PubMed1976

The formation of septate-like junctional complexes between lymphoid cells in vitro.

During the initial 48 hr of incubation in immunized Mishell-Dutton spleen cell cultures, most lymphocytes exist as single cells or an occasional pair of cells (doublets). Ultrastructural examination of the area between those cells forming doublets revealed a septate-like junctional complex occurring over large portions of the plasmalemma. This junctional zone persisted in areas where extensive cytoplasmic interdigitation among cell processes was evident. Approximately 1% of the lymphocytes isolated from the top fraction of a discontinuous fetal calf serum gradient were involved in formation of doublets. The majority of these doublets showed evidence of junctional interaction. Moreover, this septate-like zone of adhesion was seen using several different fixatives and stains and remained intact after osmotic cell disruption. The junction was not demonstrable by using freeze-fracture techniques and therefore must be limited to interactions within the surface of the cells.

Animals↗
PubMed1976

Absence of enhanced intimal thickening in the response of the carotid arterial wall to endothelial injury in hypercholesterolemic rats.

Young male Sprague-Dawley rats fed a high cholesterol, thyroid-suppressive diet were subjected to drying injury of carotid artery endothelium; animals were sacrificed at various times up to 3 months after injury, and the vessels were examined by light, scanning, and transmission electron microscopy. The diet induced marked elevation of serum cholesterol mainly present in lipoproteins of density less than 1.063. The morphology and degree of intimal thickening in the injured carotids of such animals were compared with the changes found in control groups of normolipemic rats. In the control groups, endothelium was completely regenerated between 7 and 14 days; intimal thickening was present at 14 days and at later stages and contained smooth muscle cells without lipid. In the cholesterol-fed animals, endothelial regeneration and intimal thickening occurred as in the controls with the following additional features: in the zone of intimal thickening in the injured segment, lipid was present in smooth muscle cells and, at later stages, in the extracellular matrix; undifferentiated mononuclear cells were also noted in the thickened intima and, at 3 months, were found adhering to normal and regenerated endothelium. However, no differences were found between control and hypercholesterolemic rats with respect to the degree of intimal thickening within the injured segment; enhancement of the smooth muscle proliferative response was not evident in the hypercholesterolemic rats. Our findings suggest that this form of hypercholesterolemia and its associated hyperlipoproteinemia may not be directly responsible for rat smooth muscle proliferation following endothelial denudation. They also indicate that hyperlipemia does not necessarily cause persistence of myointimal hyperplasia in arteries.

Animals↗
PubMed1976

Ultrastructural differences between rat inner medullary descending and ascending vasa recta;.

The ultrastructure of rat inner medullary vasa recta was studied by both conventional transmission and freeze-fracture electron microscopy. The identify of descending and ascending vasa recta in the inner medulla was established by tracing outer medullary descending vasa recta and ascending vasa recta into the inner medulla, as well as by the incomplete carbon labeling technique to identify isolated descending vessels or loops. As in the outer medulla, descending vessels possess thick continuous endothelium with pinocytotic vesicles budding off the luminal and basal plasma membranes (more numerous in the latter location), but no fenestrae. Ascending vasa show thin, attenuated endothelium with numerous (500 to 800 A fenestrae bridged by 40 A thick fenestral diaphragms. Intercellular junctions in both vessels are zonulae occludens with usually one, and uncommonly two or three zones of outer leaflet membrane fusion. Intramembranous particles are numerous in endothelial plasma membranes of descending vasa recta; similar particles are much fewer in corresponding ascending vasa recta endothelial plasma membranes. The ultrastructural differences between descending vasa recta and ascending vasa recta may reflect markedly different permeability properties, particularly to macromolecules, and may be relevant to recent functional studies on rat inner medullary vasa recta.

Animals↗
PubMed1976

Isolation of phagocytic cells from the rat renal glomerulus.

A method is described for the dissociation of rat glomerular cells in vitro. Isolated endothelial cells were characterized by the persistence of fenestrae. Mesangial cells showed a variety of morphologic appearances; if dissociation was performed after an intravenous injection of ferritin, such cells were identified by the presence of large ferritin-filled vacuoles. Intact epithelial cells were rarely seen, presumably because they did not survive the separation procedure. In culture, a high proportion of cells adhered to glass surfaces, were phagocytic, and had labeled heavily in vivo with ferritin; they were, thus, considered to be mesangial in origin. Receptors for immunoglobulin (Fc) and complement (c3) were also detected in such cells after 24 hours in culture, but not immediately after dissociation. Our findings suggest that a population of isolated glomerular cells, probably comprising mainly mesengial cells, is highly phagocytic and has the potential to develop Fc and C3 receptors. We propose that this technique offers a useful approach for the delineation of glomerular cell properties and functions.

Animals↗
PubMed1975

Membrane structural specialization of the toad urinary bladder revealed by the freeze-fracture technique. I. The granular cell.

Examination of the toad urinary bladder by freeze-fracture electron-microscopy demonstrates structural specialization of the granular cell's luminal membrane compared to its basal membrane. Although both membranes appear to possess about 1,700 intramembranous particles per mum2, those of the luminal membrane tend to be significantly larger in size. In addition, the fracturing properties of the two membranes are markedly different: the majority of particles are found on fracture face B (outer membrane face), in the case of the luminal membrane, and the majority are found on fracture face A (inner membrane face), in the case of the basal membrane. While the two fracture faces of the basal membrane possess a similar distribution of particle sizes, in the case of the luminal membrane the B face was found to possess particles generally larger than those found on the A face. It was established that the probability of luminal membrane particles adhering to face B instead of face A is closely correlated with the size of the particle. The structural specialization of the granular cell's luminal membrane may have an important relationship to the characteristic permeability properties of this membrane and the capacity of this cell type to respond physiologically to the hormone vasopressin.

Animals↗
PubMed1975

Degradation of abnormal proteins in Escherichia coli. Formation of protein inclusions in cells exposed to amino acid analogs.

Cells of Escherichia coli selectively degrade proteins that have incorporated amino acid analogs. Within 1 hour after exposure of cells to canavanine, 50% of the analog-containing proteins were degraded to acid-soluble form. At the same time, no net loss of canavanine-containing protein occurred from the 100,000 X g supernatant. Instead, most of the proteins containing the analog, unlike normal ones, accumulated in particulate fractions sedimenting at 10,000 X g or 100,000 X g. They were then lost from these fractions concomitant with the degradation of the abnormal proteins. The loss of such proteins from particulate fractions accounted for all of the protein degraded to acid-soluble form. Similar observations were obtained after incorporation of other analogs or puromycin. The 10,000 X g pellets correspond to amorphous dense intracellular granules visible in electron micrographs of cells exposed to canavanine. Upon removal of the analog, these granules disappeared, simultaneously with the degradation of the analog-containing proteins. These pellets do not resemble a degradative organelle, like the lysosome; they are not osmotically sensitive, do not exclude inulin, are not enclosed by a membrane, and do not show autolytic activity. The proteins in the granules could be solubilized by sodium dodecyl sulfate but not by Triton, NaC1, dithiothreitol, RNase, DNase, or phospholipase. The proteins extracted from the pellet with sodium dodecyl sulfate tend to become particulate again upon removal of this detergent. Incorporation of canavanine caused a normally soluble polypeptide, the monomer of beta-galactosidase, to be inactive and found in the sedimentable fraction. These findings suggest that (a) the presence of amino acid analogs in proteins can make them less soluble, and (b) the inclusions are formed by the spontaneous precipitation of abnormal proteins rather than by an active granule-forming process.

Amino Acids↗
PubMed1975

An ultrastructural study of the mechanisms of proteinuria in aminonucleoside nephrosis.

Sprague-Dawley rats injected i.v. with a single dose of puromycin aminonucleoside (PAN) developed massive proteinuria five days later. Electron microscopic studies of perfusion-fixed glomeruli showed that loss of epithelial foot processes and their replacement by flattened expanses of epithelial cytoplasm began at two days and was extensive by four days after the injection of PAN. At and after five days (correlating with the onset and persistence of massive proteinuria), areas of focal loss of the epithelial covering on the outside of the glomerular basement membrane (GBM) were observed in 30% of glomeruli. Intravenously administered ferritin was distributed normally in most sections of the GBM of nephrotic animals, but abnormally deep penetration of particles was observed in GBM segments that lacked an external covering of epithelium. The same changes were found following in situ fixation of superficially placed glomeruli of Munich-Wistar rats with PAN nephrosis. We propose that the massive, early proteinuria in PAN nephrosis may be primarily due to a glomerular epithelial lesion, leading to scattered focal defects in the external covering of the GBM. Increased bulk flow of glomerular filtrate across the GBM in such areas may explain the highly selective proteinuria found in this form of the nephrotic syndrome.

Animals↗
PubMed1975

The isoporous substructure of the human glomerular slit diaphragm.

The substructure of the human glomerular slit diaphragm is examined after perfusion fixation of a human kidney with tannic acid-glutaraldehyde. The present study shows that the substructure of the slit diaphragm in man is very similar to that described in rats and mice, and that the dimensions of the contained pores (50 times 120 A) in the human differ only slightly from those reported for the murine kidney. It is unlikely that rectangular pores of the dimensions of those measured in the human kidney would permit, under physiological conditions, the penetration of significant quantities of serum protein molecules. It is possible, therefore, that the slit diaphragm in the human glomerulus could function as a fine filter for serum proteins, although this remains to be definitely proved.

Basement Membrane↗
PubMed1975

Cytochemical demonstration of hydrogen peroxide in polymorphonuclear leukocyte phagosomes.

Phagocytosis by polymorphonuclear leukocytes (PMN) is accompanied by specific morphological and metabolic events which may result in the killing of internalized micro-organism. Hydrogen peroxide is produced in increased amounts during phagocytosis (17) and in combination with myeloperoxidase and halide ions constitute a potent, microbicidal mechanism (8,9,11). There can be direct iodination of micro-organisms (10), or alternatively, other intermediate reaction products, i.e. chloramines and aldehydes (21), can exert a microbicidal effect. The H2O2-peroxidase-halide system is presumed to operate within the phagocytic vacuole (12,18). Myeloperoxidase, present in the primary granules of PMN, enters the phagocytic vacuole during degranulation (1,4,7), and halide ions are probably derived from the extracellular medium or are present in the PMN (see 11, 18). For the operation of this system in intact cells, the presence of H2O2 in the phagocytic vacuole is necessary, and indeed this has been suggested by the work of several investigators (12, 18, 21). In the present investigation, the diaminobenzidine reaction of Graham and Karnovsky (5), modified to utilize endogenous myeloperoxidase and hydrogen peroxide, has been applied to actively phagocytizing PMN to demonstrate cytochemically the presence of H2O2 in the phagocytic vacuole.

Animals↗
PubMed1975

Substructure of the glomerular slit diaphragm in freeze-fractured normal rat kidney.

In the renal glomerulus, the narrow slits between adjacent epithelial podocytes are bridged by a diaphragm (2, 8, 11). In rat and mouse kidneys fixed by perfusion with tannic acid and glutaraldehyde (TAG), it has recently been discovered that this diaphragm has a highly ordered, isoporous substructure (9). It consists of a regular array of alternating cross bridges extending from the podocyte plasma membranes to a centrally running filament. This zipperlike pattern results in two rows of rectangular pores, approximately 40 X 140 A in cross section, dimensions consistent with the proposed role of the diaphragm as an important filtration barrier to plasma proteins (6). In the present study, we found in freeze-cleaved and in freeze-etched normal rat glomeruli that the surface of the slit diaphragm has an appearance conforming to the pattern found in sectioned material.

Animals↗
PubMed1975

Localization of NADH oxidase on the surface of human polymorphonuclear leukocytes by a new cytochemical method.

The ultrastructural localization of NADH oxidase, a possible enzyme in the increased oxidative activity of polymorphonuclear leukocytes (PMN) during phagocytosis, was studied. A new cytochemical technique for the localization of H2O2, a product of NADH oxidase activity, was developed. Cerous ions, in the presence of peroxide, form an electron-dense precipitate. Resting and phagocytically stimulated PMN were exposed to cerous ions at pH 7.5 to demonstrate sites of NADH-dependent, cyanide-insensitive H2O2 production. Resting PMN exhibites slight activity on the plasma membrane; phagocytizing PMN had extensive deposits of reaction product localized within the phagosome and on the plasma membrane. Peroxide involvement was demonstrated by the inhibitory effect of catalase on cerium precipitation; the surface localization of the enzyme responsible was confirmed by using nonpenetrating inhibitors of enzymatic activity. A correlative study was performed with an NADH-dependent, tetrazolium-reduction system. As with cerium, formazan deposition on the surface of the cell was NADH dependent, cyanide insensitive, and stimulated by phagocytosis. Superoxide dismutase did not inhibit tetrazolium reduction, as observed cytochemically, indicating direct enzymatic dye reduction without superoxide interposition. These findings, combined with oxygen consumption studies on resting and stimulated PMN in the presence or absence of NADH, indicate that NADH oxidase is a surface enzyme in human PMN. It is internalized during phagocytosis and retains its peroxide-generating capacity within the phagocytic vacuole.

Animals↗
PubMed1975

Early cellular responses to mitogens and adjuvants in the mouse spleen.

The purpose of this study was to evaluate the cellular events in the spleens of mice following the intravenous injection of mitogens and adjuvants. The compounds used were concanavalin A, polyadenylic polyuridylic acid, beryllium sulfate, bacterial endotoxin, tuberculin-purified protein derivative, and dextran sulfate. Nine different strains of mice (some deficient in the C5 component of complement) received a single dose of these compounds, and their spleens were studied at sequential time intervals, ranging from 1 hour to 14 days. Concanavalin A triggered marked blast activity in the T cell zones of the splenic white pulp which was maximal at 24 hours following the injection. -3H-thymidine incorporation increased significantly, but the number of immunoglobulin-negative cells did not increase, probably because of a concomitant loss of hematopoietic cells. Polyadenylic polyuridylic acid and beryllium sulfate produced an increase in the number of lymphocytes in the T cell zones by 12 to 24 hours, but mitotic activity was unremarkable. None of the above T cell zone changes was observed in neonatally thymectomized mice. Endotoxin, purified protein derivative, and dextran sulfate produced marked B cell zone hyperplasia. Similar histologic changes were seen in the thymectomized animals. -3H-thymidine incorporation and number of immunoglobulin-positive cells were significantly increased by 24 hours. Endotoxin and dextran sulfate in some strains of mice caused marked depletion of the T cell zones. The effects of concanavalin A, endotoxin, and dextran sulfate were unrelated to the presence or absence of C5 protein. These experiments show that (1) concanavalin A, bacterial endotoxin, tuberulin-purified protein derivative, and dextran sulfate trigger in vivo the same cellular components of the immune system as they do in vitro; (2) polyadenylic polyuridylic acid and beryllium sulfate may influence the immune system by increased localization of lymphocytes in the T cell zones.

Adjuvants, Immunologic↗
PubMed1975

Non-random distribution of surface immunoglobulins on murine B lymphocytes.

An indirect labeling technique was used to map the surface immunoglobulin (Ig) on murine splenic B lymphocytes by freeze-etching. Cells were labeled first with fluorescein conjugates of monovalent (papain-digested) anti-Ig antibody followed by monovalent anti-fluorescein antibody coupled to ferritin. The technique avoids cross-linking and aggregation of surface Ig. Freeze-etched replicas of cells labeled at 4 degrees C, as well as of cells prefixed with paraformaldehyde showed that surface Ig was distributed in small clusters with interconnecting networks. The observed pattern was analyzed statistically by comparing it with the expected random (Poisson) distribution and shown to be non-random to a high degree of statistical significance. The deviation from randomness could be explained by the presence of clusters and relative excesses of bare membrane. Such an observed distribution of surface Ig suggests that this membrane macromolecule may be organized in a specific manner. The distribution may also play a role in the function of surface Ig as the antigen receptor on B lymphocytes.

Animals↗
PubMed1975

Endothelial regeneration in the rat carotid artery and the significance of endothelial denudation in the pathogenesis of myointimal thickening.

A new model was developed to study endothelial regeneration and the effects of endothelial denudation in large arteries. Complete endothelial loss was produced in a sharply defined, unbranched segment of the rat common carotid artery by brief drying with a gentle stream of air along the lumen of the vessel. Platelets became attached to the denuded surface, but no polymorphonuclear or mononuclear leukocytic adherence or infiltration was observed. Regeneration occurred by the ingrowth, from each end of the denuded segment, of sheets of rapidly dividing endothelial cells. Endothelial replacement was complete by 7 to 10 days after drying. It was also noted that, by 14 days after drying, a striking myointimal thickening developed in the central region of the denuded segment. This central region was the last area to be covered with new endothelium; while still denuded, the region showed edema and signs of cellular proliferation in the innermost medial layer. The myointimal thickening consisted of smooth muscle-like cells in a fibroelastic stroma in the deeper zones, and a layer of rounded, relatively undifferentiated cells in the superficial zones. Progressive regression and condensation of the thickening was observed between 14 days and 3 months. In this model, the clear demarcation and completeness of endothelial denudation in the arterial segment allow study of the over-all process of endothelial replacement, avoiding the confusion imposed by islands of residual endothelium or contributions from branch vessels. The results suggest that the new endothelium in this situation is derived from each end of the segment. In addition, because the method of inducing denudation does not cause significant damage to the underlying media (as shown by the absence of inflammatory cell attachment or infiltration), the model offers a unique opportunity to study the relationship between endothelial loss and changes in the vessel wall. The correlation between the duration of endothelial denudation and the extent of myointimal thickening suggests that sustained insudation of certain, as yet undefined, factors from the lumen may provoke cellular proliferation in the arterial wall. Further investigation of this model should provide information relevant to the pathogenesis of the fibromuscular thickenings of human atherosclerosis.

Animals↗
PubMed1974

Ligand-induced movement of lymphocyte membrane macromolecules. V. Capping, cell movement, and microtubular function in normal and lectin-treated lymphocytes.

Capping of surface Ig by anti-Ig antibodies involves a membrane perturbation requiring an energy-dependent step. Lymphocytes treated with anti-Ig are stimulated to move. Previously, we had shown that movement was not essential for capping, although it influenced the localization of the cap. We have investigated the role of cell movement and of microtubular proteins in this phenomenon. Treatment of B lymphocytes with colchicine does not affect capping of Ig nor does it affect the increase in translational movement produced by anti-Ig antibodies. Treatment of lymphocytes with cytochalasin B stops translational movement and may affect capping to some degree under appropriate circumstances. Lymphocytes treated with both drugs are impaired in capping. We surmise that there may be two cytoplasmic events regulating directly or indirectly capping: one associated with the process of translational movement, the other associated with the activity of microtubules. Lymphocytes treated with concanavalin A do not cap Ig. Colchicine reverses this inhibition. Certain experimental procedures antagonize the colchicine effect, the most striking of which is the use of cytochalasin B. Colchicine appears to increase movement of the Con A-treated lymphocyte, and this increased movement appears responsible for the accumulation of complexes to the posterior part of the cell. Con A inhibits patching of Ig by anti-Ig, and this is not reversed by colchicine.

Animals↗