Search PubMed⌕ Search

Biomedical subjects

M J Karnovsky

Publications and source records attributed to M J Karnovsky.

At least 91 records · Page 5Linked to original sources
PubMed1987

Metabolic effects of heparin on rat cervical epithelial cells.

The glycosaminoglycan heparin inhibits the growth of a number of different cell types in vitro including smooth muscle cells, mesangial cells, fibroblasts, and rat cervical epithelial cells (RCEC). Studies investigating the antiproliferative effects of heparin on smooth muscle cells have demonstrated the site of the cell cycle block and revealed several metabolic alterations that could be causally associated with growth inhibition. We have investigated these metabolic parameters in RCEC to determine whether they are also associated with the antiproliferative effects of heparin in epithelial cells. Heparin acts rapidly to inhibit RCEC growth with inhibition detectable by autoradiography 7 h after the addition of heparin. Heparin treated RCEC begin to enter S-phase 12 h after the removal of heparin. These findings suggest that heparin blocks RCEC in the early-to-mid G1 phase of the cell cycle rather than late in G1 or early in S-phase as has previously been demonstrated for smooth muscle cells. Unlike smooth muscle cells, the uptake of thymidine and uridine is not inhibited by heparin in RCEC. Treatment of medium with heparin-Sepharose does not reduce the subsequent growth of RCEC; heparin inhibits the growth of RCEC in heparin-Sepharose treated medium in a manner identical to that in nontreated medium. Therefore the growth inhibitory effects of heparin cannot be explained by the inactivation of mitogens present in serum. In contrast to its effects on smooth muscle cells, heparin treatment of RCEC does not result in a reduction in the binding of epidermal growth factor (EGF) to the cells. These results indicate that although heparin inhibits the growth of a variety of cell types, significant differences exist in the responses of the different cells to heparin.

Animals↗
PubMed1987

Exacerbation of chronic aminonucleoside nephrosis by dietary cholesterol supplementation.

Abnormalities in lipid metabolism resulting from nephrotic syndrome may play a role in the progression of initial glomerular injury to focal and segmental glomerulosclerosis (FSGS). In order to more specifically assess this, we fed male Sprague-Dawley rats, made nephrotic with a single intravenous injection of puromycin aminonucleoside (PA), either normal rodent chow (Group 1) or the same formulation supplemented with 4% cholesterol/1% cholic acid (Group 2). This 4% cholesterol/1% cholic acid-added diet was utilized because, in normal, non-nephrotic rats, this alimentary supplement produces, for the most part, only a significant rise in fasting serum cholesterol and not fasting serum triglycerides. FSGS developed 18 weeks after PA delivery, and both groups of rats were studied functionally and morphologically. Group 2 rats had significantly higher daily urine protein excretion, lower inulin clearance, and greater blood urea nitrogen concentrations than Group 1 animals. Histologically, Group 2 animals demonstrated a significantly greater percentage of glomeruli examined with segmental areas of glomerulosclerosis/hyalinosis, mesangial cell proliferation, and mesangial "foam" cells. At 2, 4, 12, and 18 weeks after PA delivery, the fasting serum cholesterol was always significantly greater in Group 2 rats, whereas in regards to fasting serum triglycerides it was only significantly elevated in Group 2 rats at 4 and 12 weeks after PA administration.

Animals↗
PubMed1987

Cell surface dynamics of neutrophils stimulated with phorbol esters or retinoids.

Neutrophils undergo rapid morphological changes as well as metabolic perturbations when stimulated with certain phorbol esters. Stimulated cells initially exhibit pronounced projections emanating from the cell bodies, followed by rounding of the cells, reduction in granule number, and the appearance of intracellular vesicles. We show these vesicles to be derived, at least in part, from the plasmalemma. The experimental approach involved labeling stimulated and unstimulated cells with native ferritin and cationized ferritin, along with the cytochemical localization of ecto-5'-nucleotidase. The labeling patterns of the vesicles indicate that these structures are involved in both phorbol ester-stimulated adsorptive and fluid-phase endocytosis. Neutrophils stimulated with 12-O-tetradecanoyl-phorbol-13-acetate (TPA) exhibit two distinct rates of superoxide release in which the second, prolonged level is approximately 50% of the initial rate. All-trans-retinal, which we have recently shown to stimulate O2- release but not granule exocytosis or cell vesiculation, induces a single prolonged rate of maximal O2- release. Neutrophils treated with both all-trans-retinal and TPA exhibit only a single sustained rate of maximal O2- release similar to that observed with all-trans-retinal alone. Moreover, treatment of cells with all-trans-retinal blocks the vesiculation of neutrophils induced by TPA in a dose-dependent manner. This observation provides a possible explanation for the differences in the kinetics of superoxide release.

Animals↗
PubMed1987

Adhesion of polymorphonuclear leukocytes to endothelium enhances the efficiency of detoxification of oxygen-free radicals.

Polymorphonuclear leukocytes can produce active oxygen species such as hydrogen peroxide and superoxide under various conditions. Because these substances can be toxic to cells, it is possible that the interaction between the circulating leukocytes and the blood vessel wall, either in normal circulation or during the acute inflammatory response, could damage the endothelial lining. Using an in vitro system of cultured endothelial cells and isolated polymorphonuclear leukocytes, we have measured the levels of detectable superoxide when neutrophils are attached to either endothelial monolayers or to plastic. Our results show that the levels of superoxide, on a per-cell basis, are lower when the neutrophils are attached to endothelium than when attached to plastic, even if the neutrophils are stimulated with phorbol myristate acetate. This is also reflected in data showing that no injury occurs to the endothelial cells, as measured by 51Cr release, under these same conditions. When endothelial cells are pretreated with an inhibitor of superoxide dismutase, diethyldithiocarbamate, the levels of superoxide detected are the same for neutrophils stimulated on plastic and those on the endothelial monolayer, suggesting that endothelial superoxide dismutase may remove a portion of the neutrophil-generated superoxide from the detection system. Further evidence for the role of endothelium in destroying superoxide is suggested by results that show that the level of detectable superoxide released from neutrophils attached to formalin-fixed endothelial monolayers is the same as that for neutrophils attached to plastic. It is important to note that with the inhibitor of superoxide dismutase present, the endothelial monolayers do not display enhanced 51Cr release under the conditions employed. When both endothelial catalase and glutathione reductase are inhibited, we detect increased 51Cr release from endothelial cells in response to stimulated neutrophils. Our results show that the endothelial cells are important in affecting the apparent reduction of toxic oxygen products derived from polymorphonuclear leukocytes attached to their surface.

Animals↗
PubMed1987

Fate of the DNA in plasmid-containing Escherichia coli minicells ingested by human neutrophils.

Escherichia coli minicells containing the plasmid pSC101 (approximately 10 kb) or pBR322 (approximately 4 kb) were opsonized and incubated with human neutrophils. The neutrophils responded to the minicells as they would to native E coli: they ingested the minicells, discharged their granule contents into the minicell-containing phagosomes, and expressed a respiratory burst. After one hour of incubation, the fate of the ingested plasmid DNA was examined. No DNA degradation was detected by trichloroacetic acid precipitation or agarose gel electrophoresis. Moreover, when pBR322 recovered from ingested minicells was transformed into E coli, no mutations in either of the antibiotic resistance genes carried by the plasmid were detected out of many thousand transformants screened. These findings confirm the surprisingly limited effect of neutrophils on ingested DNA.

Blood Bactericidal Activity↗
PubMed1987

Ameliorative effects of dietary protein restriction in chronic aminonucleoside nephrosis.

Because many investigators have demonstrated the efficacy of dietary protein restriction in various experimental models of glomerular injury that progress to focal and segmental glomerulosclerosis (FSGS), we used this dietary maneuver in chronic aminonucleoside nephrosis. This model of glomerular disease, which uses a single intravenous injection of the puromycin aminonucleoside (PAN), slowly progresses over 18 weeks with mesangial cell proliferation and FSGS as the pathologic hallmarks. We performed renal functional and histopathologic studies in 21 rats with chronic aminonucleoside nephrosis. Group 1 rats (n = 10) were fed a standard rodent diet containing 23.4% protein, whereas group 2 animals (n = 11) were maintained with a 6% protein diet replete with electrolytes, minerals, and vitamins. In those animals subjected to dietary protein restriction, proteinuria was significantly reduced at 14, 28, 84, and 126 days after PAN administration. In rats maintained with the 6% protein diet at 126 days after PAN delivery, there was also a significant reduction in the percent of glomeruli with segmental areas of glomerulosclerosis or hyalinosis and mesangial cell proliferation. We conclude that the renal functional and histologic consequences of chronic aminonucleoside nephrosis can be blunted by dietary protein restriction.

Animals↗
PubMed1986

Retinoids stimulate the release of superoxide by neutrophils and change their morphology.

All-trans-retinal stimulated the release of superoxide by human and guinea pig neutrophils 63 +/- 14 SD and 53 +/- 5 SD nmol of O2-/min/10(7) cells, respectively. Superoxide release by unstimulated cells was negligible. All-trans-retinal also induced morphological changes (i.e., evaginations) in these cells. Other retinoids were effective in instigating these phenomena. The similarities of these effects to those instigated by cis-unsaturated fatty acids (Badwey, J.A., et al., 1984, J. Biol. Chem., 259:7870-7877) are discussed in light of possible mechanisms.

Animals↗
PubMed1986

Isolation of bovine aortic endothelial cell plasma membranes: identification of membrane-associated cytoskeletal proteins.

The plasma membrane of bovine aortic endothelium was isolated, characterized, and found to contain at least four membrane-associated cytoskeletal proteins. Exposure of the plasma membranes to salt media (up to 1M KCl) resulted in the release of 30% of the total plasma membrane-associated proteins and extraction with 1% Triton X-100, 60%. At least four heavily glycosylated bands (185-, 165-, 150-, and 130,000 mol-wt) were evident. The Triton-insoluble pellet fraction contained several major polypeptides (30-, 43-, 58-, and 240,000 mol-wt), two of which were identified by immunoblotting as cytoplasmic actin (43,000 mol-st) and vimentin (58,000 mol-wt). Strikingly, vimentin and a 240,000 mol-wt polypeptide were routinely present in approximately a mole ratio of 4:1 in more than 60% of the plasma membrane preparations. We also report the presence of a 2.1-like and a 4.1-like protein associated with plasma membranes. The 2.1-like protein demonstrated similar solubilities and apparent molecular weight (210,000) as erythroid protein 2.1. Likewise, the endothelial 4.1-like protein exhibited similar solubilities and apparent molecular weight as erythroid protein 4.1. Immunofluorescence staining of fixed and permeabilized cultures with anti-2.1 antibodies showed a fibrillar pattern. In contrast, cells stained with anti-protein 4.1 were brightly fluorescent, bearing both a diffuse and punctate pattern. This paper presents several novel observations pertaining to the composition of bovine aortic endothelial cell plasma membranes, namely: the presence of two erythroid-like cytoskeletal polypeptides; the presence of vimentin and a 240,000 mol-wt polypeptide in a 4:1 mole ratio in more than 60% of the plasma membrane preparations and the co-elution in a 4:1 mol ratio with a protein perturbant; and the inability to release actin from the plasma membrane preparations, suggesting the association of actin with other molecules in the plasma membrane preparation.

Actins↗
PubMed1986

Superoxide production by polymorphonuclear leukocytes. A cytochemical approach.

Phagocytosis by polymorphonuclear leukocytes triggers a burst of oxidative metabolism resulting in hydrogen peroxide and superoxide production, and these active oxygen species function in the killing of microorganisms. A new cytochemical technique, based on a manganese dependent diaminobenzidine oxidation, has been developed to detect superoxide in these cells. It has been shown that superoxide generation is associated with the plasma membrane in cells activated by particulate (zymosan) and non-particulate (phorbol myristate acetate) stimuli. This membrane activity is maintained during invagination such that reduced oxygen is generated within the endocytic vacuoles. Reaction product is absent from unstimulated cells; additionally, formation of precipitate is blocked by omission of Mn++, low temperature, glutaraldehyde prefixation, and the presence of superoxide dismutase in the incubation medium.

3,3'-Diaminobenzidine↗
PubMed1986

Cytochemical localization of acid phosphatase in striated muscle.

Normal skeletal and cardiac striated muscle from adult rats was incubated for the cytochemical detection of acid phosphatase activity with cerium as the capture metal. Results from these experiments show that normal striated muscle has a greater number of acid phosphatase-positive structures, which are presumed to be lysosomes, than has been indicated by several previous cytochemical studies.

Acid Phosphatase↗
PubMed1986

A role for oxygen free radicals in aminonucleoside nephrosis.

The cellular processes responsible for the proteinuria induced by the aminonucleoside of puromycin (PA) remain inadequately defined. Hypoxanthine is both a metabolic breakdown product of PA as well as a substrate for xanthine oxidase, which catalyzes its enzymatic conversion to xanthine and uric acid, yielding the superoxide anion in the process. We examined whether oxygen free radical production contributes to the development of proteinuria in this model. Seven groups of male Sprague-Dawley rats were studied. Proteinuria was quantitated and histology examined 7 days after rats were treated with PA intravenously over 5 min. PA-treated animals received either saline, dimethyl sulfoxide, superoxide dismutase, or catalase over 30 min prior to and 30 min following PA administration. Another group received allopurinol over 4 hr prior to PA. The superoxide dismutase and allopurinol treatment groups had a significant suppression of urinary protein excretion compared to the PA control group. There were also less severe glomerular morphologic changes in the superoxide dismutase group vs. the PA controls, which demonstrated a pathologic pattern that included epithelial cell blebbing, segmental mesangial cell proliferation and matrix expansion, loss of glomerular capillary lumina, and occasional adhesions between the glomerular tuft and Bowman's capsule. The allopurinol group exhibited normal glomerular morphology on light microscopy, with the exception of occasional epithelial cell blebs. All groups showed spreading of the epithelial cell cytoplasm along the glomerular basement membrane with loss of foot processes, focal areas of lifting of the epithelial cell from the glomerular basement membrane, cytoplasmic vacuolization, and protein reabsorption droplets; however, allopurinol-treated animals demonstrated these changes to a lesser extent.(ABSTRACT TRUNCATED AT 250 WORDS)

Allopurinol↗
PubMed1986

Unusual lysosomes in aortic smooth muscle cells: presence in living and rapidly frozen cells.

Unusual tubular structures have been observed in rat aortic smooth muscle cells (SMC) grown in culture. These tubular structures have several characteristics that strongly suggest that they are lysosomes: they are bounded by a single membrane bilayer, contain densely staining material, and acid phosphatase activity. Furthermore, these structures are present in living cells, as demonstrated by their ability to accumulate the membrane-impermeable fluorescent dye lucifer yellow CH. In ultrastructural preparations they are best seen in samples that are cryofixed by rapid freezing and then freeze-substituted in osmium-acetone solutions. Conventional chemical fixation did not appear to preserve these structures to as great an extent as did rapid freezing. Comparison of SMC in vitro to the same cells in situ revealed differences in lysosome number as well as morphological appearance. Thus, the culturing of rat SMC leads to the formation of unusual tubular lysosomes whose ultrastructural appearance is particularly sensitive to the methods employed for examination.

Acid Phosphatase↗
PubMed1986

Structural determinants of the capacity of heparin to inhibit the proliferation of vascular smooth muscle cells. II. Evidence for a pentasaccharide sequence that contains a 3-O-sulfate group.

Earlier work from our laboratory demonstrated that heparin inhibited the proliferation of vascular smooth muscle cells in vivo and in vitro. Both anticoagulant and non-anticoagulant heparin species were equally effective as antiproliferative agents. Previous structure-function studies indicated that hexasaccharide and larger fragments retained antiproliferative activity, whereas tetra- and disaccharides were inactive. These experiments also suggested that both N- and O-sulfates of heparin were necessary for growth inhibitory capacity. In this paper, we have further analyzed the structural determinants of the antiproliferative activity of heparin. These experiments were done using synthetically prepared and therefore chemically defined heparin oligosaccharides. We present evidence that a pentasaccharide fragment retains antiproliferative activity, and that the 3-O-sulfate on the internal glucosamine residue is critical for growth inhibitory capacity of the pentasaccharide. We also show that heparins obtained from different manufacturers differ significantly in their ability to suppress smooth muscle cell proliferation.

Animals↗
PubMed1986

Nonanticoagulant protective effect of heparin in chronic aminonucleoside nephrosis.

Aminonucleoside nephrosis progresses over an 18-week period to focal and segmental glomerulosclerosis (FSGS). Whole heparin has been shown to blunt the extent of renal injury in another model of FSGS, renal ablation; however, the precise mechanism of protection has remained uncertain. Since heparin has a variety of physiologic actions unrelated to anticoagulation, we administered three different heparin compounds, each with a distinct profile of biological properties, to groups of rats given a single intravenous dose of puromycin aminonucleoside (PA). In the absence of a prolongation of the activated partial thromboplastin time (aPTT), both whole heparin (WH) and a 7,000- to 11,000-dalton-molecular-weight nonanticoagulant heparin (NAH) ameliorated the functional and histologic abnormalities of chronic aminonucleoside nephrosis as evidenced by significant reductions in 24-hour urine protein excretion while preserving the glomerular filtration rate and blunting the rise in serum creatinine as compared to untreated PA control animals at the conclusion of the study. In addition, the NAH and WH groups exhibited significantly fewer glomeruli with either segmental mesangial proliferative areas or glomerulosclerosis/hyalinosis lesions 18 weeks after PA administration. A fragment of heparin (HF) was ineffective. We conclude that heparin may exert its beneficial effect in chronic aminonucleoside nephrosis through a biologic action, other than anticoagulation, perhaps by inhibition of mesangial cell proliferation.

Animals↗
PubMed1986

Improved isolation of rat lung alveolar type II cells. More representative recovery and retention of cell polarity.

We have developed an improved isolation procedure for rat lung alveolar Type II (ATII) cells which yields a more representative sample of ATII cells with respect to their densities. This procedure includes an initial selection on a density gradient of approximately the complete density range of rat lung ATII cells. Subsequently, to exclude contaminating macrophages and lymphocytes from this fraction, the authors have exploited the fact that the contaminating cells have leukocyte common antigen (LC) on their surfaces, whereas the ATII cells do not. Our procedure yields 2 X 10(7) ATII cells per rat in a fraction which is 90% pure; the cells are immediately available for biochemical or pharmacologic analysis and represent a 90-95% recovery of the ATII cells loaded onto the density gradient. The cells retain their in vivo morphologic characteristics, including their polarity.

Animals↗
PubMed1986

Isolation of pulmonary alveolar type I cells from adult rats.

The authors have developed a procedure for the isolation of alveolar Type I (ATI) cells from adult rat lung. After an initial selective enzymatic digestion of the lungs by lavage with 0.2% collagenase, 0.05% trypsin, 0.008% elastase, and 0.005% DNAse Type I, the cells which are released are separated by density gradient centrifugation, and a fraction which includes all ATI cells (density, 1.0177-1.0411) is harvested. Contaminating leukocytes are excluded by specific surface adsorption, exploiting the fact that these cells have leukocyte common antigen on their surfaces, whereas ATI cells do not. Similarly, contaminating alveolar Type II (ATII) cells are removed by specific surface adsorption with the use of the lectin Maclura pomifera agglutinin, which binds to freshly isolated ATII cells and not to ATI cells. Our procedure yields 5 X 10(6) ATI cells per rat in a fraction that is at least 85-88% pure; the cells are immediately available for biochemical or pharmacologic analysis and represent a 98% recovery of the ATI cells loaded onto the density gradient. The ATI cells retain their essential in vivo morphologic characteristics, including their polarity.

Animals↗