Biomedical subjects
M L Karnovsky
Publications and source records attributed to M L Karnovsky.
Production of superoxide and hydrogen peroxide by an NADH-oxidase in guinea pig polymorphonuclear leukocytes. Modulation by nucleotides and divalent cations.
Explore the source record for details and available documents.
The enzyme of granulocytes that produces superoxide and peroxide. An elusive Pimpernel.
Explore the source record for details and available documents.
Tobacco smoke and the pulmonary alveolar macrophage.
Our results indicate that tobacco smoke exposure to varying duration causes morphological, biochemical and functional alterations in pulmonary alveolar macrophages. The results of these changes is a population of alveolar macrophages made up of larger cells, with a reduced nucleus-cytoplasmic ratio, which are heavily loaded with heterolysosomes containing lipid. Though their fractional complement of mitochondria remains the same, an increase in the inner mitochondrial membrane surface area may be related to an enhanced oxidative metabolism. The cell is biochemically activated particularly following chronic exposure and is functionally impaired with respect to phagocytosis.
Tobacco smoke. Effects on pulmonary host defense.
Tobacco smoke affected both the metabolism and function of pulmonary alveolar macrophages (PAM). Phagocytosis of viable Staphylococcus aureus and inert starch particles was minimally but consistently depressed in PAM from rats exposed to tobacco smoke for six months. Oxygen consumption, superoxide and hydrogen peroxide release, and hexose monophosphate shunt activity were elevated in cells from smokers. Oxidation of glucose, labelled in the carbon-six position, remained unchanged. All observed effects of tobacco smoke on oxygen metabolism occurred during phagocytosis and did not affect the basal metabolism of the nonstimulated cell.
Hydroxyl radical formation in phagocytic cells of the rat.
Polymorphonuclear leukocytes (PMN) and macrophages, harvested from the peritoneum and lung, release superoxide (O-.2) and hydrogen peroxide (H2O2) during phagocytosis. These two agents are thought to react with each other to produce a highly active oxidative substance known as hydroxyl radical (OH.). We present evidence suggesting that these radicals are generated by phagocytic cells of the rat. Our findings are based upon an assay where ethylene gas is generated from methional by the action of this radical. Ethylene generation was shown to be inhibited by superoxide dismutase, catalase, and scavengers of OH.. Of the cells examined, PMN generated the most ethylene from methional, exhibiting a fourfold increase during phagocytosis. Pulmonary and peritoneal macrophages caused smaller amounts of this gas to be formed. Regardless of cell type, an intact cell was required for ethylene generation. Zymosan appeared to be the most effective particle for all cells in ethylene formation from methional, although opsonization was critical only for PMN. Ethylene generation was dependent on cell concentration to an extent and increased with time.
Fluoride-mediated activation of the respiratory burst in human neutrophils. A reversible process.
Fluoride ion (F-) is an effective activator of the respiratory burst in neutrophils, as indicated by its ability to induce O2- production by these cells. Other halide ions did not activate the burst, Cl-, in particular appeared to antagonize the effect of F- on O2- production. F- stimulated O2- production showed a requirement for Ca++, but was independent of other exogenous cations. Neither phagocytosis nor degranulation were necessary for respiratory burst activation by F-. The effect of F- on the respiratory burst was reversible. Washing the cells after treatment with F-, while they were still producing large amounts of O2-, returned them to the resting state. They could then be stimulated again to produce O2- in amounts equivalent to those originally produced. Our experiments indicated that restimulation did not represent the activation of a population of cells that had not been activated during the initial exposure to F-, nor did it represent serial activation of different subpopulation of the O2- forming enzyme molecules present in a given cell. Rather, our data suggest that the entire population of O2- forming enzyme molecules was activated in a reversible fashion by F-.
The effects of experimental exposure to tobacco smoke on the oxidative metabolism of alveolar macrophages.
Explore the source record for details and available documents.
Alteration of some functional and metabolic characteristics of resident mouse peritoneal macrophages by lymphocyte mediators.
Resident mouse peritoneal macrophages were incubated in Sephadex G-100 fractions of supernates from concanavalin A-stimulated lymphocytes. A significant effect of the lymphocyte supernatant fractions containing mediators on macrophage 5'-nucleotidase, glucose-1 14C oxidation, cell maintenance, and migration is reported. The 5'-nucleotidase was depressed to an extent similar to that seen in activated macrophages obtained from Listeria-infected mice. On the other hand, glucose-1-14C oxidation was enhanced, but not to the same degree as seen in the counterparts in vivo. Whereas migration inhibitory factor (MIF) and cell adherence-augmenting activity were found in a number of adjacent fractions, the metabolic effects were found predominantly in a single fraction. Resident peritoneal macrophages or those elicited by the injection of a lymphocyte-derived chemotactic factor were more responsive with respect to the biochemical changes than caseinate-elicited macrophages. On the other hand, caseinate-elicited macrophages appeared to be more sensitive with respect to the effects of mediator(s) on cell retention. A possible dissociation between MIF and cell-adherence augmenting activity, on the one hand, and the entities that stimulate glucose-1-14C oxidation is reported, based on fractionation studies, and loss of the latter activity upon storage of lymphocyte supernates.
The effect of tobacco smoke on the metabolism and function of rat alveolar macrophages.
Alveolar macrophages harvested by bronchopulmonary lavage from rats exposed to tobacco smoke for 30 days ("smokers") showed alterations in oxidative metabolism, lactate production and phagocytosis of inert starch particles when compared with control macrophages. Phagocytosis of viable Staphylococcus aureus was unaffected by tobacco smoke. Glucose oxidation measured by conversion of glucose-1-14C to 14CO2 moderately affected while oxidation of glucose-6-14C to 14CO2 was not. Smokers routinely yielded fewer cells than controls, though these cells contained approximately 17% more protein than did controls. Opsonization of particles was not necessary for macrophages from either smoker or control animals to manifest a respiratory burst and increased superoxide and hydrogen peroxide release during phagocytosis. The glycolytic inhibitors, sodium fluoride and iodoacetamide, while effectively blocking glycolysis, did not inhibit phagocytosis by macrophages from either group. The results reported clearly distinguish alveolar macrophages from other phagocytic cells (peritoneal macrophages and polymorphonuclear leukocytes) and suggest a state of non-specific activation caused by exposure to tobacco smoke.
Effect of phosphate esters, nucleotides and nucleosides on 5'-nucleotidase of cultured mouse macrophages.
Mouse peritoneal macrophages elicited by intraperitoneal injection of sodium caseinate exhibit low levels of ecto-5'-nucleotidase (E. C. 3.1.3.5) activity in contrast to macrophages obtained by peritoneal lavage. When elicited cells were cultured under standard conditions in the presence of serum, a 2.5-fold increase in 5'-nucleotidase activity was observed over a period of 48 hours. Addition of adenosine monophosphate to the culture medium led to an augmented (5-fold) increase in the specific activity (per unit cell protein) as well as an absolute increase (per culture plate) of 5'-nucleotidase. Other adenosine-containing compounds also had stimulatory effects. The levels of this enzyme thus appear to be regulated by the extracellular levels of adenosine nucleotides. The product of the enzymatic reaction--adenosine--when added to the medium exhibited a toxic effect on these cells--as did adenosine monophosphate. However, the former substance did not augment the increase in enzyme activity during culture. The toxic effect could be suppressed when the cells were cultured in the presence of uridine 5'-monophosphate. The latter substance also depressed the stimulation of enzyme activity due to AMP.
Sleep-promoting factor S: purification and properties.
Sleep-promoting factor was purified from acid/acetone extracts of whole brains of rabbits and from brainstems of slaughterhouse cattle. Intraventricular infusion of extracts purified by means of ion exchange and gel filtration induced excess slow-wave sleep in rabbits for 5-10 hr. The procedure is simple and provides material suitable for physiological studies. Further treatment by partition chromatography and electrophoresis yielded an active product that was purified at least 1 million-fold. This product was inactivated by incubation with mixed carboxypeptidases A and B. Amino acid analysis of acid hydrolysates indicated that the effective dose was less than 150 pmol per rabbit and the original concentration in brain tissue was of the order of 30 pmol/g of brain.
Biochemical criteria for activated macrophages.
Explore the source record for details and available documents.
Cerebral glucose-6-phosphatase and the movement of 2-deoxy-D-glucose across cell membranes.
Glucose-6-phosphatase (glucose-6-phosphohydrolase and its associated phosphotransferase activities) was determined in brain tissue and in several preparations derived from brain tissue. These included purified capillaries and established cell lines of neuronal or glial origin. Since it has been suggested that glucose-6-phosphatase may be involved in sugar transport, the characteristics of that process were examined in these preparations. The pattern of uptake of 2-deoxy-D-glucose in four cell lines was shown to involve transport of the analog across the cell membrane that was more rapid than the subsequent phosphorylation of the sugar in the intracellular compartment. In the remaining cell lines and in purified capillaries, phosphorylation of 2-deoxy-D-glucose was at least as rapid as uptake. No differences could be found between the cells in these two categories with respect to amount or localization of glucose-6-phosphatase, ability to phosphorylate 3-O-methyl-D-glucose, or ability to phosphorylate extracellular and intracellular 2-deoxy-D-glucose. In the course of these experiments, it was found that there was a rapid efflux of 2-deoxy-D-glucose from cells that had taken up this sugar. The efflux involves a dephosphorylation step catalyzed by intracellular phosphatase that releases free sugar in the cytoplasm. Glucose-6-phosphatase thus probably has no major role in the phosphorylation of glucose in brain cells, but acts in the more conventional sense, i.e. as a phosphohydrolase.
Uptake and metabolism of intraventricularly administered piperidine and its effects on sleep and wakefulness in the rat.
Explore the source record for details and available documents.
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.
Synthesis and release of thymidine by macrophages.
An inhibitor of DNA synthesis and cell proliferation found in macrophage supernatants was subjected to further analysis. The following conclusions were drawn: a. Biochemically, the inhibitor appeared to be thymidine, judging from the migration in three different thin-layer systems and on gel filtration. b. The inhibitor was shown to be synthesized de novo by macrophage monolayers pulsed with a radiolabeled precursor of thymidine such as 14C-formate. c. The amount of inhibitor present in tissue culture fluids proved to be sufficient to block the growth of the EL-4 leukemia cell line in a manner that could be prevented with the addition of 2'-deoxycytidine. The EL-4 line was considerably more sensitive to thymidine blockade than a number of different cells tested.
Biochemical aspects of the function of the reticulo-endothelial system.
Explore the source record for details and available documents.