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Biomedical subjects

M L Karnovsky

Publications and source records attributed to M L Karnovsky.

At least 109 records · Page 6Linked to original sources
PubMed1977

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.

Animals↗
PubMed1976

Manganese-dependent NADPH oxidation by granulocyte particles. The role of superoxide and the nonphysiological nature of the manganese requirement.

Recent work has indicated that superoxide is involved in the manganese-stimulated oxidation of NADPH by crude granule preparations of guinea pig neutrophils. The characteristics of a model manganese-requiring NADPH-oxidizing system that employs a defined O2-generator have now been compared to the original neutrophil-granule system. With respect to pH dependence, cyanide sensitivity, and reduced pyridine nucleotide specificity, the properties of the two systems are very similar. Additional information has been obtained concerning cation specificity and the kinetics of the metal-catalyzed NADPH oxidation. From the similarities between the properties of the model and neutrophil particle systems, we postulate that the manganese-dependent NADPH oxidation observed in the presence of neutrophil granules represents in large part of nonenzymatic free radical chain involving the oxidation of NADPH to NADP, with O2- as both the chain initiator and one of the propagating species. In this reaction, the neutrophil particles serve only as a source of O2-. Further, the same changes in kinetics (decrease in apparent Km for NADPH) observed previously when granules from phagocytizing rather than resting cells were employed could be mimicked by varying the rate of O2-generation by the model system. We conclude from these results that it is unnecessary to invoke a manganese-requiring enzyme as a component of the phagocytically stimulated respiratory system of the neutrophil.

Animals↗
PubMed1975

Purification of cerebral glucose-6-phosphatase. An enzyme involved in sleep.

An insoluble phosphoprotein of rat brain acquires radioactivity from inorganic phosphate more rapidly during sleep than during wakefulness. It was purified in two ways. The first was solvent delipidation of brain tissue followed by preparative sodium dodecyl sulfate polyacrylamide gel electrophoresis. The second was sucrose gradient centrifugation of a brain homogenate to remove myelin, and gel filtration on Sephadex G-100 and adsorption chromatography on DEAE-Sephadex in the presence of sodium deoxycholate. The products were homogeneous within the limits of the analytical methods used. The apparent molecular weight of the phosphoprotein was 28,000 on sodium dodecyl sulfate polyacrylamide gels, but was much higher in the presence of sodium deoxycholate. The protein had a high content of aspartic and glutamic acids compared to basic amino acids. Analysis of a base hydrolysate, as well as studies of the kinetics of hydrolysis, showed that the radioactive phosphorus was attached to histidine. The NH2-terminal residue was identified as isoleucine. The phosphoprotein purified by the second method was enzymatically active. When it was incubated in vitro with a 32P-labeled supernatant fraction from rat brain (and later with glucose [6-32P]phosphate), a radioactive phosphorylated protein intermediate was formed. Exploration of the several enzymatic activities of the preparation indicated close correspondence to those reported for the glucose-6-phosphatases of liver and kidney. Glucose-6-phosphatase activity was found in all parts of the brain in the membranous subcellular fractions of neurons. It was shown to be co-purified with the sleep-related phosphoprotein. This report constitutes, we believe, the first complete purification of glucose-6-phosphatase from any tissue and an instance in which a change in the state of a cerebral enzyme has been linked to a normal change in the physiological state of the brain.

Amino Acids↗
PubMed1975

Superoxide production by phagocytic leukocytes.

Mononuclear phagocytic leukocytes, as well as polymorphonuclear leukocytes, produce and release superoxide at rest, and this is stimulated by phagocytosis. Of the mouse monocytic cells studied, alveolar macrophages released the largest amounts of superoxide during phagocytosis, followed by normal peritoneal macrophages. Casein-elicited and "activated" macrophages released smaller quantities. In the guinea pig, polymorphonuclear leukocytes and casein-elicited macrophages were shown to release superoxide during phagocytosis whereas alveolar macrophages did not. Superoxide release accounted for only a small fraction of the respiratory burst of phagocytosis in all but the normal mouse peritoneal macrophage, the guinea pig polymorphonuclear leukocyte, and probably the mouse alveolar macrophage. There are obviously considerable species differences in O2-release by various leukocytes that might reflect both the production and/or destruction (e.g. by dismutase) of that substance.

Animals↗
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

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

Extraction of sleep-promoting factor S from cerebrospinal fluid and from brains of sleep-deprived animals.

Sleep-promoting factor (factor S) was extracted, partially purified, and concentrated from cerebrospinal fluid and from acid-acetone extracts of brain stem anc cortex of sleep-deprived goats and sheep. 2. Solutes greater than 500 daltons were largely removed by serial ultrafiltrations through molecular sieves (Amicon membranes UM10 and UM05); solutes less than 350 daltons were largely eliminated by gel filtration through Sephadex G10 columns. Sleep-promoting activity was found in a fraction eluted prior to [14C] sucrose marker. 3. Concentrated fraction were infused intraventricularly in rats (0.1 ml in 30 min just prior to 12-h dark cycle) and in rabbits (0.3 ml in 90 min in morning). Sleep-promoting activity was assayed by decrease in nocturnal locomotor activity of rats and by duration and amplitude of slow-wave cortical EEG in rabbits.

Animals↗
PubMed1974

Ecto-enzyme of granulocytes: 5'-nucleotidase.

The 5'-nucleotidase of guinea pig polymorphonuclear leukocytes is localized exclusively on the plasma membrane of intact cells. The active site of this enzyme faces the external medium, not the cytoplasm.

Adenosine Monophosphate↗
PubMed1974

Bactericidal activity of metal-mediated peroxide-ascorbate systems.

Model systems containing ascorbate, hydrogen peroxide, and divalent copper or cobalt have been shown to possess marked bactericidal activity. At equivalent concentrations, copper-containing systems were more bactericidal than the corresponding mixtures containing cobalt. Cobalt at concentrations below 10(-4) M did not appreciably augment microbicidal activity, whereas systems containing copper at concentrations as low as 5 x 10(-6) M were still capable of causing some bacterial death. Manganese was inactive. None of these systems was as potent as the well known myeloperoxidase-peroxide-halide system. The mechanisms of action of these systems are not as yet clear. The possibility that they function through the generation of superoxide (O(2) (-)), hydroxyl radical (OH.), or other free radicals was explored through the use of superoxide dismutase and several free radical scavengers. It seems likely at present that the two active metal-mediated systems function via separate mechanisms. The copper system acts with dehydroascorbate, whereas the cobalt system does not. Activity in the cobalt system appears to depend upon the generation of free radicals.

Journal Article↗
PubMed1973

Iodinating ability of various leukocytes and their bactericidal activity.

A rapid method that employs monolayers of different phagocytic cells, primarily from guinea pigs and mice, has allowed a kinetic determination of (a) ingestion by these cells of labeled particles, (b) fixation of (131)I and (c) microbicidal activity in the cells after periods as short as 5' of exposure of bacteria to phagocytes. Phagocytes so examined included polymorphonuclear leukocytes (PMN) elicited into the peritoneal cavity, elicited peritoneal mononuclear cells (monocytes) (MN), and peritoneal macrophages (MAC) obtained simply by lavage. Circulating PMN from normal human subjects and from children afflicted with chronic granulomatous disease were also studied. The potential for generation of H(2)O(2) (a key component of the iodinating system) of all the normal cells studied, gauged by their content of cyanide-insensitive NADH oxidase, seemed comparable. Peroxidase levels varied widely, and were highest in PMN and almost undetectable in MAC. Catalase was at negligible levels in all the cell types obtained from mice. The fixation of (131)I by phagocytes ingesting (14)C-labeled dead tubercle bacilli appeared to be primarily a function of the cellular peroxidase content. Thus, mouse macrophages, with virtually no peroxidase, displayed no fixation of iodide. PMN proved far more able to fix (131)I during phagocytosis than did MN. In experiments comparing PMN from normal human subjects and from children with chronic granulomatous disease (CGD), a sex-linked condition characterized by a deficiency of H(2)O(2) production during phagocytosis and low microbicidal activity, the iodination ratio of CGD cells was dramatically less than that of normal PMN (by about two orders of magnitude). Capacity for iodination was correlated with bactericidal activity toward E. coli. At low bacterial loads (ca. 5:1), phagocytes killed efficiently, and little discrepancy in ability among cell types was apparent. Under the stress of higher loads of (14)C-labeled E. coli (ca. 100:1), differences in bactericidal activity were exaggerated, and a substantial disparity between MN and PMN was observed in favor of the latter. The hierarchy for killing efficiencies therefore agreed with that for iodination, with one notable exception: mouse MAC were consistently competent in their killing activity, more so than MN, even though they virtually lack peroxidase and the ability to iodinate ingested bacteria.

Animals↗