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Biomedical subjects

L R DeChatelet

Publications and source records attributed to L R DeChatelet.

At least 19 recordsLinked to original sources
PubMed1979

Enhancement of hexose uptake in human polymorphonuclear leukocytes by activated complement component C5a.

The polymorphonuclear leukocyte (PMNL) depends on glucose as a source of energy for motility, chemotaxis, phagocytosis, and bactericidal activity. Activated complement (C5a) at low concentrations stimulates carrier-mediated carbohydrate transport in PMNLs as measured by the uptake of 2-deoxy-D-[(3)H]glucose. Human PMNLs were preincubated at 37 degrees C for 15 min with zymosan-activated human serum or various purified preparations of human C5a. A concentration-dependent increase in deoxyglucose transport (>700% of control) into PMNLs occurred with all test substances. Reaction was linear for 30 min, and uptake of deoxyglucose followed saturation kinetics. C5a caused a decrease in the K(m) for deoxyglucose, from 0.53 to 0.11 mM, without altering the V(max) (44 nmol/30 min per 5 x 10(6) PMNLs in control and 46.6 with C5a). The optimal concentration of C5a for enhanced carrier-mediated transport of deoxyglucose was similar to that which promoted optimal chemotaxis. Activated serum from C5-deficient mice had little or no effect on deoxyglucose transport whereas that from normal syngeneic mice enhanced deoxyglucose transport. C5a did not enhance deoxyglucose transport into isolated erythrocytes, platelets, or lymphocytes. The deoxyglucose within the cell was primarily in the phosphorylated form, and hexokinase activity was not increased in PMNLs stimulated with C5a, indicating that hexokinase was not rate limiting and that enhanced transport was the mechanism of the C5a activity. Insulin at physiologic concentration (10 ng/ml) had no effect on deoxyglucose transport in PMNL and did not act as a competitive inhibitor of C5a. This insulin-like bioactivity could be detected with the amount of C5a that would be present after activation of 0.1-0.5% of the C5 in 1 ml of serum. This suggests that uptake of [(3)H]deoxyglucose by PMNLs might serve as a highly sensitive test for activation of the fifth component of complement.

Biological Transport↗
PubMed1979

Dissociation of leukocyte alkaline phosphatase from the bactericidal activity of neutrophils.

We have identified an apparently healthy individual with no history of repeated infections whose neutrophils were virtually devoid of alkaline phosphatase activity by either spectrophotometric or histochemical assay. His cells showed normal bactericidal activity toward both Staphylococcus aureus and Escherichia coli when tested in vitro. We have also demonstrated that L-p-bromolevamisole, a potent inhibitor of alkaline phosphatase in many tissues, is likewise an effective inhibitor of the neutrophilic enzyme. Concentrations of this compound which caused nearly complete inhibition of the neutrophilic enzyme did not impair the ability of intact cells to kill bacteria. These results suggest that leukocyte alkaline phosphatase is not required for the normal bactericidal activity of neutrophils.

Alkaline Phosphatase↗
PubMed1979

Generation of chemiluminescence by a particulate fraction isolated from human neutrophils. Analysis of molecular events.

A particulate fraction isolated from human neutrophils by homogenization, then centrifugation at 27,000 g, was demonstrated to generate chemiluminescence. This luminescence required the addition of reduced pyridine nucleotide and was very low in fractions from resting normal cells. Stimulation of neutrophils with opsonized zymosan, phorbol myristate acetate, or ionophore A23187 resulted in marked enhancement of the chemiluminescence measured in subsequently isolated particulate fractions. Stimulation did not boost the luminescence produced by fractions from cells of patients with chronic granulomatous disease. The chemiluminescence of particulate fractions from stimulated neutrophils was linear with increasing protein concentration, had a pH optimum of 7.0, and was higher with NADPH as substrate than with NADH. These results confirm previous studies suggesting that the enzyme system responsible for the respiratory burst in neutrophils is present in this fraction. The particulate fraction was used to examine the nature and origin of neutrophil luminescence by investigating the effect on this phenomenon of certain chemical and enzymatic scavengers of oxygen metabolites. Results suggest that the energy responsible for the luminescence of particulate fractions and, presumably, the intact cell, is derived from more than one oxygen species and that luminescence is a product of the interaction of these species and excitable substrates within the cell.

Catalase↗
PubMed1978

Comparison of myeloperoxidase activity in leukocytes from normal subjects and patients with chronic granulomatous disease.

A study was undertaken to compare myeloperoxidase (MPO) activity in leukocytes from normal subjects and those with chronic granulomatous disease (CGD) and to eliminate further consideration of MPO as the oxidase responsible for the post-phagocytic respiratory burst and subsequent oxidase microbicidal sequence lacking in leukocytes from patients with CGD. With use of granule fractions isolated from a number of samples, MPO activity in several MPO-mediated biochemical systems (peroxidase assays, protein iodination, and amino acid decarboxylation) was measured. No difference was demonstrated between granule fraction preparations from normal subjects and those with CGD. These data show that MPO activity is normal in CGD leukocytes and are inconsistent with a role for MPO in initiating the post-phagocytic respiratory burst.

Granulomatous Disease, Chronic↗
PubMed1978

Allosteric transformation of reduced nicotinamide adenine dinucleotide (phosphate) oxidase induced by phagocytosis in human polymorphonuclear leukocytes.

We used sensitive isotopic and fluorometric assay procedures to investigate reduced nicotinamide adenine dinucleotide (phosphate) [NAD(P)H]oxidation in a particulate fraction derived from normal and chronic granulomatous disease leukocytes. Granules isolated from normal resting cells showed allosteric kinetics with regard to oxidation of either NADH or NADPH, so that no enzyme activity was observed at physiological concentrations of substrate. If the granules were isolated from cells that had previously phagocytized zymosan, normal hyperbolic kinetics were obtained, so that activity could now be observed at low levels of substrate. The activity towards NADPH was always substantially greater than that towards NADH at any given concentration of substrate. This alteration in kinetics with phagocytosis was not observed with the other granule enzymes, acid phosphatase or beta-glucuronidase, and thus appeared to be specific for the reduced pyridine nucleotide oxidase(s). In contrast, granules isolated from cells of patients with chronic granulomatous disease showed allosteric kinetics regardless of whether they were obtained from resting or phagocytizing cells, so that NADPH oxidation was not measurable at physiological concentrations of substrate. This defect in the oxidation of NADPH by granules isolated from phagocytizing chronic granulomatous disease cells was observed over the pH range of 4.0 to 7.0. These data suggest that initiation of the respiratory burst by pahgocytosis normally requires an allosteric transformation in a reduced pyridine nucleotide oxidase, which in turn allows expression of enzymatic activity at physiological concentrations of substrate. The defect in chronic granulomatous disease appears to lie in an inability to achieve this transformation, and the enzyme remains in the inactive, allosteric form.

Acid Phosphatase↗
PubMed1978

Isolation of enzymatically homogeneous populations of human lymphocytes, monocytes, and granulocytes by zonal centrifugation.

Human monocytes, lymphocytes, granulocytes, red cells, and platelets were completely separated from each other by zonal centrifugation on linear sucrose density gradient. The monocytes contained only one tenth the amount of myeloperoxidase, one half the amount of lysozyme, one half the amount of acid ,hosphatase, and one half the amount of beta-glucuronidase found in granulocytes; the monocytes contained no alkaline phosphatase or neutral protease. The lymphocyte fraction contained only acid phosphatase and beta-glucuronidase in amounts one half as much as in the monocytes. Fluctuations in enzyme levels of monocytes and granulocytes were noted following infection. In vitro, the isolated monocytes transformed into macrophages. The results suggest that lymphocytes, monocytes, and granulocytes may be linked biochemically in a differentiation sequence through sets of commonly shared enzymes as well as by groups of enzymes specific for each divergent cell line.

Acid Phosphatase↗
PubMed1978

Comparison of intracellular bactericidal activities of human neutrophils and eosinophils.

Eosinophils from a patient with hypereosinophilia were observed to phagocytize radiolabeled E. coli or S. aureus as effectively as neutrophils from a normal control. This was observed at a number of bacteria/cell ratios and a various time intervals following initial challenge and was confirmed by direct microscopic examination. In spite of comparables rates of phagocytosis, the eosinophils were consistently less capable of killing the bacteria. This correlates with an inability of eosinophil peroxidase to catalyze the peroxidase-H2O2-CI--mediated decarboxylation of amino acids; in contrast, both eosinophil and neutrophil peroxidases showed similar capabilities to iodinate protein in vitro. These data are compatible with the importance of the chloride-mediated mechanism in the bactericidal activity of intact phagocytes.

Blood Bactericidal Activity↗
PubMed1977

Deficiency of NADPH oxidase activity in chronic granulomatous disease.

NADPH oxidase activity was examined in paired 27,000 x g granule fractions isolated from normal polymorphonuclear leukocytes from patients with chronic granulomatous disease. At 0.17 mM NADPH, the oxidase activity was not measurable in normal resting cells but was activated by phagocytosis. This activation was absent in CGD cells. At higher levels of NADPH, activity was present in cells from patients with CGD, although it was lower than normal, and no difference in activity was found between resting and phagocytizing cells. Granule fractions from phagocytizing normal cells exhibited higher than granule fractions from resting normal cells at all levels of NADPH. These results suggest that NADPH oxidase activity is defective in chronic granulomatous disease, and further that the defect is not the absence of the enzyme but rather a failure to activate it.

Adolescent↗
PubMed1977

Magnesium-dependent adenosine triphosphatase as a marker enzyme for the plasma membrane of human polymorphonuclear leukocytes.

The adenosine triphosphatase (ATPase) activities of human polymorphonuclear leukocytes (PMNL) were studied with an assay that monitored the release of 32P-labeled inorganic pyrophosphate (32P1) from gamma-[32P]adenosine 5'-triphosphate (ATP). In cell homogenates, (Na+ + K+)-sensitive, ouabain-inhibitable ATPase comprised an insignificant fraction of the total ATPase activity. Additions of p-nitrophenyl phosphate and beta-glycerophosphate (substrates for nonspecific acid and alkaline phosphatases) and of tartrate (inhibitor of acid phosphatase) gave no indication of inhibition. This suggested that the assay was relatively specific for ATP hydrolysis. The activity was found to have a pH optimum of 8.7 and a Km for ATP of 0.6 mM. There was an absolute requirement for Mg2+, with other divalent cations substituting less efficiently. When the Mg2+-dependent ATPase activity of intact cells was compared with that in homogenized cells, no significant difference was observed. The activity in intact cells was linear with respect to incubation time up to at least l0 min. Trypan blue staining and lactate dehydrogenase assays revealed that greater than 92% of the PMNL remained intact and viable during the assay. No soluble ATPase was released from the cells under assay conditions. In following the distribution of gamma[32P]ATP and 32P2 counts became cell associated. Since the experimental evidence supports the observation that PMNL remain intact and viable and that ATP does not penetrate the cell under assay conditions, it is proposed that greater than 90% of the Mg2+-dependent ATPase of the human PMNL is associated with a plasma membrnae enzyme. This would qualify the enzyme for the role of a plasma membrane marker for future fractionation and isolation attempts.

Adenosine Triphosphatases↗