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J W Parce

Publications and source records attributed to J W Parce.

36 records · Page 2Linked to original sources
PubMed1984

Co-localization of superoxide generation and NADP formation in plasma membrane fractions from human neutrophils.

In order to resolve discrepancies in the literature concerning the subcellular localization of NADPH oxidase, we disrupted human neutrophils by nitrogen cavitation and fractionated the subcellular organelles on a discontinuous sucrose density gradient. The lightest fraction was 20- to 40-fold enriched for plasma membranes as determined by the marker enzymes alkaline phosphatase and phosphodiesterase I as well as by the ratio of lipid phosphorus to protein. There was a significant decrease in the specific activities of the granule markers myeloperoxidase, lysozyme, and beta-glucuronidase. An intermediate fraction was enriched in membrane markers but not to the extent the lightest fraction was enriched. This fraction contained more granular contamination, as shown by the marker enzymes. In contrast, the densest bands of the gradient were enriched for granule markers with little contamination by plasma membrane. Superoxide generation and NADP formation were primarily associated with the two membrane-enriched fractions from polymorphonuclear leukocytes stimulated with phorbol myristate acetate. The NADP formation associated with a dense granule fraction observed previously in our laboratory was probably due to a cyanide-stimulated oxidation of NADPH by myeloperoxidase.

Alkaline Phosphatase↗
PubMed1984

Flow cytometric quantitation of oxidative product formation by polymorphonuclear leukocytes during phagocytosis.

Stimulation of the oxidative metabolic burst of human polymorphonuclear leukocytes (PMNL) may occur by an all-or-none trigger mechanism or by a graded response to increasing stimulation of an individual cell. If the proposed all-or-none mechanism occurred during phagocytosis, a PMNL would expend all of its metabolic potential at once, yet PMNL can proceed to ingest multiple organisms. This study employed dual laser flow cytometry to correlate the number of cell-associated organisms with oxidative product formation in individual PMNL. Intracellular oxidation of nonfluorescent 2',7'-dichlorofluorescein (DCFH) to highly fluorescent 2',7'-dichlorofluorescein (DCF) provided a quantitative assay of H2O2-dependent oxidative product formation generated by the cell's oxidative metabolic burst. Staphylococcus aureus were fixed and stained with Texas red to allow simultaneous monitoring of bacteria (red fluorescence, greater than 580 nm) and DCF (green fluorescence, 510 to 550 nm) content of each cell. Computer correlation of bacterial and DCF fluorescence allowed determination of the DCF formation by PMNL containing specific numbers (0 to 15) of bacteria. Oxidative product formation was directly related to the number of bacteria ingested in a time-dependent manner (mean per cell of 6.4, 12.8, 19.1, and 24.4 attomoles (amol) DCF formed per cell per bacterium after 15, 30, 45, and 60 min, respectively. Opsonization of bacteria with fresh normal serum (primarily C3b opsonization) or with specific IgG demonstrated qualitatively similar responses, except that the response per IgG-opsonized organism was, on the average, more than twice the response to bacteria opsonized with serum. Thus, sequential phagocytosis of multiple bacteria elicits an incremental oxidative response of human PMNL.

Blood Physiological Phenomena↗
PubMed1984

Characterization of the effect of influenza virus on polymorphonuclear leukocyte membrane responses.

Depressed chemotactic activity of polymorphonuclear leukocytes (PMNL) infected with influenza virus could be due to changes occurring at the plasma membrane. The present study examined the effect of unopsonized influenza virus on chemotaxis, adherence, receptor binding, shape change, membrane fluidity, and release of specific granules from PMNL. Chemotactic activity of PMNL under-agarose to the chemoattractants, zymosan-activated serum ( ZAS ) and N-formyl-methionyl-leucyl-phenylalanine (fMLP), and adherence of PMNL to a plastic surface were markedly decreased in virus-treated cells as compared to control cells. The binding of fMLP to the PMNL was increased in virus-treated cells compared with control cells. Exposure of cells to virus, ZAS , or fMLP caused 35%-50% of the cells to become bipolar in shape, whereas less than 5% of the cells exposed to buffer became bipolar. Influenza virus did not alter membrane fluidity as measured by electron spin resonance spectroscopy with the probe 5-doxyl stearate. Virus-treated PMNL stimulated with FMLP or Staphylococcus aureus exhibited a marked decrease in the amount of lactoferrin released into phagosomes, onto the cells' outer membrane, and into the extracellular medium as compared to control cells. The possible relationship between inhibition of lysosomal enzyme degranulation and decreased chemotactic activity and adherence of PMNL is discussed.

Cell Adhesion↗
PubMed1983

Measurement of antibody-dependent binding, proteolysis, and turnover of C1s on liposomal antigens localizes the fluidity-dependent step in C1 activation.

The antibody-dependent binding and activation of the first component of human complement (C1) by liposomes containing nitroxide spin-label lipid haptens have been simultaneously measured. The liposomes were either fluid (dimyristoylphosphatidylcholine) or solid (dipalmitoylphosphatidylcholine) at the temperature of the experiments (32 degrees C). In 10 minutes fluid liposomes activate 40% of the C1 whereas solid liposomes only activate 10% of the C1. The fraction of C1 bound at the end of the activation incubation is approx. 2% for fluid liposomes and approx. 4% for solid liposomes. This binding is consistent with the relative amounts of antibody which bind to these two types of liposomes. These results demonstrate turnover of C1 or C1r2s2 on the liposome surface. It is concluded that the differential activation of C1 is due to a difference in the rate of activation of C1 after it is bound to the liposome surface. Lower limits for the activation rate constant for C1 bound to fluid and solid liposomes are estimated to be 8 X 10(-2) s-1 and 1 X 10(-2) s-1, respectively.

Animals↗
PubMed1983

Flow cytometric studies of oxidative product formation by neutrophils: a graded response to membrane stimulation.

We have developed a quantitative assay to monitor the oxidative burst (H2O2 production) of polymorphonuclear leukocytes (PMNL) using single cell analysis by flow cytometry, and have examined whether PMNL respond to membrane stimulation with an all-or-none oxidative burst. During incubation with normal neutrophils, dichlorofluorescin diacetate diffused into the cells, was hydrolyzed to 2',7'-dichlorofluorescin (DCFH) and was thereby trapped within the cells. The intracellular DCFH, a nonfluorescent fluorescein analogue, was oxidized to highly fluorescent 2',7'-dichlorofluorescein (DCF) by PMNL stimulated by phorbol myristate acetate (PMA). That the oxidative product was DCF was shown by excitation/emission spectra and by mass spectrometry of the product from PMA-stimulated PMNL. Normal resting and PMA-stimulated PMNL oxidized 6.9 +/- 0.7 and 160 +/- 13 attomoles DCF per cell, respectively, in 15 min. Absence of calcium and magnesium ions and/or addition of 2 mM EDTA did not inhibit DCF formation by PMNL stimulated by 100 ng/ml PMA. Since EDTA prevented aggregation of PMNL (even when stimulated by 100 ng/ml PMA), which would prevent accurate flow cytometric analysis, further experiments were performed with EDTA in the medium. A close correlation between average DCFH oxidation and hexose monophosphate shunt stimulation was demonstrated using cells from patients whose PMNL had oxidative metabolic defects of varying severity. Intracellular DCFH was also oxidized by reagent H2O2 or oxygen derivatives generated by glucose oxidase + glucose or by xanthine oxidase + acetaldehyde; DCFH oxidation by these systems was inhibited by catalase but unchanged by superoxide dismutase. The data indicate that the DCFH oxidation assay is quantitatively related to the oxidative metabolic burst of PMNL, and they strongly suggest that the reaction is mediated by H2O2 generated by the PMNL. Incubation of PMNL with varying concentrations of PMA caused graded responses by all PMNL present; i.e., 1 ng/ml PMA caused a mean response of 34% maximal with a single population of responding PMNL (rather than 66% resting and 34% fully stimulated as predicted by the all-or-none hypothesis). Thus, with these assay conditions, oxidative product formation by PMNL occurs as a graded response to membrane stimulation by PMA.

Flow Cytometry↗
PubMed1982

Identification and quantitation of electron-transport components in human polymorphonuclear neutrophils.

Using dithionite difference spectra we have detected cytochrome b in highly purified human neutrophils at a concentration of 0.08 nmol/mg protein. The presence of quinone was identified in lipid extracts at a concentration of approx. 0.06 nmol/mg protein. It was identified as ubiquinone-10 by mass spectrographic analysis. Simultaneous measurements of cytochrome oxidase indicated that these compounds could not be attributed to mitochondrial contamination. These results are compatible with the hypothesis that initiation of the respiratory burst in human neutrophils involves a multicomponent electron-transport system.

Cytochrome b Group↗
PubMed1982

Antibody-dependent phagocytosis of haptenated liposomes by human neutrophils is dependent on the physical state of the liposomal membrane.

In the present study we have examined the response of human neutrophils to specific antibody-dependent stimulation by spin-label haptenated fluid phase and solid phase liposomes. Both fluid and solid liposomal antigens are shown to stimulate the neutrophil respiratory burst to approximately equivalent degrees as assessed by measurement of oxygen consumption or oxidation of [1-14C]glucose to 14CO2. In contrast, release of superoxide and hydrogen peroxide from the neutrophils is stimulated to a significantly greater degree by fluid-phase liposomes than by the equivalent solid-phase liposomes. This apparent discrepancy is shown to be due to an inability of the neutrophils to phagocytose fluid-phase liposomes under conditions in which solid-phase liposomes are readily phagocytosed. A fluorescence assay, which does not depend upon binding measurements, has been developed in order to quantitate liposomal phagocytosis.

Antibodies↗
PubMed1982

H-2Kk and vesicular stomatitis virus G proteins are not extensively associated in reconstituted membranes recognized by T cells.

It is shown that liposomes containing (i) a fluorescein-labeled murine histocompatibility antigen (FITC-H-2Kk) and the G protein of vesicular stomatitis virus or (ii) H-2Kk and fluorescein-labeled viral protein (FITC-G) can elicit H-2-restricted syngeneic antiviral cytotoxic T cells as assayed by 51Cr release from appropriate virus-infected target cells. Fluorescence recovery after photobleaching was used to measure the diffusion coefficients of these reconstituted proteins in four different samples: (i) FITC-H-2Kk; (ii) FITC-H-2Kk and G; (iii) FITC-G; and (iv) FITC-G and H-2Kk. The same rate of lateral diffusion (D = 1 x 10(-8) cm2/sec at 37 degrees C in 25% cholesterol/75% dimyristoylphosphatidylcholine) was obtained in every case. Both proteins, fluorescent as well as nonfluorescent, could be patched by using specific antibodies. When G was patched with antibody, FITC-H-2Kk did not copatch. When H-2Kk was patched with antibody FITC-G did not copatch. These diffusion and patching measurements rule out the possibility that these proteins have either extensive oligomeric associations or strong specific pairwise associations.

Animals↗
PubMed1981

Effects of quercetin on magnesium-dependent adenosine triphosphatase and the metabolism of human polymorphonuclear leukocytes.

The bioflavinoid quercetin was found to exert at least three separate effects on human polymorphonuclear leukocytes. (1) Concentrations of approximately 100 microM inhibited the membrane-associated magnesium adenosine triphosphatase by 60%-80% in either broken cell preparations or intact cells. Lineweaver-Burk plots showed the inhibition to be uncompetitive in nature. (2) Similar concentrations of quercetin inhibited respiratory burst activity of the cells as measured by oxygen consumption, glucose oxidation, or iodination of protein. All inhibitions were dose-dependent and were observed with either opsonized zymosan or phorbol myristate acetate as stimulus. (3) Quercetin likewise inhibited the transport of the nonmetabolizable hexose, 3H-2-deoxyglucose. These observations are most consistent with the hypothesis that quercetin exerts a generalized effect at the level of the cell membrane of the neutrophil.

Adenosine Triphosphatases↗
PubMed1980

Deterioration of rat liver mitochondria under conditions of metabolite deprivation.

In a previous study [Parce, Cunningham & Waite (1978) Biochemistry 17, 1634-1639] changes in mitochondrial phospholipid metabolism and energy-linked functions were monitored as coupled mitochondria were aged in iso-osmotic sucrose solution at 18 degrees C. The sequence of events that occur in mitochondrial deterioration under the above conditions have been established more completely. Total adenine nucleotides are depleted early in the aging process, and their loss parallels the decline in respiratory control. Related to the loss of total adenine nucleotides is a dramatic decrease in ADP and ATP translocation (uptake). The decline of respiratory control is due primarily to a decrease in State-3 respiration; loss of this respiratory activity can be related to the decline in ADP translocation. Mitochondrial ATPase activity does not increase significantly until State-4 respiration has increased appreciably. At the time of loss of respiratory control the ATPase activity increases to equal the uncoupler-stimulated activity. The H+/O ratio and P/O ratios do not decrease appreciably until respiratory control is lost. Similarly, permeability of the membrane to the passive diffusion of protons increases only after respiratory control is lost. There observations reinforce our earlier conclusion that there are two main phases in mitochondrial aging. The first phase is characterized by loss of the ability to translocate adenine nucleotides. The second phase is characterized by a decline in the ability of the mitochondrion to conserve energy (i.e. maintain a respiration-driven proton gradient) and to synthesize ATP.

Adenine Nucleotides↗
PubMed1979

The physical state of membrane lipids modulates the activation of the first component of complement.

Activation of the first component of human complement (C1) by bilayer-embedded nitroxide spin label lipid haptens and specific rabbit antinitroxide antibody has been measured. The nitroxide spin label hapten was contained in host bilayers of either dimyristoyl phosphatidylcholine or dipalmitoyl phosphatidylcholine in the form of both liposomes and vesicles. At a temperature of 32 degrees C, which is intermediate between the hydrocarbon chain-melting temperatures of the two phospholipids, activation of C1 in such vesicles and liposomes is more efficient in the fluid membrane. Studies of C1 activation in binary mixtures of cholesterol and dipalmitoyl phosphatidylcholine indicate that the activation of C1 is not limited by the lateral diffusion of the lipid haptens in these membranes.

Cholesterol↗
PubMed1979

Effect of chronic ethanol administration on energy metabolism and phospholipase A2 activity in rat liver.

1. For a period of 31 days male rats were given a liquid diet containing 36% of its energy as ethanol. Liver mitochondria from these animals demonstrated lowered respiratory control with succinate as substrate, a diminished energy-linked anilinonaphthalene-sulphonic acid fluorescence response, and lowered endogenous ATP concentrations. The phospholipid/protein ratio in mitochondria from these animals was unchanged; only minor alterations in the phospholipid fatty acid composition were observed. 2. In experiments where mitochondria were incubated at 18 degrees C in iso-osmotic sucrose (aging experiments), the above energy-linked properties were lost at an earlier time in organelles from ethanol-fed animals. Phospholipase A2 acitivty was depressed in mitochondria from control animals until respiratory control was lost and ATP was depleted. In contrast, no lag in the expression of phospholipase activity was observed in mitochondria from ethanol-fed rats. This loss of control of the phospholipase resulted in an earlier degradation of membrane phospholipids under the conditions of the aging experiments. 3. The ATPase (adenosine triphosphatase) activities, measured in freshly prepared tightly coupled mitochondria and in organelles uncoupled with carbonyl cyanide p-trifluoromethoxyphenylhydrazone, were not significantly different in ethanol-fed and liquid-diet control animals. When the mitochondria were aged at 18 degrees C, the activity increased with time of incubation in organelles from both groups of animals. A lag was observed, however, as the ATPase activity increased in control preparations. This lag was not present as APTase activity increased in mitochondria from ethanol-fed animals. 4. The significantly lowered values observed for energy-linked functions with succinate as an energy source demonstrate that ethanol elicits an alteration in liver mitochondria that affects the site II-site III regions of the oxidative-phosphorylation system. The apparent lack of control of the phospholipase A2 and ATPase activities in mitochondria from ethanol-fed animals suggests that the membrane microenvironment of these enzymes has been altered such that they can exert their catabolic effects more readily under conditions of mild perturbation. The fatty acid analyses demonstrate that the observed alterations both in the energy-linked functions and in control of the phospholipase and ATPase are not mediated through changes in the acyl chain composition of bulk-phase phospholipids.

Adenosine Triphosphatases↗
PubMed1979

Antibodies bound to lipid haptens in model membranes diffuse as rapidly as the lipids themselves.

A pattern photobleaching method has been used to measure the rates of lateral diffusion of fluorescent-labeled specific anti-nitroxide IgG bound with both combining sites to nitroxide-containing phospholipids in liposomal membranes composed of dimyristoyl phosphatidylcholine at 28 degrees C ("fluid"), dipalmitoyl phosphatidylcholine at 32 degrees C ("Solid"), and dipalmitoyl phosphatidylcholine containing 15 or 25 mol% cholesterol ("solid" or "fluid," respectively, at 32 degrees C). The diffusion coefficients of the bound immunoglobulin were found to be the same as those of fluorescent-labeled phospholipids in each case even though these diffusion coefficients range from 10(-11) to 10(-8) cm2/sec. Hapten-containing liposomal membranes of the type studied here have previously been shown to elicit a number of antibody-dependent immune responses. Therefore, this work indicates that membrane-bound but otherwise freely diffusing antibodies are sufficient for these reponses.

Antigen-Antibody Complex↗
PubMed1978

Mitochondrial phospholipase A2 activity and mitochondrial aging.

The changes in mitochondrial phospholipid metabolism and energy-linked functions have been followed as coupled mitochondria are allowed to age in isotonic sucrose at 18 degrees C. Analysis of the aging process has provided an approach for studying the structure--function relationships within the mitochondrion without adding external agents to perturb the membrane structure. The initial event observed in this process of deterioration is a loss of respiratory control which is paralleled by diminishing levels of ATP. As ATP levels decline, so do the rates of reacylation of monoacyglycerophosphorylethanolamine and fatty acid oxidation. In most cases the previously inactive phospholipase A2 (EC 3.1.1.4, phosphatide-2-acyl-hydrolase) begins rapid hydrolysis of membrane phosphatidylethanolamine as ATP levels approach zero. The final energy-linked phenomenon observed to decline is the anilinonaphthalenesulfonic acid fluorescence response. Evidence is presented which suggests strongly that the activity of the mitochondrial phospholipase A2 on endogenous phospholipids is suppressed in tightly coupled mitochondria. This suppression is temporally linked to ATP levels in the mitochondria. Furthermore, this study demonstrates that mitochondria which are only slightly damaged have the potential to effect membrane repair through reacylation of monoacyl phospholipids.

Adenosine Triphosphate↗
PubMed1978

Specific antibody-dependent binding of complement component C1q to hapten-sensitized lipid vesicles.

The binding of a component of human complement (C1q) to membrane-bound specific anti-nitroxide antibodies was studied as a function of the physical properties (fluid vs. solid) of vesicle lipids. The antibodies were bound to spin-label lipid haptens in the vesicle membrane. The binding of C1q to the sensitized vesicles shows a maximum as a function of specific IgG concentration. The binding of antibodies and of C1q to the vesicle membrane does not depend strongly on the physical state of the membrane lipids. even at low hapten (0.05 mol%) concentrations. These results are of significance for the understanding of the previously reported effect of lipid physical states on complement depletion.

Antibodies↗
PubMed1978

Visualization of specific antibody and C1q binding to hapten-sensitized lipid vesicles.

Specific IgG antibodies directed against the spin-label nitroxide group present as a lipid hapten in single-compartment lipid vesicles have been visualized by using freeze-etch electron microscopy. Individual "particles" with diameters of the order of 20 nm are identified as single IgG molecules bound to lipid hapten. No significant aggregation of these IgG molecules was observed over a period of 1 hr in a dipalmitoyl phosphatidylcholine vesicle at 22 degrees. Binding of the human complement component C1q results in the formation of large (approximately 50-100 nm) asymmetric particles having a partially resolved substructure that may arise from individual IgG molecules bound to the membranes as well as to C1q. The binding of C1q appears to result in a clustering of membrane-bound IgG molecules. Samples containing a serum factor (perhaps anti-IgG antibodies) exhibit some IgG clustering distinct from that produced by Clq.

Complement C1↗