Biomedical subjects

J W Parce

Publications and source records attributed to J W Parce.

At least 37 records · Page 2Linked to original sources

PubMed1990

Biosensors for directly measuring cell affecting agents.

Cellular perfusion chambers have been constructed from the Light Addressable Potentiometric Sensor (LAPS) previously described. The authors have used these chambers to measure the effects of a variety of agents on the metabolic rates of cells. The chambers are used in a stopped flow mode. When flow is on, samples may be introduced to the chamber. When flow is stopped, acidification of the very small volume of medium in the chamber is used to determined the metabolic rate of the cells. Using a variety of types of mammalian cells the authors have demonstrated the following. The triggering of cellular receptors can be determined in minutes. Metabolic inhibition of normal human cells by a test compound can be correlated with the compounds in vivo ocular irritancy. And the efficacy of chemotherapeutic agents on tumor cells exhibiting multidrug resistance can be determined in a few hours.

Biosensing Techniques↗

PubMed1989

Detection of cell-affecting agents with a silicon biosensor.

Cellular metabolism is affected by many factors in a cell's environment. Given a sufficiently sensitive method for measuring cellular metabolic rates, it should be possible to detect a wide variety of chemical and physical stimuli. A biosensor has been constructed in which living cells are confined to a flow chamber in which a potentiometric sensor continually measures the rate of production of acidic metabolites. Exploratory studies demonstrate several applications of the device in basic science and technology.

Biosensing Techniques↗

PubMed1988

Light-addressable potentiometric sensor for biochemical systems.

Numerous biochemical reactions can be measured potentiometrically through changes in pH, redox potential, or transmembrane potential. An alternating photocurrent through an electrolyte-insulator-semiconductor interface provides a highly sensitive means to measure such potential changes. A spatially selectable photoresponse permits the determination of a multiplicity of chemical events with a single semiconductor device.

Electrolytes↗

PubMed1987

Possible mechanisms by which the H-2Kbm3 mutation may decrease cytotoxic T-lymphocyte recognition of vesicular stomatitis virus nucleoprotein antigen.

Spleen cells from C57BL/6 (B6) mice generate a strong in vitro cytotoxic T-lymphocyte (CTL) response specific for vesicular stomatitis virus (VSV). Spleen cells from VSV-primed B6-H-2bm3 (bm3) mice, which have a mutation in H-2Kb, require approximately 10-fold more UV-inactivated VSV to generate in vitro secondary anti-VSV CTL, compared with spleen cells from primed B6 mice. Anti-VSV CTL elicited in both bm3 and B6 mice are primarily specific for the viral nucleocapsid protein (N protein), as demonstrated by using recombinant vaccinia viruses that express the VSV N protein. bm3 CTL were found to exhibit only a very low level of lytic activity when tested against autologous VSV-infected concanavalin A spleen cell blasts as well as several H-2b tumor cell lines. The weak anti-VSV response of bm3 CTL was found to be the result of a combination of inefficient recognition of VSV-infected target cells and decreased elicitation of secondary effector cells. VSV-infected bm3 target cells were not killed as well as B6 targets by either bm3 or B6 effectors. This is because of the inefficient recognition of targets, as demonstrated by the fact that VSV-infected bm3 cells were unable to competitively inhibit the lysis of VSV-infected B6 target cells by either bm3 or B6 effectors. By using cells from recombinant mice, it was shown that the CTL response restricted by H-2Kb was low in the bm3 mice, compared with that of the B6 mice. However, the H-2Db-restricted CTL activity was similarly low in both the B6 and bm3 mice. The possibility that the low response to VSV-infected bm3 cells is caused by differences between the bm3 and B6 cells in expression of either viral antigens or H-2K was investigated by radiolabeling and immunoprecipitation. VSV-infected B6 and bm3 cells were found to express equivalent levels of both viral antigens and H-2K. These results indicate that the bm3 mutation alters a functional site on the H-2Kb molecule that is involved in the recognition of VSV-infected cells. The observation that elicitation of bm3 CTL can occur at high antigen doses further suggests that the bm3 mutation results in a lower affinity of H-2K either for viral antigen or for receptor sites on the CTL.

Animals↗

PubMed1986

Effect of phospholipids on the catalytic subunits of the mitochondrial F0.F1-ATPase.

Beef heart mitochondrial F0.F1-ATPase was reconstituted into phospholipid liposomes using the octylglucoside solubilization, discontinuous sucrose gradient centrifugation procedure described in the preceding manuscript (Laird, D., Smith Eble, K., and Cunningham, C. (1986) J. Biol. Chem. 261, 14844-14850). The influence of individual phospholipids (phosphatidylcholine (PC), phosphatidylethanolamine (PE), and diphosphatidylglycerol (DPG)) on the kinetic parameters related to ATPase activity were investigated. The specific activities for the PC, PE, and DPG reconstituted preparations were 9.8, 6.8, and 7.6 mumol of ATP hydrolyzed per min/mg of protein, respectively. The F0.F1-DPG complex demonstrated a 40% decrease in the Km for ATP. Both the F0.F1-PC and the F0.F1-PE complexes exhibited Ki values for adenyl-5'-yl imidodiphosphate and guanyl-5'-yl imidodiphosphate approximately 2.5 times lower than those obtained in the absence of exogenous phospholipid. The F0.F1-DPG complex displayed Ki values 11.7- and 1.8-fold lower for adenyl-5'-yl imidodiphosphate and guanyl-5'-yl imidodiphosphate, respectively, as compared to the lipid-depleted enzyme. The phospholipids with which F0.F1 were reconstituted also influenced the ATP-induced decrease in the fluorescence of enzyme-associated aurovertin. The rate of the ATP-elicited decrease in aurovertin fluorescence was accelerated in the presence of all three phospholipids with DPG having the most dramatic effect; the t1/2 for maximal decrease in aurovertin fluorescence was 4.3 s for lipid-deficient enzyme and 0.48 s with the F0.F1-DPG complex. The effects of phospholipids on these parameters associated with the catalytic center of the ATPase suggest that phospholipids can modulate catalytic events occurring in F1. In the intact mitochondrion the primary role of phospholipids may be to stabilize conformations of the enzyme consistent with its range of activities.

Animals↗

PubMed1986

Suppression of endocytosis in neutrophils by influenza A virus in vitro.

The effect of influenza A virus on the endocytic pathway in polymorphonuclear leukocytes (PMNLs) and the relationship of altered endocytic activity to virus-induced inhibition of other PMNL functions were examined with virus that caused decreased phagosomelysosome fusion and bacterial killing (depressing virus [DV]) and virus that did not (non-DV). Binding of both viruses to PMNL surface receptors was similar, but uptake of DV into PMNLs was decreased compared with that of non-DV. Both viruses were associated with the PMNL plasma membrane and were in endosomes. DV caused less stimulation of pinocytosis than did non-DV. The rate of exocytosis of fluoresceinated-dextran (FL-dextran) from cells stimulated with DV was significantly less than for non-DV. When PMNLs were pretreated with buffer, DV, or non-DV and then exposed to FL-dextran and N-formylmethionylleucylphenylalanine, the pinocytosis of FL-dextran was significantly less in cells pretreated with DV as compared with non-DV or buffer.

Endocytosis↗

PubMed1985

Labeling of the cytoplasmic domain of the influenza virus hemagglutinin with fluorescein reveals sites of interaction with membrane lipid bilayers.

The hemagglutinin (HA) glycoprotein of influenza virus was labeled in its cytoplasmic domain with fluorescein. Reactive amino groups in the external domain were blocked by modification of the intact virus with the membrane-impermeable reagent isethionyl acetimidate. The HA was then solubilized with the detergent octyl glucoside, and the single lysine in the cytoplasmic domain was reacted with fluorescein isothiocyanate. This protocol resulted in the incorporation of 1.3 mol of fluorescein/mol of HA. Using a virus strain lacking lysine in the cytoplasmic domain of HA, it was determined that 0.47 mol of fluorescein/mol of HA was located at an additional site(s). The fluorescein groups at both sites exist in an environment of reduced polarity as shown by a shift in excitation and emission maxima and a shift in the pKa of the fluorescein groups. The fluorescence polarization and the pKa of the fluorescein groups were greater when the HA was incorporated into liposomes than when in detergent solution. These data indicate that the fluorescein groups interact directly with the lipid bilayer, probably in the phospholipid head-group region. The fluorescence properties of the labeled HA were not responsive to the gel to liquid-crystal phase transition in the lipid bilayer. These results indicate that the boundary between the cytoplasmic domain and the hydrophobic sequence that anchors the protein to the lipid bilayer is located in the head-group region of the bilayer.

Electrophoresis, Polyacrylamide Gel↗

PubMed1985

Independent stimulation of membrane potential changes and the oxidative metabolic burst in polymorphonuclear leukocytes.

Early events of stimulus-response coupling in polymorphonuclear leukocytes (PMNL) reportedly include membrane depolarization as a necessary antecedent to oxidative responses. However, depolarization by nonspecific means (ionophores) is insufficient to elicit an oxidative burst. This apparent conflict might be related to whether depolarization is due to membrane receptor-mediated stimulation of PMNL. We used two fluorescent probes and dual laser flow cytometry to monitor both membrane potential and oxidative product formation in individual PMNL, following stimulation by phorbol myristate acetate (PMA) or formylmethionyl-leucyl-phenylalanine (fMLP). Dipentylindocarbocyanine [di-I-C5(3)] is one of a family of dyes that partition between cells and aqueous media as a function of transmembrane potential. The dye appeared stable in the presence of PMNL oxidative products. Oxidation of intracellularly trapped, nonfluorescent dichlorofluorescin (DCFH) to fluorescent dichlorofluorescein (DCF) provided a quantitative assessment of oxidative metabolism (H2O2 production) of stimulated PMNL. Incubating PMNL with both fluorochromes resulted in stable red [di-l-C5(3)] and green (DCF) fluorescence when examined on a Cytofluorograph. Upon stimulation by 0.03 to 0.1 nmol/L PMA, PMNL showed a unimodal apparent depolarization (decrease in di-l-C5(3) fluorescence). Oxidative activity (increased DCF fluorescence) was first seen at a concentration of PMA of 0.17 nmol/L, higher than that required to elicit depolarization. This oxidative burst appeared as a dose-dependent, graded response. Thus, receptor-mediated membrane depolarization, although antecedent to the onset of the oxidative burst, was not in itself sufficient to trigger the oxidative metabolic response. When PMNL were isolated by centrifugation through Ficoll-metrizoate, fMLP caused an apparent depolarization of a variable subpopulation of the cells. However, such purified PMNL appeared relatively unstable and often depolarized spontaneously. PMNL studied without centrifugation through Ficoll-metrizoate were stable. Moreover, fMLP stimulation of such cells did not cause membrane depolarization but did stimulate a two- to six-fold increase in DCFH oxidation. Apparently, membrane depolarization may antecede oxidative responses in PMNL, but appears to depend upon the method of cell preparation and the nature of the stimulus.

Carbocyanines↗

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

Specificity of lipoprotein lipase and hepatic lipase toward monoacylglycerols varying in the acyl composition.

We report here that both the hepatic lipase and lipoprotein lipase demonstrate specificity towards the acyl group present on monoacylglycerols. We found that unsaturated glycerides are more readily degraded than saturated glycerides. However, the basis for this specificity appears to be different for each enzyme. The activity of the hepatic lipase, but not the lipoprotein lipase, could be stimulated by Triton X-100 and phosphoglycerides. We interpret these results to show that while both the lipoprotein lipase and hepatic lipase are sensitive to the physical state of the substrate (as shown by fluorescence depolarization), the lipoprotein lipase also has a low affinity for monoacylglycerols that contain a saturated acyl group. In the course of this study we also obtained evidence that some type of phase separation occurs when mixtures of saturated and unsaturated monoacylglycerols are prepared.

Animals↗

PubMed1981

Pattern photobleaching of fluorescent lipid vesicles using polarized laser light.

A burst of linearly polarized laser radiation incident on a spherical lipid vesicle, liposome, or biological cell can produce a well-defined nonuniform distribution of membrane-bound fluorescent molecules, provided the absorption transition dipole moment of the fluorescent label has a nonrandom orientation relative to the membrane surface and can be photobleached by the laser radiation. The return (recovery) of fluorescent membrane-bound molecules to a uniform distribution can be monitored using the same polarized radiation source. Under appropriate conditions this recovery is characterized by a single exponential time constant tau. This time constant is related to the radius R of the vesicle and the lateral diffusion coefficient D of the fluorescent membrane-bound molecules by the equation R2 = 6D tau. In the case of vesicle membranes this result is not limited by diffraction and so should be applicable to vesicles whose radii are less than the wavelength of light. The above considerations are illustrated by the polarized light photobleaching-recovery of lipid vesicles containing a fluorescent lipid, N-4-nitro-benzo-2-oxa,1,3-diazole l-alpha-dimyristoylphosphatidylethanolamine (NBD-DMPE).

Diffusion↗