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Biomedical subjects

O T Jones

Publications and source records attributed to O T Jones.

At least 37 records · Page 2Linked to original sources
PubMed1996

Expression of phagocyte NADPH oxidase components in human endothelial cells.

Low-level generation of reactive oxygen species (ROS) by endothelial cells in response to a variety of stimuli has been observed; however, the enzyme system responsible is unknown. Using a variety of techniques, we examined for components of the phagocyte superoxide-generating NADPH oxidase to elucidate whether this enzyme could be a source of endothelial-derived ROS. Superoxide generation on addition of 100 microM NAD(P)H to human umbilical vein endothelial cell (HUVEC) sonicates (using lucigenin-enhanced chemiluminescence) was partially inhibited on addition of the flavoenzyme inhibitor diphenyliodonium (IDP). Reverse transcriptase-polymerase chain reaction (RT-PCR) demonstrated expression of gp91phox, p22phox, p67phox, and p47phox in four independent HUVEC isolates. Expression of p22phox was also confirmed by Northern blotting. RT-PCR for tumor necrosis factor-alpha was negative, indicating an absence of mononuclear cell contamination (a potential source of NADPH oxidase). Immunoperoxidase staining, using anti-p47phox (JW-1)- and anti-p67phox (JW-2)-specific antibodies, showed protein expression of these cytosolic components. However, heme spectroscopy failed to indicate the presence of the low-potential cytochrome b558. These data indicate that cultured human endothelial cells express both mRNA and protein for cytosolic components of the phagocyte superoxide-generating NADPH oxidase. However, because the cytochrome b558 heme could not be conclusively demonstrated, a contribution of the phagocyte NADPH oxidase to endothelial oxidant generation may be unlikely.

Cells, Cultured↗
PubMed1995

Long-term bFGF neuronal culture: reintroduction into serum medium yields neurons and non-neuronal cells with neuronal characteristics.

The potential use of bFGF immortalized cells as hosts for delivering foreign genes into nervous tissue led us to examine the effect of maintaining, E-18 hippocampal neurons for extended periods in bFGF culture prior to transfer into a standard, serum-containing, medium. We found: (1) many, if not most, precursors seen in bFGF, mature into glia and not into primary neurons after medium exchange; (2) the electrophysiology of the neurons which do mature after medium transfer and replating, is similar to that of neurons in standard cultures; (3) extended culture in bFGF prior to cell harvesting and replating into standard medium generates neurons from the precursors that possess proper neuronal polarization, morphology, and electrophysiology; and (4) extended bFGF also induces the expression, on transfer into standard medium, of an additional cell type with a distinct non-neuronal morphology that stains with the neuronal marker MAP-2. These results illustrate the need for additional characterization of long-term growth factor effects on maintained progenitor cells prior to their use in gene therapy and transplantation.

Action Potentials↗
PubMed1995

The expression of NADPH oxidase components in human glomerular mesangial cells: detection of protein and mRNA for p47phox, p67phox, and p22phox.

Previous work has shown that human mesangial cells (HMC) are capable of low rates of generation of reactive oxygen species for considerable periods of time. In this communication, the presence of components of an NADPH oxidase-like system, more commonly associated with phagocytic leukocytes, is shown. The ability of HMC to generate low levels of superoxide may have important implications in cellular signaling in general and may contribute to glomerular injury. Spectroscopic analysis of HMC membranes revealed a low-potential cytochrome b component, redox midpoint potential centered around -250 mV, which is present at 60 pmol/mg of membrane protein. Immunodetection studies suggested the presence of the p22phox, p47phox, and p67phox components of the NADPH oxidase, whereas the gp91phox was not detected. Further studies with oligonucleotide polymerase chain reaction primers showed that, in HMC the mRNA expression of the p67phox and p47phox was absent from growth-arrested cells but was present in HMC treated with interleukin-1 beta (1,000 pg/mL), whereas gp91phox could not be detected. Only mRNA corresponding to p22phox was present in growth-arrested cells; p47phox mRNA was induced by 2-h treatment with interleukin-1 beta but declined after 6-h treatment. These data illustrate for the first time that HMC are capable of expressing mRNA for several NADPH oxidase components. The apparent absence, or variation, of the gp91phox indicates the likelihood of an NADPH oxidase isoenzyme.

Base Sequence↗
PubMed1994

The functional expression of p47-phox and p67-phox may contribute to the generation of superoxide by an NADPH oxidase-like system in human fibroblasts.

Recent evidence suggests that a number of non-phagocytic cell types may contain a superoxide generating NADPH oxidase. Studies to data on cultured human fibroblasts have primarily concerned the identification of cytochrome b558, whilst expression of other NADPH oxidase components have not been addressed. In this study we have investigated the expression of NADPH oxidase with particular reference to the cytosolic factors p47-phox and p67-phox. Reverse transcriptase-polymerase chain reaction (RT-PCR) showed that human fibroblasts express mRNA for p47-phox, p67-phox and p22-phox. Expression of the gp91-phox transcript was not detected, indicating that human fibroblasts may possess an NADPH oxidase isoenzyme. Western blot analysis of human fibroblast cytosol, using an anti-p47-phox antibody (JW-1), identified a 47 kDa protein. Cell-free reconstitution assays showed that fibroblast cytosol could initiate superoxide generation when mixed with either human fibroblast membranes (0.16 nmol superoxide/min/microgram membrane protein), or resting human neutrophil membranes (0.20 nmol superoxide/min/microgram membrane protein). These data indicate that the expression of p47-phox and p67-phox by human fibroblasts may contribute to the cells' generation of superoxide.

Base Sequence↗
PubMed1994

The regulation of superoxide production by the NADPH oxidase of neutrophils and other mammalian cells.

Superoxide is produced by a NADPH oxidase of phagocytic cells and contributes to their microbicidal activities. The oxidase is activated when receptors in the neutrophil plasma membrane bind to the target microbe. These receptors recognise antibodies and complement fragments which coat the target cell. The oxidase electron transport chain, located in the plasma membrane, comprises a low potential cytochrome b heterodimer (gp 91-phox and p22-phox) associated with FAD. It is non-functional until at least three proteins, p67-phox, p47-phox and p21rac (and possibly others), move from the cytosol to dock on the cytochrome b. The docking involves the interaction of SH3 domains on p47-phox or p67-phox with a proline-rich sequence on the small subunit of the cytochrome b. These SH3 domains may become exposed following phosphorylation of p47-phox by protein kinase C or, in model systems, by addition of arachidonic acid to reconstitution mixtures. Following the docking process the electron-transporting component is able to transfer electrons from NADPH to oxygen. This electrogenic event is charge-compensated by the opening of a proton channel. Components of the oxidase are expressed in non-phagocytes, where their function is uncertain but could be related to some signal function of superoxide.

Animals↗
PubMed1994

N-type Ca2+ channels are located on somata, dendrites, and a subpopulation of dendritic spines on live hippocampal pyramidal neurons.

In the nervous system the influx of Ca2+ orchestrates multiple biochemical and electrical events essential for development and function. A major route for Ca2+ entry is through voltage-dependent calcium channels (VDCCs). It is becoming increasingly clear that the precise contribution VDCCs make to neuronal function depends not only upon their specific electrophysiological properties but also on their distribution over the nerve cell surface. One location where the presence of VDCCs may be critical is the dendritic spine, a structure known to be the major site of excitatory synaptic input. On spines, VDCCs are hypothesized to play an essential role in signal processing, learning, and memory. However, direct evidence for the presence of VDCCs on spines is lacking. Attempts to examine the distribution of VDCCs, or indeed any other components, on spines have been hampered since the size of many spines is close to the limits of resolution of conventional light microscopy. Using a new, biologically active, fluorescein conjugate of omega-conotoxin (Fl-omega-CgTx), a selective blocker of N-type VDCCs, and confocal microscopy, we have mapped the distributions of N-type VDCCs on live CA1 neurons in rat hippocampal slices. VDCCs were found on somata, throughout the dendritic arbor, and on dendritic spines in all hippocampal subfields. A comparison of three-dimensional reconstructions of structures labeled by Fl-omega-CgTx with those outlined by 1,1-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine (Dil) or Lucifer yellow confirmed the presence of N-type VDCCs on dendritic spines. However, spine frequency on dendrites labeled with Fl-omega-CgTx was much lower than the spine frequency on dendrites labeled with Lucifer yellow or Dil, suggesting that some spines lack N-type VDCCs. These results offer the first direct evidence for the localization of any voltage-dependent channel on dendritic spines. The presence of N-type VDCCs on dendrites and their spines argues that these channels may participate in the generation of active Ca2+ conductances in distal dendrites, and is consistent with a role for spines as specialized compartments for concentrating Ca2+.

Animals↗
PubMed1994

Involvement of phenyl radicals in iodonium inhibition of flavoenzymes.

Iodonium inhibition of the flavoenzymes neutrophil NADPH oxidase and cytochrome P450 reductase has been suggested to require reductive metabolism of the inhibitor to a phenyl radical. Inhibition would ultimately result from covalent attachment of phenyl radicals to either the flavin cofactor or adjacent amino acid side chains important in catalysis. In this paper we provide evidence, using EPR techniques, that phenyl radicals are formed during reaction of iodonium diphenyl with reduced free flavin (FMN) and protein-bound (cytochrome P450 reductase or xanthine oxidase) flavin. Kinetic analysis indicated iodonium diphenyl to be an uncompetitive inhibitor of xanthine oxidase, suggesting the need for reduced enzyme for inhibition. A study of the catalytic and structural properties of different flavoenzymes suggested that only enzymes containing flavins that function in one-electron transfer are targets for iodonium inhibition.

Animals↗
PubMed1993

N-Methyl-D-aspartate receptors are clustered and immobilized on dendrites of living cortical neurons.

The response of nerve cells to synaptic inputs and the propagation of this activation is critically dependent on the cell-surface distribution of ion channels. In the hippocampus, Ca2+ influx through N-methyl-D-aspartate receptors (NMDAR) and/or voltage-dependent calcium channels on dendrites is thought to be critically involved in long-term potentiation, neurite outgrowth, epileptogenesis, synaptogenesis, and cell death. We report that conantokin-G (CntxG), a peptide from Conus geographus venom, competitively blocked with high affinity and specificity NMDAR-mediated currents in hippocampal neurons and is a reliable probe for exploring NMDAR distribution. Fluorescent derivatives of CntxG were prepared and used to directly determine NMDAR distribution on living hippocampal neurons by digital imaging and confocal fluorescence microscopy. In hippocampal slices, the CA1 dendritic subfield was strongly labeled by CntxG, whereas the CA3 mossy fiber region was not. On CA1 hippocampal neurons in culture, dendritic CntkG-sensitive NMDAR were clustered at sites of synaptic contacts, whereas somatic NMDAR were distributed diffusely and in patches. NMDAR distribution differed from the distribution of voltage-dependent calcium channels. A significant fraction of labeled NMDAR on somata and dendrites was found to be highly mobile: rates were consistent with the possible rapid recruitment of NMDAR to specific synaptic locations. The localization of NMDAR and modulation of this distribution demonstrated here may have important implications for the events that underlie neuronal processing and synaptic remodeling during associative synaptic modification.

2-Amino-5-phosphonovalerate↗
PubMed1993

Photometric characteristics of haem proteins in erythropoietin-producing hepatoma cells (HepG2).

Erythropoietin (Epo)-producing hepatoma cells (HepG2) reveal, in addition to the cytochromes of the respiratory chain, a photometrically measurable haem signal with absorbance maxima at 559 nm and 427 nm, suggesting the presence of a b-type cytochrome. This activity exhibited a low midpoint potential, CO-binding spectra and reduction which was insensitive to both cyanide and antimycin. This haem possessed a 22 kDa subunit and might be part of an electron transfer chain similar to the NADPH oxidase, since the NADPH oxidase cytosolic activating factor (p47) could be identified by Western blot analysis. H2O2, which was detected inside the cells by confocal microscopy, might therefore be produced by the suggested electron transfer chain. This cyanide- and antimycin-insensitive but hypoxia-sensitive cytochrome b would be an attractive candidate for controlled Epo production in response to pO2.

Blotting, Western↗
PubMed1993

Studies on the inhibitory mechanism of iodonium compounds with special reference to neutrophil NADPH oxidase.

Diphenyleneiodonium (DPI) and its analogues have been previously shown to react via a radical mechanism whereby an electron is abstracted from a nucleophile to form a radical, which then adds back to the nucleophile to form covalent adducts [Banks (1966) Chem. Rev. 66, 243-266]. We propose that the inhibition of neutrophil NADPH oxidase by DPI occurs via a similar mechanism. A reduced redox centre in the oxidase could serve as electron donor to DPI, and inhibition would occur after direct phenylation of the redox cofactor, or of adjacent amino acid groups by the DPI radical. In the absence of an activatory stimulus, human neutrophil NADPH-oxidase was not inhibited by DPI. The Ki for time-dependent inhibition by DPI of human neutrophil membrane NADPH oxidase was found to be 5.6 microM. Inhibitory potency of DPI was shown to be directly related to rate of enzyme turnover, indicating the need for a reduced redox centre. Adducts were formed between photoreduced flavin (FAD or FMN) and inhibitor (DPI or diphenyliodonium). These were separated by h.p.l.c. and characterized by absorbance spectroscopy, 1H-n.m.r. and fast-atom-bombardment m.s. and found to have properties consistent with substituted 4a,5-dihydroflavins. After incubation of pig neutrophil membranes with DPI, the quantity of recoverable intact flavin was greatly diminished when NADPH was present to initiate oxidase turnover, indicating that the flavin may be the site of DPI activation. These results may provide a common mechanism of action for iodonium compounds as inhibitors of other flavoenzymes.

Animals↗
PubMed1993

Preparation and characterization of biotinylated analogs of the calcium channel blocker omega-conotoxin.

We have prepared a series of biotinylated analogs of omega-conotoxin (omega CgTx) as potent, selective markers for N-type calcium channels. At pH 9.5, reaction of omega CgTx with amidocaproylbiotin succinimidyl ester gives three biotinylated conjugates, labeled at lysines 2 or 24, or at both positions. Kinetic competition assays of 125I-omega CgTx binding to rat brain synaptic membranes show that each conjugate has a similar rate constant for association (1-1.3 x 10(6) M-1 s-1) but not dissociation (1-4 x 10(-4) s-1). Comparison with rate constants obtained for the association (1.2 x 10(7) M-1 s-1) and dissociation (5 x 10(-5) s-1) of native omega CgTx indicates that while biotinylation reduces omega CgTx potency (Kdkin = k-2/k2 = 4 pM for omega CgTx), binding of these labels to membranes is nevertheless of very high affinity (Kdkin 0.1-0.3 nM).

Amino Acid Sequence↗
PubMed1991

Functional expression of NADPH oxidase components (alpha- and beta-subunits of cytochrome b558 and 45-kDa flavoprotein) by intrinsic human glomerular mesangial cells.

Recently, we have shown that human glomerular mesangial cells (HMCs) release oxygen radicals from the plasma membrane in response to cytokines. Now we have used diphenylene iodonium, a covalent binding inhibitor of activated 45-kDa flavoprotein, in neutrophils radiolabeled with 125I and could identify a 45-kDa protein band in a separated HMC plasma membrane fraction. Low temperature difference spectroscopy showed a peak absorbance at 428 and 558 nm. Direct potentiometry of HMC membranes (-340 to -160 mV) showed the presence of a low potential cytochrome (76 pmol/mg to HMC membrane protein) identified as cytochrome b558. In slot blots, mouse monoclonal antibody (mAb) 7D5, specific for the extracellular domain of the alpha-subunit, showed a positive reaction with HMCs. In Western blots, mAb 449, directed against the cytoplasmic epitope of the alpha-subunit, identified a 23-kDa protein; and mAb 48, raised against the large (beta) subunit of cytochrome b558 of human neutrophils (Verhoeven, A. J., Bolscher, B. G. J. M., Meerhof, L. J., van Zwieten, R., Keijer, J., Weening, R. S., and Roos, D. (1989) Blood 73, 1686-1694), detected a smear between 75 and 100 kDa in denatured HMC membrane protein. These data determined with HMCs, suggest for the first time the expression of three essential components of NADPH:O2- oxidoreductase in mesenchymal cells.

Antibodies, Monoclonal↗
PubMed1991

Identification of a superoxide-generating NADPH oxidase system in human fibroblasts.

Human fibroblasts have the capacity to release superoxide radicals upon stimulation of an electron transport system similar to the NADPH oxidase of leukocytes. Two components of the NADPH oxidase system, (1) a flavoprotein of 45 kDa which binds diphenylene iodonium (a compound described as a specific inhibitor of the leukocyte NADPH oxidase), and (2) a low-potential cytochrome b, are present in fibroblast membranes. Fibroblasts exhibit these compounds at lower concentrations than do polymorphonuclear leukocytes or B-lymphocytes. The superoxide-generating system is rather uniformly associated with the outer cell membrane, as shown by light and electron microscopy. Superoxide release upon stimulation with various agents was prevented by the addition of micromolar concentrations of diphenylene iodonium, making an NADPH oxidase a likely source.

Calcimycin↗
PubMed1991

The use of diphenylene iodonium and its analogues to investigate the role of the NADPH oxidase in the tumoricidal activity of macrophages in vitro.

Lipopolysaccharide (LPS) was shown to induce tumoricidal activity in peritoneal macrophages. The optimal concentration was found to be 25 micrograms/mL. Approximately 20-h exposure to LPS was required before maximal tumor cell killing was attained. Optimal tumor killing was obtained with a ratio of tumor cell to macrophages of 1:40 with the macrophages in a confluent layer. Diphenylene iodonium (DPI) reduced the tumor cell killing in a dose dependent manner up to 1 microM. Under similar conditions DPI was shown to inhibit the superoxide production of macrophages and this supports the view that the production of oxygen radicals is important in the killing of tumor cells by macrophages and that the inhibitor DPI can be used to investigate their contribution to cytotoxicity.

Animals↗