Biomedical subjects

Matthew Whiteman

Publications and source records attributed to Matthew Whiteman.

At least 37 records · Page 2Linked to original sources

PubMed2005

Mercaptoethylguanidine inhibition of inducible nitric oxide synthase and cyclooxygenase-2 expressions induced in rats after fluid-percussion brain injury.

The present study examined the temporal expression of nitric oxide synthase (iNOS) and cyclo-oxygenase (COX)-2 in rat brains after traumatic brain injury (TBI). We studied the effects of mercaptoethylguanidine (MEG), a dual inhibitor of the inducible iNOS and COX with scavenging effect on peroxynitrite, on physiologic variables, brain pathogenesis, and neurologic performance in rats after a lateral fluid percussive-induced TBI. Mean arterial blood pressure and percentage cerebral tissue perfusion in MEG-treated TBI rats showed significant improvement when compared with TBI rats. Immunohistochemical analysis showed a marked number of iNOS and COX-2 immunopositive cells in the cerebral cortex ipsilateral to the injury in TBI rats when compared with MEG-treated TBI rats. MEG also significantly decreased the number of hyperchromatic and shrunken cortical neurons when compared with TBI rats' brain nitrate/nitrite, and prostaglandin E2 levels were attenuated in MEG-treated TBI rats when compared with TBI rats. It is therefore suggested that treatment of MEG via inhibition of iNOS and COX-2 might contribute to improved physiologic variables, neuronal cell survival, and neurologic outcome after TBI.

Animals↗

PubMed2005

Oxidative damage in mitochondrial DNA is not extensive.

Since 1988 several research groups have reported greater levels of oxidative damage in mitochondrial DNA than in nuclear DNA, while others have suggested that the greater damage in mtDNA might be due to artifactual oxidation. The popular theory that mtDNA is more heavily damaged in vivo than nDNA does not stand on firm ground. Using an improved GC-MS method and pure mtDNA, our analyses revealed that the damage level in mtDNA is not higher, and may be somewhat lower, than that in nDNA.

Animals↗

PubMed2004

Cytochrome bc(1) regulates the mitochondrial permeability transition by two distinct pathways.

The mitochondrial permeability transition (MPT) pore is a calcium-sensitive channel in the mitochondrial inner membrane that plays a crucial role in cell death. Here we show that cytochrome bc(1) regulates the MPT in isolated rat liver mitochondria and in CEM and HL60 cells by two independent pathways. Glutathione depletion activated the MPT via increased production of reactive oxygen species (ROS) generated by cytochrome bc(1). The ROS producing mechanism in cytochrome bc(1) involves movement of the "Rieske" iron-sulfur protein subunit of the enzyme complex, because inhibition of cytochrome bc(1) by pharmacologically blocking iron-sulfur protein movement completely abolished ROS production, MPT activation, and cell death. The classical inhibitor of the MPT, cyclosporine A, had no protective effect against MPT activation. In contrast, the calcium-activated, cyclosporine A-regulated MPT in rat liver mitochondria was also blocked with inhibitors of cytochrome bc(1). These results indicate that electron flux through cytochrome bc(1) regulates two distinct pathways to the MPT, one unregulated and involving mitochondrial ROS and the other regulated and activated by calcium.

Animals↗

PubMed2004

Peroxynitrite mediates calcium-dependent mitochondrial dysfunction and cell death via activation of calpains.

Chondrocyte cell death is a hallmark of inflammatory and degenerative joint diseases such as rheumatoid arthritis (RA) and osteoarthritis (OA), but the molecular and cellular mechanisms involved have yet to be elucidated. Because 3-nitrotyrosine, a marker for reactive nitrogen species such as peroxynitrite, has been observed in OA and RA cartilage and has been associated with chondrocyte cell death, we investigated the mechanisms by which peroxynitrite induces cell death in human articular chondrocytes. The earliest biochemical event observed, subsequent to treatment with either peroxynitrite or the peroxynitrite generator SIN-1, was a rapid rise in intracellular calcium that lead to mitochondrial dysfunction and cell death. Although, chondrocyte death exhibited several classical hallmarks of apoptosis, including annexin V labeling, increased fraction of cells with subG1 DNA content and DNA condensation, we did not find evidence for caspase involvement either by Western blotting, fluorimetric assays, or caspase inhibition. Additionally, peroxynitrite did not inhibit cellular caspase activity. Furthermore, using other established assays of cell viability, including the MTT assay and release of lactate dehydrogenase, we found that the predominant mode of cell death involved calcium-dependent cysteine proteases, otherwise known as calpains. Our data show, for the first time, that peroxynitrite induces mitochondrial dysfunction in cells via a calcium-dependent process that leads to caspase-independent apoptosis mediated by calpains.

Calcium↗

PubMed2004

Characterization of antioxidant and antiglycation properties and isolation of active ingredients from traditional chinese medicines.

There is considerable interest in the isolation of more potent antioxidant compounds to treat diseases involving oxidative stress. Thirty-three traditional Chinese medicine (TCM) extracts were examined for their antioxidant activity using the 2,2'-azinobis[3-ethylbenzothiazoline-6-sulfonate] (ABTS) assay. Five extracts with high activity (Cratoxylum cochinchinense, Cortex magnoliae officinalis, Psoralea corylifolia L, Curculigo orchioides Gaertn, and Glycyrrhiza uralensis Fisch.) were selected for further characterization. C. cochinchinense outperformed other extracts in most of the assays tested except phospholipid peroxidation inhibition, where P. corylifolia L showed higher activity. C. cochinchinense was particularly potent in inhibiting the formation of advanced glycation end products on proteins and strongly inhibited hypochlorous acid-induced DNA damage. We attempted to isolate the active ingredients from C. cochinchinense and obtained an extract (YCT) containing at least 90% mangiferin as identified by HPLC and mass spectrometry. However, YCT showed significantly higher activity in assays of phospholipid peroxidation, inhibition of protein glycation, and superoxide (O(2)(?-)) and peroxynitrite (ONOO(-)) scavenging, as compared with mangiferin, suggesting that the nonmangiferin constituents of YCT contribute to its additional antioxidant activities.

Antioxidants↗

PubMed2004

Mechanism of cell death induced by an antioxidant extract of Cratoxylum cochinchinense (YCT) in Jurkat T cells: the role of reactive oxygen species and calcium.

YCT is a semipurified extract from Cratoxylum cochinchinense that has antioxidant properties and contains mostly mangiferin. We show here that YCT is selectively toxic to certain cell types and investigate the mechanisms of this toxicity in Jurkat T cells. By flow cytometric analyses, we show that YCT causes intense oxidative stress and a rise in cytosolic Ca(2+). This is followed by a rise in mitochondrial Ca(2+), release of cytochrome c, collapse of Deltapsi(m), a fall in ATP levels, and eventually cell death. The mechanism(s) of intense oxidative stress may involve a plasma membrane redox system, as cell death is inhibited by potassium ferricyanide. Cell death has some features of apoptosis (propidium iodide staining, externalization of phosphatidylserine, limited caspase-3 and -9 activities), but there was no internucleosomal DNA fragmentation.

Adenosine Triphosphate↗

PubMed2004

A high-throughput and sensitive methodology for the quantification of urinary 8-hydroxy-2'-deoxyguanosine: measurement with gas chromatography-mass spectrometry after single solid-phase extraction.

8-hydroxy-2'-deoxyguanosine (8OHdG) is a widely used biomarker for the measurement of endogenous oxidative DNA damage. A sensitive method for the quantification of 8OHdG in urine by single solid-phase extraction and GC-MS (gas chromatography with MS detection) using selective ion monitoring is described in the present study. After solid-phase extraction, samples are freeze-dried, derivatized by trimethylsilylation and analysed by GC-MS. The urinary 8OHdG was quantified using heavy isotope dilution with [18O]8OHdG. The recovery of 8OHdG after the solid-phase extraction ranged from 70 to 80% for a wide range of urinary 8OHdG levels. Using 1 ml of urine, the limit of quantification was >2.5 nM (2.5 pmol/ml) and the calibration curve was linear in the range 2.5-200 nM. This method was applied to measure 8OHdG in urine samples from 12 healthy subjects. The intra- and inter-day variations were <9%. Urinary 8OHdG levels in spot urine samples from four healthy subjects were also measured for 1 week and, again, the variation was small. The presence of H2O2 in urine did not cause artifactual formation of 8OHdG. Since this assay is simple, rapid, sensitive and reproducible, it seems suitable to be used as a routine methodology for the measurement of urinary excretion of 8OHdG in large population studies.

8-Hydroxy-2'-Deoxyguanosine↗

PubMed2004

Chronic exposure to U18666A induces apoptosis in cultured murine cortical neurons.

Niemann-Pick disease type C (NPC) is a juvenile neurodegenerative disorder characterized by premature neuronal loss and altered cholesterol metabolism. Previous reports applying an 8-h exposure of U18666A, a cholesterol transport-inhibiting agent, demonstrated a dose-dependent reduction in beta-amyloid (Abeta) deposition and secretion in cortical neurons, with no significant cell injury. In the current study, we examined the chronic effect of 24-72h of U18666A treatment on primary cortical neurons and several cell lines. Our results showed caspase-3 activation and cellular injury in U18666A-treated cortical neurons but not in the cell lines, suggesting cell death by apoptosis only occurred in cortical neurons after chronic exposure to U18666A. We also demonstrated through filipin staining the accumulation of intracellular cholesterol in cortical neurons treated with U18666A, indicating the phenotypic mimic of NPC by U18666A. However, additions of 10 and 25microM pravastatin with 0.5microg/ml U18666A significantly attenuated toxicity. Taken together, these data showed for the first time that U18666A induces cell death by apoptosis and suggested an important in vitro model system to study NPC.

Androstenes↗

PubMed2004

The redox regulation of intermediary metabolism by a superoxide-aconitase rheostat.

In this article, we discuss a hypothesis to explain the preferential synthesis of the superoxide sensitive form of aconitase in mitochondria and the phenotype observed in manganese superoxide dismutase mutant mice, which show a gross over accumulation of stored fat in liver. The model proposes that intermediary metabolism is redox regulated by mitochondrial superoxide generated during mitochondrial respiration. This regulates the level of reducing equivalents (NADH) entering the electron transport chain (ETC) through the reversible inactivation of mitochondrial aconitase. This control mechanism has a dual function; firstly, it regulates levels of superoxide generated by the ETC and, secondly, it fine-tunes metabolism by channeling citrate either for the production of NADH for energy metabolism or diverting it for the synthesis of fats. In this setting, the mitochondrial redox state influences metabolic decisions via a superoxide-aconitase rheostat.

Aconitate Hydratase↗

PubMed2004

Prion protein is ubiquitinated after developing protease resistance in the brains of scrapie-infected mice.

Although the key event in the pathology of prion diseases is thought to be the conversion of cellular prion protein (PrP(C)) to the protease-resistant scrapie species termed PrP(Sc), the factors that contribute to neurodegeneration in scrapie-infected animals are poorly understood. One probable determinant could be when the accumulation of PrP(Sc) in infected brain overwhelms the ubiquitin-proteasome system and triggers the degenerative cascade. In the present study, it was found that in mouse brains infected with the ME7 scrapie strain, the level of ubiquitin protein conjugates increased significantly at approximately 144 days post-infection (pi) when clinical signs first become apparent. This elevation correlated with the detection of protease-resistant PrP(Sc) and a decline in two endopeptidase activities associated with proteasome function. However, ubiquitination of PrP was only detected at the terminal stage, 3 weeks after the development of clinical symptoms (approximately 165 days pi). These results suggest that ubiquitination of PrP is a late event phenomenon and this conjugation occurs after the formation of protease-resistant PrP(Sc). Whether this post-translational modification and the impairment of proteasome function are pivotal events in the pathogenesis of prion diseases remains to be determined.

Animals↗

PubMed2004

Increased iron staining in the cerebral cortex of cholesterol fed rabbits.

The link between hypercholesterolemia and neuronal damage is not clear. In the present study, we studied some of the possible effects of hypercholesterolemia on the brain, using the cholesterol fed New Zealand White rabbit as a model. An increase in the number of iron positive cells (i.e. oligodendrocytes) was observed in the brain parenchyma, in rabbits treated with a high cholesterol diet for 8 weeks. At this time, no neuronal death was observed, indicating that the increased iron did not occur as a consequence of neuronal injury. No heme oxygenase-1 (HO-1) or bilirubin immunoreactivity was observed in the brains in these rabbits, indicating that the iron accumulation did not occur as a consequence of increased breakdown of heme. It is postulated that cholesterol could have subtly damaged brain endothelial cells, resulting in increased iron transport across brain endothelial cells. Hypercholesterolaemia is known to be associated with increased plasma lipid peroxidation which might contribute to such damage.

Animals↗

PubMed2004

Measuring reactive species and oxidative damage in vivo and in cell culture: how should you do it and what do the results mean?

Free radicals and other reactive species (RS) are thought to play an important role in many human diseases. Establishing their precise role requires the ability to measure them and the oxidative damage that they cause. This article first reviews what is meant by the terms free radical, RS, antioxidant, oxidative damage and oxidative stress. It then critically examines methods used to trap RS, including spin trapping and aromatic hydroxylation, with a particular emphasis on those methods applicable to human studies. Methods used to measure oxidative damage to DNA, lipids and proteins and methods used to detect RS in cell culture, especially the various fluorescent "probes" of RS, are also critically reviewed. The emphasis throughout is on the caution that is needed in applying these methods in view of possible errors and artifacts in interpreting the results.

Animals↗

PubMed2004

Do mitochondria make nitric oxide? no?

Several papers have claimed that mitochondria contain nitric oxide synthase (NOS) and make nitric oxide (NO*) in amounts sufficient to affect mitochondrial respiration. However, we found that the addition of L-arginine or the NOS inhibitor L-NMMA to intact rat liver mitochondria did not have any effect on the respiratory rate in both State 3 and State 4. We did not detect mitochondrial NO* production by the oxymyoglobin oxidation assay, or electrochemically using an NO* electrode. An apparent NO* production detected by the Griess assay was identified as an artifact. NO* generated by eNOS added to the mitochondria could easily be detected, although succinate-supplemented mitochondria appeared to consume NO*. Our data show that NO* production by normal rat liver mitochondria cannot be detected in our laboratory, even though the levels of production claimed in the literature should easily have been measured by the techniques used. The implications for the putative mitochondrial NOS are discussed.

Animals↗

PubMed2004

The novel neuromodulator hydrogen sulfide: an endogenous peroxynitrite 'scavenger'?

Hydrogen sulfide (H2S) is a well-known cytotoxic gas. Recently it has been shown to stimulate N-methyl-D-aspartate (NMDA) receptors to enhance long-term potentiation suggesting a novel neuromodulatory role in vivo. Endogenous levels of H2S in the brain are reported to range between 10 and 160 microm. Considerably lower H2S levels are reported in the brains of Alzheimer's disease (AD) patients, where levels of brain protein nitration (probably mediated by peroxynitrite) are markedly increased. Activation of NMDA receptors leads to intracellular tyrosine nitration by peroxynitrite. Because H2S and peroxynitrite are important mediators in brain function and disease, we investigated the effects of the H2S 'donor', sodium hydrogen sulfide (NaSH) on peroxynitrite-mediated damage to biomolecules and to cultured human SH-SY5Y cells. H2S significantly inhibited peroxynitrite-mediated tyrosine nitration and inactivation of alpha1-antiproteinase to a similar extent to reduced glutathione at each concentration tested (30-250 microm). H2S also inhibited peroxynitrite-induced cytotoxicity, intracellular protein nitration and protein oxidation in human neuroblastoma SH-SY5Y cells. These data suggest that H2S has the potential to act as an inhibitor of peroxynitrite-mediated processes in vivo and that the potential antioxidant action of H2S deserves further study, given that extracellular GSH levels in the brain are very low.

Cell Line↗

PubMed2004

Sulfite-mediated oxidative stress in kidney cells.

BACKGROUND: Chronic renal failure has been associated with oxidative stress. Serum sulfite, sulfate, cysteine, homocysteine, cysteine sulfinic acid, and gamma-glutamylcysteine are elevated in patients on hemodialysis, suggesting an accelerated catabolism of sulfur-containing amino acids or a reduced elimination of sulfite/sulfate, or both. Administration of metabisulfite has also been shown to damage kidney cells. METHODS: Measurement of reactive oxygen species (ROS) was performed with the fluorescence of dichlorofluorescein (DCF), and that of intracellular ATP was by the luciferin-luciferase reaction. Oxidation of sulfite and succinate by isolated mitochondria from rat kidney was monitored polarographically. The fluorescent probe, 5, 5', 6, 6'-tetrachloro-1,1',3,3'-tetraethylbenzimidazolcarbocyanine iodide (JC-1) was employed to assess any loss in membrane potential in energized respiring mitochondria. Activities of glutamate and malate dehydrogenases (GDH, MDH, respectively) were assayed by the spectrophotometric measurement of NADH. Sulfite was determined by HPLC-fluorimetric measurement of monochlorobimane-sulfite and cell viability was by the MTT procedure. RESULTS: An immediate increase in ROS followed exposure of Madin-Darby canine kidney (MDCK), type II, and opossum kidney (OK) cells to 5-500 micromol/L sulfite. Depletion of intracellular ATP was also observed. A low rate of oxidation of 100 micromol/L sulfite was observed polarographically in isolated kidney mitochondria, but ADP-stimulated State 3 respiration was not apparent. ATP biosynthesis from the oxidation of glutamate in rat kidney mitochondria was significantly inhibited by coincubation with 100 micromol/L sulfite; this was not the case with malate, succinate, and TMPD/ascorbate. However, activities of both GDH and MDH in kidney mitochondrial extract were inhibited. The mitochondrial membrane potential and cell viability were not compromised. CONCLUSION: Micromolar sulfite elicited an immediate increase in ROS in MDCK, type II, and OK cells. This was accompanied by a depletion of intracellular ATP, which could be explained by its inhibitory effect on mitochondrial GDH. Although MDH was similarly inhibited, the impact was buffered by the high level of this enzyme in kidney mitochondria.

Adenosine Diphosphate↗

PubMed2003

The Coenzyme Q10 analog decylubiquinone inhibits the redox-activated mitochondrial permeability transition: role of mitcohondrial [correction mitochondrial] complex III.

The mitochondrial permeability transition (MPT) is a key event in apoptotic and necrotic cell death and is controlled by the cellular redox state. To further investigate the mechanism(s) involved in regulation of the MPT, we used diethylmaleate to deplete GSH in HL60 cells and increase mitochondrial reactive oxygen species (ROS) production. The site of mitochondrial ROS production was determined to be mitochondrial respiratory complex III (cytochrome bc1), because 1). stigmatellin, a Qo site inhibitor, blocked ROS production and prevented the MPT and cell death and 2). cytochrome bc1 activity was abolished in cells protected from the redox-dependent MPT by stigmatellin. We next investigated the effect of pretreating cells with coenzyme Q10 analogs decylubiquinone (dUb) and ubiquinone 0 (Ub0) on the redox-dependent MPT. Pretreatment of HL60 cells with dUb blocked ROS production induced by GSH depletion and prevented activation of the MPT and cell death, whereas Ub0 did not. Since we also found that dUb did not inhibit cytochrome bc1 activity, the mechanism of protection against redox-dependent MPT by dUb may depend on its ability to scavenge ROS generated by cytochrome bc1. These results indicate that dUb, like the clinically used ubiquinone analog idebenone, may serve as a candidate antioxidant compound for the development of pharmacological agents to treat diseases where there is an oxidative stress component.

Electron Transport Complex III↗

PubMed2003

Inhibition of hypochlorous acid-induced oxidative reactions by nitrite: is nitrite an antioxidant?

Acute and chronic inflammation result in increased nitrogen monoxide (z.rad;NO) formation and the accumulation of nitrite (NO(2)(-)). Neutrophils stimulated by various inflammatory mediators release myeloperoxidase to produce the cytotoxic agent hypochlorous acid (HOCl). At physiologically attainable concentrations, we found that NO(2)(-) significantly inhibits HOCl-mediated DNA strand breakage and ascorbate depletion. HOCl-mediated inactivation of pure alpha(1)-antiproteinase or of the elastase inhibitory capacity of human plasma was inhibited by the addition of NO(2)(-). NO(2)(-) was more effective than ascorbate, GSH, and urate at inhibiting HOCl-mediated toxicity to human HepG2 cells in culture. These data suggest that NO(2)(-) may act in an antioxidant manner by removing HOCl at sites of inflammation where both HOCl and z.rad;NO are overproduced.

Antioxidants↗

PubMed2003

Inhibition of peroxynitrite-mediated cellular toxicity, tyrosine nitration, and alpha1-antiproteinase inactivation by 3-mercapto-2-methylpentan-1-ol, a novel compound isolated from Allium cepa.

Peroxynitrite formation in vivo is implicated in numerous human diseases and there is considerable interest in the use of antioxidants and natural products such as thiols as "peroxynitrite scavengers". We therefore investigated the effects of a recently identified constituent of onions, 3-mercapto-2-methylpentan-1-ol (3-MP), for its ability to inhibit peroxynitrite-mediated processes in vitro and using cultured human cells and compared its effectiveness against glutathione. 3-MP significantly inhibited peroxynitrite-mediated tyrosine nitration and inactivation of alpha(1)-antiproteinase to a greater extent than glutathione at each concentration tested (15-500 microM). 3-MP also inhibited peroxynitrite-induced cytotoxicity, intracellular tyrosine nitration, and intracellular reactive oxygen species generation in human HepG2 cells in culture to a greater extent than glutathione. These data suggest that 3-MP has the potential to act as an inhibitor of ONOO(-)-mediated processes in vivo and that the antioxidant action of 3-MP deserves further study.

Drug Interactions↗