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Biomedical subjects

R A Floyd

Publications and source records attributed to R A Floyd.

At least 55 records · Page 3Linked to original sources
PubMed1998

Anoxic injury of endothelial cells causes divergent changes in protein kinase C and protein kinase A signaling pathways.

Alterations in protein kinase C (PKC) and cAMP-dependent kinase have been documented in anoxic brain injury. However, the regulation of these signaling enzymes in the cerebrovasculature has not been explored. In this study, cultured brain endothelial cells exposed to anoxic injury (anoxia--20 min/reoxygenation--40 min) showed both a significant increase (p < 0.001) in PKC and decrease (p < 0.01) in cAMP-dependent kinase activity. Analysis of PKC by Western blot indicated an increase in kinase level in response to anoxic injury, whereas there was no change in the level of cAMP-dependent protein kinase, as measured by labeled cAMP binding. Inhibition of nitric oxide synthase did not affect these changes. Addition of the nitric oxide-releasing compound sodium nitroprusside caused a dose-dependent increase in the activity of both signaling systems in endothelial cells. These data demonstrate that anoxic injury of brain endothelial cells in culture causes significant and divergent changes in signaling kinase activity. Abnormalities in brain endothelial PKC and cAMP-dependent kinase could have important consequences for the blood-brain barrier in anoxic brain injury.

Animals↗
PubMed1998

Interaction of alpha-phenyl-N-tert-butyl nitrone and alternative electron acceptors with complex I indicates a substrate reduction site upstream from the rotenone binding site.

Mitochondrial complexes I, II, and III were studied in isolated brain mitochondrial preparations with the goal of determining their relative abilities to reduce O2 to hydrogen peroxide (H2O2) or to reduce the alternative electron acceptors nitroblue tetrazolium (NBT) and diphenyliodonium (DPI). Complex I and II stimulation caused H2O2 formation and reduced NBT and DPI as indicated by dichlorodihydrofluorescein oxidation, nitroformazan precipitation, and DPI-mediated enzyme inactivation. The O2 consumption rate was more rapid under complex II (succinate) stimulation than under complex I (NADH) stimulation. In contrast, H2O2 generation and NBT and DPI reduction kinetics were favored by NADH addition but were virtually unobservable during succinate-linked respiration. NADH oxidation was strongly suppressed by rotenone, but NADH-coupled H2O2 flux was accelerated by rotenone. Alpha-phenyl-N-tert-butyl nitrone (PBN), a compound documented to inhibit oxidative stress in models of stroke, sepsis, and parkinsonism, partially inhibited complex I-stimulated H2O2 flux and NBT reduction and also protected complex I from DPI-mediated inactivation while trapping the phenyl radical product of DPI reduction. The results suggest that complex I may be the principal source of brain mitochondrial H2O2 synthesis, possessing an "electron leak" site upstream from the rotenone binding site (i.e., on the NADH side of the enzyme). The inhibition of H2O2 production by PBN suggests a novel explanation for the broad-spectrum antioxidant and antiinflammatory activity of this nitrone spin trap.

Animals↗
PubMed1998

Potential mechanisms of photodynamic inactivation of virus by methylene blue. I. RNA-protein crosslinks and other oxidative lesions in Q beta bacteriophage.

A spectrum of oxidative lesions was observed in a bacteriophage-based model system that is very sensitive to the photodynamic activity of selected dyes. When suspensions of the intact bacteriophage Q beta were exposed to methylene blue plus light (MB + L), inactivating events, or "hits" occurred that were oxygen-dependent and that were associated with the formation of several specific lesions: (1) carbonyl moieties on proteins, (2) 8-oxo-7,8-dihydroguanine (8-oxoGua), and (3) single-strand breaks (ssb) in the RNA genome and (4) RNA-protein crosslinks. Formation of carbonyl groups associated with protein in the Q beta phage preparation correlated positively with photoinactivation of the phage with increasing doses of either of the sensitizers MB or rose bengal. Strand breaks in the Q beta genomic RNA were observable at high MB concentrations but appeared not to be significant at the lower concentrations of MB, as full-length Q beta RNA was observable well beyond the 99% inactivation point in MB dosage. It was shown that the number of 8-oxoGua lesions were unlikely to be sufficient to account for the number of lethal events. Following exposure to MB + L, crosslink formation between Q beta RNA and protein was observed by virtue of the location of RNA at the interface of phenol-aqueous extractions of phage suspensions. A significant increase over background of RNA-protein complexes (including full-length Q beta RNA) was observed at the lowest concentration of MB tested (0.5 microM), which corresponded roughly to an average of 2 lethal hits per phage or approximately 13% survival compared to the zero MB control (100% survival). Due to its close correlation with Q beta inactivation and its expected lethality, RNA-protein crosslink formation may be important as an inactivating lesion in bacteriophage Q beta following MB + L exposure.

Allolevivirus↗
PubMed1997

Quantitation of protein-bound 3-nitrotyrosine and 3,4-dihydroxyphenylalanine by high-performance liquid chromatography with electrochemical array detection.

Reactive oxygen species (ROS) and reactive nitrogen species (RNS) have been implicated in myriad disease etiologies and may represent an obligate pathologic sequelus of inflammation. Unfortunately, few sensitive and specific analytical techniques exist for the routine assay of biomarkers indicative of ROS and RNS elaboration. In this study, high-performance liquid chromatography is used in conjunction with coulometric electrochemical array (HPLC-EC) detection to allow ultrasensitive determination of protein-bound 3-nitrotyrosine and 3, 4-dihydroxyphenylalanine (3-hydroxytyrosine) as specific in situ biomarkers of protein exposure to reactive nitrating and oxidizing species. Tyrosine and derivatives can be analyzed simultaneously with practical detection limits for tyrosine, 3-NT, and 3,4-Dopa being 10, 50, and 2 pmol, respectively, in as little as 20 microL of sample. HPLC-EC array detection allows two-dimensional resolution of chromatograms, greatly facilitating peak detection and confidence assignment. A method of sample preparation wherein tyrosine analogs are enzymatically hydrolyzed from protein without the need for sample extraction, concentration, or derivatization is reported.

Animals↗
PubMed1997

Glutathione/Fe3+/O2-mediated DNA strand breaks and 8-hydroxydeoxyguanosine formation. Enhancement by copper, zinc superoxide dismutase.

Oxidative DNA damage reflected by the formation of 8-hydroxy-2'-deoxyguanosine (8-OH-dG) and strand breaks caused by a glutathione mixed-function oxidation system (GSH-MFO) comprised of Fe3+, O2, and glutathione as an electron donor was enhanced by copper, zinc superoxide dismutase (CuZnSOD) in a concentration-dependent manner. Unlike CuZnSOD, manganese SOD (MnSOD) as well as iron SOD (FeSOD) did not enhance either strand breaks or 8-OH-dH formation in DNA. The capacity of CuZnSOD to enhance damage to DNA was inhibited by 5,5-dimethyl-1-pyrroline N-oxide (DMPO), a spin trapping agent. The salicylate hydroxylation assay showed that hydroxyl radicals formed in the presence of the GSH-MFO system was increased by CuZnSOD. The GSH-MFO system caused the release of free copper from CuZnSOD. Based on these results, we interpret the effects of CuZnSOD on the GSH-MFO induced DNA damage as due to reactive oxygen species, probably .OH, formed by the reaction of free Cu2+, released from oxidatively damaged CuZnSOD, and H2O2 produced by the GSH-MFO system.

8-Hydroxy-2'-Deoxyguanosine↗
PubMed1997

Spin trapping agent phenyl N-tert-butylnitrone protects against the onset of drug-induced insulin-dependent diabetes mellitus.

Insulin-dependent diabetes mellitus is an autoimmune disease believed to be caused by an inflammatory process in the pancreas leading to selective destruction of the beta-cells. Cytokines and nitric oxide (NO) have been shown to be involved in this destruction. Phenyl N-tert-butylnitrone (PBN) has demonstrated protective effects against several pathological conditions including ischemia-reperfusion injury and endotoxin-induced shock. We report here that PBN co-administration can prevent the onset of the STZ-induced diabetes in mice. PBN co-treatment inhibited the streptozotocin (STZ)-induced hyperglycemia, the elevation in the level of glycated hemoglobin and weight loss in the treated mice. Histological observations indicated destruction of B-cells in the STZ-treated animals and its prevention by PBN co-treatment. EPR spin trapping experiments in the pancreas indicated the in vivo formation of NO in STZ-treated animals and its attenuation by PBN treatment.

Animals↗
PubMed1997

Diurnal variation of TNF alpha in the rat brain.

Tumor necrosis factor-alpha (TNF alpha) is thought to play a physiological role in the brain. These studies were performed to determine whether a diurnal rhythm of TNF alpha exist in the rat brain. Samples were collected from hippocampus, hypothalamus, cerebral cortex, cerebellum, pons and midbrain at light onset and at 6 h intervals thereafter over a day. A TNF alpha bioassay was used to measure TNF alpha in each area. TNF alpha was highest at light onset in the hypothalamus, hippocampus and cerebral cortex. Levels at light onset were about 10-fold greater than minimal night-time levels. Changes in TNF alpha activity in other brain areas were also evident, but smaller. These results support the hypothesis that TNF alpha has physiological roles in the brain.

Analysis of Variance↗
PubMed1997

Nitrone-based free radical traps as neuroprotective agents in cerebral ischaemia and other pathologies.

Nitrone-based spin trapping compounds have been shown to protect experimental animals from pathology associated with ischaemia/reperfusion injury, endotoxaemia, natural and accelerated aging, certain xenobiotics, and physical trauma. Moreover, these compounds have an intriguing nootropic action. Nitrones affect pathophysiological correlates in both the central nervous system and peripheral organ systems. These compounds have been shown to affect cellular oxidation state and oxidatively sensitive enzyme systems, but the precise mode of nitrone action has not been elucidated. Recent discoveries regarding the ability of nitrones to suppress gene transcriptional events associated with pathophysiological states, particularly the elaboration of NF kappa B-regulated cytokines and inducible nitric oxide synthase, argue that nitrones may act at a proximal level to oxidatively sensitive signal amplification systems.

Animals↗
PubMed1997

Proteins but not nucleic acids are molecular targets for the free radical attack during reoxygenation of rat hepatocytes.

Isolated rat hepatocytes generate large amounts of reactive oxygen species and suffer a significant cell injury during postanoxic reoxygenation. The aim of this study was to determine whether oxidation of proteins and nucleic acids occurs during reoxygenation and whether their damage is related to the development of hepatocyte injury. Isolated perfused rat hepatocytes were exposed sequentially to 1 h of aerobic control, 2.5 h of anoxia, and 2 h of reoxygenation. Protein oxidation was determined by measuring the hepatocyte protein carbonyl content. DNA and RNA oxidation was assessed by measuring the 8-hydroxydeoxyguanosine and 8-hydroxyguanosine adducts, respectively. The control preanoxic carbonyl content was 6.48 +/- 1.03 nmol/mg protein. The preanoxic 8-8 hydroxydeoxyguanosine and 8-hydroxyguanosine levels were 4.76 +/- 1.22 pmol/ml and 14.19 +/- 2.17 pmol/ml, respectively. During anoxia, protein and nucleic acid oxidation did not change significantly. With reoxygenation, the protein carbonyl content increased significantly within 30 min, reaching a value of 10.25 +/- 1.58 nmol/mg. The nucleic acid oxidation level remained stable. Perfusion with 100 muM of during reoxygenation abolished protein oxidation. These results indicate that in rat hepatocytes during the early phase of reoxygenation: (1) the protein oxidation level increased significantly above the preanoxic aerobic values; (2) DNA and RNA oxidation does not appear to occur; and (3) free metal-mediated free radical reactions are involved in the oxidative protein damage.

8-Hydroxy-2'-Deoxyguanosine↗
PubMed1997

The effect of peroxides and free radicals on body tissues.

It is possible that peroxides used to whiten teeth cause damage if used improperly. Oxygen free radicals associated with peroxides are important etiologic agent in the development of many pathological conditions. This report provides background information about the action of peroxides on body tissues-information the dentist can use to perform peroxide bleaching safely.

Animals↗
PubMed1997

Free radical production during ethanol intoxication, dependence, and withdrawal.

Indices of free radical production and cell damage were examined in male Sprague-Dawley rats chronically exposed to either ethanol (ETOH) or water vapor. In experiment 1, rats experienced either 1 or 11 cycles of ETOH exposure and withdrawal. Brain tissue was harvested 12 hr after ETOH exposure, and 1 hr after being injected with sodium salicylate as a scavenger. Brain tissue was analyzed for the formation of salicylate hydroxylation products as a measure of .OH production during withdrawal. Significant group differences for .OH production were demonstrated for 2,3- and 2,5-dihydroxybenzoic acid in the single cycle ETOH exposed rats compared with their water cohorts. A significant between group difference for 2,5-dihydroxybenzoic acid, only, was demonstrated for the multiple cycles of ETOH exposure. Spontaneous seizures were shown to correlate with increased production of .OH in ETOH exposed rats. In experiment 2, brain tissue was harvested from different groups of rats after removal from the chambers, at 0, 2, 12, 24, 36, and 48 hr after a single exposure cycle. Tissue was analyzed for (1) salicylate hydroxylation (as above), (2) glutamine synthetase activity, (3) whole brain glutamate concentration, and (4) oxidized protein. A multiple regression analysis was conducted on the five dependent variables and found they could be predicted by specific behavioral and neurological ratings. These data suggest that cell damage during withdrawal may have multiple time-dependent components.

Alcohol Withdrawal Delirium↗
PubMed1996

Reactive oxygen species-mediated inactivation of pyruvate dehydrogenase.

Brain ischemia reperfusion causes increased formation of reactive oxygen species (ROS). Activity of the mitochondrial enzyme pyruvate dehydrogenase (PDH) has been shown to undergo a significant decrease following reperfusion of the ischemic tissue. We have examined the effect of a superoxide radical-generating system (xanthine oxidase/hypoxanthine, XO/HX) on the activity of this enzyme. Incubation of PDH in the presence of XO/HX resulted in its inactivation. The degree of the inactivation was dependent on the amount of XO present, which correlated linearly with the concentration of superoxide radical generated by this system. The activity of lactate dehydrogenase, an enzyme resistant to inactivation by ischemia reperfusion, was not affected by this system. Superoxide dismutase partially prevented and catalase exerted a nearly complete protective effect against the inactivation of PDH. Deferoxamine was partially protective. The sulfhydryl protective reagents, dithiothreitol and glutathione, prevented the inactivation of PDH, even though to varying degrees, which implicates sulfhydryl oxidation. A hydroxyl radical-generating system (hydrogen peroxide irradiated with ultraviolet radiation) effectively inactivated PDH. These results demonstrate that PDH is susceptible to damage and inactivation by ROS and point to the involvement of Fenton chemistry and hydroxyl radicals formed through it in PDH inactivation by XO/HX. A similar mechanism may be responsible for the PDH inactivation during ischemia/reperfusion.

Animals↗
PubMed1996

Damage, repair, and mutagenesis in nuclear genes after mouse forebrain ischemia-reperfusion.

To determine whether oxidative stress after cerebral ischemia-reperfusion affects genetic stability in the brain, we studied mutagenesis after forebrain ischemia-reperfusion in Big Blue transgenic mice (male C57BL/6 strain) containing a reporter lacI gene, which allows detection of mutation frequency. The frequency of mutation in this reporter lacI gene increased from 1.5 to 7.7 (per 100,000) in cortical DNA after 30 min of forebrain ischemia and 8 hr of reperfusion and remained elevated at 24 hr reperfusion. Eight DNA lesions that are characteristic of DNA damage mediated by free radicals were detected. Four mutagenic lesions (2,6-diamino-4-hydroxy-5-formamidopyrimidine, 8-hydroxyadenine, 5-hydroxycytosine, and 8-hydroxyguanine) examined by gas chromatography/mass spectrometry and one corresponding 8-hydroxy-2'-deoxyguanosine by a method of HPLC with electrochemical detection increased in cortical DNA two- to fourfold (p < 0.05) during 10-20 min of reperfusion. The damage to gamma-actin and DNA polymerase-beta genes was detected within 20 min of reperfusion based on the presence of formamidopyrimidine DNA N-glycosylase-sensitive sites. These genes became resistant to the glycosylase within 4-6 hr of reperfusion, suggesting a reduction in DNA damage and presence of DNA repair in nuclear genes. These results suggest that nuclear genes could be targets of free radicals.

Animals↗
PubMed1996

In vivo trapping of nitric oxide in the brain of neonatal rats treated with the HIV-1 envelope protein gp 120: protective effects of alpha-phenyl-tert-butylnitrone.

AIDS dementia complex is a neurological syndrome characterized by cognitive deficits and motor and behavioral dysfunction. The HIV-1 envelope glycoprotein gp 120 has been implicated in the development of AIDS dementia. This protein has been shown to be neurotoxic and to cause learning impairment and retardation of the development of complex motor behavior in rat neonates. Nitric oxide has been implicated in gp 120-induced neurotoxicity. In the present study, we report for the first time in vivo evidence for the formation of nitric oxide in the CNS as a result of multiple subcutaneous injections of gp 120 to neonatal rats. Nitric oxide was trapped in the brain of neonatal rats by N-methyl-D-glucamine dithiocarbamate-Fe and the nitric oxide content measured by electron paramagnetic resonance spectroscopy. The nitrone-based spin trap alpha-phenyl-tert-butylnitrone at 50 mg/kg was found to prevent gp 120-mediated nitric oxide formation and to also protect against gp 120-induced behavioral impairment.

Animals↗
PubMed1996

Anoxic injury of endothelial cells increases production of nitric oxide and hydroxyl radicals.

Reactive oxygen radicals have been implicated as mediators of anoxic injury in brain, but the cellular source of these radicals is unknown. In the periphery, there is evidence that endothelial cells play a fundamental role in anoxic tissue injury. The objective of the present study was to examine the response of rat brain endothelial cells to anoxia/reoxygenation injury in vitro. The results demonstrate that brain endothelial cells produce hydroxyl radicals and have increased nitric oxide synthase activity after anoxic injury. The increased production of nitric oxide in the cerebral endothelial cells does not appear to be mediated by an increase in either inducible or constitutive nitric oxide synthase. The radical trap alpha-phenyl-tert-butyl nitrone blocked hydroxyl free radical production, but not nitric oxide. These data suggest that the cerebral microcirculation may be an important site of oxygen free radical production in the brain in ischemic stroke.

Aerobiosis↗
PubMed1996

Inactivation of glutathione peroxidase by benzaldehyde.

Chronic benzaldehyde exposure is known to cause central nervous system (CNS) disturbances. Previous studies have shown that benzaldehyde causes the formation of reactive oxygen species (ROS) in rat synaptosomal fractions. Benzaldehyde has also been implicated in ROS formation in the CNS of rats treated with toluene. We have found that benzaldehyde effectively inactivates the antioxidant enzyme glutathione peroxidase (Ki approximately 15 microM), but has no effect on the other antioxidant enzymes tested: catalase, superoxide dismutase, and glutathione reductase. This effect has been found to be specific to benzaldehyde since other structurally related and unrelated aldehydes tested were found to be devoid of inactivating capacity toward glutathione peroxidase. Since glutathione peroxidase is the main enzyme responsible for removal of hydrogen peroxide and organic hydroperoxides in brain, its inactivation by benzaldehyde may be a main contributor to the observed ROS formation and the observed neurotoxicity caused by either benzaldehyde or toluene exposure.

Animals↗