Internal mammary artery smooth muscle cells resist migration and possess high antioxidant capacity

Objective: This study investigated whether differences exist in atherogen-induced migratory behaviors and basal antioxidant enzyme capacity of vascular smooth muscle cells (VSMC) from human coronary (CA) and internal mammary (IMA) arteries. Methods: Migration experiments were performed using the Dunn chemotaxis chamber. The prooxidant [NAD(P)H oxidase] and antioxidant [NOS, superoxide dismutase, catalase and glutathione peroxidase] enzyme activities were determined by specific assays. Results: Chemotaxis experiments revealed that while both sets of VSMC migrated towards platelet-derived growth factor-BB (1-50 ng/ml) and angiotensin II (1-50 nM), neither oxidized-LDL (ox-LDL, 25-100 ng/ml) nor native LDL (100 ng/ml) affected chemotaxis in IMA VSMC. However, high dose ox-LDL produced significant chemotaxis in CAVSMC that was inhibited by pravastatin (100 nM), mevastatin (10 nM), losartan (10 nM), enalapril (1 micro.M), and MnTBAP (a free radical scavenger, 50 micro.M). Microinjection experiments with isoprenoids i.e. geranylgeranylpyrophosphate (GGPP) and farnesylpyrophosphate (FPP) showed distinct involvement of small GTPases in atherogeninduced VSMC migration. Significant increases in antioxidant enzyme activities and nitrite production along with marked decreases in NAD(P)H oxidase activity and superoxide levels were determined in IMA versus CA VSMC. Conclusions: Enhanced intrinsic antioxidant capacity may confer on IMAVSMC resistance to migration against atherogenic agents. Drugs that regulate ox-LDL or angiotensin II levels also exert antimigratory effects.

Differential effects of an anti-oxidant intervention on cardiomyocyte expression of adrenomedullin and intermedin and their receptor components in chronic nitric oxide deficiency

Background: Chronic inhibition of nitric oxide (NO) synthesis is associated with hypertension, myocardial oxidative stress and hypertrophic remodeling. Up-regulation of the cardiomyocyte adrenomedullin (AM) / intermedin (IMD) receptor signaling cascade is also apparent in NO-deficient cardiomyocytes: augmented expression of AM and receptor activity modifying proteins RAMP2 and RAMP3 is prevented by blood pressure normalization while that of RAMP1 and intermedin (IMD) is not, indicating that the latter is regulated by a pressure-independent mechanism. Aims: to verify the ability of an anti-oxidant intervention to normalize cardiomyocyte oxidant status and to investigate the influence of such an intervention on expression of AM, IMD and their receptor components in NO-deficient cardiomyocytes. Methods: NO synthesis inhibitor, NG-nitro-L-arginine methyl ester (L-NAME, 35mg/kg/day) was given to rats for 8 weeks, with/without con-current administration of antioxidants (Vitamin C (25mg/kg/day) and Tempol (25mg/kg/day)). Results: In left ventricular cardiomyocytes isolated from L-NAME treated rats, increased oxidative stress was indicated by augmented (3.6 fold) membrane protein oxidation, enhanced expression of catalytic and regulatory subunits of pro-oxidant NADPH oxidases (NOX1, NOX2) and compensatory increases in expression of anti-oxidant glutathione peroxidase and Cu/Zn superoxide dismutases (SOD1, SOD3). Vitamin C plus Tempol did not reduce systolic blood pressure but normalized augmented plasma levels of IMD, but not of AM, and in cardiomyocytes: (i) abolished increased membrane protein oxidation; (ii) normalized augmented expression of prepro-IMD and RAMP1, but not prepro-AM, RAMP2 and RAMP3; (iii) attenuated (by 42%) increased width and normalized expression of hypertrophic markers, skeletal-�-actin and prepro-endothelin-1 similarly to blood pressure normalization but in contrast to blood pressure normalization did not attenuate augmented brain natriuretic peptide (BNP) expression. Conclusion: normalization specifically of augmented IMD/RAMP1 expression in NO-deficient cardiomyocytes by antioxidant intervention in the absence of blood pressure reduction indicates that these genes are likely to be induced directly by myocardial oxidative stress. Although oxidative stress contributed to cardiomyocyte hypertrophy, induction of IMD and RAMP1 is unlikely to be secondary to cardiomyocyte hypertrophy.

MEASUREMENT OF SUPEROXIDE FORMATION BY MITOCHONDRIAL COMPLEX I OF YARROWIA LIPOLYTICA

Complex I (NADH: ubiquinone oxidoreductase) is generally regarded as one of the major sources of mitochondrial reactive oxygen species (ROS). Mitochondrial membranes from the obligate aerobic yeast Yarrowia lipolytica, as well as the purified and reconstituted enzyme, can be used to measure complex I-dependent generation of superoxide (O-2(center dot-)). The use of isolated complex I excludes interference with other respiratory chain complexes and matrix enzymes during superoxide dismutase-sensitive reduction of acetylated cytochrome c. Alternately. hydrogen peroxide formation can be measured by the Amplex Red/horseradish peroxidase assay. Both methods allow the determination of complex I-generated ROS, depending on substrates (NADH, artificial ubiquinones), membrane potential, and active/deactive transition. ROS production by Yorrowia complex I in the

A competitive enzyme-linked immunosorbent assay for determination of chloramphenicol

Chloramphenicol is a broad-spectrum antibiotic shown to have specific activity against a wide variety of organisms that are causative agents of several disease conditions in domestic animals. Chloramphenicol has been banned for use in food-producing animals for its serious adverse toxic effects in humans. Due to the harmful effects of chloramphenicol residues livestock products should be free of any traces of these residues. Several analytical methods are available for chloramphenicol analysis but sensitive methods are required in order to ensure that no traces of chloramphenicol residues are present in edible animal products. In order to prevent the illegal use of chloramphenicol, regulatory control of its residues in food of animal origin is essential. A competitive enzyme-linked immunosorbent assay for chloramphenicol has been locally developed and optimized for the detection of chloramphenicol in sheep serum. In the assay, chloramphenicol in the test samples and that in chloramphenicol-horseradish peroxidase conjugate compete for antibodies raised against the drug in camels and immobilized on a microtitre plate. Tetramethylbenzidine-hydrogen peroxide (TMB/H2O2) is used as chromogen-substrate system. The assay has a detection limit of 0.1 ng/mL of serum with a high specificity for chloramphenicol. Cross-reactivity with florfenicol, thiamphenicol, penicillin, tetracyclines and sulfamethazine was not observed. The assay was able to detect chloramphenicol concentrations in normal sheep serum for at least 1 week after intramuscular injection with the drug at a dose of 25 mg/kg body weight (b.w.). The assay can be used as a screening tool for chloramphenicol use in animals.

Retinal vascular endothelial cell endocytosis increases in early diabetes.

BACKGROUND: Several physiological studies in recent years have convincingly demonstrated increased clearance of intravascular protein tracers by several different tissues, including the retina, during early diabetes and galactosemia in the rat. This change has been described as a consequence of increased permeation, although vascular leakage has not been demonstrated, and the fate of such tracers remains unelucidated. EXPERIMENTAL DESIGN: A pilot study in this laboratory showed no evidence of vascular leakage but suggested increased endocytosis of horseradish peroxidase (HRP) by retinal vascular endothelial cells (RVECs) in early diabetes. We therefore quantified RVEC endocytosis in normal, streptozotocin (STZ)-treated nondiabetic and STZ-diabetic rats using the design-based stereology method of "vertical sections." A duration of diabetes (6 weeks) was chosen to approximate the time period in which other workers have demonstrated increased protein permeation of the retina. RESULTS: After a 20-minute exposure to the tracer, HRP reaction product was observed in small vesicular and tubular endosomes and larger multivesicular bodies of the RVECs. Stereological analysis revealed a 6.5-fold increase in the volume of HRP-containing organelles in the RVECs of diabetic rats compared with STZ-treated nondiabetics or normal controls. None of the animals in this study showed HRP reaction product outside the retinal vascular endothelium. CONCLUSIONS: A highly significant increase in RVEC endocytosis occurs in early diabetes. Increased RVEC endocytosis may contribute to the observed clearance of intravascular protein tracers by the retina during early diabetes.

Increased endocytosis in retinal vascular endothelial cells grown in high glucose medium is modulated by inhibitors of nonenzymatic glycosylation

We sought to determine if hyperglycaemia is responsible for increased retinal vascular endothelial-cell (RVEC) endocytosis in diabetes and to assess the role of nonenzymatic glycosylation in mediation of this novel endothelial-cell pathology. RVECs were propagated in media containing either 5 or 25 mmol/l glucose for up to 10 days after which they were exposed to the protein tracer horseradish peroxidase for 30 min. The level of RVEC endocytosis was quantified in intact cell monolayers by electron microscopic stereology, and in cell lysates by a simple spectrophotometric method. The effect of the nonenzymatic glycosylation inhibitors, aminoguanidine and D-lysine, on high-glucose medium induced changes in RVEC endocytosis was tested by inclusion of these agents in the culture medium. RVECs exposed to 25 mmol/l glucose showed a stepwise increase in endocytosis of horseradish peroxidase culminating in a two- to threefold increase after 10 days. Endocytosis returned to normal levels after a further 10 days in 5 mmol/l glucose medium. The increase in RVEC endocytosis was markedly reduced, but not completely normalised, by aminoguanidine and D-lysine. Exposure of cultured RVECs to 25 mmol/l glucose causes an increase in endocytosis of similar magnitude to that experienced by RVEC in early diabetes, and implicates hyperglycaemia in the latter situation. A significant component of the increase in RVEC endocytosis appears to be mediated by nonenzymatic glycosylation.

Glucose-induced oxidative stress in vascular contractile cells - Comparison of aortic smooth muscle cells and retinal pericytes

Free radical-mediated damage to vascular cells may be involved in the pathogenesis of diabetic vasculopathy. The aim of this study was to compare the extent of glucose-induced oxidative stress in both vascular smooth muscle cells (VSMCs) and pericytes and the effect on antioxidant enzyme gene expression and activities. Porcine aortic VSMC and retinal pericytes were cultured in either 5 or 25 mmol/l glucose for 10 days. Intracellular malondialdehyde (MDA) was measured as a marker of peroxidative damage, and mRNA expression of CuZn-SOD, MnSOD, catalase, and glutathione peroxidase (GPX) were measured by Northern analysis. Glutathione (GSH) was also measured. There was a significant increase in MDA in VSMCs in 25 mmol/l glucose (1.34 +/- 0.11 vs. 1.88 +/- 0.24 nmol/mg protein, 5 vs. 25 mmol/l D-glucose, mean +/- SE, n = 15, P

Subdivision of the bacterioferritin comigratory protein family of bacterial peroxiredoxins based on catalytic activity

Peroxiredoxins are ubiquitous proteins that catalyze the reduction of hydroperoxides, thus conferring resistance to oxidative stress. Using high-resolution mass spectrometry, we recently reclassified one such peroxiredoxin, bacterioferritin comigratory protein (BCP) of Escherichia coli, as an atypical 2-Cys peroxiredoxin that functions through the formation of an intramolecular disulfide bond between the active and resolving cysteine. An engineered E. coli BCP, which lacked the resolving cysteine, retained enzyme activity through a novel catalytic pathway. Unlike the active cysteine, the resolving cysteine of BCP peroxiredoxins is not conserved across all members of the family. To clarify the catalytic mechanism of native BCP enzymes that lack the resolving cysteine, we have investigated the BCP homologue of Burkholderia cenocepacia. We demonstrate that the B. cenocepacia BCP (BcBCP) homologue functions through a 1-Cys catalytic pathway. During catalysis, BcBCP can utilize thioredoxin as a reductant for the sulfenic acid intermediate. However, significantly higher peroxidase activity is observed utilizing glutathione as a resolving cysteine and glutaredoxin as a redox partner. Introduction of a resolving cysteine into BcBCP changes the activity from a 1-Cys pathway to an atypical 2-Cys pathway, analogous to the E. coli enzyme. In contrast to the native B. cenocepacia enzyme, thioredoxin is the preferred redox partner for this atypical 2-Cys variant. BCP-deficient B. cenocepacia exhibit a growth-phase-dependent hypersensitivity to oxidative killing. On the basis of sequence alignments, we believe that BcBCP described herein is representative of the major class of bacterial BCP peroxiredoxins. To our knowledge, this is the first detailed characterization of their catalytic activity. These studies support the subdivision of the BCP family of peroxiredoxins into two classes based on their catalytic activity.

In vitro resistance of Burkholderia cepacia complex isolates to reactive oxygen species in relation to catalase and superoxide dismutase production

The Burkholderia cepacia complex comprises groups of genomovars (genotypically distinct strains with very similar phenotypes) that have emerged as important opportunistic pathogens in cystic fibrosis (CF) patients. The inflammatory response against bacteria in the airways of CF individuals is dominated by polymorphonuclear cells and involves the generation of oxidative stress, which leads to further inflammation and tissue damage. Bacterial catalase, catalase-peroxidase and superoxide dismutase activities may contribute to the survival of B. cepacia following exposure to reactive oxygen metabolites generated by host cells in response to infection. In the present study the authors investigated the production of catalase, peroxidase and SOD by isolates belonging to various genomovars of the B. cepacia complex. Production of both catalase and SOD was maximal during late stationary phase in almost all isolates examined. Native PAGE identified 13 catalase electrophoretotypes and two SOD electrophoretotypes (corresponding to an Fe-SOD class) in strains belonging to the six genomovars of the B. cepacia complex. Seven out of 11 strains displaying high-level survival after H(2)O(2) treatment in vitro had a bifunctional catalase/peroxidase, and included all the genomovar III strains examined. These isolates represent most of the epidemic isolates that are often associated with the cepacia syndrome. The majority of the isolates from all the genomovars were resistant to extracellular O(-)(2), while resistance to intracellularly generated O(-)(2)was highly variable and could not be correlated with the detected levels of SOD activity. Altogether the results suggest that resistance to toxic oxygen metabolites from extracellular sources may be a factor involved in the persistence of B. cepacia in the airways of CF individuals.

Mitochondrial J haplogroup is associated with lower blood pressure and anti-oxidant status: findings in octo/nonagenarians from the BELFAST Study

Mitochondria produce cellular energy but also free-radicals, which damage cells despite an array of endogenous anti-oxidants. In Northern Europe, the mitochondrial haplogroup J has been related to longevity in nonagenarians and centenarians but also with age-related disease. Hypertension is an important contributor to atherosclerotic-related diseases and its pathogenesis is associated with increased oxidative stress. In this study, we questioned whether J haplogroup octo/nonagenarians from the Belfast Elderly Longitudinal Free-living Elderly STudy (BELFAST) study showed evidence of protective blood pressure or anti-oxidant profile which might explain their longevity advantage. Briefly, in a cross-sectional study, community-living, mentally alert (Folstein >25/30), octo/nonagenarian subjects, recruited for good health, were enlisted and consented as part of the BELFAST study, for blood pressure, anthropometric measurements and blood sampling. DNA typing for mitochondrial haplotypes was carried out with measurements for enzymatic and non-enzymatic antioxidants. J haplogroup carriers showed lower systolic blood pressure and glutathione peroxidase activity (Gpx) with higher folate measurements. There was no change in urate, bilirubin, albumin or nutrition-related antioxidants-selenium or vitamins A, C and a and ß carotene. BELFAST study mtDNA J haplogroup octo/nonagenarians showed lower blood pressure and reduced glutathione peroxidase activity and higher folate, but no change for other antioxidants. These findings are of interest in view of mtDNA J haplogroup's association with increased age in some previous studies.

'False, fleeting, perjured Clarence': pseudonymity and criminality in the writings of James Clarence Mangan

This article explores the significance of the adopted partial pseudonym “Clarence” to James Clarence Mangan (1803-1849), who is increasingly regarded as the most important Irish poet before W. B. Yeats. Tracing the literary history of “Clarence” from Shakespeare to Maria Edgeworth, this essay argues that the intriguing adoption exposes a preoccupation with themes of unlawful textual copying that is at the centre of Mangan’s imagination. These tropes assume singular significance when appreciated alongside Mangan’s profession as a scrivener. While literary criticism has separated Mangan the poet from Mangan the legal scribe, his hitherto under-explored assumption of “Clarence” provides a clue to their close and crucial connection. These themes of pseudonymity, copying, and criminality combine with particular resonance in his quasi-translation “The Man in the Cloak” (1838) to open up new perspectives on Mangan’s writing and its participation in wider European cultural contexts and concerns. The essay will conclude with a salient comparison of Mangan’s story with Nikolay Gogol’s seminal story “The Overcoat”, or, “The Cloak” (1842).

A population-based association study of SNPs of GSTP1, MnSOD, GPX2 and Barrett's esophagus and esophageal adenocarcinoma

Oxidative stress appears to be important in the pathogenesis of Barrett's esophagus (BE) and esophageal adenocarcinoma (EAC). Single-nucleotide polymorphisms (SNPs) of antioxidant enzyme genes may play a part in determining individual susceptibility to these diseases. The Factors Influencing the Barrett's Adenocarcinoma Relationship (FINBAR) study is a population-based, case-control study of BE and EAC in Ireland. DNA from EAC (n = 207), BE (> or =3 cm BE at endoscopy with specialized intestinal metaplasia on biopsy, n = 189) and normal population controls (n = 223) were analyzed. Several SNPs spanning the genes for glutathione S-transferase P1 (GSTP1), manganese superoxide dismutase (MnSOD) and glutathione peroxidase 2 (GPX2) were genotyped using multiplex polymerase chain reaction and SNaPshottrade mark. The chi(2) test was used to compare genotype and allele frequencies between case and control subjects. Linkage disequilibrium between SNPs was quantified using Lewontin's D' value and haplotype frequency estimates obtained using Haploview. Eleven SNPs were genotyped (six for GSTP1, three for MnSOD and two for GPX2); all were in Hardy-Weinberg equilibrium. None was significantly associated with EAC or BE even before Bonferroni correction. Odds ratios for EAC for individual SNPs ranged from 0.68 [95% confidence interval (CI) 0.43-1.08] to 1.25 (95% CI 0.73-2.16), and for BE from 0.84 (95% CI 0.52-1.30) to 1.30 (95% CI 0.85-1.97). SNPs in all three genes were in strong linkage disequilibrium (D' > 0.887) but haplotype analysis did not show any significant association with EAC or BE. SNPs involving the GSTP1, MnSOD and GPX2 genes were not associated with BE or EAC. Further studies aimed at identifying susceptibility genes should focus on different antioxidant genes or different pathways.

Development of a solid-phase extraction coupling chemiluminescent enzyme immunoassay for determination of organophosphorus pesticides in environmental water samples

Solid-phase extraction (SPE) and direct competitive chemiluminescence enzyme immunoassay (dcCL-EIA) were combined for the detection of organophosphorus pesticides (OPs) in environmental water samples. dcCL-EIA based on horseradish peroxidase labeled with a broad-specificity monoclonal antibody against OPs was developed, and the effects of several physicochemical parameters on dcCL-EIA performance were studied. SPE was used for the pretreatment of water samples to remove interfering substances and to concentrate the OP analytes. The coupling of SPE and dcCL-EIA can detect seven OPs (parathion, coumaphos, phoxim, quinalphos, triazophos, dichlofenthion, and azinphos-ethyl) with the limit of quantitation below 0.1 ng/mL. The recoveries of OPs from spiked water samples ranged from 62.5% to 131.7% by SPE-dcCL-EIA and 69.5% to 112.3% by SPE-HPLC-MS/MS. The screening of OP residues in real-world environmental water samples by the developed SPE-dcCL-EIA and their confirmatory analysis using SPE-HPLC-MS/MS demonstrated that the assay is ideally suited as a monitoring method for OP residues prior to chromatographic analysis.

An immunohistochemical study of the distribution of biotin in tissues of pigs and chickens

An optimised indirect peroxidase-anti-peroxidase immunohistochemical technique was used to detect endogenous biotin in frozen tissue sections from biotin-supplemented and biotin-depleted pigs and chickens. A monoclonal anti-biotin antibody was used as primary antibody in this technique. Immunoreactive biotin was detected in many tissues of both species including liver, kidney, pancreas, adipose tissue, adrenal gland, testis, brain, choroid plexus, cardiac and skeletal muscle, epithelium of the respiratory and digestive systems, skin and lymphoid tissues. The specificity of immunostaining for biotin was confirmed by the finding of reduced staining intensities in tissues of biotin-depleted animals compared to those of biotin-supplemented animals. The results of this study suggest that biotin has metabolic functions in a wider range of tissues than previously known. They also indicate that endogenous tissue biotin should be considered as a source of false positive staining when immunohistochemical or histochemical techniques which use avidin or streptavidin reagents or anti-biotin antibodies as components of the detection system, are applied to tissue sections.

Growth, dye degradation and ligninolytic activity studies on Zimbabwean white rot fungi

White rot fungi were collected from Chirinda and Chimanimani hardwood forests in Zimbabwe and studied with respect to growth temperature optima and dye decolorization. Temperature optima were found to vary (between 25-37 degreesC) amongst the isolates. The isolates were screened for their ability to degrade the polymeric dyes; blue dextran and Poly R478 and the triphenylmethane dyes; cresol red, crystal violet and bromophenol blue. Semi-quantitative determination of the hydrolytic enzyme activities possessed by the white rot fungi was determined using the API ZYM system. Lignin peroxidase (LiP), manganese peroxidase (MnP) and laccase activities in the fungi were also determined. No LiP was detected in any of the isolates but all isolates showed manganese peroxidase and laccase activities. Time related decolorization studies and optimum pH determinations for Poly R478 degradation by the isolates were carried out in liquid cultures. The most significant rates of Poly R478 decolorization in liquid cultures were found with the following isolates: Trametes cingulata, Trametes versicolor, Trametes pocas, DSPM95 (a species to be identified), Datronia concentrica and Pyenoporus sanguineus. (C) 2001 Elsevier Science Inc. All rights reserved.