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Journal Article 2009; 30(Suppl 1): 148-151 PubMed PMID: 20027162 Citation  Keywords:  Animals, Antioxidants:pharmacology, Calcium-Transporting ATPases:antagonists & inhibitors, Dose-Response Relationship, Drug, Female, Flavonoids:pharmacology, Hypochlorous Acid:chemistry, Inhibitory Concentration 50, L-Lactate Dehydrogenase:metabolism, Oxi.   

OBJECTIVES: Effect of rutin and its lipophilic derivatives on Ca2+-ATPase of sarcoplasmic reticulum (SERCA) oxidized by hypochloric acid and peroxynitrite was investigated to examine the role of flavonoids in SERCA activity modulation.

METHODS: Ca2+-ATPase activity was measured spectrophotometrically at 37 degrees C using NADH-coupled enzyme pyruvate kinase/lactate dehydrogenase assay. SERCA was oxidized by HOCl (3 min) or ONOO- (30 s) after previous treatment with flavonoids (2 min) at 37 degrees C. Lipophilic rutin derivatives were prepared by lipase-catalyzed esterification of flavonoids with fatty acids.

RESULTS: Both hypochloric acid (HOCl) and peroxynitrite (ONOO-) decreased ATPase activity concentration-dependently with IC50 of 50+/-10 micromol/l and 150+/-15 micromol/l, respectively. Rutin was found to have a protective effect on SERCA activity in both oxidation systems in the concentration range 5 - 250 micromol/l. Lipophilic rutin derivatives (rutin oleate, rutin linoleate, rutin linolenate) exerted inhibitory effect on ATPase activity both in the presence and absence of oxidants.

CONCLUSION: The results suggest that selective lipophilization of the flavonoid skeleton may represent a useful tool for SERCA activity modulation....

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Journal Article 2009; 30(Suppl 1): 143-147 PubMed PMID: 20027161 Citation  Keywords:  Animals, Colostrum:chemistry, Diet, Dietary Supplements, Female, Liver:chemistry, Milk:chemistry, Muscle, Skeletal:chemistry, Myocardium:chemistry, Pregnancy, Selenium:administration & dosage, Species Specificity, Swine:metabolism,.   

OBJECTIVES: The aim of this trial was to determine selenium status in pure bred duroc sows and their progeny and to compare it to Czech Large White x Landrace breed.

DESIGN: The pregnant duroc sows (n=12) and pregnant Czech Large White x Landrace sows (n=12) were fed identical diets supplemented with sodium selenite.

RESULTS: During lactation significantly higher serum Se concentrations (p<0.001) were found in duroc piglets. Also significantly higher serum GSH-Px activities (p<0.001) were found in duroc sows and piglets. No differences in concentrations of Se in colostrums and milk were found between the two breeds.

CONCLUSION: Our findings support the hypothesis that there may be breed differences in indices of selenium status in pigs....

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Journal Article 2009; 30(Suppl 1): 137-142 PubMed PMID: 20027160 Citation  Keywords:  Adult, Alleles, Azathioprine:adverse effects, Female, Genetic Predisposition to Disease, Genotype, Humans, Leukopenia:chemically induced, Male, Methyltransferases:genetics, Polymerase Chain Reaction, Polymorphism, Genetic, Polymorphism, Single Nucleotide,.   

OBJECTIVES: Our study aims to find the relationship between metabolic enzyme thiopurine S-methyltransferase (TPMT) gene polymorphisms and clinical output of the therapy with azathioprine. We focused on patients who experienced leucopenia caused by high blood levels of active azathioprine metabolites.

DESIGN: Our group consists of 87 patients who have been treated by azathioprine. 21 individuals experienced leucopenia during treatment with standard dose of azathioprine. We have used PCR-REA and "real-time" PCR methods for genotype detection G238C, G460G and A719G substitutions in TPMT gene.

RESULTS: We have found statistical association between the presence of non-standard TPMT alleles and adverse event associated with azathioprine treatment - leucopenia (p=0.0033).

CONCLUSION: Our results confirm that TPMT genotyping prior to the treatment with azathioprine could predict patients with genetic predisposition for serious leucopenia and seems to be a useful genetic marker for individualisation of the therapy....

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Comparative Study 2009; 30(Suppl 1): 133-136 PubMed PMID: 20027159 Citation  Keywords:  Benzothiepins:pharmacology, Brompheniramine:pharmacology, Chlorpheniramine:pharmacology, Dose-Response Relationship, Drug, Extracellular Space:drug effects, Histamine H1 Antagonists:administration & dosage, Humans, Intracellular Space:drug effects, Lorata.   

OBJECTIVES: We analysed and compared the effect of five H1-antihistamines on stimulated oxidative burst at extra- and intracellular level of isolated and stimulated human polymorphonuclear leukocytes.

DESIGN: Oxidative burst of isolated human neutrophils was studied by means of luminol and isoluminol enhanced chemiluminescence.

RESULTS: The following rank order of potency for H1-antihistamines to decrease chemiluminescence was evaluated extracellularly: dithiaden> loratadine> chlorpheniramine> brompheniramine> pheniramine and at intracellular site: loratadine> dithiaden.

CONCLUSION: H1-antihistamines differ substantially according to their chemical structure in suppressing oxidative burst both at extra- and intracellular site of isolated stimulated human neutrophils....

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Journal Article 2009; 30(Suppl 1): 128-132 PubMed PMID: 20027158 Citation  Keywords:  Absorption, Animals, Cattle, Centrifugation, Collagen Type I:chemistry, Electrophoresis, Polyacrylamide Gel, Humans, Hydrogen Peroxide:chemistry, Hydroxyl Radical:chemistry, Luminescence, Oxidation-Reduction, Phagocytes:metabolism, Platelet Aggregation, P.   

OBJECTIVES: The effects of non-modified and oxidatively modified calf skin collagen type I on platelet aggregation and the oxidative burst of phagocytes were examined in the framework of a general hypothesis that collagen, platelets and phagocytes cooperate to modulate the oxidative burst of phagocytes and the extent of oxidative stress.

MATERIALS AND METHODS: Calf skin collagen type I was subjected to oxidative modification by hydrogen peroxide or hydroxyl radical. Thermal denaturation of collagen was performed in a spectrophotometer equipped with a temperature gradient device. The aggregation of isolated human platelets obtained after differential centrifugation was measured using a dual-channel aggregometer. The production of reactive oxygen species by human whole blood phagocytes was evaluated by luminol-enhanced chemiluminescence.

RESULTS: Oxidative modification of collagen samples was characterized by a decrease in denaturation transition temperature. Oxidatively modified samples showed a modified SDS-PAGE pattern, evidencing a significant destruction of the collagen. All oxidatively modified collagen samples, independent of the oxidation treatment applied, lost their platelet-aggregating and phagocyte oxidative burst-inducing activity.

CONCLUSION: The results suggest that reactive oxygen species were able to modify collagen. On the other hand, oxidatively modified collagen lost its activating properties towards platelets and phagocytes....

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Comparative Study 2009; 30(Suppl 1): 121-127 PubMed PMID: 20027157 Citation  Keywords:  3T3 Cells, Adenosine Triphosphate:metabolism, Animals, Cell Count:methods, Cell Line, Cell Proliferation, Cells, Cultured, Colorimetry, Dermis:cytology, Electric Impedance, Fibroblasts:cytology, Humans, Keratinocytes:cytology, Luminescent Measurements, Mi.   

OBJECTIVES: Determination of cell numbers is a crucial step in studies focused on cytokinetics and cell toxicity. The impedance-based analysis employing electronic sensor array system xCELLigence System allowing label-free dynamic monitoring of relative viable adherent cell amounts was compared with the most utilized methods for relative quantification of viable cell numbers based on a determination of cellular metabolism.

DESIGN: Colorimetric assay based on reduction of tetrazolium salt (MTT) by mitochondrial enzymes and chemiluminiscent assay based on intracellular adenosine triphosphate (ATP) determination were compared with the impedance-based system. Cell morphology was compared by microscopic evaluation. Normal human epidermal keratinocytes (NHEK) and normal human dermal fibroblasts (NHDF), together with 3T3 mouse fibroblast and HaCaT keratinocyte cell lines were employed.

RESULTS: The progress of cell growth curves obtained by different methods during 72 hours reflected cell type and cell seeding densities. The impedance-based method was found to be applicable for the determination of the cell proliferation of 3T3 fibroblasts, HaCaT and NHDF, since the comparison of this method with ATP and MTT determinations showed a comparable results. In contrast, the proliferation of NHEK measured by the impedance-based method did not correlate with other methodological approaches. This could be accounted to the specific morphological appearance of these cells.

CONCLUSION: The study shows the impedance-based detection of viable adherent cells is a valuable approach for cytokinetics and pharmacological studies. However, the specific morphological characteristics of cell lines have to be considered employing this method for determination of cell proliferation without using other reference methods....

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Journal Article 2009; 30(Suppl 1): 116-120 PubMed PMID: 20027156 Citation  Keywords:  Adult, Aldehydes:blood, Antioxidants:metabolism, C-Reactive Protein:metabolism, Cholesterol, HDL:blood, Chromatography, High Pressure Liquid, Female, Humans, Luminescent Measurements, Male, Malondialdehyde:blood, Middle Aged, Nitrites:blood, Oxidative Str.   

OBJECTIVES: Chronic pancreatitis (CP) is a heterogeneous disease defined as chronic inflammatory changes of the pancreatic tissue caused by variety of aetiologies. Oxidative stress accompanying the inflammatory processes has been suggested as an important factor contributing to CP development. The aim of this study was to determine levels of lipid peroxidation products malondialdehyde (MDA) and 4-hydroxynonenal (4-HNE), together with nitrites and the total antioxidant capacity in the plasma of patients with CP and control subjects.

DESIGN: One hundred and five patients with chronic pancreatitis and twenty seven healthy controls were included into this study. Levels of MDA and 4-HNE were analyzed using high-performance liquid chromatography. The total antioxidant capacity of plasma against peroxyl radicals was evaluated using chemiluminescent determination. Nitrites were determined using Griess reaction. Biochemical and haematological parameters were measured by standard methods.

RESULTS: The plasma levels of both MDA and 4-HNE, together with the plasma levels of nitrites, were significantly higher in CP patients, compared to healthy controls. The total antioxidant capacity did not differ significantly. Biochemical parameters were in the normal range. The MDA and 4-HNE levels correlated positively with the levels of high-density lipoprotein cholesterol. Nitrite levels correlated positively with C-reactive protein, total white blood cells, and triglycerides.

CONCLUSION: The significantly increased plasma levels of MDA, 4-HNE, and nitrites indicate that oxidative stress is present in patients with CP and that it may play a role in initiation and maintenance of inflammation within the pancreatic tissue in CP patients....

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Journal Article 2009; 30(Suppl 1): 112-115 PubMed PMID: 20027155 Citation  Keywords:  Animals, Aorta, Blotting, Western, Calcimycin:pharmacology, Cattle, Cells, Cultured, Endothelial Cells:drug effects, Homocysteine:metabolism, Ionophores:pharmacology, Kinetics, Microelectrodes, Nitric Oxide:metabolism, Nitric Oxide Synthase Type III:metab.   

OBJECTIVES: Elevated plasma uric acid indicates an increased risk of cardiovascular diseases associated with endothelial dysfunction. However, the role of uric acid in the pathogenesis of endothelial dysfunction is still a matter of debate. It is not clear whether uric acid is a real causative risk factor, an inert marker, or even a protective molecule with respect to its antioxidant properties. We have studied the effect of uric acid on intact endothelial cells as well as cells with homocysteine-induced endothelial dysfunction.

DESIGN: Bovine aortic endothelial cells were treated with uric acid (100 - 600 muM) and homocysteine (100 muM) or with uric acid only. After 24 hours, the cells were stimulated with 1 mug/ml of calcium ionophore A23187, and nitric oxide (NO) production was measured electrochemically with the use of a NO-sensitive microelectrode. The expression of endothelial nitric oxide synthase (eNOS) and eNOS phosphorylation at Ser1179 was estimated with the use of Western blotting. Interaction between NO and uric acid was measured with a NO electrode. Superoxide generation was measured with the use of the fluorescence dye MitoSox Red.

RESULTS: Homocysteine strongly diminished A23187-induced NO release. 100 muM uric acid slightly restored NO production; higher concentrations were ineffective. Interestingly, a dose-dependent decrease of NO release was observed in the cells treated only with uric acid. Uric acid did not scavenge NO and did not change eNOS protein expression or phosphorylation at Ser1179, but dose-dependently increased superoxide production in A23187-stimulated cells.

CONCLUSION: In conclusion, uric acid decreased NO bioavailability and enhanced superoxide generation in A23187-stimulated bovine aortic endothelial cells....

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