Biomedical subjects

Grzegorz Bartosz

Publications and source records attributed to Grzegorz Bartosz.

At least 19 recordsLinked to original sources

PubMed2006

Metal chelators react also with reactive oxygen and nitrogen species.

Popular chelators (desferrioxamine, SIH, EDTA, EGTA, DTPA, and NTA) were demonstrated to have antioxidant properties, being able to reduce ABTS radical cation and react with peroxyl radicals, peroxynitrite, and hypochlorite. Desferrioxamine and SIH were most potent antioxidants in all cases. These results point to the necessity of a careful interpretation of experiments in which the inhibition of free radical reactions by antioxidants is used as a proof of involvement of metal ions in a reaction.

Chelating Agents↗

PubMed2006

Hypothesis: cell volume limits cell divisions.

Mammalian somatic cells and also cells of the yeast Saccharomyces cerevisiae are capable of undergoing a limited number of divisions. Reaching the division limit is referred to, apparently not very fortunately, as replicative aging. A common feature of S. cerevisiae cells and fibroblasts approaching the limit of cell divisions in vitro is attaining giant volumes. In yeast cells this phenomenon is an inevitable consequence of budding so it is not causally related to aging. Therefore, reaching a critically large cell volume may underlie the limit of cell divisions. A similar phenomenon may limit the number of cell divisions of cultured mammalian cells. The term replicative (generative) aging may be therefore illegitimate.

Animals↗

PubMed2006

Relationship between the replicative age and cell volume in Saccharomyces cerevisiae.

Reaching the limit of cell divisions, a phenomenon referred to as replicative aging, of the yeast Saccharomyces cerevisiae involves a progressive increase in the cell volume. However, the exact relationship between the number of cell divisions accomplished (replicative age), the potential for further divisions and yeast cell volume has not been investigated thoroughly. In this study an increase of the yeast cell volume was achieved by treatment with pheromone alpha for up to 18 h. Plotting the number of cell divisions (replicative life span) of the pheromone-treated cells as a function of the cell volume attained during the treatment showed an inverse linear relationship. An analogous inverse relationship between the initial cell volume and replicative life span was found for the progeny of the pheromone-treated yeast. This phenomenon indicates that attaining an excessive volume may be a factor contributing to the limitation of cellular divisions of yeast cells.

Cell Division↗

PubMed2006

Expression of the human ABCC6 gene is induced by retinoids through the retinoid X receptor.

Mutations in the human ABCC6 gene are responsible for the disease pseudoxanthoma elasticum, although the physiological function or substrate of the gene product (an ABC transporter known also as MRP6) is not known. We found that the expression of this gene in cells of hepatic origin (where this gene is predominantly expressed in the body) is significantly upregulated by retinoids, acting as agonists of the retinoid X receptor (RXR) rather than the retinoid A receptor (RAR). The direct involvement of this nuclear receptor in the transcriptional regulation of ABCC6 gene expression was confirmed by transient transfection and chromatin immunoprecipitation assays. This constitutes the first direct proof of previously suggested involvement of nuclear hormone receptors in ABCC6 gene expression and the first identification of a transcription factor which may be relevant to regulation of ABCC6 level in tissues and in some PXE patients.

Animals↗

PubMed2006

Accumulation of cadmium ions in the methylotrophic yeast Hansenula polymorpha.

Intracellular cadmium (Cd(2+)) ion accumulation and the ability to produce specific Cd(2+) ion chelators was studied in the methylotrophic yeast Hansenula polymorpha. Only one type of Cd(2+) intracellular chelators, glutathione (GSH), was identified, which suggests that sequestration of this heavy metal in H. polymorpha occurs similarly to that found in Saccharomyces cerevisiae, but different to Schizosaccharomys pombe and Candida glabrata which both synthesize phytochelatins. Cd(2+) ion uptake in the H. polymorpha wild-type strains appeared to be an energy dependent process. It was found that Deltagsh2 mutants, impaired in the first step of GSH biosynthesis, are characterized by increase in net Cd(2+) ion uptake by the cells, whereas Deltagsh1/Deltamet1 and Deltaggt1 mutants impaired in sulfate assimilation and GSH catabolism, respectively, lost the ability to accumulate Cd(2+) intracellularly. Apparently H. polymorpha, similarly to S. cerevisiae, forms a Cd-GSH complex in the cytoplasm, which in turn regulates Cd(2+) uptake. Genes GSH1/MET1 and GGT1 are involved in maturation and metabolism of cellular Cd-GSH complex, respectively. Transport of [(3)H]N-ethylmaleimide-S-glutathione ([(3)H]NEM-SG) conjugate into crude membrane vesicules, purified from the wild-type cells of H. polymorpha appeared to be MgATP dependent, uncoupler insensitive and vanadate sensitive. We suggest that MgATP dependent transporter involved in Cd-GSH uptake in H. polymorpha, is similar to S. cerevisiae Ycf1-mediated vacuolar transporter responsible for accumulation of organic GS-conjugates and Cd-GSH complex.

Adenosine Triphosphate↗

PubMed2006

Accumulation of oxidative damage during replicative aging of the yeast Saccharomyces cerevisiae.

Comparison of senescent yeast obtained by the "baby machine" technique with 2-day-old stationary phase cells revealed decreased activities of glutathione reductase, glutathione S-transferase, glutathione peroxidase and alcohol dehydrogenase, reduction of total antioxidant capacity, protein glycation and accumulation of products of oxidative damage: protein carbonyls and DNA damage assessed by augmented content of 8-oxoguanine and increased tail momentum of cellular DNA in the comet assay. These results are consistent with a role for oxidative damage during replicative senescence of Saccharomyces cerevisiae.

Antioxidants↗

PubMed2006

Use of spectroscopic probes for detection of reactive oxygen species.

The detection and quantitation of reactive oxygen species (ROS) receives a great deal of interest because of their importance in a wide range of physiological and pathogenic events. Probe-assisted spectroscopy (electron spin resonance, spectrophotometry, fluorescence and luminescence) is the main tool for this application. This review discusses the properties of spectroscopic probes most commonly used for ROS detection and highlights their limitations in cellular systems. These include poor stability of some probes and/or products that may be subjected to cellular metabolism and lack of specificity in their reactions with oxidants or reductants. Additional problems often arise from undesired reactions of the probes and from their non-homogeneous distribution in the studied system, production of ROS by the probes themselves, perturbation of the systems under investigation by the probes, and artifacts due to the presence of ROS in the reaction medium. The limits imposed by these difficulties on the precise evaluation of the amounts and rates of formation of ROS are discussed critically.

Animals↗

PubMed2006

Production of nitric oxide, lipid peroxidation and oxidase activity of ceruloplasmin in blood of elderly patients with primary hypertension. Effects of perindopril treatment.

BACKGROUND AND AIMS: One of the factors playing a role in both the aging process and the etiopathogenesis of primary hypertension is oxidative stress. The purpose of this work was to estimate the production of nitric oxide (NO), oxidase activity of ceruloplasmin (Cp) and level of malondialdehyde (MDA), the end-product of lipid peroxidation in the blood of elderly patients with primary hypertension. The influence of treatment with the angiotensin-converting enzyme inhibitor perindopril (P) on the above parameters was also ascertained. METHODS: The study was carried out on 37 patients (69-91 yrs) with primary hypertension, treated with perindopril for 6 weeks, and normotensive subjects who constituted the two control groups: 25 (65-96 yrs--K1) and 23 (22-41 yrs--K2). Cp oxidase activity in serum, MDA level expressed as the concentration of substances reacting to thiobarbituric acid in red blood cells, and production of NO (concentration of nitrite/nitrate in plasma) by the Griess reaction were all determined. RESULTS: In the group of patients with hypertension, Cp activity was significantly higher than that in the control groups (K1 and K2). MDA concentrations in the erythrocytes of patients with hypertension were significantly higher than in the control groups and hypertensive patients had a lower NO (nitrite/nitrate) levels in plasma when compared with controls. In patients with hypertension, after only 7 days of therapy a substantial decrease in Cp oxidative activity was found, although the lowest value was observed after 6 weeks of therapy. Nevertheless, significantly lower MDA was observed after 6 weeks of therapy, corresponding to the values obtained in controls (K1 and K2). After 6 weeks of perindopril therapy, NO (nitrite/nitrate) was statistically higher in comparison with the values obtained before therapy. CONCLUSIONS: Results confirm intensification of oxidative stress and disorders in the production of NO in elderly patients with primary hypertension, and indicate the beneficial antioxidant effect of P.

Adult↗

PubMed2006

Yeast flavohemoglobin protects against nitrosative stress and controls ferric reductase activity.

The role of Saccharomyces cerevisiae flavohemoglobin (Yhb1) is controversial and far from understood. This study compares the effects of nitrosative and oxidative challenge on the yeast mutant lacking the YHB1 gene. Growth of the mutant was impaired by nitrosoglutathione and peroxynitrite, whereas increased sensitivity to reactive oxygen species was not observed. Increased levels of intracellular NO(*) after incubation with NO(*) donors were found in the mutants cells as compared to the wild-type cells. Deletion of the YHB1 gene was found to augment the reduction of Fe(3+) by yeast cells which suggests that flavohemoglobin participates in regulation of the activity of plasma membrane ferric reductase(s).

Cell Division↗

PubMed2006

The influence of ferrylhemoglobin and methemoglobin on the human erythrocyte membrane.

The aim of the study was to examine and compare the effects of methemoglobin (metHb) and ferrylhemoglobin (ferrylHb) on the erythrocyte membrane. Kinetic studies of the decay of ferrylhemoglobin (*HbFe(IV)=O denotes ferryl derivative of hemoglobin present 5 min after initiation of the reaction of metHb with H(2)O(2); ferrylHb) showed that autoredecay of this derivative is slower than its decay in the presence of whole erythrocytes and erythrocyte membranes. It provides evidence for interactions between ferrylHb and the erythrocyte membrane. Both hemoglobin derivatives induced small changes in the structure and function of the erythrocyte membrane which were more pronounced for ferrylHb. The amount of ferrylHb bound to erythrocyte membranes increased with incubation time and, after 2 h, was twice that of membrane-bound metHb. The incubation of erythrocytes with metHb or ferrylHb did not influence osmotic fragility and did not initiate peroxidation of membrane lipids in whole erythrocytes as well as in isolated erythrocyte membranes. Membrane acetylcholinesterase activity increased by about 10% after treatment of whole erythrocytes with both metHb and ferrylHb. ESR spectra of membrane-bound maleimide spin label demonstrated minor changes in the conformation of label-binding proteins in ferrylHb-treated erythrocyte membranes. The fluidity of the membrane surface layer decreased slightly after incubation of erythrocytes and isolated erythrocyte membranes with ferrylHb and metHb. In whole erythrocytes, these changes were not stable and disappeared during longer incubation.

Acetylcholinesterase↗

PubMed2005

Does yeast shmooing mean a commitment to apoptosis?

Treatment of yeast Saccharomyces cerevisiae with alpha-pheromone has been reported to lead to massive apoptosis of cells finding no conjugation partner [Severin FF, Hyman AA. Pheromone induces programmed cell death in S. cerevisiae. Curr Biol 2002;12:R233-5]. We report here that this effect is not common in yeast. Using different yeast strains, we demonstrate that identical treatment results in a low mortality even after prolonged treatment with the pheromone. These findings are followed by a general discussion of the biological relevance of apoptosis in yeast.

Apoptosis↗

PubMed2005

Replicative aging of the yeast does not require DNA replication.

Mating pheromone treatment resulting in shmoo formation is a physiologically relevant model for separation of cell growth and division processes in the yeast Saccharomyces cerevisiae. Using this attitude we demonstrate that yeast loses its capacity for division at a faster rate when engaged in intensive growth and metabolism without cell divisions (in the shmoo state) than during normal reproductive growth. These results suggest that limitation of the division potential in the yeast is not due to a counter of cell divisions but is of growth/metabolic nature, perhaps involving attaining a limitation of cell volume.

Aging↗

PubMed2005

Induction of apoptosis and modulation of production of reactive oxygen species in human endothelial cells by diphenyleneiodonium.

Diphenyleneiodonium (DPI) inhibits activity of flavoenzymes like NADPH oxidase, the major source of superoxide anion in cardiovascular system, but affects also other oxidoreductases. Contradictory data have been published concerning the effect of diphenyleneiodonium on the production of reactive oxygen species in cells, both inhibitory and stimulatory action of DPI being reported. We have examined the effect of DPI on the cellular production of reactive oxygen and nitrogen species (ROS/RNS) and on the proliferation and apoptosis of human vascular endothelial cells. We found increased oxidation of ROS-sensitive probes (dihydrorhodamine 123 and 2',7'-dichlorodihydrofluorescein diacetate) when DPI (20 microM-100 microM) was present in the treated cells. However, oxidation of the fluorogenic probes was inhibited if DPI (20 microM-100 microM) was removed from the reaction medium after cell preincubation. These results suggest an artifactual oxidation of the fluorogenic probes by DPI or its metabolites. A similar pattern of influence of DPI on the production of NO (measured with 4-amino-5-methylamino-2',7'-difluorofluorescein diacetate) was observed. Modulation of generation of reactive oxygen and nitrogen species in DPI-treated cells influenced the nitration of tyrosine residues of cellular proteins, estimated by Western blotting. Decreased level of nitration generally paralleled the lowered production of ROS. A decreased 3-(4,5-dimethylthiazolyl)-3-3(4-sulphophenyl) tetrazolium (MTT) reducing activity of cells for was observed immediately after 1h treatment of human endothelial cells with DPI (1 microM-100 microM), in spite of lack of changes in cell viability estimated by other methods. These results point to a next limitation of MTT in estimation of viability of cells treated with oxidoreductase inhibitors. DPI inhibited the proliferation of HUVECs as well as immortalized cell line HUVEC-ST, as assessed by acid phosphatase activity test and measurement of total nucleic acid content. Proapoptotic action of DPI was observed 12 h after incubation with this compound.

Acid Phosphatase↗

PubMed2005

On the specificity of 4-amino-5-methylamino-2',7'-difluorofluorescein as a probe for nitric oxide.

The specificity of 4-amino-5-methylamino-2',7'-difluorofluorescein (DAF-FM) for nitric oxide was evaluated in in vitro systems. The probe was found fairly specific for nitric oxide. Potential sources of artifacts include the autoxidation of DAF-FM, potentiated by light, and its oxidation by sources of superoxide and peroxyl radicals, leading to fluorescence spectra indistinguishable from those of the nitric oxide adduct. Although DAF-FM reacts with peroxynitrite, this reaction seems to be of secondary importance under quasi-physiological conditions. On the other hand, a simultaneous presence of a nitric oxide source and a superoxide or hydrogen peroxide decreases or increases the fluorescence of DAF-FM, respectively, resulting in biased estimates of nitric oxide production.

Artifacts↗

PubMed2005

Ascorbate abolishes auxotrophy caused by the lack of superoxide dismutase in Saccharomyces cerevisiae. Yeast can be a biosensor for antioxidants.

Yeast (Saccharomyces cerevisiae) mutants lacking cytoplasmic superoxide dismutase (CuZnSOD) show Lys and Met auxotrophy under aerobic conditions. This metabolic defect can be ameliorated by exogenous ascorbate as well as other antioxidants (glutathione, cysteine and N-acetylcysteine). Restoration of growth of CuZnSOD- yeast mutants on media devoid of Met and/or Lys may therefore be a simple and useful means to detect and quantify antioxidants. The protective effect of antioxidants is oxygen-dependent: the lower the oxygen content of the atmosphere, the lower antioxidant concentrations are required to restore prototrophy. Therefore, the sensitivity of the test can be augmented by growing the yeast under lowered partial oxygen pressure. While 6 mM, 10 mM and 30 mM ascorbate was necessary to restore the growth in the absence of Met, in the absence of Lys, and in the absence of Lys and Met, respectively, under 21% oxygen, 3 mM and 6 mM ascorbate was sufficient for growth restoration in the absence of Lys and in the absence of Lys and Met, respectively, under 3% oxygen. The protective effects of cysteine and N-acetylcysteine peaked at 0.5 mM and 6 mM, respectively, disappearing at higher concentrations of these compounds, pointing to the detection of not only protective but also toxic cellular effects of the compounds studied by the test proposed.

Antioxidants↗

PubMed2005

A novel test for identifying genes involved in aldehyde detoxification in the yeast. Increased sensitivity of superoxide-deficient yeast to aldehydes and their metabolic precursors.

A novel test for the identification of genes involved in aldehyde metabolism is proposed, based on detection of altered sensitivity of the yeast to corresponding alcohols, metabolic precursors of the aldehydes. This attitude enabled to an unexpected detection increased sensitivity of mutants devoid of CuZn-superoxide dismutase (CuZnSOD) to allyl alcohol (precursor of acrolein) and nonenol. We interpret this finding as due to inactivation of some important element of aldehyde detoxification by increased flux of superoxide in DeltaCuZnSOD mutants.

Acrolein↗

PubMed2005

Differential regulation of the human MRP2 and MRP3 gene expression by glucocorticoids.

Multidrug resistance proteins, which catalyse the detoxification of xenobiotics and excretion of metabolites, are very often controlled at the transcriptional level by interaction of exogenous compounds or hormones with nuclear receptors. Since synthetic glucocorticoids have found extensive use as anti-inflammatory drugs, also in the inhaled form in the treatment of asthma, lung cancer is potentially highly prone to transcriptional induction of multidrug resistance proteins by these steroids. MRP3 and MRP2 are major active anionic conjugate transporters in human cells and play a significant role in clinical multidrug resistance in cancer. A549 cells (non-small-cell lung cancer cell line) were challenged with glucocorticoids (dexamethasone, hydrocortisone and prednisone) at physiologically and therapeutically relevant concentrations for 24h and changes in MRP2 and MRP3 expression were followed on four levels: promoter regulation (luciferase reporter constructs), mRNA level (semi-quantitative real-time PCR), protein level (Western blotting) and activity (drug resistance and cellular transport of the model substrate calcein). DEX and HCT in the submicromolar concentration range caused a 2-fold induction of transcriptional activity at the MRP3 promoter construct, while MRP2 expression was not activated. All investigated glucocorticoids caused a modest stimulation of organic anion transport activity. We conclude that glucocorticoids used in clinical practice have the ability to transcriptionally upregulate human MRP3 gene expression in lung-derived cells where this protein is a major component of the organic anion extrusion system. This phenomenon has to be taken into account when designing treatments for lung cancer, especially for patients treated simultaneously with glucocorticoids against inflammatory symptoms.

Dexamethasone↗

PubMed2005

Antioxidants protect the yeast Saccharomyces cerevisiae against hypertonic stress.

Yeast (Saccharomyces cerevisiae) mutants lacking CuZnSOD have been reported to be hypersensitive to hypertonic media and to show increased oxidative damage. This study demonstrates that hypertonic medium (containing 0.8 M NaCl) increases the generation of superoxide and other reactive species in yeast cells. Other sequelae of exposure to hypertonic medium include oxidation of cellular low-molecular weight thiols and decrease in total antioxidant capacity of cellular extracts. deltasod1 mutant is more sensitive than a wild-type strain to colony growth inhibition on a hypertonic medium. Anaerobic conditions, ascorbate, glutathione, cysteine and dithiothreitol are able to ameliorate this growth inhibition but a range of other antioxidants does not protect. The protective ability of the antioxidants does not correlate with the rate of their reactions with superoxide but seems to be conditioned by low redox potential for one-electron oxidation of free radicals of the antioxidants. It suggests that repair of low-redox potential targets rather than prevention of their damage by superoxide is important in the antioxidant protection against oxidative stress induced by hypertonic conditions.

Animals↗