Dusan Dobrota

Free radical-induced protein modification and inhibition of Ca2+-ATPase of cardiac sarcoplasmic reticulum

The effect of oxidative stress on the Ca2+-ATPase activity, lipid peroxidation and protein modification of cardiac sarcoplasmic reticulum (SR) membranes was investigated. Isolated SR vesicles were exposed to FeSO4/EDTA (0.2 μmol Fe2+ per mg of protein) at 37°C for 1 h in the presence or absence of antioxidants. FeSO4/EDTA decreased the maximum velocity of Ca2+-ATPase reaction without a change of affinity for Ca2+ or Hill coefficient. Treatment with radical-generating system led also to conjugated diene formation, loss of sulfhydryl groups, changes in tryptophan and bityrosine fluorescences and to production of lysine conjugates with lipid peroxidation end-products. Lipid antioxidants butylated hydroxytoluene (BHT) and stobadine partially prevented inhibition of Ca2+-ATPase and decrease in tryptophan fluorescence, while the loss of –SH groups and formation of bityrosines or lysine conjugates were completely prevented. Glutathione also partially protected Ca2+-ATPase activity and decreased formation of bityrosine, but it was not able to prevent oxidative modification of tryptophan and lysine. These findings suggest that combination of amino acid modifications, rather than oxidation of amino acids of one kind, is responsible for inhibition of SR Ca2+-ATPase activity.

Study of the oxidative stress in a rat model of chronic brain hypoperfusion

A multiple analysis of the cerebral oxidative stress was performed on a physiological model of dementia accomplished by three-vessel occlusion in aged rats. The forward rate constant of creatine kinase, k(for), was studied by saturation transfer (31)P magnetic resonance spectroscopy in adult and aged rat brain during chronic hypoperfusion. In addition, free radicals in aging rat brain homogenates before and/or after occlusion were investigated by spin-trapping electron paramagnetic resonance spectroscopy (EPR). Finally, biochemical measurements of oxidative phosphorylation parameters in the above physiological model were performed. The significant reduction of k(for) in rat brain compared to controls 2 and 10 weeks after occlusion indicates a disorder in brain energy metabolism. This result is consistent with the decrease of the coefficient of oxidative phosphorylation (ADP:O), and the oxidative phosphorylation rate measured in vitro on brain mitochondria. The EPR study showed a significant increase of the ascorbyl free radical concentration in this animal model. Application of alpha-phenyl-N-tert-butylnitrone (PBN) and 5,5-dimethyl-1-pyrroline N-oxide (DMPO) spin traps revealed formation of highly reactive hydroxyl radical (.OH) trapped in DMSO as the .CH(3) adduct. It was concluded that the ascorbate as a major antioxidant in brain seems to be useful in monitoring chronic cerebral hypoperfusion.

Effect of Hypoxia/Ischemia and Hypoxic Preconditioning/Reperfusion on Expression of Some Amyloid‐Degrading Enzymes

Abstract: Alzheimers disease (AD) is linked to certain common brain pathologies (e.g., ischemia, stroke, and trauma) believed to facilitate its development and progression. One of the logical approaches to this problem is to study the effects of ischemia and hypoxia on the metabolism of amyloid precursor protein, which plays one of the key roles in the pathogenesis of AD. This involves an analysis of (1) proteases, which participate in proteolysis of amyloid precursor protein either by the nonamyloidogenic route (α‐secretase) or the amyloidogenic pathway and lead to formation of toxic β‐amyloid peptides (β‐ and γ‐secretases) and (2) several metallopeptidases that might play a role in degradation of β‐amyloid peptide (Aβ). The study of the effects of prenatal hypoxia and acute hypoxia in adult animals allowed us to conclude that oxygen deprivation results not only in an increase of amyloid precursor protein expression in the brain but also in a decrease in the activity of α‐secretase. In some brain structures involved in AD pathology (the cortex and striatum), we also observed a decrease in the expression of two of the Aβ degrading enzymes, neprilysin and endothelin‐converting enzyme, after hypoxia. A decrease in expression of these metalloproteases was also observed in the model of four‐vessel occlusion ischemia in rats with their restoration to the control levels after reperfusion. Preconditioning to mild hypoxia both in the prenatal period and in adults appeared to have a neuroprotective effect restoring, in particular, the levels of amyloid precursor protein, activity of a‐secretase, and expression of neprilysin and endothelin‐converting enzyme to their control values.

Na/K-ATPase under Oxidative Stress: Molecular Mechanisms of Injury

Abstract1. The authors compare oxidative injury to brain and kidney Na/K-ATPase using in vitro and in vivo approaches. The substrate dependence of dog kidney Na/K-ATPase was examined both before and after partial hydrogen peroxide modification. A computer simulation model was used for calculating kinetic parameters.2. The substrate dependence curve for the unmodified endogenous enzyme displayed a typical curve with an intermediate plateau, adequately described by the sum of hyperbolic and sigmoidal components.3. The modified enzyme demonstrated a dependent curve that closely approximates normal hyperbola. The estimated ATP Km value for the endogenous enzyme was about 85 μM; the Kh was equal to 800 μM. The maximal number of protomers interacting was 8. Following oxidative modification, the enzyme substrate dependence curve did not show a significant change in the maximal protomer rate Vm, while the Km was increased slightly and interprotomer interaction was abolished.4. Na/K-ATPase from an ischemic gerbil brain showed a 22% decrease in specific activity. The maximal rate of ATP hydrolysis by an enzyme protomer changed slightly, but the sigmoidal component, characterizing the enzymes ability to form oligomers was abolished completely. The Km value was almost unchanged, but the Hill coefficient fell to 1. These data show that Na/K-ATPase molecules isolated from the ischemic brain have lost the ability to interact with one another.5. We suggest that the most important consequence of oxidative modification is Na/K-ATPase oligomeric structure formation and subsequent hydrolysis rate suppression.

Kinetic parameters of Na/K-ATPase modified by free radicals in vitro and in vivo.

NdK-ATPase is characterized by complex kinetic behavior reflected in abnormal substrate dependence and described as a curve with an intermediary plateau.’ This feature does not depend on the source of the enzyme studied,2 but is closely connected to interprotomer interaction of the enzyme and the modulating effect of ATP on the activity. SoIubilization of the enzyme into a monomeric state by nonionic detergents results in transformation of the complex substrate dependence curve into one resembling a hyperb~la.’.~ From these observations we concluded that ATP stimulates enzyme activity modifying interprotomer interactions within oligomeric ensembles of NdKATPase. NdK-ATPase is a key enzyme regulating ionic homeostasis of the cell. Unfavorable conditions such as oxidative stress accompanied by increased generation of reactive oxygen species result in inhibition of NdK-ATPase in V ~ V O . ~ The same result can be achieved using different oxidants in vitro.6 In our experiments we compared the kinetic properties of Na/K-ATPase after oxidative modification by hydrogen peroxide or hypochlorous anion in vitro and after experimental brain ischemia in vivo. Hydrogen peroxide inhibited NdK-ATPase very slowly; 5 mM H202 led to 25-30% inhibition after 30 minutes of preincubation. Hypochlorous anion provided the same inhibiting effect much faster and at as low a concentration as 5 pM. Experimental brain ishemia for 15 minutes in rats or gerbils was also accompanied by pronounced (24-28%) inhibition of brain NdK-ATPase. To elucidate the mechanism of inhibition, we compared the kinetic properties of highly purified membrane-bound enzyme2 before and after oxidative attack. Kinetic

Rat tau proteome consists of six tau isoforms: implication for animal models of human tauopathies

Human brain encompasses six tau isoforms, containing either three (3R) or four (4R) repeat domains, all of which participate in the pathogenesis of human tauopathies. To investigate the role of tau protein in the disease, transgenic rat models have been created. However, unlike humans, it has been suggested that rat brain expresses only three 4R tau isoforms. Because of the significance of the number of tau isoforms for faithful reproducibility of neurofibrillary pathology in transgenic rat models, we reopened this issue. Surprisingly, our results showed that adult rat brain contains six tau isoforms like humans. Protein expression of 4R tau isoforms was ninefold higher than 3R isoforms. Furthermore, the protein levels of tau isoforms with none, one or two N‐terminal inserts were 30%, 35%, and 35% of total tau, respectively. Moreover, amount and ratio of tau isoforms were developmentally regulated. The levels of 4R tau isoforms progressively increased from early postnatal period until adulthood, whereas the expression of 3R tau isoforms reached maximum at P10 and then gradually declined. Our results show that rat brain encompasses full tau proteome similar to humans. These findings support the use of rat as an animal model in human tauopathies research.

Gene polymorphisms in bladder cancer

In Europe, cancer of the bladder is the fourth most common cancer among men, accounting for 7% of total cancers. In the USA, bladder cancer is the fifth most common cancer in men and seventh in women. This disease is three times more common in men than in women. Several risk factors, such as cigarette smoking and occupational chemical exposure, contribute to bladder cancer development. The balance between activation and detoxification of carcinogens affects the amount of DNA damage that accumulates in cells. The entire process leading to DNA damage and subsequent repair of the damage involves a host of enzymes, many of which are polymorphic. Polymorphisms in metabolic enzyme genes and repair genes may cause alterations in protein product functions that can finally lead to genomic instability and carcinogenesis. In this article, we review the polymorphisms in a number of genes that have been found to be the modulators of bladder cancer risk. Improved understanding of the molecular biology of urothelial malignancies is helping to more clearly define the role of new prognostic indices and multidisciplinary treatment for this disease.

In vitro assays for the evaluation of drug resistance in tumor cells

Oncologic diseases are among leading cause of mortality in developed countries. Despite significant progress, the use of standard cytotoxic chemotherapy has reached a therapeutical plateau. Currently, the process of selecting chemotherapy represents a trial and error method neglecting biological individuality of tumor and its bearer. The improvement of treatment results is expected from ex vivo drug sensitivity testing which may allow to choose the most effective drug for individual patient and to exclude agents to which the tumor cells exert resistance. New techniques and rapidly increasing knowledge about the molecular basis of malignant diseases provide important opportunities for the future of chemotherapy. This paper reviews current methods used to test the resistance of tumor cells to a panel of anticancer agents in vitro. In addition, we focused on the in vitro MTT assay which represents one of major technique for testing of tumor cell resistance to anticancer agents.

Carnosine protects rats under global ischemia

Rat brain subjected to 45-min global ischemia is characterized by decreased activity of K-p-nitrophenyl phosphatase and monoamine oxidase B and a disordering of the membrane bilayer by reactive oxygen species attack, the latter being monitored by the fluorescence of the membrane fluorescent probe, 1-anilino, 8-naphtalene sulphonate (ANS). Ischemic injury resulted in 67% mortality of the animals. In the group of animals pre-treated with the neuropeptide carnosine the mortality was only 30%. At the same time, carnosine protected both the activity of the above-mentioned enzymes and the brain membrane disordering, which was also tested by ANS fluorescence. The conclusion was made that carnosine protects the brain against oxidative injury and thereby increases the survival of the animals.

CARNOSINE : AN ENDOGENOUS NEUROPROTECTOR IN THE ISCHEMIC BRAIN

Abstract1. The biological effects of carnosine, a natural hydrophilic neuropeptide, on the reactive oxygen species (ROS) pathological generation are reviewed.2. We describe direct antioxidant action observed in the in vitro experiments.3. Carnosine was found to effect metabolism indirectly. These effects are reflected in ROS turnover regulation and lipid peroxidation (LPO) processes.4. During brain ischemia carnosine acts as a neuroprotector, contributing to better cerebral blood flow restoration, electroencephalography (EEG) normalization, decreased lactate accumulation, and enyzmatic protection against ROS.5. The data presented demonstrate that carnosine is a specific regulator of essential metabolic pathways in neurons supporting brain homeostasis under unfavorable conditions.