Protein Carbonyl Derivatives Research Articles

P-27 - Oxidazed proteins in blood of patients with nephropathies of various origins

The main purpose of our study was to investigate the oxidative modifications of proteins in blood of patients with nephropathies of various origins. There were 80 patients divided into 4 groups depending on the type of nephropathy. 1st group was represented by patients with acute alcoholic nephropathy; the 2nd group - by patients with nephropathy caused by acute poisoning with alcohol surrogates; in the 3th group pregnant women with gestosis were included; the 4th group – was represented by patients with acute medication nephropathy (acute drug poisoning). The control group consisted of 15 healthy subjects. The reactive protein carbonyl derivatives were detected in plasma and erythrocytes (Levine et.al, 1990), advanced oxidation protein products (AOPP) were detected in serum (Witko-Sarsat et al., 1996). In blood plasma of patients of 1–4 groups the concentration of reactive protein carbonyl derivatives was lower in comparison with control ones (p

Mechanistic investigation of visible light driven photocatalytic inactivation of E. coli by Ag-AgCl/ZnFe2O4.

In this study, photocatalytic inactivation of Escherichia coli was investigated over magnetic nanocomposite Ag-AgCl/ZnFe2O4. The nanocomposite demonstrated efficient photocatalytic activity by complete inactivation of the bacteria within 60min of visible light irradiation. The anions HPO42- and SO42- were found to play the most important role in the inhibition of photocatalytic inactivation of E. coli. A systematic investigation of mechanism of photocatalytic bacterial inactivation was carried out based on cell membrane injury test, scanning electron microscopy (SEM) of bacterial morphology changes, Fourier transform infrared (FTIR) spectroscopy of E. coli cells before and after treatment, superoxide dismutase (SOD) and catalase (CAT) activity assay, and role of various reactive oxygen species (ROS). The activities of SOD and CAT enzymes were found to decrease due to the ROSs attacks during photocatalytic inactivation. The ROS produced in the photocatalytic disinfection severely altered the bacterial permeability and led to protein fragmentation, release of ions, and generation of protein carbonyl derivatives. The leaked cytoplasmic substances and cell debris were further degraded and, ultimately, mineralized with prolonged photocatalytic treatment.

The Role of the Glutathione System in Oxidative Modification of Proteins and Dysregulation of Apoptosis in Jurkat Tumor Cells.

We compared changes in the redox status and intensity of oxidative modification of proteins in intact Jurkat tumor cells and cells cultured with buthionine sulfoximine, an inhibitor of the key enzyme of glutathione synthesis γ-glutamylcysteine synthetase. The glutathione system components play a role in modulation of the content of protein-bound glutathione, protein carbonyl derivatives, bityrosine, and oxidized tryptophan, and in dysregulation of apoptosis in Jurkat tumor cells. Inhibition of de novo synthesis of glutathione in Jurkat tumor cells was followed by accumulation of hydroxyl radical, a reduction in the level of protein-bound glutathione and oxidized tryptophan, and a rise in the concentration of protein carbonyl derivatives. These changes were accompanied by activation of programmed cell death.

Assessment of Oxidative Status of the Brain and Blood Plasma in Rats with Modeled Focal Cerebral Ischemia/Reperfusion Injury.

Parameters of the oxidative status of the brain and blood plasma were measured in rats 24 h after 1-h focal cerebral ischemia. In the brain of rats exposed to cerebral ischemia, activities of superoxide dismutase and catalase were elevated. Ischemia reduced the total antioxidant activity of the brain and the levels of malonic dialdehyde and protein carbonyl derivatives. In the blood plasma of experimental rats, superoxide dismutase activity and malonic dialdehyde level increased and total antioxidant activity decreased, i.e. the shifts were similar to those in the brain. The ischemia-induced changes in the brain and blood were not always co-directed.

КАРБОНИЛИРОВАНИЕ БЕЛКОВ КАК МЕХАНИЗМ РЕГУЛЯЦИИ ПРОЛИФЕРАЦИИ ОПУХОЛЕВЫХ КЛЕТОК ЛИНИИ МCF-7

Tumor progression is accompanied by dysregulation of cell proliferation and apoptosis, which plays an essential role in breast cancer pathogenesis and cell resistance to chemotherapy. The role of protein carbonylation in molecular mechanisms of regulating MCF-7 breast cancer cell proliferation under the effect of roscovitine, a selective inhibitor of cyclin-dependent kinases, was evaluated. The cells were grown in adherent cell culture with or without roscovitine. The levels of reduced/oxidized glutathione and the concentration of protein carbonyl derivatives were determined by spectrophotometry. The cell cycle was evaluated by the flow cytometry; the same technique was used to measure the reactive oxygen species (ROS) concentration. Cell culture with roscovitine resulted in a decrease in the redox potential of the glutathione system and a rise in the ROS and protein carbonyl derivative concentrations. Roscovitine contributed to the G 0 /G 1 and G 2 /М phase arrest due to its interaction with ATP-binding sites of cyclin-dependent kinases. Roscovitine could also promote enzyme carbonylation. The obtained results can be further used for development of personalized approaches to breast cancer therapy.

Hyperthermia and extracellular hyperosmolality affect sarcolemmal damage and protein oxidation in mammalian skeletal muscle in vitro

In the US Armed Services, the incidence of rhabdomyolysis is 5–7 fold higher in peak summer months, suggesting that muscle membrane injury could be amplified by heat or to secondary dehydration. In this study we tested the extent each of these variables influences damage by evaluating sarcolemmal integrity and ROS‐induced protein oxidation. Extensor digitorum longus (EDL) and soleus (SOL) muscles were dissected from 24 adult male C57BL6J mice and placed at optimal length in tissue baths containing 4 ml of Krebs (95%O2/5%CO2). Muscles were intermittently stimulated to perform non‐fatiguing twitches for 30 min. Buffer temperature and osmolality were manipulated to provide four experimental conditions: normothermia/isotonic (35°C and 285 mOsm/kg), normothermia/hypertonic (35°C and 300 mOsm/kg), hyperthermia/isotonic (41°C and 285 mOsm/kg), and hyperthermia/hypertonic (41°C and 300 mOsm/kg). We quantified the integrity of the sarcolemma using both procion orange leakage into the fibers and a fluorometric assay to detect leakage out of double‐stranded cell‐free DNA (cfDNA). To assess protein oxidation, we performed immunoblot assays for protein carbonyl derivatives. For SOL we did not observe a significant effect of hyperthermia and/or hyperosmolality on procion orange leakage, but we observed an increase in cfDNA (p = 0.03) and protein carbonyls with hyperthermia (p = 0.02). Interestingly, these effects were absent when hyperosmolality was superimposed on hyperthermia. For EDL, an increase in procion orange leakage was observed with hyperthermia alone (p = 0.04) and when hyperthermia and hyperosmolality were combined (p = 0.03), but the combination did not elicit changes in cfDNA in the buffer. EDL protein carbonyls were independently augmented with hyperthermia (p = 0.01) and hyperosmolality (p = 0.02), but also did not increase when hyperthermia was combined with hyperosmolality. In conclusion, hyperthermia caused sarcolemma damage in EDL muscle, both alone and when combined with hyperosmolality. SOL was more resistant to hyperthermia‐induced procion orange leakage, despite an increase release of extracellular cfDNA. Furthermore, physiological increases in extracellular osmolality prevented protein oxidation in hyperthermia in both muscle types. These observations suggest that different muscle types may respond differently to hyperthermic and osmotic challenges. The compensatory effect of hyperosmolality on hyperthermia‐induced oxidative stress is consistent with a role for ROS production in compensatory responses to cell swelling in the heat.Support or Funding InformationSupported by the BK and Betty Stevens Endowment and the Brazilian Council for the Development of Science and Technology (CNPq no. 249094/2013‐4).

Absence of DJ-1 causes age-related retinal abnormalities in association with increased oxidative stress

Oxidative stress alters physiological function in most biological tissues and can lead to cell death. In the retina, oxidative stress initiates a cascade of events leading to focal loss of RPE and photoreceptors, which is thought to be a major contributing factor to geographic atrophy. Despite these implications, the molecular regulation of RPE oxidative stress under normal and pathological conditions remains largely unknown. A better understanding of the mechanisms involved in regulating RPE and photoreceptors oxidative stress response is greatly needed. To this end we evaluated photoreceptor and RPE changes in mice deficient in DJ-1, a protein that is thought to be important in protecting cells from oxidative stress.Young (3 months) and aged (18 months) DJ-1 knockout (DJ-1 KO) and age-matched wild-type mice were examined. In both group of aged mice, scanning laser ophthalmoscopy (SLO) showed the presence of a few autofluorescent foci. The 18 month-old DJ-1 KO retinas were also characterized by a noticeable increase in RPE fluorescence to wild-type. Optical coherence tomography (OCT) imaging demonstrated that all retinal layers were present in the eyes of both DJ-1 KO groups. ERG comparisons showed that older DJ-1 KO mice had reduced sensitivity under dark- and light-adapted conditions compared to age-matched control. Histologically, the RPE contained prominent vacuoles in young DJ-1 KO group with the appearance of enlarged irregularly shaped RPE cells in the older group. These were also evident in OCT and in whole mount RPE/choroid preparations labeled with phalloidin. Photoreceptors in the older DJ-1 KO mice displayed decreased immunoreactivity to rhodopsin and localized reduction in cone markers compared to the wild-type control group. Lower levels of activated Nrf2 were evident in retina/RPE lysates in both young and old DJ-1 KO mouse groups compared to wild-type control levels. Conversely, higher levels of protein carbonyl derivatives and iNOS immunoreactivity were detected in retina/RPE lysates from both young and old DJ-1 KO mice.These results demonstrate that DJ-1 KO mice display progressive signs of retinal/RPE degeneration in association with higher levels of oxidative stress markers. Collectively this analysis indicates that DJ-1 plays an important role in protecting photoreceptors and RPE from oxidative damage during aging.

КАРБОНИЛЬНЫЕ ПРОИЗВОДНЫЕ БЕЛКОВ МОЛОКА И КИСЛОМОЛОЧНЫХ ПРОДУКТОВ

In the study on spontaneous oxidative degradation of ryazhenka and varenets proteins the increase in content of carbonyl derivatives of ryazhenka proteins in comparison with milk is revealed at the wavelengths of 356 nm, 370 nm, 430 nm, 530 nm in 1.51; 1,64; 1,43; 1.95 times, respectively. The maximum value of products with spontaneous oxidative modification of ryazhenka proteins is at a wavelength of 530 nm, the minimum is at 430 nm. The increase of carbonyl derivatives of varenets proteins in comparison with indicators of milk is found at a wavelength of 356 nm in 1.60 times, at a wavelength of 370 nm in 1.78 times, at a wavelength of 430 nm in 1.52 times, at a wavelength of 530 nm in 1.69 times. The maximum value of the spontaneous oxidative modification of varenets proteins is at a wavelength of 370 nm, the minimum is similar to the ryazhenka, i.e. 430 nm. In the study of metal-catalyzed oxidative destruction an increase in protein carbonyl derivatives of this product is revealed at a wavelengths of 356 nm, 370 nm, 430 nm, 530 nm in 2.11; 2,08; 1,86; 1.73 times compared with parameters of milk, respectively. The similar increase is found while examining metal-catalyzed oxidative degradation of varenets proteins at all wavelengths: in 2.01 times at a wavelength of 356 nm, in 2.24 times at a wavelength of 370 nm, in 2.01 times at a wavelength of 430 nm, and in 1.42 times at a wavelength of 530 nm in comparison with milk parameters. The maximum value of metal-catalyzed oxidative modification of ryazhenka proteins is established at a wavelength of 356 nm and the minimum at a wavelength of 530 nm. In case of varenets the maximum value of metal-catalyzed oxidative modification of proteins is at a wavelength of 370 nm, the minimum is at a wavelength of 530 nm. The indicators of carbonyl derivatives of proteins, as the findings of chemiluminescence analysis on dairy products indicate the most intensive processes of free radical oxidation in ryazhenka and varenets compared to milk.

Oxidative damage, ultrastructural alterations and gene expressions of hemocytes in the freshwater crab Sinopotamon henanense exposed to cadmium

Toxicity of Cd was tested with the hemocytes of the freshwater crab, Sinopotamon henanense, which were exposed to concentrations of 0, 0.725, 1.450, and 2.900mgL−1 Cd for 7, 14 and 21 d. We investigated the effects of Cd on the total antioxidant capacity (TAC), and oxidative damage of biomarkers, such as malondialdehyde (MDA), protein carbonyl derivates (PCO), and DNA–protein crosslink (DPC). Transmission electron microscopy (TEM) was applied to assess ultrastructural changes of hemocytes. The mRNA expression levels of prophenoloxidase (proPO), lysozyme (LSZ), metallothionein (MT), and the activity of phenoloxidase (PO) were also determined. Our results showed that TAC was inhibited by Cd, resulting in an increase of MDA contents, PCO contents, and DPC levels in hemocytes, respectively. Ultrastructural observations revealed that chromatin condensation, nucleus deformation, mitochondrial dilation, rough endoplasmatic reticulum (rER) degranulation and secondary or tertiary lysosomes were observed in hemocytes of crabs exposed to Cd. Meanwhile, the expression levels of proPO were down-regulated, while the activity of PO was up-regulated in hemocytes. The expression levels of LSZ and MT were up-regulated to some extent. Our findings suggest these parameters could be used as biomarkers in the monitoring of heavy metal pollution and quantitative risk assessments of pollutant exposure.

Mediterranean diets supplemented with virgin olive oil and nuts enhance plasmatic antioxidant capabilities and decrease xanthine oxidase activity in people with metabolic syndrome: The PREDIMED study.

This study assessed plasmatic antioxidant capabilities and xanthine oxidase (XOX) activity in metabolic syndrome patients after 5 years intervention with Mediterranean diet (MeDiet) supplemented with extra-virgin olive oil or with nuts or with low-fat diet (the PREDIMED [PREvención con Dieta MEDiterránea] study). Seventy-five participants were randomly selected. Daily energy and nutrient intake were assessed with a validated 137-item food frequency questionnaire, and adherence to the MeDiet was assessed using a 14-item questionnaire. Catalase, superoxide dismutase (SOD), myeloperoxidase, XOX activities and protein levels, and protein carbonyl derivatives, nitrotyrosine, nitrite and nitrate levels were determined in overnight fasting venous blood samples. The plasma activity and protein levels of SOD and catalase were significantly higher and XOX activity was lower in MeDiet supplemented with extra-virgin olive oil and MeDiet supplemented with nuts than in the control group. Participants in both MeDiet groups showed higher plasma nitrate levels than in the control group. Adherence to the MeDiet showed a positive correlation with SOD and catalase plasma antioxidant activities. A MeDiet enriched with either virgin olive oil or nuts enhances the plasma antioxidant capabilities and decreases XOX activity in patients with the metabolic syndrome but we did not observe changes in myeloperoxidase or markers of oxidative damage.

Oxidative metabolism of neutrophils in patients with community-acquired pneumonia

Abstract At the present time, available views show our limited knowledge of the peculiarities of the functional status of neutrophils and their metabolism in patients with community-acquired pneumonia (CAP). The studying of changes of metabolic status of neutrophils can broaden our views about pneumonia pathogenesis and define datum points of therapeutic effect. Purpose of our research: to define oxidative stress activity and the level of oxidative modification of proteins of neutrophils in CAP patients. Materials and methods: neutrophils obtained from 23 patients with community-acquired pneumonia. Control group consisting of 19 healthy volunteers. The reactive carbonyl derivatives of proteins and advanced oxidation protein products were defined so as to assess the oxidative damage of proteins. The malondialdehyde and nitrite ions were assessed as being indicators of the oxidative stress. The neutrophils of CAP patients with moderate severity were characterized by a tendency of evidencing decreasing content of advanced oxidation protein products, along with the statistically important enhanced levels of carbonyl derivatives and nitrite ions, while their malondialdehyde status practically leveled off with the control and had only an insignificant trend towards growth. We have demonstrated the accumulation of carbonyl derivatives and nitrite ions in the peripheral neutrophils of CAP patients. These results give evidence of an oxidative misbalance in the cells which contributes to the aggravation of the disease.

THE ROLE OF PROTEIN OXIDATIVE MODIFICATION IN REDOX-REGULATION OF CASPASE-3 ACTIVITY IN BLOOD LYMPHOCYTES DURING OXIDATIVE STRESS IN VITRO

The formation of oxidative stress lies at the heart of many frequent and socially-important diseases. Blood lymphocytes are the cells which provide immunological control of our organism. As a result of their function implementation blood lymphocytes contact with different endogenic and exogenic factors, which can lead to active oxygen species production activation, macromolecules oxidative modification and to cell survival alteration. At the present time it is essential to expand and deepen the fundamental knowledge of blood lymphocytes apoptosis regulation peculiarities. The research objective was to establish the interaction among alterations of glutathione system condition, carbonylation level, protein glutathionylation and caspase-3 activity in blood lymphocytes during oxidative stress in vitro. Material and Methods. The material for research was blood lymphocytes cultivated with addition of hydrogen peroxide in final concentration of 0,5 mmol and/or protein SH-group inhibitor N- ethylmaleimide – 5 mmol, protector – 5 mmol – 1,4-dithioerythritol. Reduced, oxidized and protein-bound glutathione concentration was measured by method of spectropho-tometry, additionally, the ratio size of reduced to oxidized thiol fraction was estimated. With help of enzymoimmunoassay the level of protein carbonyl derivatives was evaluated; caspase-3 activity was registered by spectrofluorometric method. Results . Protein SH-group blocking in blood lymphocytes during oxidative stress in vitro was accompanied by protein-bound glutathione concentration rapid decrease in connection with increase of protein carbonyl derivatives content and caspase-3 activity. Protein SH-group protection in blood lymphocytes during oxidative stress in vitro was accompanied by concentration increase of protein-bound glutathione and protein carbonyl derivatives under comparable values of enzyme activity under study. Conclusion . The carried out research shows that caspase-3 and protein-bound glutathione are the molecular targets of selective control over programmed cell death. The received indices of caspase-3 activity change and protein-bound glutathione concentration alteration in blood lymphocytes during oxidative stress in vitro can be used when elaborating target therapy approaches to diseases accompanied by apoptosis disregulation.

THE ROLE OF PROTEIN OXIDATIVE MODIFICATION IN REDOX-REGULATION OF CASPASE-3 ACTIVITY IN BLOOD LYMPHOCYTES DURING OXIDATIVE STRESS IN VITRO

The formation of oxidative stress lies at the heart of many frequent and socially-important diseases. Blood lymphocytes are the cells which provide immunological control of our organism. As a result of their function implementation blood lymphocytes contact with different endogenic and exogenic factors, which can lead to active oxygen species production activation, macromolecules oxidative modification and to cell survival alteration. At the present time it is essential to expand and deepen the fundamental knowledge of blood lymphocytes apoptosis regulation peculiarities. The research objective was to establish the interaction among alterations of glutathione system condition, carbonylation level, protein glutathionylation and caspase-3 activity in blood lymphocytes during oxidative stress in vitro.Material and Methods. The material for research was blood lymphocytes cultivated with addition of hydrogen peroxide in final concentration of 0,5 mmol and/or protein SH-group inhibitor N-ethylmaleimide – 5 mmol, protector – 5 mmol – 1,4-dithioerythritol. Reduced, oxidized and protein-bound glutathione concentration was measured by method of spectropho-tometry, additionally, the ratio size of reduced to oxidized thiol fraction was estimated. With help of enzymoimmunoassay the level of protein carbonyl derivatives was evaluated; caspase-3 activity was registered by spectrofluorometric method.Results. Protein SH-group blocking in blood lymphocytes during oxidative stress in vitro was accompanied by protein-bound glutathione concentration rapid decrease in connection with increase of protein carbonyl derivatives content and caspase-3 activity. Protein SH-group protection in blood lymphocytes during oxidative stress in vitro was accompanied by concentration increase of protein-bound glutathione and protein carbonyl derivatives under comparable values of enzyme activity under study.Conclusion. The carried out research shows that caspase-3 and protein-bound glutathione are the molecular targets of selective control over programmed cell death. The received indices of caspase-3 activity change and protein-bound glutathione concentration alteration in blood lymphocytes during oxidative stress in vitro can be used when elaborating target therapy approaches to diseases accompanied by apoptosis disregulation.

Effect of freezing rate for cryopreservation of Persian sturgeon (Acipenser persicus) spermatozoa

This study examined the effect of freezing rate (−10 °C, −15 °C, −20 °C, −30 °C, and −40 °C/min) on motility parameters, rates of fertilization and hatching, ATP content, and indices of oxidative stress including thiobarbituric acid reactive substances and carbonyl derivatives of proteins in Persian sturgeon (Acipenser persicus) sperm. After sampling, sperm was diluted in an extender composed of 23.4-mM sucrose, 0.25-mM KCl, and 30-mM Tris-HCl, pH 8.0, containing 10% methanol and subsequently frozen in a programmable freezer. For postthaw sperm that were frozen at a rate of −40 °C/min, sperm motile duration (134 ± 27.01 seconds), sperm motile percent (60 ± 4.1%), fertilizability (72 ± 8.36% for fertilization rate and 65 ± 7.58% for hatching rate), and ATP content (4.8 ± 0.57 nmol/108 sperm) were significantly higher than for sperm frozen at any of the four slower rates (P < 0.05). Moreover, sperm cryopreserved using the fastest freezing rate had significantly lower levels of thiobarbituric acid reactive substances (0.5 ± 0.05 nmol/108 sperm) and carbonyl derivatives of proteins (41.3 ± 4.9 nmol/108 sperm) than sperm cryopreserved using all other freezing rates (P < 0.05). In addition, there is a significant difference (P < 0.05) between fresh sperm and the recovery of cryopreserved Persian sturgeon sperm using programmable freezing with −40 °C/min being the optimal freezing rate among those tested.

The study of cardiomyocytes mitochondrial dysfunction in hyperhomocysteinemia and nitric oxyde deficiency

Aim. To study the influence of nitric oxide metabolism disturbance on the development of mitochondrial dysfunction in case of hyperhomocysteinemia.&#x0D; Methods. The research was conducted on 32 Wistar male rats. Hyperhomocysteinemia was simulated by intragastric injection of methionine suspension prepared using starch and Tween-80 with addition of this amino acid into the drinking water. The nitric oxide deficiency was induced by intraperitoneal injection of L-Nω-nitroarginine methyl ester (L-NAME) solution.&#x0D; Results. Hyperhomocysteinemia is accompanied by dysfunction of cardiac cells mitochondria, manifesting in growth of cytoplasmic lactate level and development of oxidative stress with increased mitochondrial proteins carbonylation. Oxidative stress is largely compensated by the activation of the antioxidant defense system (including superoxide dismutase), as evidenced by a slight decrease of succinate dehydrogenase and H+-ATPase activity, the absence of statistically significant changes of cytoplasmic lactate dehydrogenase activity. Tween-80 showed antioxidant properties, reducing the content of protein carbonyl derivatives and superoxide dismutase activity. Nitric oxide deficiency caused by the L-NAME injection was accompanied by an inhibition of aerobic oxidation processes in cardiomyocytes mitochondria, which was proved by a significant decrease in succinate dehydrogenase activity as well as slight reduction of lactate dehydrogenase activity and lactate accumulation in the cytoplasm, and an oxidative phosphorylation reduction which manifested with a decrease of H+-ATPase activity. One reason for these changes is increased carbonylation of proteins due to high production of reactive oxygen species, which is not sufficiently compensated by increased activity of superoxide dismutase.&#x0D; Conclusion. Since hyperhomocysteinemia is associated with reduced concentrations of nitric oxide metabolites in cardiomyocytes mitochondria, and changes in these organelles after the administering of methionine have some similarities with those after injection of L-NAME, it can be argued that nitric oxide deficiency plays an important role in the pathogenesis of mitochondrial dysfunction of cardiomyocytes in case of hyperhomocysteinemia.

The influence of cryoprotectants on sturgeon (Acipenser ruthenus) sperm quality, DNA integrity, antioxidant responses, and resistance to oxidative stress

This study examined the effect of cryoprotectants on DNA integrity, antioxidant defense, and resistance to oxidative stress in cryopreserved sterlet Acipenser ruthenus sperm. The freeze-thaw process significantly influenced sperm motility, with significant differences among cryoprotectants. In vitro exposure of cryopreserved sperm to the xanthine–xanthine oxidase (X–XO) system as a model reactive oxygen species inducer resulted in a lesser motility rate and velocity compared to the control, and there was a decrease in these variables in a time- and dose-dependent manner. The greatest X (0.6mM)–XO (0.05U/mL) concentration and incubation period (30min) was associated with 62% DNA fragmentation in sperm cryopreserved with 10% ethylene glycol (EG). The maximum lipid peroxidation (LPO) and carbonyl derivatives of proteins (CP) was also observed in sperm cryopreserved with 10% EG and exposed to the X–XO system at a concentration of 0.6mM X–0.05U/mL XO. The frozen/thawed sperm containing 10% EG and that with 10% dimethyl sulfoxide (DMSO) had a significant enhancement of superoxide dismutase (SOD) and glutathione reductase (GR) activity. The current study confirms that EG is not effective for cryopreservation, and sterlet sperm were highly sensitive to free radicals after cryopreservation with EG.

Oxidation as an important factor of protein damage: Implications for Maillard reaction.

Protein oxidation, the process caused especially by reactive oxygen and nitrogen species, is thought to play a major role in various oxidative processes within cells and is implicated in the development of many human diseases. This review provides a brief overview of the protein oxidation with the emphasis on the types of oxidation (oxidation of protein backbone and amino acid residues side chains, site-specific metal-catalysed protein oxidation), oxidationdependent generation of protein hydroperoxides, carbonyl derivatives and protein-protein cross-linkages. Nonenzymatic glycoxidation (also known as Maillard reaction) as an important factor of protein damage, consequences of oxidative protein impairment and related diseases as well as means of monitoring and assessment of protein modifications are discussed.

Protein carbonylation during natural leaf senescence in winter wheat, as probed by fluorescein-5-thiosemicarbazide.

Leaf senescence is characterised by a massive degradation of proteins in order to recycle nitrogen to other parts of the plant, such as younger leaves or developing grain/seed. Protein degradation during leaf senescence is a highly regulated process and it is suggested that proteins to be degraded are marked by an oxidative modification (carbonylation) that makes them more susceptible to proteolysis. However, there is as yet no evidence of an increase in protein carbonylation level during natural leaf senescence. The aim of our study was thus to monitor protein carbonylation level during the process of natural senescence in the flag leaf of field-grown winter wheat plants. For this purpose, we adapted a fluorescence-based method using fluorescein-5-thiosemicarbazide (FTC) as a probe for detecting protein carbonyl derivatives. As used for the first time on plant material, this method allowed the detection of both quantitative and qualitative modifications in protein carbonyl levels during the last stages of wheat flag leaf development. The method described herein represents a convenient, sensitive and reproducible alternative to the commonly used 2,4-dinitrophenylhydrazine (DNPH)-based method. In addition, our analysis revealed changes in protein carbonylation level during leaf development that were associated with qualitative changes in protein abundance and carbonylation profiles. In the senescing flag leaf, protein carbonylation increased concomitantly with a stimulation of endoproteolytic activity and a decrease in protein content, which supports the suggested relationship between protein oxidation and proteolysis during natural leaf senescence.

Effects of competition on acute phase proteins and lymphocyte subpopulations - oxidative stress markers in eventing horses.

The aim of the study was to evaluate markers of the acute phase response (APR) in eventing horses by measuring acute phase proteins (APP) (haptoglobin, Hp, and serum amyloid A, SAA), lysozyme, protein adducts such as pentosidine-like adducts (PENT), malondialdehyde adducts (MDA), hydroxynonenal adducts (HNE) and total advanced glycation/glycoxidation end products (AGEs), complete blood count and lymphocyte subpopulations (CD4+, CD8+ and CD21+) both at rest and at the end of an eventing competition. Blood samples were collected from eight Warmblood horses (medium age 10±3) during an official national 2-day event competition at rest (R) and 10min after the arrival of the cross-country test on the second day. Exercise caused a significant increase in red blood cell number, haemoglobin, packed cell volume, neutrophils, white blood cell and lymphocyte number; however, these values remained within the normal range. The CD4+ and CD8+ cells significantly increased, whereas the CD21+ lymphocytes decreased; a significant increase in serum SAA, lysozyme and protein carbonyl derivates was also observed. Two-day event causes significant changes in APR markers such as lysozyme, protein carbonyl derivates (HNE, AGEs, PENT) and lymphocyte subpopulations. The data support the hypothesis that 2-day event may alter significantly APR markers. Limitations of the study were the relatively small sample size and sampling time conditioned by the official regulations of the event. Therefore, further studies are needed to investigate the time required for recovery to basal values in order to define the possible effects on the immune function of the athlete horse.

P43 - The modified proteins in erythrocytes at hemodialysis patients

The main goal of our research was to study the modified proteins and activity of integral band 3 protein in erythrocytes of patients with end-stage renal failure during hemodialysis. Depending on the starting nosological variant 2 groups were formed. The first group included 27 patients with end-stage renal failurein the outcome of chronic pyelonephritis, the second – 20 patients with end-stage renal failure, developed on a background of chronic glomerulonephritis. The control group consisted of 15 healthy subjects. The patients of 1-th and 2-nd groups were treated with hemodialysis. The concentration of reactive protein carbonyl derivates, membrane-binding hemoglobin were detected in erythrocytes. Activity of band 3 protein was detected following the protocol of I. Mindukshev et al. (2010). Comparison the results obtained was performed using non-parametric Mann-Whitney U-test (for independent variables).Before hemodialysis the significant increasing of concentration of reactive protein carbonyl derivates and membrane-binding hemoglobin in erythrocytes of both group patients in comparison with healthy ones was observed. During hemodialysis the further augmentation of modified proteins in erythrocytes took place.Different changes in the activity of the protein band 3 in erythrocytes of patients during hemodialysis were found. The similar type of protein band 3 activity changing was determined in erythrocytes of 1-th group patients. There was positive pair correlation between concentration of the reactive protein carbonyl derivates and protein band 3 activity (r= 0,31, p<0,05). Two different types of protein band 3 activity changing were determined in erythrocytes of 2-th group patients. Thus, the differences in modified proteins concentration and protein band 3 activity that occurred in the erythrocytes of patients during hemodialysis were established.