Oxidative stress and nitrite dynamics under maximal load in elite athletes: relation to sport type

Maximal workload in elite athletes induces increased generation of reactive oxygen/nitrogen species (RONS) and oxidative stress, but the dynamics of RONS production are not fully explored. The aim of our study was to examine the effects of long-term engagement in sports with different energy requirements (aerobic, anaerobic, and aerobic/anaerobic) on oxidative stress parameters during progressive exercise test. Concentrations of lactates, nitric oxide (NO) measured through stabile end product-nitrites (NO₂ ^?), superoxide anion radical (O₂ ^??), and thiobarbituric reactive substances (TBARS) as index of lipid peroxidation were determined in rest, after maximal workload, and at 4 and 10th min of recovery in blood plasma of top level competitors in rowing, cycling, and taekwondo. Results showed that sportmen had similar concentrations of lactates and O₂ ^?? in rest. Nitrite concentrations in rest were the lowest in taekwondo fighters, while rowers had the highest levels among examined groups. The order of magnitude for TBARS level in the rest was bicycling > taekwondo > rowing. During exercise at maximal intensity, the concentration of lactate significantly elevated to similar levels in all tested sportsmen and they were persistently elevated during recovery period of 4 and 10 min. There were no significant changes in O₂ ^??, nitrite, and TBARS levels neither at the maximum intensity of exercise nor during the recovery period comparing to the rest period in examined individuals. Our results showed that long term different training strategies establish different basal nitrites and lipid peroxidation levels in sportmen. However, progressive exercise does not influence basal nitrite and oxidative stress parameters level neither at maximal load nor during the first 10 min of recovery in sportmen studied.     

Phytoextraction of Cd and Zn from agricultural soils by Salix ssp. and intercropping of Salix caprea and Arabidopsis halleri

Contamination of agricultural topsoils with Cd above guideline values is of concern in many countries throughout the world. Extraction of metals from contaminated soils using high-biomass, metal-accumulating Salix sp. has been proposed as a low-cost, gentle remediation strategy, but reasonable phytoextraction rates remain to be demonstrated. In an outdoor pot experiment we assessed the phytoextraction potential for Cd and Zn of four willow species (Salix caprea, S. fragilis, S. × smithiana, S. × dasyclados) and intercropping of S. caprea with the hyperaccumulator Arabidopsis halleri on three moderately contaminated, agricultural soils. Large concentrations of Cd (250 mg kg⁻¹) and Zn (3,300 mg kg⁻¹) were determined in leaves of Salix × smithiana grown on a soil containing 13.4 mg kg⁻¹ Cd and 955 mg kg⁻¹ Zn, resulting in bioaccumulation factors of 27 (Cd) and 3 (Zn). Total removal of up to 20% Cd and 5% Zn after three vegetation periods were shown for Salix × smithiana closely followed by S. caprea, S. fragilis and S. × dasyclados. While total Cd concentrations in soils were reduced by up to 20%, 1 M NH₄NO₃-extractable metal concentrations did not significantly decrease within 3 years. Intercropping of S. caprea and A. halleri partly increased total removal of Zn, but did not enhance total Cd extraction compared to single plantings of S. caprea after two vegetation periods.     

Chronic effects of platinum(IV) complex and its diamine ligand on rat heart function: comparison with cisplatin

Mitochondrial dysfunction plays crucial role in the pathologenesis of myocardial infarction (MI). The present study evaluated the protective effect of α-bisabolol against isoproterenol (ISO)-induced mitochondrial dysfunction and apoptosis in rats. Male albino Wistar rats were pre- and co-treated with intraperitoneal injection of α-bisabolol (25 mg/kg body weight) daily for 10 days. To induce experimental MI, ISO (85 mg/kg body weight) was injected subcutaneously to the rats at an interval of 24 h for 2 days (9th and 10th day). ISO-induced MI was indicated by the decreased activities of heart creatine kinase and lactate dehydrogenase in rats. ISO administration also enhanced the concentrations of heart mitochondrial lipid peroxidation products and decreased the activities/concentrations of mitochondrial antioxidants, Kreb’s cycle dehydrogenases and mitochondrial electron transport chain complexes I, II?+?III and IV in rats. Furthermore, ISO triggers calcium overload and ATP depletion in the rat’s heart mitochondria followed by the mitochondrial cytochrome-C release and the activation of intrinsic pathway of apoptosis by upregulating the myocardial pro-apoptotic Bax, P⁵³, APAF-1, active caspase-3, active caspase-9 and down regulating the expressions of anti-apoptotic Bcl-2. α-Bisabolol pre and co-treatment showed considerable protective effects on all the biochemical and molecular parameters studied. Transmission electron microscopic study and mitochondrial swelling assay confirmed our biochemical and molecular findings. The in vitro study on hydroxyl radical also revealed the potent free radical scavenging activity of α-bisabolol. Thus, α-bisabolol attenuates mitochondrial dysfunction and intrinsic pathway of apoptosis in ISO-induced myocardial infarcted rats.

     

Exopolysaccharide Production and Ropy Phenotype Are Determined by Two Gene Clusters in Putative Probiotic Strain Lactobacillus paraplantarum BGCG11

Lactobacillus paraplantarum BGCG11, a putative probiotic strain isolated from a soft, white, artisanal cheese, produces a high-molecular-weight heteropolysaccharide, exopolysaccharide (EPS)-CG11, responsible for the ropy phenotype and immunomodulatory activity of the strain. In this study, a 26.4-kb region originating from the pCG1 plasmid, previously shown to be responsible for the production of EPS-CG11 and a ropy phenotype, was cloned, sequenced, and functionally characterized. In this region 16 putative open reading frames (ORFs), encoding enzymes for the production of EPS-CG11, were organized in specific loci involved in the biosynthesis of the repeat unit, polymerization, export, regulation, and chain length determination. Interestingly, downstream of the eps gene cluster, a putative transposase gene was identified, followed by an additional rfb gene cluster containing the rfbACBD genes, the ones most probably responsible for dTDP-l-rhamnose biosynthesis. The functional analysis showed that the production of the high-molecular-weight fraction of EPS-CG11 was absent in two knockout mutants, one in the eps and the other in the rfb gene cluster, as confirmed by size exclusion chromatography analysis. Therefore, both eps and rfb genes clusters are prerequisites for the production of high-molecular-weight EPS-CG11 and for the ropy phenotype of strain L. paraplantarum BGCG11.     

Exercise-induced changes in redox status of elite karate athletes

Regular training has been claimed to increase the activity of antioxidant enzymes and, consequently, augments the resistance to oxidative stress; however, large volumes of training performed by elite sportsmen could lead to a chronic oxidative stress state. The aim of our study was to assess the oxidative status of elite athletes at the beginning of the preparatory and the beginning of the competition training phases, so that the influence of three months of programmed physical activity on redox status could be determined. The chronic effects of exercise on the redox state of the athletes were compared to the effects of a single bout of karate training. Thirty elite karate athletes, 16-30 years old, were subjected to maximal graded exercise test to estimate their aerobic capacity; blood sampling was also performed to measure levels of superoxide anion radical (O??), hydrogen peroxide (H?O?), superoxide dismutase activity (SOD) and catalase activity (CAT). The only significant change after the three-month training process was found in the significantly decreased CAT activity (X ± SE: 7.95 ± 0.13 U/g Hb × 103 in the preparatory period, 6.65 ± 0.28 U/g Hb × 103 in the competition stage; P < 0.01). After a single karate training session, there was statistically significant decrease of O??(X ± SE: 32.7 ± 4.9 nmol/ml in the preparatory period, 24.5 ± 2.5 nmol/ml in the competition stage; P < 0.05) and increase of H?O?(X ± SE: 11.8 ± 1.0 nmol/ml in the preparatory period, 14.2 ± 0.9 nmol/ml in the competition stage; P < 0.01), as well as significant CAT increase (X ± SE: 6.6 ± 0.6 U/g Hb × 103 in the preparatory period, 8.5 ± 0.5 U/g Hb × 103 in the competition stage; P < 0.05). Although the three-month training process induced, at the first sight, negative changes in the redox state, expressed through the decrease in CAT activity, adequate response of the antioxidant system of our athletes to acute exercise was preserved.     

Mim1, a protein required for the assembly of the TOM complex of mitochondria

The translocase of the outer mitochondrial membrane (TOM complex) is the general entry site for newly synthesized proteins into mitochondria. This complex is essential for the formation and maintenance of mitochondria. Here, we report on the role of the integral outer membrane protein, Mim1 (mitochondrial import), in the biogenesis of mitochondria. Depletion of Mim1 abrogates assembly of the TOM complex and results in accumulation of Tom40, the principal constituent of the TOM complex, as a low-molecular-mass species. Like all mitochondrial beta-barrel proteins, the precursor of Tom40 is inserted into the outer membrane by the TOB complex. Mim1 is likely to be required for a step after this TOB-complex-mediated insertion. Mim1 is a constituent of neither the TOM complex nor the TOB complex; rather, it seems to be a subunit of another, as yet unidentified, complex. We conclude that Mim1 has a vital and specific function in the assembly of the TOM complex.     

Exploration of factors driving incorporation of unnatural dNTPS into DNA by Klenow fragment (DNA polymerase I) and DNA polymerase alpha

In order to further understand how DNA polymerases discriminate against incorrect dNTPs, we synthesized two sets of dNTP analogues and tested them as substrates for DNA polymerase α (pol α) and Klenow fragment (exo⁻) of DNA polymerase I (Escherichia coli). One set of analogues was designed to test the importance of the electronic nature of the base. The bases consisted of a benzimidazole ring with one or two exocyclic substituent(s) that are either electron-donating (methyl and methoxy) or electron-withdrawing (trifluoromethyl and dinitro). Both pol α and Klenow fragment exhibit a remarkable inability to discriminate against these analogues as compared to their ability to discriminate against incorrect natural dNTPs. Neither polymerase shows any distinct electronic or steric preferences for analogue incorporation. The other set of analogues, designed to examine the importance of hydrophobicity in dNTP incorporation, consists of a set of four regioisomers of trifluoromethyl benzimidazole. Whereas pol α and Klenow fragment exhibited minimal discrimination against the 5- and 6-regioisomers, they discriminated much more effectively against the 4- and 7-regioisomers. Since all four of these analogues will have similar hydrophobicity and stacking ability, these data indicate that hydrophobicity and stacking ability alone cannot account for the inability of pol α and Klenow fragment to discriminate against unnatural bases. After incorporation, however, both sets of analogues were not efficiently elongated. These results suggest that factors other than hydrophobicity, sterics and electronics govern the incorporation of dNTPs into DNA by pol α and Klenow fragment.     

Kinetic Origin of Substrate Specificity in Post-Transfer Editing by Leucyl-tRNA Synthetase

The intrinsic editing capacities of aminoacyl-tRNA synthetases ensure a high-fidelity translation of the amino acids that possess effective non-cognate aminoacylation surrogates. The dominant error-correction pathway comprises deacylation of misaminoacylated tRNA within the aminoacyl-tRNA synthetase editing site. To assess the origin of specificity of Escherichia coli leucyl-tRNA synthetase (LeuRS) against the cognate aminoacylation product in editing, we followed binding and catalysis independently using cognate leucyl- and non-cognate norvalyl-tRNA^Leu and their non-hydrolyzable analogues. We found that the amino acid part (leucine versus norvaline) of (mis)aminoacyl-tRNAs can contribute approximately 10-fold to ground-state discrimination at the editing site. In sharp contrast, the rate of deacylation of leucyl- and norvalyl-tRNA^Leu differed by about 10⁴-fold. We further established the critical role for the A76 3′-OH group of the tRNA^Leu in post-transfer editing, which supports the substrate-assisted deacylation mechanism. Interestingly, the abrogation of the LeuRS specificity determinant threonine 252 did not improve the affinity of the editing site for the cognate leucine as expected, but instead substantially enhanced the rate of leucyl-tRNA^Leu hydrolysis. In line with that, molecular dynamics simulations revealed that the wild-type enzyme, but not the T252A mutant, enforced leucine to adopt the side-chain conformation that promotes the steric exclusion of a putative catalytic water. Our data demonstrated that the LeuRS editing site exhibits amino acid specificity of kinetic origin, arguing against the anticipated prominent role of steric exclusion in the rejection of leucine. This feature distinguishes editing from the synthetic site, which relies on ground-state discrimination in amino acid selection.     

Essential role for the d-Asb11 cul5 Box domain for proper notch signaling and neural cell fate decisions in vivo

ECS (Elongin BC-Cul2/Cul5-SOCS-box protein) ubiquitin ligases recruit substrates to E2 ubiquitin-conjugating enzymes through a SOCS-box protein substrate receptor, an Elongin BC adaptor and a cullin (Cul2 or Cul5) scaffold which interacts with the RING protein. In vitro studies have shown that the conserved amino acid sequence of the cullin box in SOCS-box proteins is required for complex formation and function. However, the in vivo importance of cullin boxes has not been addressed. To explore the biological functions of the cullin box domain of ankyrin repeat and SOCS-box containing protein 11 (d-Asb11), a key mediator of canonical Delta-Notch signaling, we isolated a zebrafish mutant lacking the Cul5 box (Asb11(Cul)). We found that homozygous zebrafish mutants for this allele were defective in Notch signaling as indicated by the impaired expression of Notch target genes. Importantly, asb11(Cul) fish were not capable to degrade the Notch ligand DeltaA during embryogenesis, a process essential for the initiation of Notch signaling during neurogenesis. Accordingly, proper cell fate specification within the neurogenic regions of the zebrafish embryo was impaired. In addition, Asb11(Cul) mRNA was defective in the ability to transactivate a her4::gfp reporter DNA when injected in embryos. Thus, our study reporting the generation and the characterization of a metazoan organism mutant in the conserved cullin binding domain of the SOCS-box demonstrates a hitherto unrecognized importance of the SOCS-box domain for the function of this class of cullin-RING ubiquitin ligases and establishes that the d-Asb11 cullin box is required for both canonical Notch signaling and proper neurogenesis.