Persistent S-nitrosation of complex I and other mitochondrial membrane proteins by S-nitrosothiols but not nitric oxide or peroxynitrite: implications for the interaction of nitric oxide with mitochondria

S-nitrosation of mitochondrial proteins has been proposed to contribute to the pathophysiological interactions of nitric oxide (NO) and its derivatives with mitochondria but has not been shown directly. Furthermore, little is known about the mechanism of formation or the fate of these putative S-nitrosothiols. Here we have determined whether mitochondrial membrane protein thiols can be S-nitrosated on exposure to free NO from 3,3-bis(aminoethyl)-1-hydroxy-2-oxo-1-triazene (DETA-NONOate) by interaction with S-nitrosoglutathione or S-nitroso-N-acetylpenicillamine (SNAP) and by the NO derivative peroxynitrite. S-Nitrosation of protein thiols was measured directly by chemiluminescence detection. S-Nitrosoglutathione and S-nitroso-N-acetylpenicillamine led to extensive protein thiol oxidation, with about 30% of the modified protein thiols persistently S-nitrosated. In contrast, there was no protein thiol oxidation or S-nitrosation on exposure to 3,3-bis (aminoethyl)-1-hydroxy-2-oxo-1-triazene. Peroxynitrite extensively oxidized protein thiols but produced negligible amounts of S-nitrosothiols. Therefore, mitochondrial membrane protein thiols are S-nitrosated by preformed S-nitrosothiols but not by NO or by peroxynitrite. These S-nitrosated protein thiols were readily reduced by glutathione, so S-nitrosation will only persist when the mitochondrial glutathione pool is oxidized. Respiratory chain complex I was S-nitrosated by S-nitrosothiols, consistent with it being an important target for S-nitrosation during nitrosative stress. The S-nitrosation of complex I correlated with a significant loss of activity that was reversed by thiol reductants. S-Nitrosation was also associated with increased superoxide production from complex I. These findings point to a significant role for complex I S-nitrosation and consequent dysfunction during nitrosative stress in disorders such as Parkinson disease and sepsis.     

Learning from Small Fry: The Zebrafish as a Genetic Model Organism for Aquaculture Fish Species

In recent years, the zebrafish has become one of the most prominent vertebrate model organisms used to study the genetics underlying development, normal body function, and disease. The growing interest in zebrafish research was paralleled by an increase in tools and methods available to study zebrafish. While zebrafish research initially centered on mutagenesis screens (forward genetics), recent years saw the establishment of reverse genetic methods (morpholino knock-down, TILLING). In addition, increasingly sophisticated protocols for generating transgenic zebrafish have been developed and microarrays are now available to characterize gene expression on a near genome-wide scale. The identification of loci underlying specific traits is aided by genetic, physical, and radiation hybrid maps of the zebrafish genome and the zebrafish genome project. As genomic resources for aquacultural species are increasingly being generated, a meaningful interaction between zebrafish and aquacultural research now appears to be possible and beneficial for both sides. In particular, research on nutrition and growth, stress, and disease resistance in the zebrafish can be expected to produce results applicable to aquacultural fish, for example, by improving husbandry and formulated feeds. Forward and reverse genetics approaches in the zebrafish, together with the known conservation of synteny between the species, offer the potential to identify and verify candidate genes for quantitative trait loci (QTLs) to be used in marker-assisted breeding. Moreover, some technologies from the zebrafish field such as TILLING may be directly transferable to aquacultural research and production.     

Age shall not weary us: deleterious effects of self-regulation depletion are specific to younger adults

Self-regulation depletion (SRD), or ego-depletion, refers to decrements in self-regulation performance immediately following a different self-regulation-demanding activity. There are now over a hundred studies reporting SRD across a broad range of tasks and conditions. However, most studies have used young student samples. Because prefrontal brain regions thought to subserve self-regulation do not fully mature until 25 years of age, it is possible that SRD effects are confined to younger populations and are attenuated or disappear in older samples. We investigated this using the Stroop color task as an SRD induction and an autobiographical memory task as the outcome measure. We found that younger participants (<25 years) were susceptible to depletion effects, but found no support for such effects in an older group (40-65 years). This suggests that the widely-reported phenomenon of SRD has important developmental boundary conditions casting doubt on claims that it represents a general feature of human cognition.     

Differences in Colonization and Shedding Patterns after Oral Challenge of Cattle with Three Escherichia coli O157:H7 Strains

Experimental oral challenge studies with three different genotypes of Escherichia coli O157:H7 were conducted in cattle to determine the genotype-specific variability in shedding frequencies and concentrations and the frequency and extent of contamination of the environment. The results indicated that the E. coli O157:H7 genotype and ecological origin maybe important factors for the occurrence and concentration in the cattle host. Four groups of six young Holstein steers each were orally challenged with 10⁶ CFU of one of three E. coli O157:H7 strains: FRIK 47 (groups 1 and 2), FRIK 1641 (group 3), and FRIK 2533 (group 4). Recto-anal mucosal swabs (RAMS) and environmental samples were taken on alternate days over 30 days. The numbers of E. coli O157:H7 cells and generic E. coli cells per sample were determined. Also, the presence and absence of 28 gene targets were determined for 2,411 isolates using high-throughput real-time PCR. Over the study period, strains FRIK 47, FRIK 1641, and FRIK 2533 were detected in 52%, 42%, and 2% of RAMS, respectively. Environmental detection of the challenge strains was found mainly in samples of the hides and pen floors, with strains FRIK 47, FRIK 1641, and FRIK 2533 detected in 22%, 27%, and 0% of environmental samples, respectively. Based on the panel of 28 gene targets, genotypes of enterohemorrhagic E. coli (EHEC) and generic E. coli from the experimental samples were clustered into three subgroups. In conclusion, the results suggested that the type and intensity of measures to control this pathogen at the preharvest level may need to be strain specific.     

4-O-α-d-galactopyranosyl-d-galactose: Efficient synthetic routes from “polygalacturonic acid”

Enzymic hydrolysis of “polygalacturonic acid” gave a mixture of oligomers which was fractionated by ion-exchange chromatography. The resulting di- and tri-saccharides were treated, respectively, with methanol and ethylene oxide, and the resulting esters were reduced with sodium borohydride. Treatment of the products with acetic anhydride and sulfuric acid, followed by deacetylation, produced the title compound.     

Combination of capillary electrophoresis, PCR and physiological assays in differentiation of clinical strains of Staphylococcus aureus

Fast, sensitive and cheap determination of pathogenic bacteria is extremely important in many branches, for example biotechnology, quality control, analysis of samples and antimicrobial therapy. The development and application of analytical techniques in practice could provide new possibilities in this regard. The bacterial pathogen Staphylococcus aureus is responsible for a significant amount of human morbidity and mortality. Rapid and sensitive determination is therefore very important. In the present study, novel methods, based on capillary zone electrophoresis and (as confirmation of these results) molecular analysis of a part of the coag gene, were developed for identification and differentiation of three S. aureus strains. The electrophoretic measurements rely on the differential mobility of bacteria in the fused silica capillary under the direct current electric field. To perform coagulase gene typing, the repeated units encoding hypervariable regions of the S. aureus gene were amplified using the PCR technique followed by restriction enzyme digestion and analysis of restriction fragment length polymorphism patterns as well as sequencing. Finally, the results of electrophoretic measurements with molecular analysis were compared.     

Coupled biogeochemical and hydrological responses of streams and rivers to drought

• 1 Severe or extreme droughts occurred about 10% of the time over a 105‐year record from central New Mexico, U.S.A., based on the Palmer Drought Severity Index.
• 2 Drought lowers water tables, creating extensive areas of groundwater recharge and fragmenting reaches of streams and rivers. Deeper groundwater inputs predominate as sources of surface flows during drought. Nutrient inputs to streams and rivers reflect the biogeochemistry of regional ground waters with longer subsurface residence times.
• 3 Inputs of bioavailable dissolved organic carbon to surface waters decrease during drought, with labile carbon limitation of microbial metabolism a byproduct of drought conditions.
• 4 Decreased inputs of organic forms of carbon, nitrogen and phosphorus and a decrease in the organic : inorganic ratio of nutrient inputs favours autotrophs over heterotrophs during drought.
• 5 The fate of autotrophic production during drought will be strongly influenced by the structure of the aquatic food web within impacted sites.