Model for the exceptional reactivity of peroxiredoxins 2 and 3 with hydrogen peroxide: a kinetic and computational study

Peroxiredoxins (Prx) are thiol peroxidases that exhibit exceptionally high reactivity toward peroxides, but the chemical basis for this is not well understood. We present strong experimental evidence that two highly conserved arginine residues play a vital role in this activity of human Prx2 and Prx3. Point mutation of either ArgI or ArgII (in Prx3 Arg-123 and Arg-146, which are ∼3–4 Å or ∼6–7 Å away from the active site peroxidative cysteine (C_p), respectively) in each case resulted in a 5 orders of magnitude loss in reactivity. A further 2 orders of magnitude decrease in the second-order rate constant was observed for the double arginine mutants of both isoforms, suggesting a cooperative function for these residues. Detailed ab initio theoretical calculations carried out with the high level G4 procedure suggest strong catalytic effects of H-bond-donating functional groups to the C_p sulfur and the reactive and leaving oxygens of the peroxide in a cooperative manner. Using a guanidinium cation in the calculations to mimic the functional group of arginine, we were able to locate two transition structures that indicate rate enhancements consistent with our experimentally observed rate constants. Our results provide strong evidence for a vital role of ArgI in activating the peroxide that also involves H-bonding to ArgII. This mechanism could explain the exceptional reactivity of peroxiredoxins toward H₂O₂ and may have wider implications for protein thiol reactivity toward peroxides.     

NDUFB7 and NDUFA8 are located at the intermembrane surface of complex I

Complex I (NADH:ubiquinone oxidoreductase) is the first and largest protein complex of the oxidative phosphorylation. Crystal structures have elucidated the positions of most subunits of bacterial evolutionary origin in the complex, but the positions of the eukaryotic subunits are unknown. Based on the analysis of sequence conservation we propose intra-molecular disulfide bridges and the inter-membrane space localization of three Cx(9)C-containing subunits in human: NDUFS5, NDUFB7 and NDUFA8. We experimentally confirm the localization of the latter two, while our data are consistent with disulfide bridges in NDUFA8. We propose these subunits stabilize the membrane domain of complex I.     

PCR and real time PCR for the detection of Cryptosporidium parvum oocyst DNA

Three DNA extraction kits were used, all without preliminary procedures, then DNA extraction was preceded with freeze/thaw cycles in three versions. A lack of desired effect resulted in the application of liquid nitrogen/water bath cycles before the use of the extractions in further experiments. The effectiveness of DNA extraction was measured by PCR signal and C(T) values of real time PCR. A comparison of the efficiency of various Cryptosporidium parvum undiluted oocyst treatments prior to DNA extraction with the use of three kits has shown that the best results were obtained after extraction of DNA with the QIAamp DNA Tissue Mini Kit (T kit), preceded by triple liquid nitrogen/water bath in 100 degrees C for 2 minutes and with overnight proteinase K digestion. After extraction with the T kit, the detection limit was 50 oocysts per 200 microl when effectiveness was evaluated with PCR and 10 oocysts in the case of real time PCR.     

Density-dependent and inter-specific interactions affecting European eel settlement in freshwater habitats

Identifying the factors influencing the settlement of European eel (Anguilla anguilla) juveniles in continental habitats is crucial to designing effective management and conservation measures for this endangered species. A long-term data series (1993–2008) of European eel and European catfish (Silurus glanis) abundance in a freshwater canal of the Camargue water system (southern France), along with parallel data on water salinity and glass eel abundance in the adjacent Vaccarès lagoon, was analysed to identify the possible causes of decline in eel abundance observed in the canal during the last two decades. A model including glass eel recruitment and catfish abundance as covariates explained 78% of the observed variation in eel settlement success. Results suggest that (1) salinity does not play a significant role in determining the fraction of eels moving from the brackish lagoon to the canal; (2) density dependence affects settlement success, possibly through a reduction of juvenile survival in the adjacent lagoon; and (3) catfish abundance is negatively correlated with eel settlement. We discuss this latter point in terms of possible predation of catfish upon eels and/or inter-specific competition between the two species.     

An anti-inflammatory role for carbon monoxide and heme oxygenase-1 in chronic Th2-mediated murine colitis

Cigarette smoking is a significant environmental factor in the human inflammatory bowel diseases, remarkably, conferring protection in ulcerative colitis. We previously demonstrated that a prominent component of cigarette smoke, CO, suppresses Th17-mediated experimental colitis in IL-10(-/-) mice through a heme oxygenase (HO)-1-dependent pathway. In this study, homeostatic and therapeutic effects of CO and HO-1 were determined in chronic colonic inflammation in TCR-α-deficient ((-/-)) mice, in which colitis is mediated by Th2 cytokines, similar to the cytokine milieu described in human ulcerative colitis. TCRα(-/-) mice exposed to CO or treated with the pharmacologic HO-1 inducer cobalt protoporphyrin demonstrated amelioration of active colitis. CO and cobalt protoporphyrin suppressed colonic IL-1β, TNF, and IL-4 production, whereas IL-10 protein secretion was increased. CO induced IL-10 expression in macrophages and in vivo through an HO-1-dependent pathway. Bacterial products regulate HO-1 expression in macrophages through MyD88- and IL-10-dependent pathways. CO exposure and pharmacologic HO-1 induction in vivo resulted in increased expression of HO-1 and IL-10 in CD11b(+) lamina propria mononuclear cells. Moreover, induction of the IL-10 family member IL-22 was demonstrated in CD11b(-) lamina propria mononuclear cells. In conclusion, CO and HO-1 induction ameliorated active colitis in TCRα(-/-) mice, and therapeutic effects correlated with induction of IL-10. This study provides further evidence that HO-1 mediates an important homeostatic pathway with pleiotropic anti-inflammatory effects in different experimental models of colitis and that targeting HO-1, therefore, is a potential therapeutic strategy in human inflammatory bowel diseases.     

Cutting edge: Tissue-retentive lung memory CD4 T cells mediate optimal protection to respiratory virus infection

We identify in this article a new class of lung tissue-resident memory CD4 T cells that exhibit tissue tropism and retention independent of Ag or inflammation. Tissue-resident memory CD4 T cells in the lung did not circulate or emigrate from the lung in parabiosis experiments, were protected from in vivo Ab labeling, and expressed elevated levels of CD69 and CD11a compared with those of circulating memory populations. Importantly, influenza-specific lung-resident memory CD4 T cells served as in situ protectors to respiratory viral challenge, mediating enhanced viral clearance and survival to lethal influenza infection. By contrast, memory CD4 T cells isolated from spleen recirculated among multiple tissues without retention and failed to mediate protection to influenza infection, despite their ability to expand and migrate to the lung. Our results reveal tissue compartmentalization as a major determining factor for immune-mediated protection in a key mucosal site, important for targeting local protective responses in vaccines and immunotherapies.     

Avian coronavirus in wild aquatic birds

We detected a high prevalence (12.5%) of novel avian coronaviruses in aquatic wild birds. Phylogenetic analyses of these coronaviruses suggest that there is a diversity of gammacoronaviruses and deltacoronaviruses circulating in birds. Gammacoronaviruses were found predominantly in Anseriformes birds, whereas deltacoronaviruses could be detected in Ciconiiformes, Pelecaniformes, and Anseriformes birds in this study. We observed that there are frequent interspecies transmissions of gammacoronaviruses between duck species. In contrast, deltacoronaviruses may have more stringent host specificities. Our analysis of these avian viral and host mitochondrial DNA sequences also suggests that some, but not all, coronaviruses may have coevolved with birds from the same order.     

Nostophycin biosynthesis is directed by a hybrid polyketide synthase-nonribosomal peptide synthetase in the toxic cyanobacterium Nostoc sp. strain 152

Cyanobacteria are a rich source of natural products with interesting pharmaceutical properties. Here, we report the identification, sequencing, annotation, and biochemical analysis of the nostophycin (npn) biosynthetic gene cluster. The npn gene cluster spans 45.1 kb and consists of three open reading frames encoding a polyketide synthase, a mixed polyketide nonribosomal peptide synthetase, and a nonribosomal peptide synthetase. The genetic architecture and catalytic domain organization of the proteins are colinear in arrangement, with the putative order of the biosynthetic assembly of the cyclic heptapeptide. NpnB contains an embedded monooxygenase domain linking nonribosomal peptide synthetase (NRPS) and polyketide synthase (PKS) catalytic domains and predicted here to hydroxylate the nostophycin during assembly. Expression of the adenylation domains and subsequent substrate specificity assays support the involvement of this cluster in nostophycin biosynthesis. Biochemical analyses suggest that the loading substrate of NpnA is likely to be a phenylpropanoic acid necessitating deletion of a carbon atom to explain the biosynthesis of nostophycin. Biosyntheses of nostophycin and microcystin resemble each other, but the phylogenetic analyses suggest that they are distantly related to one another.