Hydroxylation of aromatic compounds as indices of hydroxyl radical production: A cautionary note revisited

While setting up an intracerebral microdialysis system to estimate the extent of oxidative stress induced by the neurotoxin, N-methylphenylpyridinium ion (MPP⁺), we encountered a problem in the use of hydroxybenzoic acids as traps of hydroxyl radicals. Using either 2-hydroxybenzoate (salicylate) or 4-hydroxybenzoate as trapping agents, we observed a nonspecific, that is, nontissue derived, production of hydroxyl radicals as measured by the hydroxylation products, 2,3- and 2,5-dihydroxybenzoate from 2-hydroxybenzoate and 3,4-dihydroxybenzoate from 4-hydroxybenzoate. This production of dihydroxybenzoates was 10 times that expected due to the administration of MPP⁺, thus making it impossible to interpret our results. Careful investigation of the various components of the microdialysis system indicated that contact of the microdialysate with metal surfaces resulted in dihydroxybenzoic acid formation. These results should serve as a reminder to perform stringent tests of the experimental system prior to experiments with biological tissues to evaluate the contribution of hydroxyl radical production from nonbiological sources. Therefore, along with the possibility of enzymatic production of dihydroxybenzoates, artefactual production by components of the experimental apparatus must be considered before assuming that one is measuring hydroxyl radical production by a biological system.     

Risk Factors for Sporadic Domestically Acquired Campylobacter Infections in Norway 2010–2011: A National Prospective Case-Control Study

BackgroundCampylobacteriosis is the most frequently reported food- and waterborne infection in Norway. We investigated the risk factors for sporadic Campylobacter infections in Norway in order to identify areas where control and prevention measures could be improved.MethodsA national prospective case-control study of factors associated with Campylobacter infection was conducted from July 2010 to September 2011. Cases were recruited from the Norwegian Surveillance System of Communicable Diseases (MSIS). Controls were randomly selected from the Norwegian Population Registry. Cases and controls were mailed a paper questionnaire with a prepaid return envelope. Univariable analyses using logistic regression were conducted for all exposures. A final parsimonious multivariable model was developed using regularized/penalized logistic regression, and adjusted odds ratios were calculated.ResultsA total of 995 cases and 1501 controls were included in the study (response proportion 55% and 30%, respectively). Exposures that had significant increases in odds of Campylobacter infection in multivariable analysis were drinking water directly from river, stream, or lake (OR: 2.96), drinking purchased bottled water (OR: 1.78), eating chicken (1.69), eating meat that was undercooked (OR: 1.77), eating food made on a barbecue (OR: 1.55), living on a farm with livestock (OR: 1.74), having a dog in the household (OR: 1.39), and having household water supply serving fewer than 20 houses (OR: 1.92).ConclusionsConsumption of poultry and untreated water remain important sources of Campylobacter infection in Norway, despite ongoing control efforts. The results justify the need for strengthening education for consumers and food handlers about the risks of cross-contamination when preparing poultry and with consuming raw or undercooked chicken. The public should also be reminded to take precautions when drinking untreated water in nature and ensure continued vigilance in order to protect and maintain the quality of water from small-scale water supply systems.     

In vivo protein cyclization promoted by a circularly permuted Synechocystis sp. PCC6803 DnaB mini-intein.

A synthetic Synechocystis sp. PCC6803 DnaB split mini-intein gene was constructed for the in vivo cyclization of recombinant proteins expressed in Escherichia coli. The system was used to cyclize the NH(2)-terminal domain of E. coli DnaB, the structure of which had been determined previously by NMR spectroscopy. Cyclization was found to proceed efficiently, with little accumulation of precursor, and the product was purified in high yield. The solution structure of cyclic DnaB-N is not significantly different from that of linear DnaB-N and it unfolds reversibly at temperatures approximately 14 degrees C higher. Improved hydrogen bonding was observed in the first and last helices, and the length of the last helix was increased, while the 9-amino acid linker used to join the NH(2) and COOH termini was found to be highly mobile. The measured thermodynamic stabilization of the structure (Delta Delta G approximately 2 kcal/mol) agrees well with the value estimated from the reduced conformational entropy in the unfolded form. Simple polymer theory can be used to predict likely free energy changes resulting from protein cyclization and how the stabilization depends on the size of the protein and the length of the linker used to connect the termini.     

Construction,expression, purification and antigenicity of recombinant Campylobacter jejuni flagellar proteins

Campylobacter jejuni, a flagellated, spiral-rod Gram-negative bacterium, is the leading etiologic agent of human acute bacterial gastroenteritis worldwide. The source of this microorganism for human infection has been implicated as consumption and handling of poultry meat where this microorganism is a commensal in the gut. Because the genomes of many C. jejuni isolates have been sequenced, our ultimate goal is to develop protein arrays for exploring this microorganism and host interactions. In this communication, we report cloning, expression and purification of C. jejuni flagellar proteins in a bacterial expression system. Twelve recombinant proteins were purified, which were confirmed by SDS–PAGE analysis and a His tag detection kit. The FlgE1, FlgG, FlgK, FliE, FlgH/FliH and FlaA recombinant proteins were further confirmed by LC–ESI-MS/MS. The purified recombinant proteins were tested whether they were immunogenic using antibodies from several sources. BacTrace anti-Campylobacter species antibody reacted to the FlaA recombinant protein, but not others. Rabbit anti-MOMP1 peptide antibody reacted strongly to FliE and weakly to FlaA, but not others. Rabbit anti-MOMP2 peptide antibody reacted strongly to the FlaA, FliG, FliE, FlhF, FlgG, FlgE1 and FliD recombinant proteins, less to FlgK and FlgH/FliH, and did not react to the FliY, FliS and FliH recombinant proteins. These antibody studies suggest that these recombinant flagellar proteins have potential for novel targets for vaccine development. It is also anticipated that these recombinant proteins provide us a very useful tool for investigating host immune response to C. jejuni.     

Gender differences in Salix myrsinifolia at the pre‐reproductive stage are little affected by simulated climatic change

Females of dioecious species are known often to prioritize defense, while males grow faster. As climatic change is known to influence both growth and defense in plants, it would be important to know whether it affects the sexes of dioecious species differently. This could have impacts on future sex ratios in nature. We grew four clones of each sex of Salix myrsinifolia in greenhouse chambers under ambient conditions, enhanced temperature, enhanced CO₂ or enhanced temperature + enhanced CO₂. The females had the greatest growth and also the highest levels of phenolic compounds in twigs, while in leaves some compounds were higher in males, some in females. Enhanced CO₂ increased growth equally in both sexes, while growth was not affected by elevated temperature. Phenolic compounds in twigs were, however, lowered under elevated temperature. The gender differences were not strongly affected by the simulated climatic changes, but the effects seen on some highly concentrated compounds may be important. We interpret the intensive growth at pre‐reproductive phase as a strategy in females to get an initial advantage before later periods with fewer resources available for growth.     

Is the rapid post-mating inhibition of pheromone response triggered by ecdysteroids or other factors from the sex accessory glands in the male moth Agrotis ipsilon?

In many animals, male copulation is dependent on the detection and processing of female-produced sex pheromones, which is generally followed by a sexual refractory post-ejaculatory interval (PEI). In the male moth, Agrotis ipsilon, this PEI is characterized by a transient post-mating inhibition of behavioral and central nervous responses to sex pheromone, which prevents males from re-mating until they have refilled their reproductive tracts for a potential new ejaculate. However, the timing and possible factors inducing this rapid olfactory switch-off are still unknown. Here, we determined the initial time delay and duration of the PEI. Moreover, we tested the hypothesis that the brain, the testis and/or the sex accessory glands (SAGs) could produce a factor inducing the PEI. Lastly, we investigated the possible involvement of ecdysteroids, hormones essential for development and reproduction in insects, in this olfactory plasticity. Using brain and SAG cross-injections in virgin and newly-mated males, surgical treatments, wind tunnel behavioral experiments and EIA quantifications of ecdysteroids, we show that the PEI starts very shortly after the onset of copulation, and that SAGs contain a factor, which is produced/accumulated after copulation to induce the PEI. Moreover, SAGs were found to be the main source of ecdysteroids, whose concentration decreased after mating, whereas it increased in the haemolymph. 20-Hydroxyecdysone (20E) was identified as the major ecdysteroid in SAGs of A. ipsilon males. Finally, 20E injections did not reduce the behavioral pheromone response of virgin males. Altogether our data indicate that 20E is probably not involved in the PEI.     

Purification of the Neurite Outgrowth Promoting Fragment of Mouse Laminin

Abstract

A method for isolation of the neurite outgrowth promoting fragment of mouse laminin (fragment 8) is described in this paper. Besides producing excellent yields, this method was shown to be fast and practical, since it is based on a single step which consists in an ion exchange chromatography of elastase digested laminin.     

Sprint interval running increases insulin sensitivity in young healthy subjects

High intensity cycling training increases oxidative capacity in skeletal muscles and improves insulin sensitivity. The present study compared the effect of eight weeks of sprint interval running (SIT) and continuous running at moderate intensity (CT) on insulin sensitivity and cholesterol profile in young healthy subjects (age 25.2 ± 0.7; VO(2max) 49.3 ± 1.2 ml·kg(-1)·min(-1)). SIT and CT increased maximal oxygen uptake by 5.3 ± 1.8 and 3.8 ± 1.6%, respectively (p < 0.05 for both). Oral glucose tolerance test (OGTT) was performed before and 60 h after the last training session. SIT, but not CT, reduced glucose area under curve and improved HOMA β-cell index (p < 0.05). Insulin area under curve did not decrease significantly in any group. SIT, but not CT, reduced LDL and total cholesterol. In conclusion, sprint interval running improves insulin sensitivity and cholesterol profile in healthy subjects, and sprint interval running may be more effective to improve insulin sensitivity than continuous running at moderate intensity.     

A New Approach for Tracking Vegetation Change after Restoration: A Case Study with Peatlands

Developing objective tools for tracking progress of restored sites is of general concern. Here, we present an innovative approach based on principal response curves (PRC) and species classification according to their preferential habitats to monitor changes in community composition. Following large‐scale restoration of a cut‐over peatland, vegetation was surveyed biannually over 8 years. We evaluated whether the establishing plant communities fell within the range of natural variation. We used both general diversity curves and PRC applied on plant species grouped by preferred habitat to compare restored sites and unrestored sites to a reference ecosystem. After 8 years, diversity and richness differed between the sites, with Forest and Ruderal species more prominent in unrestored sites, and Peatland, Forest, and Wetland species dominant in restored sites. The PRC revealed that the restored site became rapidly dominated by typical peatland plants, the main drivers of temporal changes being Sphagnum rubellum, Pohlia nutans, and Mylia anomala. Some differences remained between the restored and the undisturbed species pools: the former had more herbaceous species associated with wetlands such as Calamagrostis canadensis and Typha latifolia and the latter had more forested species like Kalmia angustifolia throughout the study. PRC revealed to be an efficient tool identifying species driving changes at the community level after restoration. In our case study, examining PRC scores after classifying species according to their preferred habitat allowed to illustrate objectively how restoration promotes target species (associated to peatlands) and how lack of intervention benefits ruderal species.