Crystal structure of the CN-hydrolase SA0302 from the pathogenic bacterium Staphylococcus aureus belonging to the Nit and NitFhit Branch of the nitrilase superfamily

The nitrilases include a variety of enzymes with functional specificities of nitrilase, amidase, and hydrolase reactions. The crystal structure of the uncharacterized protein SA0302 from the pathogenic microorganism Staphylococcus aureus is solved at 1.7?Å resolution. The protein contains 261 amino acids and presents a four-layer αββα sandwich with a chain topology similar to that of a few known CN-hydrolase folds. In the crystal, the proteins are arranged as dimers whose monomers are related by a pseudo twofold rotation symmetry axis. Analysis of the sequences and structures of CN-hydrolases with known 3D structures shows that SA0302 definitely is a member of Branch 10 (Nit and NitFhit) of the nitrilase superfamily. Enzyme activities and substrate specificities of members of this branch are not yet characterized, in contrast to those of the members of Branches 1–9. Although the sequence identities between Branch 10 members are rather low, less than 30%, five conserved regions are common in this subfamily. Three of them contain functionally important catalytic residues, and the two other newly characterized ones are associated with crucial intramolecular and intermolecular interactions. Sequence homology of the area near the active site shows clearly that the catalytic triad of SA0302 is Glu41-Lys110-Cys146. We suggest also that the active site includes a fourth residue, the closely located Glu119. Despite an extensive similarity with other Nit-family structural folds, SA0302 displays an important difference. Protein loop 111–122, which follows the catalytic Lys110, is reduced to half the number of amino acids found in other Nit-family members. This leaves the active site fully accessible to solvent and substrates. We have identified conservative sequence motifs around the three core catalytic residues, which are inherent solely to Branch 10 of the nitrilase superfamily. On the basis of these new sequence fingerprints, 10 previously uncharacterized proteins also could be assigned to this hydrolase subfamily.

An animated interactive 3D complement (I3DC) is available in Proteopedia at http://proteopedia.org/w/Journal:JBSD:19     

Application of Abscisic Acid (S-ABA) to ‘Crimson Seedless’ Grape Berries in a Mediterranean Climate: Effects on Color, Chemical Characteristics, Metabolic Profile, and S-ABA Concentration

‘Crimson Seedless’ is a table grape cultivar that often fails to develop adequate red color in Mediterranean climates. Application of abscisic acid (S-ABA) may be an aid for improving color, but its potential effects on overall quality and S-ABA concentration of the berry should be also considered. We tested two concentrations (200 and 400 mg/L) and different times of application (from 1 week after veraison up to 9 days before harvest) of a commercial formulation of S-ABA (ProTone^®) to verify the effect on harvestable bunches, color, chemical characteristics, metabolic profile, and S-ABA concentration in the berry. It was found that either the application of S-ABA at 400 mg/L one week after veraison or the application of S-ABA at 400 mg/L one week and four weeks after veraison positively affected the berry skin color, shifting the hue (h°) from 20 to a more red-violet hue (h° = 11–12). In general, the application of S-ABA, with the exception of the late treatments, enhanced coloration of the berries and increased the amount of harvestable bunches at the first pick because it promoted the skin-coloring process. S-ABA did not affect berry firmness but reduced the berry detachment force. Nevertheless, the values remained sufficiently high and the general quality of the bunch was not compromised. Ripening parameters (°Brix, pH, titratable acidity) were not affected by S-ABA applications, and even the primary metabolite profile was not influenced by the treatments as ascertained by multivariate statistical analyses [principal component analyses (PCA) and partial least squares discriminant analysis (PLS-DA)] applied to nuclear magnetic resonance (NMR) data. The S-ABA concentration in the berry, when treatments were performed around veraison, was within the natural range for grape (10–400 ng/g f.w.), whereas when late treatments were applied (few days before harvest), the concentration was higher (more than 1,000 ng/g f.w.). The best results for yield, quality, and S-ABA concentration in the berry were observed for the treatments performed a few days after veraison at the dose of 400 mg/L. This study gives new information about the positive effects of S-ABA on color without any particular change in the metabolic profile of the berry.     

Effects of reduced seawater pH on fertilisation, embryogenesis and larval development in the Antarctic seastar Odontaster validus

The effects of ocean acidification will be pronounced in high-latitude marine communities, although little is known on how reproduction in free-spawning polar invertebrates will respond. Using the circum-Antarctic sea star Odontaster validus, we examined fertilisation, larval survival and development under a controlled seawater treatment (temperature = ?0.5 °C, pH 8.1, pCO₂(aq) = 326.6 μatm, TA = 2,274.2 μmol kg soln^?1), two near-future pH treatments (pH 7.8 and 7.6) and an extreme treatment (pH 7.0). At a sperm concentration of 3.5 × 10⁵ sperm ml^?1, percentage of fertilisation was 98–90 % across a pH 8.1–7.0 range. At near-future pH ranges (pH 7.8 and 7.6), fertilisation was not significantly lower than in the control pH 8.1 except at the lowest sperm concentration (2.2 × 10³ sperm ml^?1) where fertilisation was reduced to 60 and 61 % in pH 7.6 and 7.8, respectively. Larval survival was not significantly affected by a decrease in pH of 0.3 units, but at pH 7.6 survival was significantly reduced. This difference was apparent at 9 days, and at the end of the experiment at 58 days, survival was 55 % compared with 85 % in the ambient treatment. Near-future changes to pH yielded smaller larvae, a result of both subtle differences in their morphology and slowed development rates, while larvae at pH 7.0 showed evidence of abnormal development. O. validus fertilisation and larval success declines in seawater pH conditions expected in coastal Antarctica over the coming decades, although the responses observed are within the range observed in warmer-water echinoderms.     

Knock down of caveolin‐1 affects morphological and functional hallmarks of human endothelial cells

Caveolin‐1 (CAV1) is the principal structural component of caveolae which functions as scaffolding protein for the integration of a variety of signaling pathways. In this study, we investigated the involvement of CAV1 in endothelial cell (EC) functions and show that siRNA‐induced CAV1 silencing in the human EC line EA.hy926 induces distinctive morphological changes, such as a marked increase in cell size and formation of stress fibers. Design‐based stereology was employed in this work to make unbiased quantification of morphometric properties such as volume, length, and surface of CAV1 silenced versus control cells. In addition, we showed that downregulation of CAV1 affects cell cycle progression at G1/S phase transition most likely by perturbation of AKT signaling. With the aim to assess the contribution of CAV1 to typical biological processes of EC, we report here that CAV1 targeting affects cell migration and matrix metalloproteinases (MMPs) activity, and reduces angiogenesis in response to VEGF, in vitro. Taken together our data suggest that the proper expression of CAV1 is important not only for maintaining the appropriate morphology and size of ECs but it might represent a prospective molecular target for studying key biological mechanisms such as senescence and tumorigenesis. J. Cell. Biochem. 114: 1843–1851, 2013. © 2013 Wiley Periodicals, Inc.     

Assessing changes in oral health‐related quality of life and its factors in community‐dwelling older Brazilians

doi: 10.1111/j.1741‐2358.2012.00656.x Assessing changes in oral health‐related quality of life and its factors in community‐dwelling older Brazilians Objective: To describe changes in oral health‐related quality of life and to evaluate the associations of these changes in community‐dwelling older people. Materials and methods: In this longitudinal study a representative sample of 872 older people, living in Brazil, was evaluated during 2004. The follow‐up was carried out during 2008, with 587 older persons evaluated. A questionnaire assessing socio‐demographic information, health history, oral health‐impact profile and number of natural teeth was used. Changes in oral health‐related quality of life were categorized as improvement or deterioration. Data analysis was performed using a hierarchical approach based in a conceptual framework. A hierarchal approach was carried out using Poisson regressions. Results: Older persons living in rural areas, those who reported brushing once a day or less and older persons with fewer natural teeth had an increased chance of reporting deterioration in oral health‐related quality of life. Women and participants who received a minimum wage of less than US$219.50 were more likely to report improvement in oral health‐related quality of life. Conclusion: The results of this study suggest that changes in the oral health‐related quality of life are influenced by many of the variables that were included in the conceptual framework.     

Two Steady‐Entrainment Phases and Graded Masking Effects by Light Generate Different Circadian Chronotypes in Octodon degus

Processes involved in the operation of the circadian pacemaker are well characterized; however; little is known about what mechanisms drive the overt diurnal, nocturnal, or crepuscular behavior in a species. In this context, dual‐phasing rodents, such as Octodon degus, emerge as a useful model to decipher these keys. Two main chronotypes, nocturnal and diurnal, have been traditionally described in laboratory‐housed degus based on the percentage of activity displayed by the animals during the scotophase or photophase. However, if one considers also the entrainment phase angle during the first days following a change from LD to DD conditions, a third chronotype (intermediate)—or more properly, a continuous grading of circadian expressions between diurnal and nocturnal chronotype—can be observed. Our experiments suggest the pacemaker of the diurnal animal is entrained to the photophase, and light does not exert a negative masking effect. The pacemaker of the nocturnal degus, on the other hand, is entrained to the scotophase, and light exerts a strong negative masking effect. Finally, the intermediate chronotype is characterized by variable negative masking effect of light overlapping a pacemaker entrained to the photophase. The phase shift inversion from diurnal to nocturnal chronotype is related to the availability of a wheel in the cage, and the effect may be located downstream from the clock. However, body temperature rhythm recordings, less affected by masking effects, point to an involvement of the circadian pacemaker in chronotype differentiation, as transient entrainment cycles, and not an abrupt phase shift, were detected after providing access to the wheel. The diurnality of degus seems to be the result of a variety of mechanisms, which may explain how different processes can lead to similar chronotypes.     

Factors Affecting Work Ability in Day and Shift‐Working Nurses

Satisfactory work ability is sustained and promoted by good physical and mental health and by favorable working conditions. This study examined whether favorable and rewarding work‐related factors increased the work ability among European nurses. The study sample was drawn from the Nurses' Early Exit Study and consisted of 7,516 nursing staff from seven European countries working in state‐owned and private hospitals. In all, 10.8% were day, 4.2% were permanent night, 20.9% were shift without night shift, and 64.1% were shift workers with night shifts. Participants were administered a composite questionnaire at baseline (Time 0) and 1 yr later (Time 1). The Work Ability Index (WAI) at Time 1 was used as the outcome measure, while work schedule, sleep, rewards (esteem and career), satisfaction with pay, work involvement and motivation, and satisfaction with working hours at Time 0 were included as potential determinants of work ability. Univariate and multivariate analyses were conducted after adjusting for a number of confounders (i.e., country, age, sex, type of employment, family status, and other job opportunities in the same area). Work schedule was not related to Time 1 changes in WAI. Higher sleep quality and quantity and more favorable psychosocial factors significantly increased work ability levels. Higher sleep quality and quantity did not mediate the effect of work schedule on work ability. No relevant interaction effects on work ability were observed between work schedule and the other factors considered at Time 0. As a whole, sleep and satisfaction with working time were gradually reduced from day work to permanent night work. However, scores on work involvement, motivation, and satisfaction with pay and rewards were the highest in permanent night workers and the lowest in rotating shift workers that included night shifts.