Exploiting the natural poly(3-hydroxyalkanoates) production capacity of Antarctic Pseudomonas strains: from unique phenotypes to novel biopolymers

Journal of Industrial Microbiology & Biotechnology - Extreme environments are a unique source of microorganisms encoding metabolic capacities that remain largely unexplored. In this work, we...     

Acoustic emissions of Sorex unguiculatus (Mammalia: Soricidae): Assessing the echo‐based orientation hypothesis

Shrew species have been proposed to utilize an echo‐based orientation system to obtain additional acoustic information while surveying their environments. This system has been supported by changes in vocal emission rates when shrews encounter different habitats of varying complexity, although detailed acoustic features in this system have not been reported. In this study, behavioral experiments were conducted using the long‐clawed shrew (Sorex unguiculatus) to assess this orientation system. Three experimental conditions were set, two of which contained obstacles. Short‐click, noisy, and different types of tonal calls in the audible‐to‐ultrasonic frequency range were recorded under all experimental conditions. The results indicated that shrews emit calls more frequently when they are facing obstacles or exploring the experimental environment. Shrews emitted clicks and several different types of tonal calls while exploring, and modified the use of different types of calls for varying behavior. Furthermore, shrews modified the dominant frequency and duration of squeak calls for different types of obstacles, that is, plants and acrylic barriers. The vocalizations emitted at short inter‐pulse intervals could not be observed when shrews approached these obstacles. These results are consistent with the echo‐based orientation hypothesis according to which shrews use a simple echo‐orientation system to obtain information from their surrounding environments, although further studies are needed to confirm this hypothesis.     

The ATPase activity of the DNA transporter TrwB is modulated by protein TrwA: implications for a common assembly mechanism of DNA translocating motors

Conjugative systems contain an essential integral membrane protein involved in DNA transport called the Type IV coupling protein (T4CP). The T4CP of conjugative plasmid R388 is TrwB, a DNA-dependent ATPase. Biochemical and structural data suggest that TrwB uses energy released from ATP hydrolysis to pump DNA through its central channel by a mechanism similar to that used by F1-ATPase or ring helicases. For DNA transport, TrwB couples the relaxosome (a DNA-protein complex) to the secretion channel. In this work we show that TrwA, a tetrameric oriT DNA-binding protein and a component of the R388 relaxosome, stimulates TrwBDeltaN70 ATPase activity, revealing a specific interaction between the two proteins. This interaction occurs via the TrwA C-terminal domain. A 68-kDa complex between TrwBDeltaN70 and TrwA C-terminal domain was observed by gel filtration chromatography, consistent with a 1:1 stoichiometry. Additionally, electron microscopy revealed the formation of oligomeric TrwB complexes in the presence, but not in the absence, of TrwA protein. TrwBDeltaN70 ATPase activity in the presence of TrwA was further enhanced by DNA. Interestingly, maximal ATPase rates were achieved with TrwA and different types of dsDNA substrates. This is consistent with a role of TrwA in facilitating the interaction between TrwB and DNA. Our findings provide a new insight into the mechanism by which TrwB recruits the relaxosome for DNA transport. The process resembles the mechanism used by other DNA-dependent molecular motors, such as the RuvA/RuvB system, to be targeted to the DNA followed by hexamer assembly.     

Adiposity, physical activity, and physical fitness among children from Aragón, Spain

Objective: The main purpose of this study was to determine the relationship between physical activity (PA) levels and adiposity. The secondary purpose was to assess the effect of physical fitness and living area on adiposity. Research Methods and Procedures: A cross‐sectional study was carried out in a regional representative sample of 1068 children 7 to 12 years of age. Anthropometric and physical fitness values (including BMI, aerobic capacity, strength levels, velocity assessment, and flexibility) were measured in all children. Results: The prevalence of being overweight and obese in the entire sample was 31% and 6%, respectively. No difference between urban and rural children was found. The proportion of boys who were classified as overweight and obese was similar in physically active and sedentary (non‐physically active) groups. However, physically active girls tended to show lower obesity prevalence compared with their sedentary counterparts (p = 0.06). In girls, the sum of the 6 skinfolds thickness (SSF) measurements was lower in the physically active group when compared with the non‐physically active group (p < 0.05); however, this effect was not observed in boys. Multiple regression analysis revealed that the level of physical activity (PA) had a significant effect on BMI and SSF in boys but not in girls, while maximal oxygen uptake (Vo _2max) was significantly related to adiposity in both sexes. Discussion: Regular participation in at least 2 hours per week of sports activities on top of the compulsory education program is associated with better physical fitness and lower whole body adiposity. In the children included in our study, among all physical fitness variables, Vo _2max showed the strongest relationship with BMI and fat mass assessed by means of skinfold measurements.     

Insights into the activation of brain serine racemase by the multi-PDZ domain glutamate receptor interacting protein, divalent cations and ATP

Brain serine racemase contains pyridoxal phosphate as a prosthetic group and is known to become activated by divalent cations such as Ca(2+) and Mg(2+), as well as by ATP and ADP. In vivo, brain serine racemase is also activated by a multi-PSD-95/discs large/ZO-1 (PDZ) domain glutamate receptor interacting protein (GRIP) that is usually coupled to the GluR2/3 subunits of the alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid Ca(2+) channel. In the present study, we analysed the mechanisms by which serine racemase becomes activated by GRIP, divalent cations and ATP. We show that binding of PDZ6 of GRIP to serine racemase does not result in increased d-serine production. However, full-length GRIP does augment significantly enzymatic activity. We expressed various GRIP shorter constructs to map down the regions within GRIP that are necessary for serine racemase activation. We observed that, whereas recombinant proteins containing PDZ4-PDZ5-PDZ6 are unable to activate serine racemase, other constructs containing PDZ4-PDZ5-PDZ6-GAP2-PDZ7 significantly augment its activity. Hence, activation of serine racemase by GRIP is not a direct consequence of the translocation towards the calcium channel but rather a likely conformational change induced by GRIP on serine racemase. On the other hand, the observed activation of serine racemase by divalent cations has been assumed to be a side-effect associated with ATP binding, which is known to form a complex with Mg(2+) ions. Because no mammalian serine racemase has yet been crystallized, we used molecular modelling based on yeast and bacterial homologs to demonstrate that the binding sites for Ca(2+), ATP and the PDZ6 domain of GRIP are spatially separated and modulate the enzyme through distinct mechanisms.     

A proteomics analysis of cell signaling alterations in colorectal cancer

To gain further insight into alterations in cellular pathways, tumor profiling, and marker discovery in colorectal cancer (CRC) we used a new antibody microarray specific for cell signaling. Soluble protein extracts were prepared from paired tumor/normal biopsies of 11 patients diagnosed with colorectal carcinoma at different stages; four liver carcinomas were used as a reference. Antibody microarray analysis identified 46 proteins that were differentially expressed between normal colorectal epithelium and adenocarcinoma. These proteins gave a specific signature for CRC, different from other tumors, as well as a panel of novel markers and potential targets for CRC. Twenty-four proteins were validated by using a specific colorectal cancer tissue microarray and immunoblotting analysis. Together with some previously well known deregulated proteins in CRC (beta-catenin, c-MYC, or p63), we found new potential markers preferentially expressed in CRC tumors: cytokeratin 13, calcineurin, CHK1, clathrin light chain, MAPK3, phospho-PTK2/focal adhesion kinase (Ser-910), and MDM2. CHK1 antibodies were particularly effective in discriminating between tumoral and normal mucosa in CRC. Moreover a global picture of alterations in signaling pathways in CRC was observed, including a significant up-regulation of different components of the epidermal growth factor receptor and Wnt/beta-catenin pathways and the down-regulation of p14(ARF). The experimental approach described here should be applicable to other pathologies and neoplastic processes.     

Competition for alfalfa nodulation under metal stress by the metal‐tolerant strain Ochrobactrum cytisi Azn6.2

Legume plants, in association with rhizobia, are gaining increasing interest for heavy metal rhizoremediation. This symbiotic interaction combines the advantages of rhizoremediation and soil nitrogen enrichment. In metal polluted soils, Ochrobactrum cytisi can elicit non‐fixing nodules on legumes, including Medicago sativa. Nodulation kinetics was much slower when M. sativa plants were inoculated with O. cytisi Azn6.2 compared with the natural symbiont Ensifer meliloti 1021 and nodules were ineffective in nitrogen fixation. A competition experiment was performed using alfalfa grown on heavy metals, and co‐inoculated with equal amounts of the metal‐sensitive E. meliloti 1021 and the metal‐resistant O. cytisi Azn6.2. When plants were inoculated in non‐polluted substrates, all nodules were formed by E. meliloti 1021. Nevertheless, under increasing metal concentrations, the number of nodules occupied by O. cytisi grew. At the highest metal concentration, all nodules were elicited by O. cytisi, suggesting that the resistant species can take the place of the natural symbiont. This fact has important ecological and environmental implications when proposing legume–rhizobia symbioses for rhizoremediation and highlights the need of selecting highly resistant rhizobia in order to be competitive in polluted soils.     

Molecular Insights on the Release of Avibactam from the Acyl-Enzyme Complex

Avibactam is a non-β-lactam β-lactamase inhibitor for treating complicated urinary tract and respiratory infections caused by multidrug-resistant bacterial pathogens, a serious public health threat. Despite its importance, the release mechanism of avibactam from the enzyme-inhibitor complex has been scarcely studied from first principles, considering the total protein environment. This information at the molecular level is essential for the rational design of new antibiotics and inhibitors. In this article, we addressed the release of avibactam from the complex CTX-M-15 by means of molecular dynamics simulations and quantum mechanics/molecular mechanics calculations. This study provides molecular information not available earlier, including exploration of the potential energy surfaces, characterization of the observed intermediate, and their critical points, as well. Our results show that unlike that observed in the acylation reaction, the residues Glu166 and Lys73 would be in their neutral forms. Release of avibactam follows a stepwise mechanism in which the first stage corresponds to the formation of a tetrahedral intermediate, whereas the second stage corresponds to the cleavage of the Ser70-C7 bond, mediated by Lys73, either directly or through Ser130.