Perturbing the linker regions of the alpha-subunit of transducin: a new class of constitutively active GTP-binding proteins

The GDP-GTP exchange activity of the retinal G protein, transducin, is markedly accelerated by the photoreceptor rhodopsin in the first step of visual transduction. The x-ray structures for the alpha subunits of transducin (alpha(T)) and other G proteins suggest that the nucleotide-binding (Ras-like) domain and a large helical domain form a "clam shell" that buries the GDP molecule. Thus, receptor-promoted G protein activation may involve "opening the clam shell" to facilitate GDP dissociation. In this study, we have examined whether perturbing the linker regions connecting the Ras-like and helical domains of Galpha subunits gives rise to a more readily exchangeable state. The sole glycine residues in linkers 1 and 2 were individually changed to proline residues within an alpha(T)/alpha(i1) chimera (designated alpha(T)(*)). Both alpha(T)(*) linker mutants showed significant increases in their basal rates of GDP-GTP exchange when compared either to retinal alpha(T) or recombinant alpha(T)(*). The alpha(T)(*) linker mutants were responsive to aluminum fluoride, which binds to alpha-GDP complexes and induces changes in Switch 2. Although both linker mutants were further activated by light-activated rhodopsin together with the betagamma complex, their activation was not influenced by betagamma alone, arguing against the idea that the betagamma complex helps to pry apart the helical and Ras-like domains of Galpha subunits. Once activated, the alpha(T)(*) linker mutants were able to stimulate the cyclic GMP phosphodiesterase. Overall, these findings highlight a new class of activated Galpha mutants that constitutively exchange GDP for GTP and should prove valuable in studying different G protein-signaling systems.     

X-ray analysis of Mycobacterium smegmatis Dps and a comparative study involving other Dps and Dps-like molecules

The structure of the DNA binding protein from starved cells from Mycobacterium smegmatis has been determined in three crystal forms and has been compared with those of similar proteins from other sources. The dodecameric molecule can be described as a distorted icosahedron. The interfaces among subunits are such that the dodecameric molecule appears to have been made up of stable trimers. The situation is similar in the proteins from Escherichia coli and Agrobacterium tumefaciens, which are closer to the M.smegmatis protein in sequence and structure than those from other sources, which appear to form a dimer first. Trimerisation is aided in the three proteins by the additional N-terminal stretches that they possess. The M.smegmatis protein has an additional C-terminal stretch compared to other related proteins. The stretch, known to be involved in DNA binding, is situated on the surface of the molecule. A comparison of the available structures permits a delineation of the rigid and flexible regions in the molecule. The subunit interfaces around the molecular dyads, where the ferroxidation centres are located, are relatively rigid. Regions in the vicinity of the acidic holes centred around molecular 3-fold axes, are relatively flexible. So are the DNA binding regions. The crystal structures of the protein from M.smegmatis confirm that DNA molecules can occupy spaces within the crystal without disturbing the arrangement of the protein molecules. However, contrary to earlier suggestions, the spaces do not need to be between layers of protein molecules. The cubic form provides an arrangement in which grooves, which could hold DNA molecules, criss-cross the crystal.     

FAIR: A server for internal sequence repeats

An Internet computing server has been developed to identify all the occurrences of the internal sequence repeats in a protein and DNA sequences. Further, an option is provided for the users to check the occurrence(s) of the resultant sequence repeats in the other sequence and structure (Protein Data Bank) databases. The databases deployed in the proposed computing engine are up-to-date and thus the users will get the latest information available in the respective databases. The server is freely accessible over the World Wide Web (WWW). AVAILABILITY: http://bioserver1.physics.iisc.ernet.in/fair/     

Sex differences in the rate of fatigue development and recovery

Background

Many musculoskeltal injuries in the workplace have been attributed to the repetitive loading of muscle and soft tissues. It is not disputed that muscular fatigue is a risk factor for musculoskeltal injury, however the disparity between gender with respect to muscular fatigability and rate of recovery is not well understood. Current health and safety guidelines do not account for sex differences in fatiguability and may be predisposing one gender to greater risk. The purpose of this study was to quantify the sex differences in fatigue development and recovery rate of lower and upper body musculature after repeated bouts of sustained isometric contractions.

Methods

Twenty-seven healthy males (n = 12) and females (n = 15) underwent bilateral localized fatigue of either the knee extensors (male: n = 8; female: n = 8), elbow flexors (male: n = 8; female: n = 10), or both muscle groups. The fatigue protocol consisted of ten 30-second sub-maximal isometric contractions. The changes in maximum voluntary contraction (MVC), electrically evoked twitches, and motor unit activation (MUA) were assessed along with the ability to control the sustained contractions (SLP) during the fatigue protocol using a mixed four-factor repeated measures ANOVA (gender × side × muscle × time) design with significance set at p < 0.05.

Results

There was a significant loss of MVC, MUA, and evoked twitch amplitude from pre- to post-fatigue in both the arms and legs. Males had greater relative loss of isometric force, a higher rate of fatigue development, and were less capable of maintaining the fatiguing contractions in the legs when compared to the females.

Conclusion

The nature of the induced fatigue was a combination of central and peripheral fatigue that did not fully recover over a 45-minute period. The results appear to reflect sex differences that are peripheral, and partially support the muscle mass hypothesis for explaining differences in muscular fatigue.

     

A meta-analysis of the traits affecting dispersal ability in butterflies: can wingspan be used as a proxy?

1. Dispersal ability of a species is a key ecological characteristic, affecting a range of processes from adaptation, community dynamics and genetic structure, to distribution and range size. It is determined by both intrinsic species traits and extrinsic landscape-related properties. 2. Using butterflies as a model system, the following questions were addressed: (i) given similar extrinsic factors, which intrinsic species trait(s) explain dispersal ability? (ii) can one of these traits be used as a proxy for dispersal ability? (iii) the effect of interactions between the traits, and phylogenetic relatedness, on dispersal ability. 3. Four data sets, using different measures of dispersal, were compiled from published literature. The first data set uses mean dispersal distances from capture-mark-recapture studies, and the other three use mobility indices. Data for six traits that can potentially affect dispersal ability were collected: wingspan, larval host plant specificity, adult habitat specificity, mate location strategy, voltinism and flight period duration. Each data set was subjected to both unifactorial, and multifactorial, phylogenetically controlled analyses. 4. Among the factors considered, wingspan was the most important determinant of dispersal ability, although the predictive powers of regression models were low. Voltinism and flight period duration also affect dispersal ability, especially in case of temperate species. Interactions between the factors did not affect dispersal ability, and phylogenetic relatedness was significant in one data set. 5. While using wingspan as the only proxy for dispersal ability maybe problematic, it is usually the only easily accessible species-specific trait for a large number of species. It can thus be a satisfactory proxy when carefully interpreted, especially for analyses involving many species from all across the world.     

Burden of rare sarcomere gene variants in the framingham and jackson heart study cohorts

Rare sarcomere protein variants cause dominant hypertrophic and dilated cardiomyopathies. To evaluate whether allelic variants in eight sarcomere genes are associated with cardiac morphology and function in the community, we sequenced 3,600 individuals from the Framingham Heart Study (FHS) and Jackson Heart Study (JHS) cohorts. Out of the total, 11.2% of individuals had one or more rare nonsynonymous sarcomere variants. The prevalence of likely pathogenic sarcomere variants was 0.6%, twice the previous estimates; however, only four of the 22 individuals had clinical manifestations of hypertrophic cardiomyopathy. Rare sarcomere variants were associated with an increased risk for adverse cardiovascular events (hazard ratio: 2.3) in the FHS cohort, suggesting that cardiovascular risk assessment in the general population can benefit from rare variant analysis.     

Bacterial water quality and network hydraulic characteristics: a field study of a small, looped water distribution system using culture-independent molecular methods

Aims: To determine the spatial and temporal variability in the abundance, structure and composition of planktonic bacterial assemblages sampled from a small, looped water distribution system and to interpret results with respect to hydraulic conditions. Methods and Results: Water samples were collected from five sampling points, twice a day at 06:00 h and 09:00 h on a Monday (following low weekend demand) and a Wednesday (higher midweek demand). All samples were fully compliant with current regulated parameter standards. This study did not show obvious changes in bacterial abundance (DAPI count) or community structure Denaturing gradient gel electrophoresis analysis with respect to sample site and hence to water age; however, the study did show temporal variability with respect to both sampling day and sample times. Conclusions: Data suggests that variations in the bacterial assemblages may be associated with the local system hydraulics: the bacterial composition and numbers, over short durations, are governed by the interaction of the bulk water and the biofilm influenced by the hydraulic conditions. Significance and Impact of the Study: This study demonstrates general stability in bacterial abundance, community structure and composition within the system studied. Trends and patterns supporting the transfer of idealized understanding to the real world were evident. Ultimately, such work will help to safeguard potable water quality, fundamental to public health.     

Differential activities of the two closely related withanolides, withaferin a and withanone: bioinformatics and experimental evidences

BACKGROUND AND PURPOSE: Withanolides are naturally occurring chemical compounds. They are secondary metabolites produced via oxidation of steroids and structurally consist of a steroid-backbone bound to a lactone or its derivatives. They are known to protect plants against herbivores and have medicinal value including anti-inflammation, anti-cancer, adaptogenic and anti-oxidant effects. Withaferin A (Wi-A) and Withanone (Wi-N) are two structurally similar withanolides isolated from Withania somnifera, also known as Ashwagandha in Indian Ayurvedic medicine. Ashwagandha alcoholic leaf extract (i-Extract), rich in Wi-N, was shown to kill cancer cells selectively. Furthermore, the two closely related purified phytochemicals, Wi-A and Wi-N, showed differential activity in normal and cancer human cells in vitro and in vivo. We had earlier identified several genes involved in cytotoxicity of i-Extract in human cancer cells by loss-of-function assays using either siRNA or randomized ribozyme library. METHODOLOGY/PRINCIPAL FINDINGS: In the present study, we have employed bioinformatics tools on four genes, i.e., mortalin, p53, p21 and Nrf2, identified by loss-of-function screenings. We examined the docking efficacy of Wi-N and Wi-A to each of the four targets and found that the two closely related phytochemicals have differential binding properties to the selected cellular targets that can potentially instigate differential molecular effects. We validated these findings by undertaking parallel experiments on specific gene responses to either Wi-N or Wi-A in human normal and cancer cells. We demonstrate that Wi-A that binds strongly to the selected targets acts as a strong cytotoxic agent both for normal and cancer cells. Wi-N, on the other hand, has a weak binding to the targets; it showed milder cytotoxicity towards cancer cells and was safe for normal cells. The present molecular docking analyses and experimental evidence revealed important insights to the use of Wi-A and Wi-N for cancer treatment and development of new anti-cancer phytochemical cocktails.     

TPX: Biomedical literature search made easy

TPX is a web-based PubMed search enhancement tool that enables faster article searching using analysis and exploration features. These features include identification of relevant biomedical concepts from search results with linkouts to source databases, concept based article categorization, concept assisted search and filtering, query refinement. A distinguishing feature here is the ability to add user-defined concept names and/or concept types for named entity recognition. The tool allows contextual exploration of knowledge sources by providing concept association maps derived from the MEDLINE repository. It also has a full-text search mode that can be configured on request to access local text repositories, incorporating entity co-occurrence search at sentence/paragraph levels. Local text files can also be analyzed on-the-fly. Availability: http://tpx.atc.tcs.com