Automatically extracting functionally equivalent proteins from SwissProt

Background  

There is a frequent need to obtain sets of functionally equivalent homologous proteins (FEPs) from different species. While it is usually the case that orthology implies functional equivalence, this is not always true; therefore datasets of orthologous proteins are not appropriate. The information relevant to extracting FEPs is contained in databanks such as UniProtKB/Swiss-Prot and a manual analysis of these data allow FEPs to be extracted on a one-off basis. However there has been no resource allowing the easy, automatic extraction of groups of FEPs – for example, all instances of protein C.     

Characterization of Frex as an NADH sensor for in vivo applications in the presence of NAD+ and at various pH values

Photosynthesis Research - The fluorescent biosensor Frex, recently introduced as a sensitive tool to quantify the NADH concentration in living cells, was characterized by time-integrated and...     

Investigation of the NADH/NAD+ ratio in Ralstonia eutropha using the fluorescence reporter protein Peredox

Ralstonia eutropha is a hydrogen-oxidizing (“Knallgas”) bacterium that can easily switch between heterotrophic and autotrophic metabolism to thrive in aerobic and anaerobic environments. Its versatile metabolism makes R. eutropha an attractive host for biotechnological applications, including H₂-driven production of biodegradable polymers and hydrocarbons. H₂ oxidation by R. eutropha takes place in the presence of O₂ and is mediated by four hydrogenases, which represent ideal model systems for both biohydrogen production and H₂ utilization. The so-called soluble hydrogenase (SH) couples reversibly H₂ oxidation with the reduction of NAD⁺ to NADH and has already been applied successfully in vitro and in vivo for cofactor regeneration. Thus, the interaction of the SH with the cellular NADH/NAD⁺ pool is of major interest. In this work, we applied the fluorescent biosensor Peredox to measure the [NADH]:[NAD⁺] ratio in R. eutropha cells under different metabolic conditions. The results suggest that the sensor operates close to saturation level, indicating a rather high [NADH]:[NAD⁺] ratio in aerobically grown R. eutropha cells. Furthermore, we demonstrate that multicomponent analysis of spectrally-resolved fluorescence lifetime data of the Peredox sensor response to different [NADH]:[NAD⁺] ratios represents a novel and sensitive tool to determine the redox state of cells.     

Dynamics of Correlated Genetic Systems. V. Rates of Decay of Linkage Disequilibria in Experimental Populations of DROSOPHILA MELANOGASTER

The dynamic behavior of four-locus gametic frequency distributions was studied in five replicate cage populations of Drosophila melanogaster for up to 50 generations. The joint frequency distributions were resolved into gene frequencies and various disequilibrium measures. In addition, F statistics for marginal single-locus genotypic frequency distributions were followed through time. The gene frequency, disequilibrium and F statistics were obtained for four chromosome 3 enzyme marker loci [isocitrate dehydrogenase (3–27.1), esterase-6 (3–36.8), phosphoglucomutase (3–43.4) and esterase-C (3–49.0)]. The initial structure of the experimental populations featured random mating proportions, and two complementary gametic types with respect to the marker loci, thus assuring complete pairwise linkage disequilibrium among the markers.——The experimental results indicate: (1) the between-replicate variance in gene frequency varied substantially among loci, with isocitrate dehydrogenase showing the greatest between-replicate variance, and esterase-C the least. (2) The F statistics initially were strongly negative but decayed to the neighborhood of zero for all marker loci except esterase-C. The rate at which the F statistics approached zero varied among the marker loci, indicating substantial differences in the distribution of selective effects along the chromosome. The centromeric region, marked by esterase-C, shows the strongest selective effects. (3) The rate of decay of linkage disequilibrium was much faster than expected for pairs of neutral loci, averaging 1.82 times the neutral rate over all replicates and pairs of loci. This acceleration, which was observed for all six pairwise combinations of loci, was interpreted as resulting from the interaction between selection and recombination. Our experimental results are consistent with many investigations of linkage disequilibrium in natural populations of Drosophila melanogaster that show little or no disequilibrium among enzyme loci. (4) A fortuitous contamination of two cages revealed an apparent regulatory interaction between the migrant and nonmigrant chromosomes at the esterase-C locus. The migrant chromosomes were very rapidly absorbed into the recipient populations, despite this interaction. This result suggests that the dynamics of migration in populations may be phenomenologically richer than anticipated by simple theory.     

Mesenchymal stem cells promote lymphangiogenic properties of lymphatic endothelial cells

Lymphatic metastasis is one of the main prognostic factors concerning long‐term survival of cancer patients. In this regard, the molecular mechanisms of lymphangiogenesis are still rarely explored. Also, the interactions between stem cells and lymphatic endothelial cells (LEC) in humans have not been well examined. Therefore, the main objective of this study was to assess the interactions between mesenchymal stem cells (MSC) and LEC using in vitro angiogenesis assays. Juvenile LEC were stimulated with VEGF‐C, bFGF, MSC‐conditioned medium (MSC‐CM) or by co‐culture with MSC. LEC proliferation was assessed using a MTT assay. Migration of the cells was determined with a wound healing assay and a transmigration assay. To measure the formation of lymphatic sprouts, LEC spheroids were embedded in collagen or fibrin gels. The LEC's capacity to form capillary‐like structures was assessed by a tube formation assay on Matrigel^®. The proliferation, migration and tube formation of LEC could be significantly enhanced by MSC‐CM and by co‐culture with MSC. The effect of stimulation with MSC‐CM was stronger compared to stimulation with the growth factors VEGF‐C and bFGF in proliferation and transmigration assays. Sprouting was stimulated by VEGF‐C, bFGF and by MSC‐CM. With this study, we demonstrate the potent stimulating effect of the MSC secretome on proliferation, migration and tube formation of LEC. This indicates an important role of MSC in lymphangiogenesis in pathological as well as physiological processes.