Effect of carbon source, growth and temperature on the expression of the sec genes of Streptomyces lividans 1326

The mRNA level in sec genes of Streptomyces lividans was studied as a function of growth temperature, glucose effect, and growth using two different carbon sources. Glucose and xylan, a complex hemicellulose, were used as carbon sources for the growth of S. lividans. For both substrates, the mRNA levels of secA, secD, secE, secF, and secY genes were almost constant during the early and log phases, but showed a marked decrease at the beginning of the stationary phase followed by a full recovery of mRNA level in the late stationary phase. This indicates that the sec genes are actively transcribed during the differentiation process. The mRNA level in xylan was generally from 1.5- to 2-fold that in glucose. At growth temperatures of 28 degrees C, 34 degrees C, or 40 degrees C, there was no significant difference in the sec gene mRNA levels.     

The influence of spatial inhomogeneities on neutral models of geographical variation IV. Discontinuities in the population density and migration rate

The equilibrium structure of the infinite, one-dimensional stepping-stone model with coincident discontinuities in the population density and migration rate is investigated in the diffusion approximation. The monoecious, diploid population is subdivided into an infinite linear array of equally large, panmictic colonies that exchange gametes isotropically. The population density and the migration rate have a discontinuity at the origin, but are elsewhere uniform. Generations are discrete and nonoverlapping; the analysis is restricted to a single locus without selection; every allele mutates to new alleles at the same rate. The three dimensionless parameters in the theory are alpha=(rho(+)/rho(-))(2) (V(+)/V(-))(3/2), and beta(+/-)=4rho(+/-) 2uV(+/-), where rho(+) (rho(-)) and V(+) (V(-)) designate the population density and variance of gametic dispersion per generation to the right (left) of the discontinuity, respectively, and u denotes the mutation rate. The characteristic length on the right (left) is V(+)/(2u) (V(-)/(2u)). The probability of identity is continuous at the origin, but its partial derivatives have a discontinuity unless migration is conservative (rho(-) V(-)=rho(+) V(+)). At least for nonconservative migration, the probability of identity (including the expected homozygosity) can be nonmonotonic even if the migration rate is uniform and the population density is monotonic. Thus, there can be a nonmonotonic genetic response in a neutral model to a monotonic environment.     

The crystal structure of a 2-fluorocellotriosyl complex of the Streptomyces lividans endoglucanase CelB2 at 1.2 A resolution

Glycoside hydrolases have been classified into over 66 families on the basis of amino acid sequence. Recently a number of these families have been grouped into "clans" which share a common fold and catalytic mechanism [Henrissat, B., and Bairoch, A. (1996) Biochem. J. 316, 695-696]. Glycoside hydrolase Clan GH-C groups family 11 xylanases and family 12 cellulases, which share the same jellyroll topology, with two predominantly antiparallel beta-sheets forming a long substrate-binding cleft, and act with net retention of anomeric configuration. Here we present the three-dimensional structure of a family 12 endoglucanase, Streptomyces lividans CelB2, in complex with a 2-deoxy-2-fluorocellotrioside. Atomic resolution (1.2 A) data allow clear identification of two distinct species in the crystal. One is the glycosyl-enzyme intermediate, with the mechanism-based inhibitor covalently linked to the nucleophile Glu 120, and the other a complex with the reaction product, 2-deoxy-2-fluoro-beta-D-cellotriose. The active site architecture of the complex provides insight into the double-displacement mechanism of retaining glycoside hydrolases and also sheds light on the basis of the differences in specificity between family 12 cellulases and family 11 xylanases.     

The effect of in-season, high-intensity interval training in soccer players

The effects of in-season, high-intensity interval training on professional male soccer players' running performances were investigated. Twenty-two subjects participated in 2 consecutive training periods of 10 weeks. The first period was considered a control period and was compared with a period where 2 high-intensity interval training exercises were included in the usual training program. Intermittent runs consisted of 12-15 runs lasting 15 seconds at 120% of maximal aerobic speed alternated with 15 seconds of rest. Sprint repetitions consisted of 12-15 all-out 40-m runs alternated with 30 seconds of rest. Results from the high-intensity interval training have shown that maximal aerobic speed was improved (+8.1 +/- 3.1%; p < 0.001) and that the time of the 40-m sprint was decreased (-3.5 +/- 1.5%; p < 0.001), whereas no change in either parameters were observed during the control period. This study shows that improvements in physical qualities can be made during the in-season period.     

Expression and functional role of peroxisome proliferator-activated receptor-gamma in ovarian folliculogenesis in the sheep

Peroxisome proliferator-activated receptor (PPARgamma) is a nuclear receptor that is activated by fatty acids and derivatives and the antidiabetic glitazones, which plays a role in the control of lipid and glucose homeostasis. In the present work, we tested the hypothesis that PPARgamma plays a role in reproductive tissues by studying its expression and function in the hypothalamo-pituitary-ovary axis in the sheep. PPARgamma 1 and PPARgamma 2 proteins and mRNAs were detected in whole ovine pituitary and ovary but not in hypothalamic extracts. In situ hybridization on ovarian section localized PPARgamma mRNA in the granulosa layer of follicles. Interestingly, PPARgamma expression was higher in small antral (1-3 mm diameter) than in preovulatory follicles (>5 mm diameter) (P < 0.001) and was not correlated with healthy status. To assess the biological activity of ovarian PPARgamma, ovine granulosa cells were transfected with a reporter construct driven by PPARgamma-responsive elements. Addition of rosiglitazone, a PPARgamma ligand, stimulated reporter gene expression, showing that endogenous PPARgamma is functional in ovine granulosa cells in vitro. Moreover, rosiglitazone inhibited granulosa cell proliferation (P < 0.05) and increased the secretion of progesterone in vitro (P < 0.05). This stimulation effect was stronger in granulosa cells from small than from large follicles. In contrast, rosiglitazone had no effect on LH, FSH, prolactin and growth hormone secretion by ovine pituitary cells in vitro. Overall, these data suggest that PPARgamma ligands might stimulate follicular differentiation in vivo likely through a direct action on granulosa cells rather than by modulating pituitary hormone secretion.     

Ca2+ oscillations in hepatocytes do not require the modulation of InsP3 3-kinase activity by Ca2+

Vacuole fusion requires Sec18p-dependent acylation of the armadillo-repeat protein Vac8p that has been isolated with cis-SNARE complexes. To gain more insight into the mechanism of acylation, we analyzed the palmitoylation reaction on isolated vacuoles or in vacuolar detergent extracts. Recombinant Vac8p is palmitoylated when added to vacuoles and is anchored to membranes after modification. The palmitoyl acyltransferase (PAT) extracted from vacuolar membranes is functional in detergent extracts and shows all characteristics of an enzymatic activity: It modifies exogenous Vac8p in a temperature-, dose- and time-dependent manner, and is sensitive to bromo-palmitate, a known inhibitor of protein palmitoylation in vivo. Importantly, PAT is specific for palmitoyl-CoA, since myristoyl- and stearyl-CoA can compete with labeled Pal-CoA only at 10-fold higher amounts.     

Filling of a wetland (Seine estuary,France): natural eutrophication or anthropogenic process? A sedimentological and geochemical study of wetland organic sediments

Hydrobiologia - For over a century the Seine estuary has been highly affected by human activities, resulting in a reduction of the surface of wetland habitat. Several ponds of the Vernier Marsh,...     

Development of a cell model for functional and structural analysis of connexin co-expression: achieving homogeneous and inducible expression of multiple connexins in stable transfectants

We set out to develop an in vitro cell model in which connexins 43, 40 and 45 are co-expressed in the same combinations as found in different sub-types of cardiomyocyte in vivo, using inducible promoters of the Tet-Off and Ecdysone systems. In initial studies, a heterogeneous pattern of gene expression was observed. To achieve homogeneous expression, an Internal Ribosome Entry Site (IRES) sequence was employed, ensuring that a single mRNA coded for connexin and antibiotic resistance. We then constructed plasmids that combine the inducibility of the Tet-Off and Ecdysone systems with the homogeneous expression given by the IRES constructs. These were demonstrated to give inducible and homogeneous expression. By using the reporter gene, Enhanced Green Fluorescent Protein (EGFP), it was further shown in the Tet-Off system that expression of the transfected gene was modulated homogeneously in all cells when induction was repressed. The cell model is now at a suitable stage of development for investigation of the functional correlates of the distinctive connexin co-expression found in different regions of the heart.