Kinetic and metabolic effects of nitrogen, magnesium and sulphur restriction in Xanthomonas campestris batch cultures

By reducing the concentration of nitrogen (from 5.0 to 2.5 mmol 1^-1), batch cultures of Xanthomonas campestris induced the enzyme UDP-glucose dehydrogenase and stimulated the Entner-Doudoroff pathway enzyme glucose-6-P dehydrogenase. The surplus energy generation was directed to xanthan biosynthesis resulting in a 10% polysaccharide increase. The nitrogen restriction led to a higher consumption of nitrogen (93%) whereas glucose consumption did not surpass 75% utilization. Low concentrations of both magnesium and sulphur exerted a negative effect on xanthan formation. Both restrictions reduced the phosphomannose isomerase enzyme activity by 10-fold turning the mannose transference presumably into the rate-limiting step for xanthan biosynthesis. Conversely, the rate of synthesis of glucuronic acid residues did not affect the rate of xanthan biosynthesis. Polysaccharide synthesis in magnesium and sulphur cultures was negatively affected in comparison with cell formation as the cell volumetric production rate increased from 0.037 to 0.091 g 1^-1 h^-1 and the xanthan volumetric production rate dropped from 0.133 g 1^-1 h^-1 to the minimum obtained at 0.083 g 1^-1 h^-1. The efficiency of the carbon substrate conversion was also greatly changed.     

Expression of Caveolin 1 Is Enhanced by DNA Demethylation during Adipocyte Differentiation. Status of Insulin Signaling

Caveolin 1 (Cav-1) is an essential constituent of adipocyte caveolae which binds the beta subunit of the insulin receptor (IR) and is implicated in the regulation of insulin signaling. We have found that, during adipocyte differentiation of 3T3-L1 cells the promoter, exon 1 and first intron of the Cav-1 gene undergo a demethylation process that is accompanied by a strong induction of Cav-1 expression, indicating that epigenetic mechanisms must have a pivotal role in this differentiation process. Furthermore, IR, PKB-Akt and Glut-4 expression are also increased during the differentiation process suggesting a coordinated regulation with Cav-1. Activation of Cav-1 protein by phosphorylation arises during the differentiation process, yet in fully mature adipocytes insulin is no longer able to significantly increase Cav-1 phosphorylation. However, these long-term differentiated cells are still able to respond adequately to insulin, increasing IR and PKB-Akt phosphorylation and glucose uptake. The activation of Cav-1 during the adipocyte differentiation process could facilitate the maintenance of insulin sensitivity by these fully mature adipocytes isolated from additional external stimuli. However, under the influence of physiological conditions associated to obesity, such as chronic inflammation and hypoxia, insulin sensitivity would finally be compromised.     

Comparative Assessment of Genetic and Morphological Variation at an Extensive Hybrid Zone between Two Wild Cats in Southern Brazil

Increased attention towards the Neotropical cats Leopardus guttulus and L. geoffroyi was prompted after genetic studies identified the occurrence of extensive hybridization between them at their geographic contact zone in southern Brazil. This is a region where two biomes intersect, each of which is associated with one of the hybridizing species (Atlantic Forest with L. guttulus and Pampas with L. geoffroyi). In this study, we conducted in-depth analyses of multiple molecular markers aiming to characterize the magnitude and spatial structure of this hybrid zone. We also performed a morphological assessment of these species, aiming to test their phenotypic differentiation at the contact zone, as well as the correlation between morphological features and the admixture status of the individuals. We found strong evidence for extensive and complex hybridization, with at least 40% of the individuals sampled in Rio Grande do Sul state (southernmost Brazil) identified as hybrids resulting from post-F1 generations. Despite such a high level of hybridization, samples collected in this state still comprised two recognizable clusters (genetically and morphologically). Genetically pure individuals were sampled mainly in regions farther from the contact zone, while hybrids concentrated in a central region (exactly at the interface between the two biomes). The morphological data set also revealed a strong spatial structure, which was correlated with the molecular results but displayed an even more marked separation between the clusters. Hybrids often did not present intermediate body sizes and could not be clearly distinguished morphologically from the parental forms. This observation suggests that some selective pressure may be acting on the hybrids, limiting their dispersal away from the hybrid zone and perhaps favoring genomic combinations that maintain adaptive phenotypic features of one or the other parental species.     

Desulfovibrio vulgaris bacterioferritin uses H(2)O(2) as a co-substrate for iron oxidation and reveals DPS-like DNA protection and binding activities

β?-GPI (β?-glycoprotein I) is a plasma glycoprotein ascribed with an anti-angiogenic function; however, the biological role and molecular basis of its action in cell migration remain unknown. The aim of the present study was to assess the contribution of β?-GPI to HAEC (human aortic endothelial cell) migration and the details of its underlying mechanism. Using wound healing and Boyden chamber assays, we found that β?-GPI inhibited endothelial cell migration, which was restored by its neutralizing antibody. NF-κB (nuclear factor κB) inhibitors and lentiviral siRNA (small interfering RNA) silencing of NF-κB significantly attenuated the inhibitory effect of β?-GPI on cell migration. Moreover, β?-GPI was found to induce IκBα (inhibitor of NF-κB) phosphorylation and translocation of p65 and p50. We further demonstrated that mRNA and protein levels of eNOS [endothelial NO (nitric oxide) synthase] and NO production were all increased by β?-GPI and these effects were remarkably inhibited by NF-κB inhibitors and siRNAs of p65 and p50. Furthermore, β?-GPI-mediated inhibition of cell migration was reversed by eNOS inhibitors and eNOS siRNAs. The findings of the present study provide novel insight into the ability of β?-GPI to inhibit endothelial cell migration predominantly through the NF-κB/eNOS/NO signalling pathway, which indicates a potential direction for clinical therapy in vascular diseases.     

Cellular characterisation of Candida tropicalis presenting fluconazole-related trailing growth

We assessed fluconazole susceptibility in 52 Candida tropicalis clinical strains using seven antifungal susceptibility methods, including broth microdilution (BMD) [standard M27 A3 (with neutral and acid pH), ATB Fungus 3, Vitek 2 system and flow cytometric analysis] and agar-based methods (disk diffusion and E-test). Trailing growth, detection of cell-associated secreted aspartic proteases (Saps) and morphological and ultrastructural traits of these clinical strains were also examined. The ranges of fluconazole 24 h-minimum inhibitory concentration (MIC) values were similar among all methods. The essential agreement among the methods used for MIC determinations was excellent and all methods categorised all strains as susceptible, except for one strain that showed a minor error. The presence of the trailing effect was assessed by six methods. Trailing positivity was observed for 86.5-100% of the strains. The exception was the BMD-Ac method where trailing growth was not observed. Morphological and ultrastructural alterations were detected in C. tropicalis trailing cells, including mitochondrial swelling and cell walls with irregular shapes. We tested the production of Saps in 13 C. tropicalis strains expressing trailing growth through flow cytometry. Our results showed that all of the C. tropicalis strains up-regulated surface Sap expression after 24 h or 48 h of exposure to fluconazole, which was not observed in untreated yeast strains. We concluded that C. tropicalis strains expressing trailing growth presented some particular features on both biological and ultrastructural levels.     

Genetic polymorphisms of glutathione S-transferases and cytochrome P450 enzymes as susceptibility factors to systemic lupus erythematosus in southern Brazilian patients

Systemic lupus erythematosus (SLE) is an autoimmune chronic inflammatory disease that presents several clinical manifestations, affecting multiple organs and systems. Immunological, environmental, hormonal and genetic factors may contribute to disease. Genes and proteins involved in metabolism and detoxification of xenobiotics are often used as susceptibility markers to diseases with environmental risk factors. Cytochrome P450 (CYP) enzymes activate the xenobiotic making it more reactive, while the Glutathione S-transferases (GST) enzymes conjugate the reduced glutathione with electrophilic compounds, facilitating the toxic products excretion. CYP and GST polymorphisms can alter the expression and catalytic activity of enzymes. This study aimed to investigate the role of genetic variants of CYP and GST in susceptibility and clinical expression of SLE, through the analysis of GSTM1 null, GSTT1 null, GSTP1*Ile105Val, CYP1A1*2C and CYP2E1*5B polymorphisms. 371 SLE patients from Hospital de Clínicas de Porto Alegre and 522 healthy blood donors from southern Brazil were evaluated. GSTP1 and CYP variants were genotyped using PCR–RFLP and GSTT1 and GSTM1 variants were analyzed by multiplex PCR. Among European-derived individuals, a lower frequency of GSTP1*Val heterozygous genotypes was found in SLE patients when compared to controls (p = 0.005). In African-derived SLE patients, the CYP2E1*5B allelic frequency was higher in relation to controls (p = 0.054). We did not observe any clinical implication of the CYP and GST polymorphisms in patients with SLE. Our data suggest a protective role of the GSTP1*Ile/Val heterozygous genotype against the SLE in European-derived and a possible influence of the CYP2E1*5B allele in SLE susceptibility among African-derived individuals.     

The composition of arbuscular mycorrhizal fungal communities in the roots of a ruderal forb is not related to the forest fragmentation process

Land‐use changes and forest fragmentation have strong impact on biodiversity. However, little is known about the influence of new landscape configurations on arbuscular mycorrhizal fungal (AMF) community composition. We used 454 pyrosequencing to assess AMF diversity in plant roots from a fragmented forest. We detected 59 virtual taxa (VT; phylogenetically defined operational taxonomic units) of AMF – including 10 new VT – in the roots of Euphorbia acerensis. AMF communities were mainly composed of members of family Glomeraceae and were similar throughout the fragmented landscape, despite variation in forest fragment size (i.e. small, medium and large) and isolation (i.e. varying pairwise distances). AMF communities in forest fragments were phylogenetically clustered compared with the global, but not regional and local AMF taxon pools. This indicates that non‐random community assembly processes possibly related to dispersal limitation at a large scale, rather than habitat filtering or biotic interactions, may be important in structuring the AMF communities. In this system, forest fragmentation did not appear to influence AMF community composition in the roots of the ruderal plant. Whether this is true for AMF communities in soil and the roots of other ecological groups of host plants or in other habitats deserves further study.