Development of glycopeptide-intermediate resistance by Staphylococcus aureus leads to attenuated infectivity in a rat model of endocarditis.

Glycopeptide-intermediate resistant Staphylococcus aureus (GISA) are characterized by multiple changes in the cell wall and an altered expression of global virulence regulators. We investigated whether GISA are affected in their infectivity in a rat model of experimental endocarditis. The glycopeptide-susceptible, methicillin-resistant S. aureus M1V2 and its laboratory-derived GISA M1V16 were examined for their ability to (i) adhere to fibrinogen and fibronectin in vitro, (ii) persist in the bloodstream after intravenous inoculation, (iii) colonize aortic vegetations in rats, and (iv) compete for valve colonization by co-inoculation. Both GISA M1V16 and M1V2 adhered similarly to fibrinogen and fibronectin in vitro. In rats, GISA M1V16 was cleared faster from the blood (P < 0.05) and required 100-times more bacteria than parent M1V2 (10(6) versus 10(4)CFU) to infect 90% of vegetations. GISA M1V16 also had 100 to 1000-times lower bacterial densities in vegetations. Moreover, after co-inoculation with GISA M1V16 and M1V2Rif, a rifampin-resistant variant of M1V2 to discriminate them in organ cultures, GISA M1V16 was out-competed by the glycopeptide-susceptible counterpart. Thus, in rats with experimental endocarditis, GISA showed an attenuated virulence, likely due to a faster clearance from the blood and a reduced fitness in cardiac vegetations. The GISA phenotype appeared globally detrimental to infectivity.

Glutamate Transport Decreases Mitochondrial pH and Modulates Oxidative Metabolism in Astrocytes.

During synaptic activity, the clearance of neuronally released glutamate leads to an intracellular sodium concentration increase in astrocytes that is associated with significant metabolic cost. The proximity of mitochondria at glutamate uptake sites in astrocytes raises the question of the ability of mitochondria to respond to these energy demands. We used dynamic fluorescence imaging to investigate the impact of glutamatergic transmission on mitochondria in intact astrocytes. Neuronal release of glutamate induced an intracellular acidification in astrocytes, via glutamate transporters, that spread over the mitochondrial matrix. The glutamate-induced mitochondrial matrix acidification exceeded cytosolic acidification and abrogated cytosol-to-mitochondrial matrix pH gradient. By decoupling glutamate uptake from cellular acidification, we found that glutamate induced a pH-mediated decrease in mitochondrial metabolism that surpasses the Ca(2+)-mediated stimulatory effects. These findings suggest a model in which excitatory neurotransmission dynamically regulates astrocyte energy metabolism by limiting the contribution of mitochondria to the metabolic response, thereby increasing the local oxygen availability and preventing excessive mitochondrial reactive oxygen species production.

RasGAP-derived fragment N increases the resistance of beta cells towards apoptosis in NOD mice and delays the progression from mild to overt diabetes.

The caspase-3-generated RasGAP N-terminal fragment (fragment N) inhibits apoptosis in a Ras-PI3K-Akt-dependent manner. Fragment N protects various cell types, including insulin-secreting cells, against different types of stresses. Whether fragment N exerts a protective role during the development of type 1 diabetes is however not known. Non-obese diabetic (NOD) mice represent a well-known model for spontaneous development of type 1 diabetes that shares similarities with the diseases encountered in humans. To assess the role of fragment N in type 1 diabetes development, a transgene encoding fragment N under the control of the rat insulin promoter (RIP) was back-crossed into the NOD background creating the NOD-RIPN strain. Despite a mosaic expression of fragment N in the beta cell population of NOD-RIPN mice, islets isolated from these mice were more resistant to apoptosis than control NOD islets. Islet lymphocytic infiltration and occurrence of a mild increase in glycemia developed with the same kinetics in both strains. However, the period of time separating the mild increase in glycemia and overt diabetes was significantly longer in NOD-RIPN mice compared to the control NOD mice. There was also a significant decrease in the number of apoptotic beta cells in situ at 16 weeks of age in the NOD-RIPN mice. Fragment N exerts therefore a protective effect on beta cells within the pro-diabetogenic NOD background and this prevents a fast progression from mild to overt diabetes.

Antifungal resistance and new strategies to control fungal infections.

Despite improvement of antifungal therapies over the last 30 years, the phenomenon of antifungal resistance is still of major concern in clinical practice. In the last 10 years the molecular mechanisms underlying this phenomenon were extensively unraveled. In this paper, after a brief overview of currently available antifungals, molecular mechanisms of antifungal resistance will be detailed. It appears that major mechanisms of resistance are essential due to the deregulation of antifungal resistance effector genes. This deregulation is a consequence of point mutations occurring in transcriptional regulators of these effector genes. Resistance can also follow the emergence of point mutations directly in the genes coding antifungal targets. In addition we further describe new strategies currently undertaken to discover alternative therapy targets and antifungals. Identification of new antifungals is essentially achieved by the screening of natural or synthetic chemical compound collections. Discovery of new putative antifungal targets is performed through genome-wide approaches for a better understanding of the human pathogenic fungi biology.

Smad3 deficiency in mice protects against insulin resistance and obesity induced by a high-fat diet.

OBJECTIVE-Obesity and associated pathologies are major global health problems. Transforming growth factor-beta/Smad3 signaling has been implicated in various metabolic processes, including adipogenesis, insulin expression, and pancreatic beta-cell function. However, the systemic effects of Smad3 deficiency on adiposity and insulin resistance in vivo remain elusive. This study investigated the effects of Smad3 deficiency on whole-body glucose and lipid homeostasis and its contribution to the development of obesity and type 2 diabetes.RESEARCH DESIGN AND METHODS-We compared various metabolic profiles of Smad3-knockout and wild-type mice. We also determined the mechanism by which Smad3 deficiency affects the expression of genes involved in adipogenesis and metabolism. Mice were then challenged with a high-fat diet to study the impact of Smad3 deficiency on the development of obesity and insulin resistance.RESULTS-Smad3-knockout mice exhibited diminished adiposity with improved glucose tolerance and insulin sensitivity. Chromatin immunoprecipitation assay revealed that Smad3 deficiency increased CCAAT/enhancer-binding protein beta-C/EBP homologous protein 10 interaction and exerted a differential regulation on proliferator-activated receptor beta/delta and proliferator-activated receptor gamma expression in adipocytes. Focused gene expression profiling revealed an altered expression of genes involved in adipogenesis, lipid accumulation, and fatty acid beta-oxidation, indicative of altered adipose physiology. Despite reduced physical activity with no modification in food intake, these mutant mice were resistant to obesity and insulin resistance induced by a high-fat diet.CONCLUSIONS-Smad3 is a multifaceted regulator in adipose physiology and the pathogenesis of obesity and type 2 diabetes, suggesting that Smad3 may be a potential target for the treatment of obesity and its associated disorders.

Resistance of the internal mammary artery to restenosis: a histomorphologic study of various porcine arteries.

BACKGROUND/AIMS: Restenosis after percutaneous transluminal angioplasty (PTA) of the internal mammary artery (IMA) grafts is much less pronounced than in other arteries and venous grafts. The aim of the study was to test whether various arteries respond differently to dilatation. METHODS: PTA of the IMA, carotid, renal and circumflex coronary (RCx) arteries was performed in 9 pigs (balloon to artery ratio of 1:1.5). After 8 weeks, angiography was repeated and vessels prepared for histological analysis. Immunohistochemical staining was done to examine proliferative activity (Ki67) and to identify the vasa vasorum of the adventitia (F VIII-RA). RESULTS: The intima-media ratio after PTA was lowest in the IMA (0.06), followed by the carotid (0.27) and renal arteries (0.49) and the RCx (0.69). Proliferation of the intima was seen at 287 degrees of the vessel circumference in the RCx, at 286 degrees in the renal and at 166 degrees in the carotid artery. No proliferative activity was seen in the IMA. The intima-adventitia ratio was lower in the IMA than in the RCx and renal arteries (p &lt; 0.05). CONCLUSION: Intima proliferation after PTA varies between the different vessels, with best results seen in the IMA. There are differences in remodeling after PTA between muscular, muscular/elastic and elastic arteries.

Protease-activated receptor 2 signalling promotes dendritic cell antigen transport and T-cell activation in vivo.

Deficiency of protease-activated receptor-2 (PAR2) modulates inflammation in several models of inflammatory and autoimmune disease, although the underlying mechanism(s) are not understood. PAR2 is expressed on endothelial and immune cells, and is implicated in dendritic cell (DC) differentiation. We investigated in vivo the impact of PAR2 activation on DCs and T cells in PAR2 wild-type (WT) and knockout (KO) mice using a specific PAR2 agonist peptide (AP2). PAR2 activation significantly increased the frequency of mature CD11c(high) DCs in draining lymph nodes 24 hr after AP2 administration. Furthermore, these DCs exhibited increased expression of major histocompatibility complex (MHC) class II and CD86. A significant increase in activated (CD44(+) CD62(-)) CD4(+) and CD8(+) T-cell frequencies was also observed in draining lymph nodes 48 hr after AP2 injection. No detectable change in DC or T-cell activation profiles was observed in the spleen. The influence of PAR2 signalling on antigen transport to draining lymph nodes was assessed in the context of delayed-type hypersensitivity. PAR2 WT mice that were sensitized by skin-painting with fluorescein isothiocyanate (FITC) to induce delayed-type hypersensitivity possessed elevated proportion of FITC(+) DCs in draining lymph nodes 24 hr after FITC painting when compared with PAR2 KO mice (0.95% versus 0.47% of total lymph node cells). Collectively, these results demonstrate that PAR2 signalling promotes DC trafficking to the lymph nodes and subsequent T-cell activation, and thus provides an explanation for the pro-inflammatory effect of PAR2 in animal models of inflammation.

CYP3A5 and ABCB1 genes influence blood pressure and response to treatment, and their effect is modified by salt.

The permeability-glycoprotein efflux-transporter encoded by the multidrug resistance 1 (ABCB1) gene and the cytochromes P450 3A4/5 encoded by the CYP3A4/5 genes are known to interact in the transport and metabolism of many drugs. Recent data have shown that the CYP3A5 genotypes influence blood pressure and that permeability-glycoprotein activity might influence the activity of the renin-angiotensin system. Hence, these 2 genes may contribute to blood pressure regulation in humans. We analyzed the association of variants of the ABCB1 and CYP3A5 genes with ambulatory blood pressure, plasma renin activity, plasma aldosterone, endogenous lithium clearance, and blood pressure response to treatment in 72 families (373 individuals; 55% women; mean age: 46 years) of East African descent. The ABCB1 and CYP3A5 genes interact with urinary sodium excretion in their effect on ambulatory blood pressure (daytime systolic: P=0.05; nighttime systolic and diastolic: P<0.01), suggesting a gene-gene-environment interaction. The combined action of these genes is also associated with postproximal tubular sodium reabsorption, plasma renin activity, plasma aldosterone, and with an altered blood pressure response to the angiotensin-converting enzyme inhibitor lisinopril (P<0.05). This is the first reported association of the ABCB1 gene with blood pressure in humans and demonstration that genes encoding for proteins metabolizing and transporting drugs and endogenous substrates contribute to blood pressure regulation.

A genome-wide association study of resistance to HIV infection in highly exposed uninfected individuals with hemophilia A.

Human genetic variation contributes to differences in susceptibility to HIV-1 infection. To search for novel host resistance factors, we performed a genome-wide association study (GWAS) in hemophilia patients highly exposed to potentially contaminated factor VIII infusions. Individuals with hemophilia A and a documented history of factor VIII infusions before the introduction of viral inactivation procedures (1979-1984) were recruited from 36 hemophilia treatment centers (HTCs), and their genome-wide genetic variants were compared with those from matched HIV-infected individuals. Homozygous carriers of known CCR5 resistance mutations were excluded. Single nucleotide polymorphisms (SNPs) and inferred copy number variants (CNVs) were tested using logistic regression. In addition, we performed a pathway enrichment analysis, a heritability analysis, and a search for epistatic interactions with CCR5 Δ32 heterozygosity. A total of 560 HIV-uninfected cases were recruited: 36 (6.4%) were homozygous for CCR5 Δ32 or m303. After quality control and SNP imputation, we tested 1 081 435 SNPs and 3686 CNVs for association with HIV-1 serostatus in 431 cases and 765 HIV-infected controls. No SNP or CNV reached genome-wide significance. The additional analyses did not reveal any strong genetic effect. Highly exposed, yet uninfected hemophiliacs form an ideal study group to investigate host resistance factors. Using a genome-wide approach, we did not detect any significant associations between SNPs and HIV-1 susceptibility, indicating that common genetic variants of major effect are unlikely to explain the observed resistance phenotype in this population.

Transport and metabolism of [214-C]abscisic acid in maize root

The tips of intact maize (cv. LG 11) roots, maintained vertically, were pretreated with a droplet of buffer solution or a bead of anion exchange resin, both containing [214-C]abscisic acid (ABA). A significant basipetal ABA movement was observed and two metabolites of ABA (possibly phaseic acid and dihydrophaseic acid) were found. ABA pretreatment enhanced the gravireaction of 10 mm apical root segments kept both in the dark and in the light. The possibility that ABA could be one of the endogenous growth inhibitors produced or released by the cap cells is discussed.

Aldosterone paradox: differential regulation of ion transport in distal nephron.

The mechanisms through which aldosterone promotes apparently opposite effects like salt reabsorption and K(+) secretion remain poorly understood. The identification, localization, and physiological analysis of ion transport systems in distal nephron have revealed an intricate network of interactions between several players, revealing the complex mechanism behind the aldosterone paradox. We review the mechanisms involved in differential regulation of ion transport that allow the fine tuning of salt and K(+) balance.