Taken together, these data suggest that daily fluctuations in GSH may promote the health of the nervous MLN4924 system more efficiently than if GSH is maintained at constitutively elevated levels. Another important point is that while per01 exhibits constant high GSH levels, the expression of the GSH-conjugating enzyme GstD1 is significantly reduced in this mutant. This suggests that dysregulation between GSH supply and utilization may occur in clock-deficient flies. One important question that remains to be addressed is whether rhythms in GSH-biosynthesis are controlled cell-autonomously or systemically. The circadian system in fly heads consists of several clusters of central pacemaker neurons forming a circuit responsible for circadian rhythms of locomotor activity. In addition, retinal photoreceptors, sensory neurons, glia, and other cells contain a molecular clock mechanism, which can function independently of the central pacemaker. Transcriptional rhythms that are detected in whole heads may be generated in peripheral oscillators. Nevertheless, at least some central pacemaker neurons appear to be among the cells showing transcriptional Gclc and Gclm rhythms, based on microarray analysis of isolated pacemaker cells. While the range of cells displaying rhythmic GSH biosynthesis remains to be determined, it is likely to be broad. A recent genome-wide study suggests that circadian expression of Gclc may occur in isolated fly brains, and our data suggest that Gclc and Gclm expression is also rhythmic in fly bodies. What is the biological advantage of adding a circadian level of regulation to GSH biosynthesis? While excessive ROS levels are detrimental to cell function, some levels of ROS are necessary in the organism, as these molecules are responsible for essential processes including cell signaling cascades and immune response. Thus, GSH acts not only as an antioxidant, but also plays a critical role in a plethora of redox-sensitive cellular functions. While over-expression of GCLc in Drosophila neuronal tissue, and thus increased GSH levels, correlated with protection against oxidative stress and extension of lifespan, recent findings suggests that GSH may rather function via affecting specific metabolic and defense pathways. An array of connections has been recently established between circadian clocks and metabolism in mammals and in flies. Our present study adds an important novel link to this array by demonstrating circadian control of glutathione, a compound that is critically involved in maintaining human health. The pathogenesis of diabetic heart disease is multi-factorial and complex. Putative mechanisms include metabolic disturbances, myocardial fibrosis and small vessel disease. High dietary intake of free fatty acids may result in intracellular accumulation of potentially toxic intermediates of the lipid metabolism, all of which lead to impaired myocardial performance and morphological changes. At the late stage of the disease myocyte loss and replacement fibrosis is increased, indicating cardiac remodeling in patients with type-2 diabetes mellitus. In accordance, assessment of cardiac lipid metabolism by means of magnetic resonance spectroscopy in obese patients with T2DM and non-ischemic cardiomyopathy demonstrated increased intramyocardial lipid content. However, to date contradictive results have been published concerning the short term effects of MYCL accumulation on cardiac function. Growing evidence indicates a potential relationship between chronic hyperinsulinemia in pre-diabetic patients and structural changes of the heart leading to myocardial fibrosis. A crucial role in the pathogenesis of myocardial hypertrophy has been identified for insulin-related cell signaling pathways including the insulin/PI3k/PKB/Akt axis.

The F6LL motif in the Cterminus, the RRR motif in the third intracellular loop, and an isolated Leu residue in the ICL1, which are essential for export trafficking of a2B-AR. In a continuing effort to search for the structural determinants of a2-AR transport, we expanded our studies to define the role of the ICL1 in the cell-surface expression of a2A-AR. Surprisingly we found that, in addition to Leu residue, a neighboring Lys residue specifically modulates the ER export and cell-surface expression of a2A-AR and this function is likely dictated by its positively charged property. These data provide the first evidence indicating that the ICL1 may possess multiple signals that use distinct mechanisms to control the processing of a2AAR. The molecular mechanisms underlying export from the ER and subsequent transport to the cell surface of the GPCR superfamily remain poorly elucidated. It has been significant efforts to define the structural determinants for GPCR export and several highly conserved hydrophobic and charged sequences which are required for GPCR export from the ER to the cell surface have been identified. We have previously investigated the role of the ICL1 in the cell-surface transport of GPCRs and identified a Leu residue essential for ER export of a group of GPCRs, including a2B-AR. This Leu residue is extremely conserved amongst the family A GPCRs and indeed, as Adriamycin demonstrated in this manuscript, Leu64 in the ICL1 also plays an obligatory role in a2A-AR export from the ER and transport to the cell surface. Thus, this isolated Leu may provide a common signal directing anterograde transport of multiple nascent GPCRs. The most important finding described in this manuscript is that different positively charged residues on the ICL1 have differential impacts on the cell-surface transport of distinct GPCRs. During the studies on the function of the ICL1. First, intact cell ligand binding and flow cytometry to quantify the cell-surface receptors showed that mutation of Lys65 to Ala reduced a2A-AR cell-surface number by more than 50%. Second, the significant reduction of receptor expression at the cell surface was supported by direct visualization of intracellular localization of the mutated receptors. As revealed by confocal microscopy, GFP-tagged K65A was extensively expressed inside the cell which is in contrast to robust cell-surface expression of its wild-type counterpart. Third, the function of Lys65 in controlling a2A-AR transport is also supported by receptor-mediated signal propagation as measured by ERK1/2 activation. The reduction of ERK1/2 activation in cells expressing K65A as compared with cells expressing wild-type a2A-AR could be simply due to less expression of the mutated a2AAR at the cell surface, which are available for binding to the ligand, indicating that mutation of Lys65 not only reduced the cellsurface expression of a2A-AR but also attenuated receptormediated signal propagation. These data strongly indicate that, in addition to Leu64, Lys65 in the ICL1 also plays a crucial role in regulating cell-surface transport of a2A-AR. By contrast, mutation of the positively charged residue at the same position, Arg49, did not influence the cell-surface transport of a2B-AR. These data demonstrated for the first time that a single positively charged residue on the short ICL1 is involved in the regulation of export trafficking of GPCRs in a receptor subtype-specific fashion. It is likely that Lys65 residue modulates a2A-AR transport at the level of the ER. We found that the a2A-AR mutant K65A was extensively co-localized with the ER marker DsRed2-ER, suggesting that the mutant was unable to exit from the ER where they are synthesized. Therefore, these studies provide another novel regulatory mechanism for the ER export of nascent a2A-AR.

Profiles from twelve volunteers have 100% similarity between day -6 and day 0, just before AMC exposure. Stability of microbiota was not absolute. In another study, monthly monitoring of Bifidobacterium species in 6 adults during 8 months, using specific qPCR, showed a very stable bifidobacterial microbiota for 5 volunteers and high variations in the population and composition for the last one. Using bifidobacterial DGGE on samples collected during a 6-week period, Vanhoutte and co-workers showed a close profile grouping for 3 subjects with similarity values ranging from 81.4% to 100%, and one subject with a mean similarity value of 70%. At day 5, in our study, mean similarity percentage of TTGE profiles versus the reference period was 55.8%67.6%. The microbiota were modified for the most, except for four volunteers, three resistant to AMC and the last perturbed only at day 8. The resistance of some microbiota has been clearly demonstrated. In the composition of their fecal microbiota than those observed in subjects without such activity. This was thought to be because the beta-lactamase activity destroyed the antibiotic residues that reach the colon during treatment, thus reducing alterations in the microbiota to a minimum. In spite of clavulanic acid presence which is a beta-lactamase inhibitor, remaining intestinal beta-lactamases from individual microbiota could influence the amount of beta-lactam present in the feces during AMC exposure and explain the resistance to changes of some microbiota. Similarity percentages of TTGE profiles at day 33 and day 64 were 59.6% and 62.3% respectively, showing that microbiota did not return to baseline. Cloning and sequencing were performed to identify bands of interest and to evaluate if the changes of bands corresponded to changes of species or of strains within the same species. The same identifications were obtained for bands with identical Rf. In agreement with previous studies, B. adolescentis, B. longum and the B. pseudocatenulatum/B. catenulatum group were the most frequent predominant bifidobacterial species present in adult microbiota followed by B. bifidum. The mean number of Bifidobacterium species per sample MLN4924 cost harbored in dominant microbiota is significantly lower at day 5 compared to reference period. In another study, the average number of species detected per individual were 2.861.2 in healthy adults. Furthermore, at day 5, significant alterations for some Bifidobacterium species were observed: for example, occurrence of B. adolescentis decreased significantly. In some cases, species not present at day 0 and probably belonging to the subdominant microbiota, became dominant, eg B. longum or B. breve. The occurrence of B. longum remained stable after the antibiotherapy. As enlightened in previous studies, the antimicrobial effect is dose-dependent and amoxicillin showed variable MIC depending on species or strains tested. Generally, B. adolescentis, B. bifidum and B. pseudocatenulatum seemed to be more susceptible in vitro than was B. longum. Thus, our results could be explained by MIC values, as well as intestinal beta-lactamases from individual microbiota. Similar results were previously obtained within microbiota of infants treated with a 7day-amoxicillin treatment, but long-term impact was not monitored. Jaccard’s similarity coefficients indicated that differences between TTGE profiles corresponded to species changes and not only to strains changes. B. bifidum was not entirely recovered at day 33 or day 64. In a previous study, a molecular monitoring of intestinal Bifidobacterium strains in four adults using RFLP and ribotyping, showed little variations 30 days and 90 days after an AMC exposure.

Able to protect cells from oxidative stress, regulate activity of detoxification enzymes and mediate redox-sensitive PD325901 signaling. Previous studies reported daily changes in GSH levels in different mammalian organs but the role of circadian mechanism in these fluctuations has not been addressed. Genome-wide analyses of circadian transcriptome in fly heads by microarray, or RNA-seq suggests that the expression of some genes comprising glutathionesynthesizing and conjugating systems may occur in a circadian manner. Here we utilized the genetic tools available in Drosophila to determine whether there is a causal relationship between circadian clocks and GSH-related pathways. We uncovered daily oscillations in glutathione levels in fly heads and investigated the molecular mechanisms underlying this rhythm. We report that the circadian clock is involved in regulating de novo glutathione biosynthesis. This study advanced our understanding of the effects of circadian clocks on cellular homeostasis. We found that the circadian system regulates de novo synthesis of glutathione by direct transcriptional control of the genes encoding GCL subunits, as well as modulation of the activity of the GCL holoenzyme and hence, its end-point product, GSH. Given the conserved nature of the circadian clock and that many metabolites linked to redox show diurnal oscillations in mammals the molecular connections we established here between the circadian clock and GSH biosynthesis may be conserved across different phyla. We show that GSH undergoes circadian fluctuations in Drosophila heads, reaching its highest levels in the morning. While diurnal GSH variations were previously reported in different mammalian organs, such as the liver, the underlying molecular mechanism was not elucidated. A critical finding of our study is that the generation of the GSH rhythm in Drosophila heads involves transcriptional regulation of genes that encode subunits comprising GCL, the first and rate limiting enzyme in glutathione production. Daily rhythms for both Gclm and Gclc mRNA were discerned in LD with peak expression in the early and late night, respectively. However, Gclc mRNA did not show significant fluctuations in DD, suggesting that the rhythm may have dampened or is modulated by LD. On the other hand, the expression of both genes was significantly altered in mutants with defects in the positive or negative arm of the clock loop. Namely, expression of Gclc and Gclm was lower at the expected peak in cyc01 flies, which have a disrupted CLK/CYC complex, and higher at the expected trough in per01 mutants lacking periodic repression of CLK/CYC activity. Thus, our functional genetic data suggest that Gclc and Gclm may be activated by the CLK/CYC complex. This conclusion is consistent with a recent genome-wide study suggesting that CLK/CYC binds chromatin in the vicinity of the Gclc and Gclm gene promoters in a time dependent manner. Since CLK binding could not be unambiguously mapped because of its occurrence near transcription start sites of genes adjacent to Gclc and Gclm, we investigated the expression of these neighboring genes and found them to be non-rhythmic. Because GSH biosynthesis is critical for cellular health, transcriptional regulation of Gclc and Gclm have been studied intensively in mammals. These genes are known to be induced by oxidative stress and electrophiles through the binding of stress responsive transcription factors to AP-1 and electrophile response elements. Analysis of DNA regulatory regions revealed the presence of such consensus motifs in the Drosophila Gclc and Gclm promoters. In mammals, Gclc is induced via Keap1/Nrf2 signaling; thus we examined the transcriptional profiles of cncC,, and Keap1.

Considering the number of samples to analyse, molecular inventories were not possible. Pyrosequencing could have been used. Nevertheless we decided to use PCR-TTGE since it was less expensive and allowed a greater number of samples to be analysed. Indeed, methods such as TTGE and DGGE based on sequence-specific separation of 16S rRNA gene amplicons, are among the best methods for rapid high throughput comparison of bacterial communities or bifidobacterial species over time. In the present study, we explored, on a 76-day period, the quantitative and qualitative changes occurring in total microbiota and also in the Bifidobacterium genus, in 18 adult men after a 5-day amoxicillin-clavulanic acid treatment, using specific real-time PCR and PCR-TTGE combined with cloned sequence analysis. The intraindividual similarity indices of the TTGE profiles were calculated, considering day -12, -6 and 0 as reference period. Mean similarity percentage within reference period was 84.5%64.1%. Only seven subjects Wortmannin presented a 100% similarity between day-12, day-6 and day 0, but twelve subjects presented a 100% similarity between day -6 and day 0 and little variations occurred for the others. At day 5, mean similarity percentages were 55.8%67.6%. The microbiota of three subjects did not respond to AMC treatment, whereas there was a marked change of TTGE profiles for the others. One or two months after the end of AMC treatment, the mean similarity percentages of TTGE profiles were 59.6% and 62.3% respectively, showing that microbiota was still modified. To improve comparisons, two groups were constituted. The first gathered volunteers with Dice’s similarity coefficients at day 64$80% ) and the others. The microbiota of three individuals within the 5 showed marked alterations of TTGE profiles at day 5 and returned to similarity percentages $90% at day 64. The last two microbiota were resistant to AMC. In this group, the microbiota studied in the reference period was very stable. Among the other volunteers microbiota, a Dice similarity coefficient between 0 and 78% was found on day 64. The TTGE profiles from Bifidobacterium species of first or second groups were not specific to one group. To check if this profile variation corresponded only to strain change or to species change, identification of bands were realised. Most fragments co-migrated to the same position as reference strains or clones but a few migrated to different positions and could not be identified in this way. Many cultured collection bifidobacteria and many clones are present in our data bank. Recently, twenty six bands were cloned and sequenced. A previous study on the same 18 volunteers focusing only on bacterial resistance showed that the administration of AMC increased the amoxicillin resistant Escherichia coli and enterococci. From a baseline of less than 1%, more than 30% of resistant E. coli and 6% of resistant enterococci were observed and progressively decreased to baseline at day 12. In the present study, the impact of a 5-day AMC treatment was quantitatively and qualitatively assessed at short- and long-term on total microbiota and on fecal Bifidobacterium species in 18 healthy men. Bifidobacteria were detected in all fecal microbiota from the 18 subjects before exposure to AMC. The counts of total bacteria and total bifidobacteria were significantly altered by AMC at day 5 and returned to baseline at day 8. Numerous publications report modifications of fecal microbiota after AMC treatment but only at genus or phylogenetic group level.