Repression of Ppargc1a Gene in Liver of Hyperglycemic Rats Induced with High Fat Diet Combined with Streptozotocin

Type 2 diabetes mellitus implies deregulation of multiple metabolic processes, being the maintenance of glycemia one of the most important. Many genes are involved in the deregulation of this particular process. Therefore, the aim of this study was to evaluate gene expression of genes related to type 2 diabetes mellitus, in the liver and pancreas of rats with hyperglycemia induced by high fat diet along with a low single dose of streptozotocin. Ahsg and Ppargc1a genes were studied in liver, whereas Kcnj11 and Slc2a2 genes were analyzed in pancreas. For this purpose, 210-240 g female rats were fed a high fat diet or a control diet for three weeks. At day 14, animals fed with high fat diet were injected with a single low dose of streptozotocin (35 mg/kg) and the control group rats were injected only with the vehicle. Plasmatic glucose, triglycerides and total cholesterol levels were measured at the beginning, day 14 and end of treatment. Body weight was also measured. Once the treatment was complete, rats were appropriately euthanized and then, pancreas and liver were surgically removed and frozen in liquid nitrogen. Total RNA was isolated using TRIzol reagent, treated with DNase land reversely transcribed to cDNA. Gene expression analysis was performed using SYBR Green - Real time PCR and comparative Cq method, using three reference genes. Rats fed with high fat diet and treated with streptozotocin showed higher values of plasmatic glucose (17.09 +/- 0.43 vs. 5.91 +/- 0.29 mmol/L, p < 0.01) and a minor expression of Ppargc1a versus the control group (2-fold less expressed, p < 0.05) in liver. We conclude that repression of Ppargc1a gene may be an important process in the establishment of chronic hyperglycemia, probably through deregulation of hepatic gluconeogenesis. However, further studies need to be performed in order to clarify the role of Ppargc1a deregulation in liver glucose homeostasis.

Repression of osteocyte Wnt/beta-catenin signaling is an early event in the progression of renal osteodystrophy

Chronic kidney diseasemineral bone disorder (CKD-MBD) is defined by abnormalities in mineral and hormone metabolism, bone histomorphometric changes, and/or the presence of soft-tissue calcification. Emerging evidence suggests that features of CKD-MBD may occur early in disease progression and are associated with changes in osteocyte function. To identify early changes in bone, we utilized the jck mouse, a genetic model of polycystic kidney disease that exhibits progressive renal disease. At 6 weeks of age, jck mice have normal renal function and no evidence of bone disease but exhibit continual decline in renal function and death by 20 weeks of age, when approximately 40% to 60% of them have vascular calcification. Temporal changes in serum parameters were identified in jck relative to wild-type mice from 6 through 18 weeks of age and were subsequently shown to largely mirror serum changes commonly associated with clinical CKD-MBD. Bone histomorphometry revealed progressive changes associated with increased osteoclast activity and elevated bone formation relative to wild-type mice. To capture the early molecular and cellular events in the progression of CKD-MBD we examined cell-specific pathways associated with bone remodeling at the protein and/or gene expression level. Importantly, a steady increase in the number of cells expressing phosphor-Ser33/37-beta-catenin was observed both in mouse and human bones. Overall repression of Wnt/beta-catenin signaling within osteocytes occurred in conjunction with increased expression of Wnt antagonists (SOST and sFRP4) and genes associated with osteoclast activity, including receptor activator of NF-?B ligand (RANKL). The resulting increase in the RANKL/osteoprotegerin (OPG) ratio correlated with increased osteoclast activity. In late-stage disease, an apparent repression of genes associated with osteoblast function was observed. These data confirm that jck mice develop progressive biochemical changes in CKD-MBD and suggest that repression of the Wnt/beta-catenin pathway is involved in the pathogenesis of renal osteodystrophy. (C) 2012 American Society for Bone and Mineral Research.

Huckebein is part of a combinatorial repression code in the anterior blastoderm

The hierarchy of the segmentation cascade responsible for establishing the Drosophila body plan is composed by gap, pair-rule and segment polarity genes. However, no pair-rule stripes are formed in the anterior regions of the embryo. This lack of stripe formation, as well as other evidence from the literature that is further investigated here, led us to the hypothesis that anterior gap genes might be involved in a combinatorial mechanism responsible for repressing the cis-regulatory modules (CRMs) of hairy (h), even-skipped (eve), runt (run), and fushi-tarazu (ftz) anterior-most stripes. In this study, we investigated huckebein (hkb), which has a gap expression domain at the anterior tip of the embryo. Using genetic methods we were able to detect deviations from the wild-type patterns of the anterior-most pair-rule stripes in different genetic backgrounds, which were consistent with Hkb-mediated repression. Moreover, we developed an image processing tool that, for the most part, confirmed our assumptions. Using an hkb misexpression system, we further detected specific repression on anterior stripes. Furthermore, bioinformatics analysis predicted an increased significance of binding site clusters in the CRMs of h 1, eve 1, run 1 and ftz 1 when Hkb was incorporated in the analysis, indicating that Hkb plays a direct role in these CRMs. We further discuss that Hkb and Slp1, which is the other previously identified common repressor of anterior stripes, might participate in a combinatorial repression mechanism controlling stripe CRMs in the anterior parts of the embryo and define the borders of these anterior stripes. (C) 2011 Elsevier Inc. All rights reserved.

Dynamic Activation and Repression of the Plasmodium falciparum rif Gene Family and Their Relation to Chromatin Modification

The regulation of variant gene expression in Plasmodium falciparum is still only partially understood. Regulation of var genes, the most studied gene family involved in antigenic variation, is orchestrated by a dynamic pattern of inherited chromatin states. Although recent evidence pointed to epigenetic regulation of transcribed and repressed rif loci, little is known about specific on/off associated histone modifications of individual rif genes. To investigate the chromatin marks for transcribed and repressed rif loci, we cultivated parasites and evaluated the transcriptional status of chosen rif targets by qRT-PCR and performed ChIP assays using H3K9ac and H3K9me3 antibodies. We then monitored changes in the epigenetic patterns in parasites after several reinvasions and also evaluated the "poised'' mark in trophozoites and schizonts of the same erythrocytic cycle by ChIP using H3K4me2 specific antibodies. Our results show that H3K9 is acetylated in transcribed rif loci and trimethylated or even unmodified in repressed rif loci. These transcriptional and epigenetic states are inherited after several reinvasions. The poised modification H3K4me2 showed a tendency to be more present in loci in trophozoites that upon progression to schizonts strongly transcribe the respective locus. However, this effect was not consistently observed for all monitored loci. While our data show important similarities to var transcription-associated chromatin modifications, the observed swiftly occurring modifications at rif loci and the absence of H3K9 modification point to a different dynamic of recruitment of chromatin modifying enzymes.

miR-21 may acts as an oncomir by targeting RECK, a matrix metalloproteinase regulator, in prostate cancer

Background: Prognosis of prostate cancer (PCa) is based mainly in histological aspects together with PSA serum levels that not always reflect the real aggressive potential of the neoplasia. The micro RNA (miRNA) mir-21 has been shown to regulate invasiveness in cancer through translational repression of the Metaloproteinase (MMP) inhibitor RECK. Our aim is to investigate the levels of expression of RECK and miR-21 in PCa comparing with classical prognostic factors and disease outcome and also test if RECK is a target of miR-21 in in vitro study using PCa cell line. Materials and methods: To determine if RECK is a target of miR-21 in prostate cancer we performed an in vitro assay with PCa cell line DU-145 transfected with pre-miR-21 and anti-miR-21. To determine miR-21 and RECK expression levels in PCa samples we performed quantitative real-time polymerase chain reaction (qRT-PCR). Results: The in vitro assays showed a decrease in expression levels of RECK after transfection with pre-miR-21, and an increase of MMP9 that is regulated by RECK compared to PCa cells treated with anti-miR-21. We defined three profiles to compare the prognostic factors. The first was characterized by miR-21 and RECK underexpression (N = 25) the second was characterized by miR-21 overexpression and RECK underexpression (N = 12), and the third was characterized by miR-21 underexpression and RECK overexpression (N = 16). From men who presented the second profile (miR-21 overexpression and RECK underexpression) 91.7% were staged pT3. For the other two groups 48.0%, and 46.7% of patients were staged pT3 (p = 0.025). Conclusions: Our results demonstrate RECK as a target of miR-21. We believe that miR-21 may be important in PCa progression through its regulation of RECK, a known regulator of tumor cell invasion.

NOTES ON THE FREUDIAN CONCEPTION OF ANXIETY

NOTES ON THE FREUDIAN CONCEPTION OF ANXIETY This article belongs to the field of history and epistemology of psychoanalysis, specifically to research on the conceptual genealogy pertaining to the construction of Freudian metapsychology of anguish. It aims to present the main arguments that allow us to discuss that theories contained in Freud's work on anxiety are not exclusive and may be considered, as a whole, a single explanatory model that would encompass the various stages of production on anxiety This paper intends to examine the barriers that can be seized in the Freudian formulations, under what is conceived, since Freud, as a "second theory of anxiety".

Football in the dock: the case of homophobia.

This essay discusses some issues related to homophobia in football. To this was discussed through official documents and literature of the difficulties and challenges in the fight against homophobia in Brazil. From these initial discussions sought to relate the world of "male" and the reproduction of discrimination in football. As a form of analysis came from two authors of the social sciences: Durkheim's concept of anomie, which offers an explanation of why the repression of different (idea of the sacred and profane), and Habermas with the idea of civil society as a construction of mechanisms to fight for a transformation of reality and possibility of dialogue.

Molecular characterization of the Aspergillus nidulans fbxA encoding an F-box protein involved in xylanase induction

The filamentous fungus Aspergillus nidulans has been used as a fungal model system to study the regulation of xylanase production. These genes are activated at transcriptional level by the master regulator the transcriptional factor XInR and repressed by carbon catabolite repression (CCR) mediated by the wide-domain repressor CreA. Here, we screened a collection of 42 A. nidulans F-box deletion mutants grown either in xylose or xylan as the single carbon source in the presence of the glucose analog 2-deoxy-D-glucose, aiming to identify mutants that have deregulated xylanase induction. We were able to recognize a null mutant in a gene (fbxA) that has decreased xylanase activity and reduced xInA and xInD mRNA accumulation. The Delta fbxA mutant interacts genetically with creAd-30, creB15, and creC27 mutants. FbxA is a novel protein containing a functional F-box domain that binds to Skp1 from the SCF-type ligase. Blastp analysis suggested that FbxA is a protein exclusive from fungi, without any apparent homologs in higher eukaryotes. Our work emphasizes the importance of the ubiquitination in the A. nidulans xylanase induction and CCR. The identification of FbxA provides another layer of complexity to xylanase induction and CCR phenomena in filamentous fungi. (C) 2011 Elsevier Inc. All rights reserved.

Vectorial signalling mechanism required for cell-cell communication during sporulation in Bacillus subtilis

Spore formation in Bacillus subtilis takes place in a sporangium consisting of two chambers, the forespore and the mother cell, which are linked by pathways of cellcell communication. One pathway, which couples the proteolytic activation of the mother cell transcription factor sE to the action of a forespore synthesized signal molecule, SpoIIR, has remained enigmatic. Signalling by SpoIIR requires the protein to be exported to the intermembrane space between forespore and mother cell, where it will interact with and activate the integral membrane protease SpoIIGA. Here we show that SpoIIR signal activity as well as the cleavage of its N-terminal extension is strictly dependent on the prespore fatty acid biosynthetic machinery. We also report that a conserved threonine residue (T27) in SpoIIR is required for processing, suggesting that signalling of SpoIIR is dependent on fatty acid synthesis probably because of acylation of T27. In addition, SpoIIR localization in the forespore septal membrane depends on the presence of SpoIIGA. The orchestration of sE activation in the intercellular space by an acylated signal protein provides a new paradigm to ensure local transmission of a weak signal across the bilayer to control cellcell communication during development.

Co-metabolic models of Streptococcus thermophilus in co-culture with Lactobacillus bulgaricus or Lactobacillus acidophilus

To shed light on the interactions occurring in fermented milks when using co-cultures of Streptococcus thermophilus with Lactobacillus bulgaricus (StLb) or Lactobacillus acidophilus (StLa), a new co-metabolic model was proposed and checked either in the presence of Inulin as a prebiotic or not. For this purpose, the experimental data of concentrations of substrates and fermented products were utilized in balances of carbon, reduction degree and ATP. S. thermophilus exhibited always quicker growth compared to the other two microorganisms, while the percentage of lactose fermented to lactic acid, that of galactose metabolized, and the levels of diacetyl and acetoin formed strongly depended on the type of co-culture and the presence of inulin. The StLb co-culture led to higher acetoin and lower diacetyl levels compared to StLa, probably because of more reducing conditions or limited acetoin dehydrogenation. Inulin addition to StLa suppressed acetoin accumulation and hindered that of diacetyl, suggesting catabolite repression of alpha-acetolactate synthase expression in S. thermophilus. Both co-cultures showed the highest ATP requirements for biomass growth and maintenance at the beginning of fermentation, consistently with the high energy demand of enzyme induction during lag phase. Inulin reduced these requirements making biomass synthesis and maintenance less energy-consuming. Only a fraction of galactose was released from lactose, consistently with the galactose-positive phenotype of most dairy strains. The galactose fraction metabolized without inulin was about twice that in its presence, which suggests inhibition of the galactose transport system of S. thermophilus by fructose released from partial inulin hydrolysis. (C) 2012 Elsevier B.V. All rights reserved.

Global transcriptional response of Caulobacter crescentus to iron availability

Abstract Background In the alpha subclass of proteobacteria iron homeostasis is controlled by diverse iron responsive regulators. Caulobacter crescentus, an important freshwater α-proteobacterium, uses the ferric uptake repressor (Fur) for such purpose. However, the impact of the iron availability on the C. crescentus transcriptome and an overall perspective of the regulatory networks involved remain unknown. Results In this work we report the identification of iron-responsive and Fur-regulated genes in C. crescentus using microarray-based global transcriptional analyses. We identified 42 genes that were strongly upregulated both by mutation of fur and by iron limitation condition. Among them, there are genes involved in iron uptake (four TonB-dependent receptor gene clusters, and feoAB), riboflavin biosynthesis and genes encoding hypothetical proteins. Most of these genes are associated with predicted Fur binding sites, implicating them as direct targets of Fur-mediated repression. These data were validated by β-galactosidase and EMSA assays for two operons encoding putative transporters. The role of Fur as a positive regulator is also evident, given that 27 genes were downregulated both by mutation of fur and under low-iron condition. As expected, this group includes many genes involved in energy metabolism, mostly iron-using enzymes. Surprisingly, included in this group are also TonB-dependent receptors genes and the genes fixK, fixT and ftrB encoding an oxygen signaling network required for growth during hypoxia. Bioinformatics analyses suggest that positive regulation by Fur is mainly indirect. In addition to the Fur modulon, iron limitation altered expression of 113 more genes, including induction of genes involved in Fe-S cluster assembly, oxidative stress and heat shock response, as well as repression of genes implicated in amino acid metabolism, chemotaxis and motility. Conclusions Using a global transcriptional approach, we determined the C. crescentus iron stimulon. Many but not all of iron responsive genes were directly or indirectly controlled by Fur. The iron limitation stimulon overlaps with other regulatory systems, such as the RpoH and FixK regulons. Altogether, our results showed that adaptation of C. crescentus to iron limitation not only involves increasing the transcription of iron-acquisition systems and decreasing the production of iron-using proteins, but also includes novel genes and regulatory mechanisms.

Functional characterisation of the non-essential protein kinases and phosphatases regulating Aspergillus nidulans hydrolytic enzyme production

Abstract Background Despite recent advances in the understanding of lignocellulolytic enzyme regulation, less is known about how different carbon sources are sensed and the signaling cascades that result in the adaptation of cellular metabolism and hydrolase secretion. Therefore, the role played by non-essential protein kinases (NPK) and phosphatases (NPP) in the sensing of carbon and/or energetic status was investigated in the model filamentous fungus Aspergillus nidulans. Results Eleven NPKs and seven NPPs were identified as being involved in cellulase, and in some cases also hemicellulase, production in A. nidulans. The regulation of CreA-mediated carbon catabolite repression (CCR) in the parental strain was determined by fluorescence microscopy, utilising a CreA: GFP fusion protein. The sensing of phosphorylated glucose, via the RAS signalling pathway induced CreA repression, while carbon starvation resulted in derepression. Growth on cellulose represented carbon starvation and derepressing conditions. The involvement of the identified NPKs in the regulation of cellulose-induced responses and CreA derepression was assessed by genome-wide transcriptomics (GEO accession 47810). CreA:GFP localisation and the restoration of endocellulase activity via the introduction of the ∆creA mutation, was assessed in the NPK-deficient backgrounds. The absence of either the schA or snfA kinase dramatically reduced cellulose-induced transcriptional responses, including the expression of hydrolytic enzymes and transporters. The mechanism by which these two NPKs controlled gene transcription was identified, as the NPK-deficient mutants were not able to unlock CreA-mediated carbon catabolite repression under derepressing conditions, such as carbon starvation or growth on cellulose. Conclusions Collectively, this study identified multiple kinases and phosphatases involved in the sensing of carbon and/or energetic status, while demonstrating the overlapping, synergistic roles of schA and snfA in the regulation of CreA derepression and hydrolytic enzyme production in A. nidulans. The importance of a carbon starvation-induced signal for CreA derepression, permitting transcriptional activator binding, appeared paramount for hydrolase secretion.

The intronic long noncoding RNA ANRASSF1 recruits PRC2 to the RASSF1A promoter, reducing the expression of RASSF1A and increasing cell proliferation

The down-regulation of the tumor-suppressor gene RASSF1A has been shown to increase cell proliferation in several tumors. RASSF1A expression is regulated through epigenetic events involving the polycomb repressive complex 2 (PRC2); however, the molecular mechanisms modulating the recruitment of this epigenetic modifier to the RASSF1 locus remain largely unknown. Here, we identify and characterize ANRASSF1, an endogenous unspliced long noncoding RNA (lncRNA) that is transcribed from the opposite strand on the RASSF1 gene locus in several cell lines and tissues and binds PRC2. ANRASSF1 is transcribed through RNA polymerase II and is 5'-capped and polyadenylated; it exhibits nuclear localization and has a shorter half-life compared with other lncRNAs that bind PRC2. ANRASSF1 endogenous expression is higher in breast and prostate tumor cell lines compared with non-tumor, and an opposite pattern is observed for RASSF1A. ANRASSF1 ectopic overexpression reduces RASSF1A abundance and increases the proliferation of HeLa cells, whereas ANRASSF1 silencing causes the opposite effects. These changes in ANRASSF1 levels do not affect the RASSF1C isoform abundance. ANRASSF1 overexpression causes a marked increase in both PRC2 occupancy and histone H3K27me3 repressive marks, specifically at the RASSF1A promoter region. No effect of ANRASSF1 overexpression was detected on PRC2 occupancy and histone H3K27me3 at the promoter regions of RASSF1C and the four other neighboring genes, including two well-characterized tumor suppressor genes. Additionally, we demonstrated that ANRASSF1 forms an RNA/DNA hybrid and recruits PRC2 to the RASSF1A promoter. Together, these results demonstrate a novel mechanism of epigenetic repression of the RASSF1A tumor suppressor gene involving antisense unspliced lncRNA, in which ANRASSF1 selectively represses the expression of the RASSF1 isoform overlapping the antisense transcript in a location-specific manner. In a broader perspective, our findings suggest that other non-characterized unspliced intronic lncRNAs transcribed in the human genome might contribute to a location-specific epigenetic modulation of genes.

Global transcriptional response of Caulobacter crescentus to iron availability

BACKGROUND: In the alpha subclass of proteobacteria iron homeostasis is controlled by diverse iron responsive regulators. Caulobacter crescentus, an important freshwater α-proteobacterium, uses the ferric uptake repressor (Fur) for such purpose. However, the impact of the iron availability on the C. crescentus transcriptome and an overall perspective of the regulatory networks involved remain unknown. RESULTS: In this work we report the identification of iron-responsive and Fur-regulated genes in C. crescentus using microarray-based global transcriptional analyses. We identified 42 genes that were strongly upregulated both by mutation of fur and by iron limitation condition. Among them, there are genes involved in iron uptake (four TonB-dependent receptor gene clusters, and feoAB), riboflavin biosynthesis and genes encoding hypothetical proteins. Most of these genes are associated with predicted Fur binding sites, implicating them as direct targets of Fur-mediated repression. These data were validated by β-galactosidase and EMSA assays for two operons encoding putative transporters. The role of Fur as a positive regulator is also evident, given that 27 genes were downregulated both by mutation of fur and under low-iron condition. As expected, this group includes many genes involved in energy metabolism, mostly iron-using enzymes. Surprisingly, included in this group are also TonB-dependent receptors genes and the genes fixK, fixT and ftrB encoding an oxygen signaling network required for growth during hypoxia. Bioinformatics analyses suggest that positive regulation by Fur is mainly indirect. In addition to the Fur modulon, iron limitation altered expression of 113 more genes, including induction of genes involved in Fe-S cluster assembly, oxidative stress and heat shock response, as well as repression of genes implicated in amino acid metabolism, chemotaxis and motility. CONCLUSIONS: Using a global transcriptional approach, we determined the C. crescentus iron stimulon. Many but not all of iron responsive genes were directly or indirectly controlled by Fur. The iron limitation stimulon overlaps with other regulatory systems, such as the RpoH and FixK regulons. Altogether, our results showed that adaptation of C. crescentus to iron limitation not only involves increasing the transcription of iron-acquisition systems and decreasing the production of iron-using proteins, but also includes novel genes and regulatory mechanisms