International Union of Pharmacology. LXI. Peroxisome proliferator-activated receptors.

The three peroxisome proliferator-activated receptors (PPARs) are ligand-activated transcription factors of the nuclear hormone receptor superfamily. They share a high degree of structural homology with all members of the superfamily, particularly in the DNA-binding domain and ligand- and cofactor-binding domain. Many cellular and systemic roles have been attributed to these receptors, reaching far beyond the stimulation of peroxisome proliferation in rodents after which they were initially named. PPARs exhibit broad, isotype-specific tissue expression patterns. PPARalpha is expressed at high levels in organs with significant catabolism of fatty acids. PPARbeta/delta has the broadest expression pattern, and the levels of expression in certain tissues depend on the extent of cell proliferation and differentiation. PPARgamma is expressed as two isoforms, of which PPARgamma2 is found at high levels in the adipose tissues, whereas PPARgamma1 has a broader expression pattern. Transcriptional regulation by PPARs requires heterodimerization with the retinoid X receptor (RXR). When activated by a ligand, the dimer modulates transcription via binding to a specific DNA sequence element called a peroxisome proliferator response element (PPRE) in the promoter region of target genes. A wide variety of natural or synthetic compounds was identified as PPAR ligands. Among the synthetic ligands, the lipid-lowering drugs, fibrates, and the insulin sensitizers, thiazolidinediones, are PPARalpha and PPARgamma agonists, respectively, which underscores the important role of PPARs as therapeutic targets. Transcriptional control by PPAR/RXR heterodimers also requires interaction with coregulator complexes. Thus, selective action of PPARs in vivo results from the interplay at a given time point between expression levels of each of the three PPAR and RXR isotypes, affinity for a specific promoter PPRE, and ligand and cofactor availabilities.

Cognate microRNA-Gene Regulatory Networks Mediating Glioblastoma Oncogenic Properties

Malignant gliomas, including the most common and fatal form glioblastoma (GBM, WHO grade IV astrocytoma), remain a challenge to treat. In the United States and Europe, more than 30,000 patients per year are newly diagnosed with GBM. Despite ongoing trials, the best currently available multimodal treatment approaches include surgical resection followed by concomitant and adjuvant radiation (RT) and temozolomide (TMZ) therapy, resulting in a low median overall survival (OS) rate ranging from 12.2 - 15.9 months. The important role of genetic and epigenetic changes in DNA, RNA, and protein alteration as well as epigenetic changes secondary to the tumor microenvironment and outside selection pressure (therapeutic interventions), are increasingly being recognized. In GBM treatment, the focus is shifting toward a more patient-centered (personalized) therapy. In this regard, in particular, microRNAs are being increasingly studied. MicroRNAs are non¬protein coding small RNAs that serve as negative gene regulators by binding to a specific sequence in the promoter region of a target gene, thus regulating gene expression. A single microRNA potentially targets hundreds of genes; thus, microRNAs and their cognate target genes have important roles as tumor suppressors and oncogenes as well as regulators of various cancer- specific cellular features, such as proliferation, apoptosis, invasion, and metastasis. The identification of distinct microRNA-gene regulatory networks in GBM patients can be expected to provide novel therapeutic insights by identifying candidate patients for targeted therapies. To this end, in this work we identified and validated clinically relevant and meaningful novel gene- microRNA regulatory networks that correlated with MR tumor phenotypes, histopathology, and patient survival and response rates to therapy. - Le traitement des gliomes malins, y compris sous leur forme la plus commune et meurtrière, le glioblastome (GBM, ou astrocytome de grade IV selon l'OMS), demeure à ce jour un défi. Aux États-Unis et en Europe, un nouveau diagnostic de GBM est prononcé dans plus de 30Ό00 cas par an. En dépit de tests en cours, les meilleures approches thérapeutiques combinées actuellement disponibles comprennent la résection chirurgicale de la tumeur, suivie d'une radiothérapie adjuvante ainsi que d'un traitement au temozolomide (RT/TMZ), thérapies dont résulte une médiane de survie globale basse (overall survival, OS), comprise entre 12.2 et 15.9 mois. On reconnaît de plus en plus le rôle majeur de l'ADN, de l'ARN et de l'altération des protéines ainsi que des modifications épigénétiques, secondaires par rapport au microenvironnement de la tumeur et à la pression de sélection extérieure (les interventions thérapeutiques). Dans le traitement du GBM, le centre d'intérêt se déplace vers une thérapie centrée sur le cas individuel du patient. Dans ce but, en particulier les microARN sont de plus en plus analysés. Les microARN sont de petits ARN non-codants (les protéines) qui servent de régulateurs négatifs de gènes en s'attachant à une séquence spécifique dans la région promotrice d'un gène-cible, régulant ainsi l'expression du gène. Un seul microARN cible potentiellement des centaines de gènes; on a ainsi découvert que les microARN et leurs gènes-cibles apparentés ont une fonction importante en tant que suppresseurs de tumeurs et d'oncogènes, ainsi que comme régulateurs de diverses caractéristiques cellulaires spécifiques du cancer, comme la prolifération, l'apoptose, l'invasion et la métastase. On peut s'attendre à ce que l'identification de réseaux microARN régulateurs de gènes, distincts selon les patients de GBM, fournisse une approche thérapeutique inédite par la détermination des patients susceptibles de réagir favorablement à des thérapies ciblées.

Valproate treatment of human cord blood CD4-positive effector T cells confers on them the molecular profile (microRNA signature and FOXP3 expression) of natural regulatory CD4-positive cells through inhibition of histone deacetylase.

Regulatory T cells (Tregs) play a key role in immune system homeostasis and tolerance to antigens, thereby preventing autoimmunity, and may be partly responsible for the lack of an appropriate immune response against tumor cells. Although not sufficient, a high expression of forkhead box P3 (FOXP3) is necessary for their suppressive function. Recent reports have shown that histones deacetylase inhibitors increased FOXP3 expression in T cells. We therefore decided to investigate in non-Tregs CD4-positive cells, the mechanisms by which an aspecific opening of the chromatin could lead to an increased FOXP3 expression. We focused on binding of potentially activating transcription factors to the promoter region of FOXP3 and on modifications in the five miRs constituting the Tregs signature. Valproate treatment induced binding of Ets-1 and Ets-2 to the FOXP3 promoter and acted positively on its expression, by increasing the acetylation of histone H4 lysines. Valproate treatment also induced the acquisition of the miRs Tregs signature. To elucidate whether the changes in the miRs expression could be due to the increased FOXP3 expression, we transduced these non-Tregs with a FOXP3 lentiviral expression vector, and found no changes in miRs expression. Therefore, the modification in their miRs expression profile is not due to an increased expression of FOXP3 but directly results from histones deacetylase inhibition. Rather, the increased FOXP3 expression results from the additive effects of Ets factors binding and the change in expression level of miR-21 and miR-31. We conclude that valproate treatment of human non-Tregs confers on them a molecular profile similar to that of their regulatory counterpart.

NLRC5 deficiency impairs MHC class I-dependent lymphocyte killing by cytotoxic T cells

Purpose/Objective: NLRs are intracellular proteins involved in sensing pathogen- and danger-associated molecular patterns, thereby initiating inflammatory responses or cell death. The function of the family member NLRC5 remains a matter of debate, particularly with respect to NF-jB activation, type I IFN, and MHC class I expression. Materials and methods: To study the function of this NLR in vivo, we generated Nlrc5-deficient mice. Results: We found that NLRC5 deletion led to a mild reduction in MHC class I expression on DCs and an intermediate decrease on B cells, while MHC class I levels were dramatically lowered on T, NKT, and NK cells. Nlrc5-/- lymphocytes showed decreased H-2 gene transcript abundance and, accordingly, NLRC5 was sufficient to drive MHC class I expression in a human lymphoid cell line. Moreover, endogenous NLRC5 localized to the nucleus and occupied the proximal promoter region of H-2 genes. Notably, cytotoxic T cell-mediated elimination of Nlrc5-/- lymphocytes was markedly reduced. In addition, we observed low NLRC5 expression in several murine and human lymphoid-derived tumor cell lines. Conclusions: We found that NLRC5 acts as a key transcriptional regulator of MHC class I genes, in particular in lymphocytes. Loss of NLRC5 expression represents an advantage for evading CD8+ T cellmediated elimination by downmodulation of MHCI levels * a mechanism transformed cells may take advantage of. Therefore, our data support an essential role for NLRs in directing not only innate, but also adaptive immune responses (Staehli F et al. J Immunol 2012).

PAX5 activates the transcription of the human telomerase reverse transcriptase gene in B cells.

Telomerase is an RNA-dependent DNA polymerase that synthesizes telomeric DNA. Its activity is not detectable in most somatic cells but it is reactivated during tumorigenesis. In most cancers, the combination of hTERT hypermethylation and hypomethylation of a short promoter region is permissive for low-level hTERT transcription. Activated and malignant lymphocytes express high telomerase activity, through a mechanism that seems methylation-independent. The aim of this study was to determine which mechanism is involved in the enhanced expression of hTERT in lymphoid cells. Our data confirm that in B cells, some T cell lymphomas and non-neoplastic lymph nodes, the hTERT promoter is unmethylated. Binding sites for the B cell-specific transcription factor PAX5 were identified downstream of the ATG translational start site through EMSA and ChIP experiments. ChIP assays indicated that the transcriptional activation of hTERT by PAX5 does not involve repression of CTCF binding. In a B cell lymphoma cell line, siRNA-induced knockdown of PAX5 expression repressed hTERT transcription. Moreover, ectopic expression of PAX5 in a telomerase-negative normal fibroblast cell line was found to be sufficient to activate hTERT expression. These data show that activation of hTERT in telomerase-positive B cells is due to a methylation-independent mechanism in which PAX5 plays an important role.

APOBEC3G genetic variants and their influence on the progression to AIDS.

The cytosine deaminase APOBEC3G, in the absence of the human immunodeficiency virus type 1 (HIV-1) accessory gene HIV-1 viral infectivity factor (vif), inhibits viral replication by introducing G-->A hypermutation in the newly synthesized HIV-1 DNA negative strand. We tested the hypothesis that genetic variants of APOBEC3G may modify HIV-1 transmission and disease progression. Single nucleotide polymorphisms were identified in the promoter region (three), introns (two), and exons (two). Genotypes were determined for 3,073 study participants enrolled in six HIV-AIDS prospective cohorts. One codon-changing variant, H186R in exon 4, was polymorphic in African Americans (AA) (f = 37%) and rare in European Americans (f < 3%) or Europeans (f = 5%). For AA, the variant allele 186R was strongly associated with decline in CD4 T cells (CD4 slope on square root scale: -1.86, P = 0.009), The 186R allele was also associated with accelerated progression to AIDS-defining conditions in AA. The in vitro antiviral activity of the 186R enzyme was not inferior to that of the common H186 variant. These studies suggest that there may be a modifying role of variants of APOBEC3G on HIV-1 disease progression that warrants further investigation.

Polymorphisms in candidate genes and their association with carcass traits and meat quality in Nellore cattle

The objective of this work was to estimate the allele polymorphism frequencies of genes in Nellore cattle and associate them with meat quality and carcass traits. Six hundred males were genotyped for the following polymorphisms: DGAT1 (VNTR with 18 nucleotides at the promoter region); ANK1, a new polymorphism, identified and mapped here at the gene regulatory region NW_001494427.3; TCAP (AY428575.1:g.346G>A); and MYOG (NW_001501985:g.511G>C). In the association study, phenotype data of hot carcass weight, ribeye area, backfat thickness, percentage of intramuscular fat, shear force, myofibrillar fragmentation index, meat color (L*, a*, b*), and cooking losses were used. Allele B from the ANK1 gene was associated with greater redness (a*). Alleles 5R, 6R, and 7R from the DGAT1 VNTR gene were associated with increased intramuscular fat, reduced cooking losses and increased ribeye area, respectively. The single nucleotide polymorphism (SNP) of the TCAP gene was not polymorphic, and MYOG alleles were not associated with any of the evaluated characteristics. These results indicate that ANK1 and DGAT1 genes can be used in the selection of Nellore cattle for carcass and meat quality.

Meta-analysis and imputation refines the association of 15q25 with smoking quantity.

Smoking is a leading global cause of disease and mortality. We established the Oxford-GlaxoSmithKline study (Ox-GSK) to perform a genome-wide meta-analysis of SNP association with smoking-related behavioral traits. Our final data set included 41,150 individuals drawn from 20 disease, population and control cohorts. Our analysis confirmed an effect on smoking quantity at a locus on 15q25 (P = 9.45 x 10(-19)) that includes CHRNA5, CHRNA3 and CHRNB4, three genes encoding neuronal nicotinic acetylcholine receptor subunits. We used data from the 1000 Genomes project to investigate the region using imputation, which allowed for analysis of virtually all common SNPs in the region and offered a fivefold increase in marker density over HapMap2 (ref. 2) as an imputation reference panel. Our fine-mapping approach identified a SNP showing the highest significance, rs55853698, located within the promoter region of CHRNA5. Conditional analysis also identified a secondary locus (rs6495308) in CHRNA3.

Regulation of bistable ICEclc transfer in pseudomonas knackmussii B13

L'élément génétique intégratif et conjugatif auto-transférable de 103 kb qui se trouve dans le génome de Pseudomonas knackmussii B13 (ICEc/c) confère la capacité de dégrader le 3-chlorobenzoate et le 2-aminophénol. L'élément ICE c/c peut être transféré par conjugaison de la souche B13 à diverses bêta- et gamma- protéobactéries. Seule une sous-population de 3 à 5% des cellules transfère l'élément, les cellules dites "compétentes pour le transfert". L'acquisition de la compétence pour le transfert est vraisemblablement la conséquence d'une régulation bistable, conduisant une partie des cellules au transfert de l'élément ICE c/c tandis que, dans les autres, l'élément reste quiescent et ne se transfère pas. À ce jour, les mécanismes et les acteurs moléculaires qui régulent l'activation bistable de l'élément sont restés inconnus. Mon travail de doctorat visait à identifier les éléments bistables du régulon de la compétence pour le transfert et d'analyser les fondements moléculaires de la bistabilité de l'élément ICE c/c chez P. knackmussii. Le premier chapitre introduit le thème du transfert génétique horizontal avec un accent particulier sur les éléments intégratifs et conjugatifs (ICE) et ICEcIc. L'état actuel des connaissances sur l'organisation génétique, la régulation, l'intégration et le transfert de différents modèles de ICEs est exposé en détail. En outre, je m'étends sur les phénomènes d'hétérogénéité et de bistabilité phénotyplques, qu'on peut distinguer dans une population isogénique dans des conditions de culture homogènes, et qui sont susceptibles de jouer un rôle dans le transfert de l'élément ICE c/c, dans la mesure où il ne s'active et n'est transférable que dans une très petite sous-population de cellules. Dans le chapitre 2, je présente une analyse globale des régions promotrices minimales des gènes appartenant au régulon de la compétence pour le transfert de l'élément ICE c/c. Nous avons étudié les caractéristiques d'expression des promoteurs et, s'ils s'avéraient bistables, leur activation dans le temps par comparaison avec le mutant lntB13. Pour ce faire, nous avons utilisé des fusions de promoteurs avec des gènes rapporteurs et testé l'expression bistable chez P. knackmussii par microscopie à épifluorescence. Pour six promoteurs présentant une expression bistable, nous avons employé de la microscopie temporelle pour déterminer la chronologie de leur expression par rapport à Pint et PinR. Parmi eux, nous avons identifié deux gènes exprimés précocement et trois gènes exprimés tardivement dans le processus d'acquisition de la compétence de transfert. Dans le chapitre 3, j'expose une analyse d'expression génétique pour l'un des groupes de gènes dont la transcription est la plus élevée dans la région conservée de ICE c/c, les gènes orf81655-orf68241 contenus dans une région de 14 kb. Nous montrons d'abord que cet opéron fait partie du même régulon bistable que intB13 et inrR et analysons les caractéristiques génétiques qui conduisent à une transcription élevée. Nous étudions les fonctions biologiques de ce groupe de gènes par des délétlons ciblées et montrons que certaines d'entre elles empêchent le transfert de l'élément. Nous approfondissons la caractérlsatlon de I'orf8l655 en construisant une fusion transcrlptionnelle avec le gène codant pour la protéine fluorescente verte (egfp) (en utilisant le système minl-Tn5). L'expression de Vorf81655 dans des cellules individuelles est comparée au signal mesuré par hybridation in situ en fluorescence (FISH) sur le ARN messager du gène. En utilisant FISH, des délétlons du promoteur et de l'analyse directe de transcription, nous avons localisé la région promotrice du groupe de gènes. En outre, nous avons utilisé des mutations dirigées pour comprendre la bistabilité de cette région promotrice, caractérisée par une transcription très élevée et une traduction lente de l'ARN messager.  Dans le chapitre 4, nous nous efforçons de comprendre comment la bistabilité est générée au sein du régulon te de l'élément ICE c/c. Pour ce faire, nous avons tenté de reconstituer une expression bistable, dans un hôte qui ne présente pas de bistabilité naturellement, à partir d'éléments génétiques individuels. L'hôte choisi est Pseudomonas putida dans lequel nous avons introduit une copie unique de Pint, PinR ou PaipA fusionnés à la egfp, construits qui permettent d'observer l'apparition de bistabilité. Nous avons ensuite construit différents assemblages de composants génétiques de l'élément ICE c/c, en nous concentrant sur la région parA-inrR. En effet, nous avons pu démontrer qu'une expression bistable apparaît dans P. putida grâce à ces éléments en l'absence de l'élément ICE c/c complet. À noter que la plupart des construits génétiques activent PaipA ou P|,,R, mais qu'un seul recrée la bistabilité de Pint, ce qui suggère que la région parA-inrR permet à la fois d'engendrer la bistabilité et d'opérer la transition entre les promoteurs précoces et les promoteurs tardifs du régulon de la bistabilité. Dans le chapitre 5, nous concluons sur une discussion de la pertinence de nos résultats et sur de futures perspectives de recherche. -- The 103-kb self-transmissible integrative and conjugative element (ICE) of Pseudomonas knackmussii B13 (ICEc/c) confers the capacity to degrade 3- chlorobenzoate and 2-aminophenol. ICEc/c can be conjugated from strain B13 to a variety of Beta- and Gammaproteobacteria. Interestingly, ICE c/c transfer is observed in a subpopulatlon of cells (3-5%) only, the so-called 'transfer competent' cells. The formation of transfer competence (tc) is thought to be the consequence of a 'bistable' decision, which forces those cells to follow the developmental path which leads to ICEc/c transfer, whereas in others ICE c/c remains silent and does not transfer. So far, the mechanisms and molecular partners generating this bistable transfer activation in cells of P. knackmussii B13 remain mostly unidentified. This thesis aimed at understanding the extent of the tc bistability regulon and to dissect the molecular basis of bistabillty formation of ICEc/c in P. knackmussii. The first chapter is a general Introduction on horizontal gene transfer (HGT) with particular emphasis on ICEs and ICE c/c. The emphasis is made on the current knowledge about the HGT gene organization, regulation and specific integration and transfer aspects of the different ICEs models. Furthermore, I focus on the phenomena of phenotypic heterogeneity and bistability (the property of two distinguishable phenotypes existing within an isogenic population under homogeneous conditions), which may play a particular role in ICEc/c behaviour, since ICE activation and transfer only occurs in a very small subpopulation of cells. In Chapter Two, I focus on a global analysis of the different core promoters that might belong to the ICEc/c tc pathway regulon. We studied both expression patterns of ICEc/c promoters and, once being identified as "bistable", their temporal activation compared to that of intB13. In order to do this, we used promoter reporter fusions and tested blstability expression in P. knackmussii using epifluorescence microscopy. For the 6 promoters that showed bistable expression, we used time-lapse microscopy to study the timing of promoter expression in comparison to that of P,,,t or PlnR. We could establish two "early" and 3 "late" phase promoters in the process of transfer competence. In Chapter Three, I focused my attention on analysis of gene expression of one of the most highly transcribed gene clusters in the conserved core region of ICEc/c, a 14-kb gene cluster formed by the genes orf81655-orf68241. First we showed that this operon is part of the same bistability 'regulon' as intB13 and inrR, and analysed the genetic features that lead to high transcription. We studied the potential biological function of this cluster for ICE c/c by making specific gene deletions, showing that some interrupt ICEc/c transfer. We further analysed the orfdl655 promoter by constructing transcriptional egfp fusion reporter strains using the miniTn5 delivery system. Expression of the orf81655 promoter in single cells was compared to signals measured by Fluorescence In Situ Hybridization (FISH) on orfSl655 mRNA. We localized the promoter region of the gene cluster using FISH, promoter deletions, and by direct transcript analysis. We further used site-directed mutagenesis to understand the bistability character of the promoter region and the extremely high transcription but low translation from this mRNA. In Chapter Four, we set out to understand how bistability is generated in the tc pathway of ICEc/c. For this we tried rebuilding bistable expression from ICEc/c individual gene components in a host, which normally does not display bistability. As host we used P. putida without ICEc/c but with a single copy Pint-, PlnR- or PalpA- egfp fusion that enabled us to verify bistability formation. Subsequently, we built different assemblages of ICEc/c gene components, focusing on the parA-inrR region. Indeed, we found that bistable expression can be build from those components in P. putida without ICEc/c. Interestingly, most genetic constructs activated PaipA or PlnR, but only one resulted in bistable activation of PinT. This suggests that the parA-inrR region acts as a bistability "generator", but also as a bistability "relay" from early to late promoters in the tc pathway hierarchy. In the final fifth chapter, we conclude with a discussion of the relevance of the present thesis and the resulting perspectives for future studies.

Influence of MCHR2 and MCHR2-AS1 Genetic Polymorphisms on Body Mass Index in Psychiatric Patients and In Population-Based Subjects with Present or Past Atypical Depression.

Obesity development during psychotropic treatments represents a major health issue in psychiatry. Melanin-concentrating hormone receptor 2 (MCHR2) is a central receptor involved in energy homeostasis. MCHR2 shares its promoter region with MCHR2-AS1, a long antisense non-coding RNA. The aim of this study was to determine whether tagging single nucleotide polymorphisms (tSNPs) of MCHR2 and MCHR2-AS1 are associated with the body mass index (BMI) in the psychiatric and in the general population. The influence of MCHR2 and MCHR2-AS1 tSNPs on BMI was firstly investigated in a discovery psychiatric sample (n1 = 474). Positive results were tested for replication in two other psychiatric samples (n2 = 164, n3 = 178) and in two population-based samples (CoLaus, n4 = 5409; GIANT, n5 = 113809). In the discovery sample, TT carriers of rs7754794C>T had 1.08 kg/m2 (p = 0.04) lower BMI as compared to C-allele carriers. This observation was replicated in an independent psychiatric sample (-2.18 kg/m2; p = 0.009). The association of rs7754794C>T and BMI seemed stronger in subjects younger than 45 years (median of age). In the population-based sample, a moderate association was observed (-0.17 kg/m2; p = 0.02) among younger individuals (<45y). Interestingly, this association was totally driven by patients meeting lifetime criteria for atypical depression, i.e. major depressive episodes characterized by symptoms such as an increased appetite. Indeed, patients with atypical depression carrying rs7754794-TT had 1.17 kg/m2 (p = 0.04) lower BMI values as compared to C-allele carriers, the effect being stronger in younger individuals (-2.50 kg/m2; p = 0.03; interaction between rs7754794 and age: p-value = 0.08). This study provides new insights on the possible influence of MCHR2 and/or MCHR2-AS1 on obesity in psychiatric patients and on the pathophysiology of atypical depression.

Purification and binding analysis of the nitrogen fixation regulatory NifA protein from Azospirillum brasilense

NifA protein activates transcription of nitrogen fixation operons by the alternative sigma54 holoenzyme form of RNA polymerase. This protein binds to a well-defined upstream activator sequence (UAS) located at the -200/-100 position of nif promoters with the consensus motif TGT-N10-ACA. NifA of Azospirillum brasilense was purified in the form of a glutathione-S-transferase (GST)-NifA fusion protein and proteolytic release of GST yielded inactive and partially soluble NifA. However, the purified NifA was able to induce the production of specific anti-A. brasilense NifA-antiserum that recognized NifA from A. brasilense but not from K. pneumoniae. Both GST-NifA and NifA expressed from the E. coli tac promoter are able to activate transcription from the nifHDK promoter but only in an A. brasilense background. In order to investigate the mechanism that regulates NifA binding capacity we have used E. coli total protein extracts expressing A. brasilense nifA in mobility shift assays. DNA fragments carrying the two overlapping, wild-type or mutated UAS motifs present in the nifH promoter region revealed a retarded band of related size. These data show that the binding activity present in the C-terminal domain of A. brasilense NifA protein is still functional even in the presence of oxygen.

Main features of DNA-based immunization vectors

DNA-based immunization has initiated a new era of vaccine research. One of the main goals of gene vaccine development is the control of the levels of expression in vivo for efficient immunization. Modifying the vector to modulate expression or immunogenicity is of critical importance for the improvement of DNA vaccines. The most frequently used vectors for genetic immunization are plasmids. In this article, we review some of the main elements relevant to their design such as strong promoter/enhancer region, introns, genes encoding antigens of interest from the pathogen (how to choose and modify them), polyadenylation termination sequence, origin of replication for plasmid production in Escherichia coli, antibiotic resistance gene as selectable marker, convenient cloning sites, and the presence of immunostimulatory sequences (ISS) that can be added to the plasmid to enhance adjuvanticity and to activate the immune system. In this review, the specific modifications that can increase overall expression as well as the potential of DNA-based vaccination are also discussed.

Glial fibrillary acidic protein (GFAP): modulation by growth factors and its implication in astrocyte differentiation

Intermediate filament (IF) proteins constitute an extremely large multigene family of developmentally and tissue-regulated cytoskeleton proteins abundant in most vertebrate cell types. Astrocyte precursors of the CNS usually express vimentin as the major IF. Astrocyte maturation is followed by a switch between vimentin and glial fibrillary acidic protein (GFAP) expression, with the latter being recognized as an astrocyte maturation marker. Levels of GFAP are regulated under developmental and pathological conditions. Upregulation of GFAP expression is one of the main characteristics of the astrocytic reaction commonly observed after CNS lesion. In this way, studies on GFAP regulation have been shown to be useful to understand not only brain physiology but also neurological disease. Modulators of GFAP expression include several hormones such as thyroid hormone, glucocorticoids and several growth factors such as FGF, CNTF and TGFß, among others. Studies of the GFAP gene have already identified several putative growth factor binding domains in its promoter region. Data obtained from transgenic and knockout mice have provided new insights into IF protein functions. This review highlights the most recent studies on the regulation of IF function by growth factors and hormones.

Role of ACTH receptor in adrenocortical tumor formation

Adrenocorticotrophin (ACTH) is the major regulatory hormone of steroid synthesis and secretion by adrenocortical cells. The actions of ACTH are mediated by its specific membrane receptor (ACTH-R). The human ACTH-R gene was recently cloned, allowing systematic determination of its sequence, expression and function in adrenal tumorigenesis. The presence of oncogenic mutations of the ACTH-R gene in adrenocortical tumors has been reported. Direct sequencing of the entire coding region of the ACTH-R gene of sporadic adrenocortical adenomas and carcinomas did not reveal constitutive activating mutations, indicating that this mechanism is not frequent in human adrenocortical tumorigenesis. Recent studies demonstrated allelic loss of the ACTH-R gene in a subset of sporadic adrenocortical tumors using a PstI polymorphism located in the promoter region of the ACTH-R gene. Loss of heterozygosity of the ACTH-R was analyzed in 20 informative patients with a variety of benign and malignant adrenocortical tumors. Three of them showed loss of heterozygosity of the ACTH-R gene. In addition, Northern blot experiments demonstrated reduced expression of ACTH-R mRNA in these three tumors with loss of heterozygosity, suggesting the functional significance of this finding at the transcriptional level. Deletion of the ACTH-R gene seems to be involved in a subset of human adrenocortical tumors, contributing to cellular dedifferentiation.

Association studies between -1185A/G von Willebrand factor gene polymorphism and coronary artery disease

High levels of von Willebrand factor (vWF) have been associated with cardiovascular disease. The A allele of the -1185A/G polymorphism in the 5'-regulatory region of the vWF gene was associated with the highest plasma vWF levels in a normal population. To examine the association between -1185A/G polymorphism and coronary artery disease (CAD), 173 Brazilian Caucasian subjects submitted to coronary angiography were studied. Of these, 57 (33%) had normal coronary arteries (control group) and 116 (67%) had CAD (patient group). Plasma vWF levels were higher in patients (145 U/dl) than in controls (130 U/dl), but the differences were significant only for O blood group subjects. Polymerase chain reaction amplification of the 864-bp vWF promoter region followed by AccII restriction digestion was used to identify the -1185A/G genotypes. The -1185A allele frequency was 43.1% in patients and 44.7% in controls. Allele and genotype frequencies were not significantly different between patients and controls. No association was observed between the -1185A/G genotypes and plasma vWF levels in patients or controls. These results suggest that -1185A/G polymorphism is not an independent risk factor for CAD.