Schistosomiasis mansoni in Bananal (State of São Paulo, Brazil): II. Intermediate hosts

We conducted monthly snail captures in Bananal, State of São Paulo, Brazil, between March 1998 and February 2001, to identify Schistosoma mansoni vectors, estimate seasonal population changes, and delimit foci. We also evaluated the impact of improvements in city water supply and basic sanitation facilities. We identified 28,651 vector specimens, 28,438 as Biomphalaria tenagophila, 49 of them (0.2%) infected with S. mansoni, and 213 as B. straminea, none of the latter infected. Vectors predominated in water bodies having some vegetation along their banks. Neither population density nor local vegetation could be linked to vector infection. We found the first infected snails in 1998 (from March to May). Further captures of infected snails ocurred, without exception, from July to December, when rainfall was least. Irrespective of season, overall temperature ranged from 16.5ºC to 21ºC; pH values, from 6.0 to 6.8. Neither factor was associated with snail population density. Frequent contact of people with the river result from wading across it, extracting sand from its bottom, fishing, washing animals, etc. Despite a marked reduction in contamination, cercaria shedding persists. Whatever the location along its urban course, contact with river Bananal, particularly of the unprotected skin, entails risks of infection.

Molecular mechanisms for the transcriptional regulation of the Connexin36 gene expression in insulin-secreting cells

Résumé Régulation de l'expression de la Connexin36 dans les cellules sécrétrices d'insuline La communication intercellulaire est en partie assurée via des jonctions communicantes de type "gap". Dans la cellule ß pancréatique, plusieurs observations indiquent que le couplage assuré par des jonctions gap formées parla Connexine36 (Cx36) est impliqué dans le contrôle de la sécrétion de l'insuline. De plus, nous avons récemment démontré qu'un niveau précis d'expression de la Cx36 est nécessaire pour maintenir une bonne coordination de l'ensemble des cellules ß, et permettre ainsi une sécrétion synchrone et contrôlée d'insuline. Le développement du diabète et du syndrome métabolique est partiellement dû à une altération de la capacité des cellules ß à sécréter de l'insuline en réponse à une augmentation de la glycémie. Cette altération est en partie causée par l'augmentation prolongée des taux circulant de glucose, mais aussi de lipides, sous la forme d'acides gras libres, et de LDL (Low Density Lipoproteins), particules assurant le transport des acides gras et du cholestérol dans le sang. Nous avons étudié la régulation de l'expression de la Cx36 dans différentes conditions reflétant la physiopathologie du diabète de type 2 et du syndrome métabolique et démontré qu'une exposition prolongée à des concentrations élevées de glucose, de LDL, ainsi que de palmitate (acide gras saturé le plus abondant dans l'organisme), inhibent l'expression de la Cx36 dans les cellules ß. Cette inhibition implique l'activation de la PKA (Proteine Kinase A), qui stimule à son tour l'expression du facteur de transcription ICER-1 (Inductible cAMP Early Repressor-1). Ce puissant répresseur se fixe spécifiquement sur un motif CRE (cAMP Response Element), situé dans le promoteur du gène de la Cx36, inhibant ainsi son expression. Nous avons de plus démontré que des cytokines pro-inflammatoires, qui pourraient contribuer au développement du diabète, inhibent également l'expression de la Cx36. Cependant, les cytokines agissent indépendamment du répresseur ICER-1, mais selon un mécanisme requérant l'activation de l'AMPK (AMP dependant protein kinase). Sachant qu'un contrôle précis des niveaux d'expression de la Cx36 est un élément déterminant pour une sécrétion optimale de l'insuline, nos résultats suggèrent que la Cx36 pourrait être impliquée dans l'altération de la sécrétion de l'insuline contribuant à l'apparition du diabète de type 2. Summary A particular way by which cells communicate with each other is mediated by gap junctions, transmembrane structures providing a direct pathway for the diffusion of small molecules between adjacent cells. Gap junctional communication is required to maintain a proper functioning of insulin-secreting ß-cells. Moreover, the expression levels of connexin36 (Cx36), the sole gap junction protein expressed in ß-cells, are critical in maintaining glucose-stimulated insulin secretion. Chronic hyperglycemia and hyperlipidemia exert deleterious effects on insulin secretion and may contribute to the progressive ß-cell failure linked to the development of type 2 diabetes and metabolic syndrome. Since modulations of the Cx36 levels might impair ß-cell function, the general aim of this work was to elucidate wether elevated levels of glucose and lipids affect Cx36 expression. The first part of this work was dedicated to the study of the effect of high glucose concentrations on Cx36 expression. We demonstrated that glucose transcriptionally down-regulates the expression of Cx36 in insulin-secreting cells through activation of the protein kinase A (PKA), which in turn stimulates the expression of the inducible cAMP early repressor-1 (ICER-1). This repressor binds to a highly conserved cAMP response element (CRE) located in the Cx36 promoter, thereby inhibiting Cx36 expression. The second part of this thesis consisted in studying the effects of sustained exposure to free fatty acids (FFA) and human lipoproteins on Cx36 levels. The experiments revealed that the most abundant FFA, palmitate, as well as the atherogenic low density lipoproteins (LDL), also stimulate ICER-1 expression, resulting in Cx36 down-regulation. Finally, the third part of the work focused on the consequences of long-term exposure to proinflammatory cytokines on Cx36 content. Interleukin-1 ß (IL-1 ß) inhibits Cx36 expression and its effect is potentialized by tumor necrosis factor α (TNFα) and interferon γ (IFNγ). We further unveiled that the cytokines effect on Cx36 levels requires activation of the AMP dependent protein kinase (AMPK). Prolonged exposures to glucose, palmitate, LDL, and pro-inflammatory cytokines have all been proposed to contribute to the development of diabetes and metabolic syndrome. Since Cx36 expression levels are critical to maintain ß-cell function, Cx36 down-regulation by glucose, lipids, and cytokines might participate to the ß-cell failure associated with diabetes development.

I. Identification and characterisation of epigenetically altered tumor supressor genes by proteomic and genomic approches / II. Studies of genes regualted by MeCP2 union to its promoter regions by proteomic and genmomic approches

DNA methylation has an important impact on normal cell physiology, thus any defects in this mechanism may be related to the development of various diseases In this project we are interested in identifying epigeneticaliy modified genes, in general controlled by processes related to the DNA methylation, by means of a new strategy combining protomic and genomic analyses. First, the two Dimensional-Difference Gel Electrophoresis (2-DIGE) protein analyses of extracts obtained from HCT-116 wt and double knockout for DNMT1 and DNMT3b (DKO) cells revealed 34 proteins overexpressed in the condition of DNMTs depletion. From five genes with higher transcript lavels in DKO cells, comparing with HCT-116 wt. oniy AKR1B1, UCHLl and VIM are melhylated in HCT-116. As expected. the DNA methvlation 1s lost in DKO cells. The rneth,vl ation of VIM and UCHLl promoters in some cancer samples has already been repaired, thus further studies has been focused on AKRlBI. AKR1B1 expression due lo DNA methyiaton of promoter region seems to occur specilfically in the colon cancer cell Iines. which was confirmed in the DNA rnethylation status and expression analyses. performed on 32 different cancer cell lines (including colon, breast, lymphoma, leukemia, neuroblastoma, glioma and lung cancer cell Iines) as well as normal colon and normal lymphocytes samples. AKRIBI expression after treatments with DNA demethvlating agent (AZA) was rescued in 5 coloncancer cell lines (including genetic regulation of the candidate gene. The methylation status of the rest of the genes identified in proteomic analysis was checked by methylation specific PCR (MSP) experiment and all appeared to be unmethylated. The similar research has been done also bv means of Mecp2-null mouse model For 14 selected candidate genes the analyses of expression leveis, methylation Status and MeCP2 interaction with promoters are currently being performed.

Astrocyte dense-core vesicle gliosecretion is governed by rest/nrsf

Astrocytes are the brain non-nerve cells competent for the expression of clear and dense-core vesicles (DCVs) and for their regulated exocytosis. This process, called gliosecretion, nearly resembles the neurosecretion occurring in neurons and neurosecretory cells. REST/NRSF is a transcription repressor known to orchestrate nerve-cell differentiation, governing the expression of hundreds of neuron-specific genes through their repression in the non-nerve and their fine modulation in the nerve cells. Our previous studies in neurosecretory rat PC12 cells identified REST as the critical factor for the expression not only of individual genes, but also of the whole neurosecretory process via multiple, direct and indirect mechanisms (D'Alessandro et al., J. Neurochem., 2008; Klajn et al., J. Neurosci., 2009). Therefore we wondered whether gliosecretion was governed by REST. We investigated rat astrocyte primary cultures: they exhibited high REST, which directly represses the transcription of at least one target gene, and expressed neither DCVs nor their markers (granins, peptides, membrane proteins). Transfection of a dominant-negative construct of REST (REST/ DBD-GFP) induced the appearance of DCVs filled with secretogranin2 and NPY that are distinct from other intracellular organelles. TIRF analysis of astrocytes co-transfected with REST/DBD-GFP and NPY-mRFP constructs revealed NPY-mRFP-positive DCVs undergoing Ca2þ-dependent exocytosis, largely prevented by BoNT/B. Immunohistochemistry of the I-II layers of the human temporal brain cortex showed all neurons and microglia exhibiting the expected inappreciable and high levels of REST, respectively. In contrast astrocyte RESTwas variable, going from inappreciable to high, accompanied by variable expression of DCVs. In this work it has been demonstrated that astrocyte DCV expression and gliosecretion are governed by REST (Prada et al., 2011 in press). The variable in situ REST levels may contribute to the well known structural/functional heterogeneity of astrocytes and this new observation might be of great interest for the understanding of both astrocyte physiology and pathology.

Positive and negative roles of the trans-acting T cell factor-1 for the acquisition of distinct Ly-49 MHC class I receptors by NK cells.

Members of the Ly-49 gene family code for class I MHC-specific receptors that regulate NK cell function. Due to a combinatorial distribution of Ly-49 receptors, NK cells display considerable clonal heterogeneity. The acquisition of one Ly-49 receptor, Ly-49A is strictly dependent on the transcriptional trans-acting factor T cell-specific factor-1 (TCF-1). Indeed, TCF-1 binds to two sites in the Ly-49a promoter and regulates its activity, suggesting that the Ly-49a gene is a direct TCF-1 target. TCF-1 deficiency resulted in the altered usage of additional Ly-49 receptors. We show in this study, using TCF-1 beta(2)-microglobulin double-deficient mice, that these repertoire alterations are not due to Ly-49/MHC class I interactions. Our findings rather suggest a TCF-1-dependent, cell autonomous effect on the acquisition of multiple Ly-49 receptors. Besides reduced receptor usage (Ly-49A and D), we also observed no effect (Ly-49C) and significantly expanded (Ly-49G and I) receptor usage in the absence of TCF-1. These effects did not in all cases correlate with the presence of TCF binding sites in the respective proximal promoter. Therefore, besides TCF-1 binding to the proximal promoter, Ly-49 acquisition may also be regulated by TCF-1 binding to more distant cis-acting elements and/or by regulating the expression of additional trans-acting factors. Consistent with the observed differential, positive or negative role of TCF-1 for Ly-49 receptor acquisition, reporter gene assays revealed the presence of an inducing as well as a repressing TCF site in certain proximal Ly-49 promoters. These findings reveal an important role of TCF-1 for the formation of the NK cell receptor repertoire.

Similarity between the association factor of ribosomal subunits and the protein Stm1p from Saccharomyces cerevisiae

A ribosome association factor (AF) was isolated from the yeast Sacchharomyces cerevisiae. Partial amino acid sequence of AF was determined from its fragment of 25 kDa isolated by treating AF with 2-(2-nitrophenylsulfenyl)-3-methyl-3'-Bromoindolenine (BNPS-skatole). This sequence has a 86% identity to the product of the single-copy S. cerevisiae STM1 gene that is apparently involved in several events like binding to quadruplex and triplex nucleic acids and participating in apoptosis, stability of telomere structures, cell cycle, and ribosomal function. Here we show that AF and Stm1p share some characteristics: both bind to quadruplex and Pu triplex DNA, associates ribosomal subunits, and are thermostable. These observations suggest that these polypeptides belong to a family of proteins that may have roles in the translation process.

Post-genomic update on a classical candidate gene for coronary artery disease: ESR1.

BACKGROUND: After age, sex is the most important risk factor for coronary artery disease (CAD). The mechanism through which women are protected from CAD is still largely unknown, but the observed sex difference suggests the involvement of the reproductive steroid hormone signaling system. Genetic association studies of the gene-encoding Estrogen Receptor α (ESR1) have shown conflicting results, although only a limited range of variation in the gene has been investigated. METHODS AND RESULTS: We exploited information made available by advanced new methods and resources in complex disease genetics to revisit the question of ESR1's role in risk of CAD. We performed a meta-analysis of 14 genome-wide association studies (CARDIoGRAM discovery analysis, N=≈87,000) to search for population-wide and sex-specific associations between CAD risk and common genetic variants throughout the coding, noncoding, and flanking regions of ESR1. In addition to samples from the MIGen (N=≈6000), WTCCC (N=≈7400), and Framingham (N=≈3700) studies, we extended this search to a larger number of common and uncommon variants by imputation into a panel of haplotypes constructed using data from the 1000 Genomes Project. Despite the widespread expression of ERα in vascular tissues, we found no evidence for involvement of common or low-frequency genetic variation throughout the ESR1 gene in modifying risk of CAD, either in the general population or as a function of sex. CONCLUSIONS: We suggest that future research on the genetic basis of sex-related differences in CAD risk should initially prioritize other genes in the reproductive steroid hormone biosynthesis system.

Inducible gene expression in fetal thymic epithelium: A new BAC transgenic model.

The thymus is the site of T cell development. Several stromal and hematopoietic cell types are necessary for the proper function of thymic selection and eventually peripheral immunity. Thymic epithelial cells (TECs) are essential for T cell lineage commitment, expansion, and maturation in the thymus. We were interested in developing an in vivo model in which exogenous gene expression could be transiently induced in embryonic TEC (Tet-On system). To this end, we have generated a bacterial artificial chromosome (BAC) transgenic mouse line in which the reverse tetracycline-dependent transactivator (rtTA) is expressed under the control of the Foxn1 promoter, a transcriptional factor indispensable for TEC development. To analyze the expression pattern and efficiency of this novel mouse model, we crossed the Foxn1-rtTA founder with a Tet-Responsive Element (TRE)-LacZ GFP mouse reporter to obtain a double transgenic mouse. In the presence of doxycycline, rtTA can interact with TRE and induce the expression of GFP and LacZ. In this double transgenic mouse, we observed that GFP expression was high, inducible and limited to TEC in fetal thymus. In contrast, in adult thymus, when TEC development and maturation is completed, GFP was barely detectable. Therefore, Foxn1-rtTA represents a new and efficient transgenic mouse model to induce genes of interest specifically in fetal thymic epithelium. genesis 51:717-724. © 2013 Wiley Periodicals, Inc.

Regulating inflammation through the anti-inflammatory enzyme platelet-activating factor-acetylhydrolase

Platelet-activating factor (PAF) is one of the most potent lipid mediators involved in inflammatory events. The acetyl group at the sn-2 position of its glycerol backbone is essential for its biological activity. Deacetylation induces the formation of the inactive metabolite lyso-PAF. This deacetylation reaction is catalyzed by PAF-acetylhydrolase (PAF-AH), a calcium independent phospholipase A2 that also degrades a family of PAF-like oxidized phospholipids with short sn-2 residues. Biochemical and enzymological evaluations revealed that at least three types of PAF-AH exist in mammals, namely the intracellular types I and II and a plasma type. Many observations indicate that plasma PAF AH terminates signals by PAF and oxidized PAF-like lipids and thereby regulates inflammatory responses. In this review, we will focus on the potential of PAF-AH as a modulator of diseases of dysregulated inflammation.

Regulation of stem cell factor expression in inflammation and asthma

Stem cell factor (SCF) is a major mast cell growth factor, which could be involved in the local increase of mast cell number in the asthmatic airways. In vivo, SCF expression increases in asthmatic patients and this is reversed after treatment with glucocorticoids. In vitro in human lung fibroblasts in culture, IL-1beta, a pro-inflammatory cytokine, confirms this increased SCF mRNA and protein expression implying the MAP kinases p38 and ERK1/2 very early post-treatment, and glucocorticoids confirm this decrease. Surprisingly, glucocorticoids potentiate the IL-1beta-enhanced SCF expression at short term treatment, implying increased SCF mRNA stability and SCF gene transcription rate. This potentiation involves p38 and ERK1/2. Transfection experiments with the SCF promoter including intron1 also confirm this increase and decrease of SCF expression by IL-1beta and glucocorticoids, and the potentiation by glucocorticoids of the IL-1beta-induced SCF expression. Deletion of the GRE or kappaB sites abolishes this potentiation, and the effect of IL-1beta or glucocorticoids alone. DNA binding of GR and NF-kappaB are also demonstrated for these effects. In conclusion, this review concerns new mechanisms of regulation of SCF expression in inflammation that could lead to potential therapeutic strategy allowing to control mast cell number in the asthmatic airways.

Precursor-product relationship between vitellogenin and the yolk proteins as derived from the complete sequence of a Xenopus vitellogenin gene.

In Xenopus laevis four estrogen-responsive genes are expressed simultaneously to produce vitellogenin, the precursor of the yolk proteins. One of these four genes, the gene A2, was sequenced completely, as well as cDNAs representing 75% of the coding region of the gene. From this data the exon-intron structure of the gene was established, revealing 35 exons that give a transcript of 5,619 bp without the poly A-tail. This A2 transcript encodes a vitellogenin of 1,807 amino acids, whose structure is discussed with respect to its function. At the nucleic acid as well as at the protein level no extensive homologies with any sequences other than vitellogenin were observed. Comparison of the amino acid sequence of the vitellogenin A2 molecule with biochemical data obtained from the different yolk proteins allowed us to localize the cleavage products on the vitellogenin precursor as follows: NH2 - lipovitellin I - phosvitin (or phosvette II - phosvette I) - lipovitellin II - COOH.

Extracellular protease of Pseudomonas fluorescens CHA0, a biocontrol factor with activity against the root-knot nematode Meloidogyne incognita.

In Pseudomonas fluorescens CHA0, mutation of the GacA-controlled aprA gene (encoding the major extracellular protease) or the gacA regulatory gene resulted in reduced biocontrol activity against the root-knot nematode Meloidogyne incognita during tomato and soybean infection. Culture supernatants of strain CHA0 inhibited egg hatching and induced mortality of M. incognita juveniles more strongly than did supernatants of aprA and gacA mutants, suggesting that AprA protease contributes to biocontrol.

Deregulated MHC class II transactivator expression leads to a strong Th2 bias in CD4+ T lymphocytes.

The MHC class II (MHC-II) transactivator (CIITA) is the master transcriptional regulator of genes involved in MHC-II-restricted Ag presentation. Fine tuning of CIITA gene expression determines the cell type-specific expression of MHC-II genes. This regulation is achieved by the selective usage of multiple CIITA promoters. It has recently been suggested that CIITA also contributes to Th cell differentiation by suppressing IL-4 expression in Th1 cells. In this study, we show that endogenous CIITA is expressed at low levels in activated mouse T cells. Importantly CIITA is not regulated differentially in murine and human Th1 and Th2 cells. Ectopic expression of a CIITA transgene in multiple mouse cell types including T cells, does not interfere with normal development of CD4(+) T cells. However, upon TCR activation the CIITA transgenic CD4(+) T cells preferentially differentiate into IL-4-secreting Th2-type cells. These results imply that CIITA is not a direct Th1-specific repressor of the IL-4 gene and that tight control over the expression of CIITA and MHC-II is required to maintain the normal balance between Th1 and Th2 responses.

Myelin gene expression during demyelination and remyelination in aggregating brain cell cultures.

Remyelination can be studied in aggregating rat brain cell cultures after limited demyelination. Demyelination was induced using a monoclonal antibody against myelin/oligodendrocyte glycoprotein (MOG mAb), in the presence of complement. De- and remyelination were assessed by measuring myelin basic protein (MBP). Two days after removing the MOG mAb, MBP levels reached 50% of controls and after 7 days 93%. During this period, cell proliferation determined by [14C]thymidine incorporation was similar in remyelinating and control cultures. Hormones and growth factors were tested for possible stimulatory effect on remyelinating cultures. Bovine growth hormone (bGH), triiodothyronine (T3), basic fibroblast growth factor (bFGF) and platelet-derived growth factor (PDGF) did not improve remyelination. Only epidermal growth factor (EGF) increased the level of remyelination. PDGF increased the rate of cell proliferation in both control and remyelinating cultures. A significant proportion of oligodendrocytes entered the cell division cycle and were not available for remyelination. The results obtained with PDGF and FGF (inhibition) support the idea that a pool of progenitor cells was still present and able to proliferate and differentiate into myelinating oligodendrocytes. The levels of myelin protein mRNAs were investigated during de- and remyelination. During demyelination, myelin protein mRNA levels decreased to approximately 50% of control cultures and returned to normal during remyelination. These preliminary results indicate that normal levels of gene transcription are sufficient to meet the increased need for newly synthesized myelin proteins during remyelination.