Distinct ASIC currents are expressed in rat putative nociceptors and are modulated by nerve injury.

The H(+)-gated acid-sensing ion channels (ASICs) are expressed in dorsal root ganglion (DRG) neurones. Studies with ASIC knockout mice indicated either a pro-nociceptive or a modulatory role of ASICs in pain sensation. We have investigated in freshly isolated rat DRG neurones whether neurones with different ASIC current properties exist, which may explain distinct cellular roles, and we have investigated ASIC regulation in an experimental model of neuropathic pain. Small-diameter DRG neurones expressed three different ASIC current types which were all preferentially expressed in putative nociceptors. Type 1 currents were mediated by ASIC1a homomultimers and characterized by steep pH dependence of current activation in the pH range 6.8-6.0. Type 3 currents were activated in a similar pH range as type 1, while type 2 currents were activated at pH < 6. When activated by acidification to pH 6.8 or 6.5, the probability of inducing action potentials correlated with the ASIC current density. Nerve injury induced differential regulation of ASIC subunit expression and selective changes in ASIC function in DRG neurones, suggesting a complex reorganization of ASICs during the development of neuropathic pain. In summary, we describe a basis for distinct cellular functions of different ASIC types in small-diameter DRG neurones.

Analysis of expressed sequence tags from Trypanosoma cruzi amastigotes

A total of 880 expressed sequence tags (EST) originated from clones randomly selected from a Trypanosoma cruzi amastigote cDNA library have been analyzed. Of these, 40% (355 ESTs) have been identified by similarity to sequences in public databases and classified according to functional categorization of their putative products. About 11% of the mRNAs expressed in amastigotes are related to the translational machinery, and a large number of them (9% of the total number of clones in the library) encode ribosomal proteins. A comparative analysis with a previous study, where clones from the same library were selected using sera from patients with Chagas disease, revealed that ribosomal proteins also represent the largest class of antigen coding genes expressed in amastigotes (54% of all immunoselected clones). However, although more than thirty classes of ribosomal proteins were identified by EST analysis, the results of the immunoscreening indicated that only a particular subset of them contains major antigenic determinants recognized by antibodies from Chagas disease patients.

Emotional behavior in acute stroke : the Lausanne emotion in stroke study

Résumé : Objectif: Etudier les comportements émotionnels chez les patients ayant souffert d'un accident vasculaire cérébral (AVC) Contexte: Les modifications du comportement émotionnel après un AVC sont actuellement bien reconnues mais ont été très peu étudiées dans la phase aigüe de l'AVC. Méthode: Tous les patients présentant un AVC datant de moins de 24 heures ont été inclus prospectivement. Nous avons validé une échelle (the Emotional Behavior Index (EBI)), comprenant 38 qualificatifs, représentant la tristesse, l'agressivité, la désinhibition, l'adaptation, la passivité, l'indifférence et le déni. Les informations cliniques et radiologiques (CT et IRM) ont été obtenues par le biais de notre Registre des AVC. L'analyse statistique a été faite au moyen de tests uni- et multi-variés Résultats: Des 254 patients inclus, 40% présentaient de la tristesse, 49% de la passivité, 17% de l'agressivité, 53% de l'indifférence, 76% de la désinhibition, 18% un manque d'adaptation et 44% une réaction de déni. Plusieurs interactions statistiquement significatives ont été mises en évidence. En effet, la tristesse semble corréler à des antécédents d'alcoolisme (r = p< 0.037), au sexe féminin (r = p<0.028) et à la nature hémorragique de l'AVC (r = p<0.063). L'agressivité corrèle à des antécédents de troubles dépressifs (r = p<0.046) et à la nature hémorragique de l'AVC (r = p<0.06). Le déni corrèle plutôt au sexe masculin (r = p<0.035) et aux lésions hémorragiques (r = p<0.05). Le comportement émotionnel ne corrèle ni au degré et type d'atteinte neurologique, ni à la localisation de l'AVC mais une association entre Les lésions hémorragiques et un comportement agressif a été mis en évidence (p<0.001) de même qu'avec un manque d'adaptation (r = p<0.015), une certaine indifférence (r = p<0.018) et une réaction de déni (r = p<0.045). Conclusion: L'observation systématique des modifications du comportement émotionnel après un AVC suggère que les altérations émotionnelles sont indépendantes de la thymie et de l'atteinte physique et représentent donc probablement des séquelles à part entière de l'AVC. Abstract: Objective: To study emotional behaviors in an acute stroke population. Background: Alterations in emotional behavior after stroke have been recently recognized, but little attention has been paid to these changes in the very acute phase of stroke. Methods: Adult patients presenting with acute stroke were prospectively recruited and studied. We validated the Emotional Behavior Index (EBI), a 38-item scale designed to evaluate behavioral aspects of sadness, aggressiveness, disinhibition, adaptation, passivity, indifference, and denial. Clinical, historical, and imaging (computed tomography/magnetic resonance imaging) data were obtained on each subject through our Stroke Registry. Statistical analysis was performed with both univariate and multivariate tests. Results: Of the 254 patients, 40% showed sadness, 49% passivity, 17% aggressiveness, 53% indifference, 76% disinhibition, 18% lack of adaptation, and 44% denial reactions. Several significant correlations were identified. Sadness was correlated with a personal history of alcohol abuse (r = P < 0.037), female gender (r = P < 0.028), and hemorrhagic nature of the stroke (r = P < 0.063). Aggressiveness was correlated with a personal history of depression (r = P < 0.046) and hemorrhage (r = P < 0.06). Denial was correlated with male gender (r= P < 0.035) and hemorrhagic lesions (r = P < 0.05). Emotional behavior did not correlate with either neurologic impairment or lesion localization, but there was an association between hemorrhage and aggressive behavior (P < 0.001), lack of adaptation (r = P < 0.015), indifference (r = P < 0.018), and denial (r = P < 0.045). Conclusions: Systematic observations of acute emotional behaviors after stroke suggest that emotional alterations are independent of mood and physical status and should be considered as a separate consequence of stroke.

Production of full-length cDNA sequences by sequencing and analysis of expressed sequence tags from Schistosoma mansoni

The number of sequences generated by genome projects has increased exponentially, but gene characterization has not followed at the same rate. Sequencing and analysis of full-length cDNAs is an important step in gene characterization that has been used nowadays by several research groups. In this work, we have selected Schistosoma mansoni clones for full-length sequencing, using an algorithm that investigates the presence of the initial methionine in the parasite sequence based on the positions of alignment start between two sequences. BLAST searches to produce such alignments have been performed using parasite expressed sequence tags produced by Minas Gerais Genome Network against sequences from the database Eukaryotic Cluster of Orthologous Groups (KOG). This procedure has allowed the selection of clones representing 398 proteins which have not been deposited as S. mansoni complete CDS in any public database. Dedicated sequencing of 96 of such clones with reads from both 5' and 3' ends has been performed. These reads have been assembled using PHRAP, resulting in the production of 33 full-length sequences that represent novel S. mansoni proteins. These results shall contribute to construct a more complete view of the biology of this important parasite.

Ki-67 is expressed in multiplying forms of Schistosoma mansoni, but not in snail host tissues

Ki-67 is a protein expressed in the nucleus of several species during cell-division, being absent during the GO resting phase of the cellular cycle. During attempts to disclose mitosis in the so-called " amebocyte-producing organ " in Biomphalaria glabrata infected with Schistosoma mansoni, the parasite multiplying forms appeared strongly marked for Ki-67, while the snail tissues were completely negative. These data are worth registering to complement general data on Ki-67, and to help future studies on the relationship of the parasite and of its intermediate host.

Analysis and chromosomal mapping of Leishmania (Leishmania) amazonensis amastigote expressed sequence tags

The characterization of expressed sequence tags (ESTs) generated from a cDNA library of Leishmania (Leishmania) amazonensis amastigotes is described. The sequencing of 93 clones generated new L. (L.) amazonensis ESTs from which 32% are not related to any other sequences in database and 68% presented significant similarities to known genes. The chromosome localization of some L. (L.) amazonensis ESTs was also determined in L. (L.) amazonensis and L. (L.) major. The characterization of these ESTs is suitable for the genome physical mapping, as well as for the identification of genes encoding cysteine proteinases implicated with protective immune responses in leishmaniasis.

A novel neutrophil elastase inhibitor prevents elastase activation and surface cleavage of the epithelial sodium channel expressed in Xenopus laevis oocytes.

The amiloride-sensitive epithelial sodium channel (ENaC) constitutes a limiting step in sodium reabsorption across distal airway epithelium and controlling mucociliary clearance. ENaC is activated by serine proteases secreted in the extracellular milieu. In cystic fibrosis lungs, high concentrations of secreted neutrophil elastase (NE) are observed. hNE could activate ENaC and contribute to further decreased mucociliary clearance. The aims of this study were (i) to test the ability of an engineered human neutrophil elastase inhibitor (EPI-hNE4) to specifically inhibit the elastase activation of ENaC-mediated amiloride-sensitive currents (I(Na)) and (ii) to examine the effect of elastase on cell surface expression of ENaC and its cleavage pattern (exogenous proteolysis). Oocytes were exposed to hNE (10-100 microg/ml) and/or trypsin (10 microg/ml) for 2-5 min in the presence or absence of EPI-hNE4 (0.7 microm). hNE activated I(Na) 3.6-fold (p < 0.001) relative to non-treated hENaC-injected oocytes. EPI-hNE4 fully inhibited hNE-activated I(Na) but had no effect on trypsin- or prostasin-activated I(Na). The co-activation of I(Na) by hNE and trypsin was not additive. Biotinylation experiments revealed that cell surface gamma ENaC (but not alpha or beta ENaC) exposed to hNE for 2 min was cleaved (as a 67-kDa fragment) and correlated with increased I(Na). The elastase-induced exogenous proteolysis pattern is distinct from the endogenous proteolysis pattern induced upon preferential assembly, suggesting a causal relationship between gamma ENaC cleavage and ENaC activation, taking place at the plasma membrane.

Identification of tumor-associated antigens by large-scale analysis of genes expressed in human colorectal cancer.

Despite the high prevalence of colon cancer in the world and the great interest in targeted anti-cancer therapy, only few tumor-specific gene products have been identified that could serve as targets for the immunological treatment of colorectal cancers. The aim of our study was therefore to identify frequently expressed colon cancer-specific antigens. We performed a large-scale analysis of genes expressed in normal colon and colon cancer tissues isolated from colorectal cancer patients using massively parallel signal sequencing (MPSS). Candidates were additionally subjected to experimental evaluation by semi-quantitative RT-PCR on a cohort of colorectal cancer patients. From a pool of more than 6000 genes identified unambiguously in the analysis, we found 2124 genes that were selectively expressed in colon cancer tissue and 147 genes that were differentially expressed to a significant degree between normal and cancer cells. Differential expression of many genes was confirmed by RT-PCR on a cohort of patients. Despite the fact that deregulated genes were involved in many different cellular pathways, we found that genes expressed in the extracellular space were significantly over-represented in colorectal cancer. Strikingly, we identified a transcript from a chromosome X-linked member of the human endogenous retrovirus (HERV) H family that was frequently and selectively expressed in colon cancer but not in normal tissues. Our data suggest that this sequence should be considered as a target of immunological interventions against colorectal cancer.

MicroRNA Profile of Circulating CD4-positive Regulatory T Cells in Human Adults and Impact of Differentially Expressed MicroRNAs on Expression of Two Genes Essential to Their Function.

Regulatory T cells (Tregs) are characterized by a high expression of IL-2 receptor α chain (CD25) and of forkhead box P3 (FOXP3), the latter being essential for their development and function. Another major player in the regulatory function is the cytotoxic T-lymphocyte associated molecule-4 (CTLA-4) that inhibits cytotoxic responses. However, the regulation of CTLA-4 expression remains less well explored. We therefore studied the microRNA signature of circulating CD4(+) Tregs isolated from adult healthy donors and identified a signature composed of 15 differentially expressed microRNAs. Among those, miR-24, miR-145, and miR-210 were down-regulated in Tregs compared with controls and were found to have potential target sites in the 3'-UTR of FOXP3 and CTLA-4; miR-24 and miR-210 negatively regulated FOXP3 expression by directly binding to their two target sites in its 3'-UTR. On the other hand, miR-95, which is highly expressed in adult peripheral blood Tregs, positively regulated FOXP3 expression via an indirect mechanism yet to be identified. Finally, we showed that miR-145 negatively regulated CTLA-4 expression in human CD4(+) adult peripheral blood Tregs by binding to its target site in CTLA-4 transcript 3'-UTR. To our knowledge, this is the first identification of a human adult peripheral blood CD4(+) Treg microRNA signature. Moreover, unveiling one mechanism regulating CTLA-4 expression is novel and may lead to a better understanding of the regulation of this crucial gene.

The ability of haemolysins expressed by atypical enteropathogenic Escherichia coli to bind to extracellular matrix components

Typical and atypical enteropathogenic Escherichia coli (EPEC) are considered important bacterial causes of diarrhoea. Considering the repertoire of virulence genes, atypical EPEC (aEPEC) is a heterogeneous group, harbouring genes that are found in other diarrheagenic E. coli pathotypes, such as those encoding haemolysins. Haemolysins are cytolytic toxins that lyse host cells disrupting the function of the plasma membrane. In addition, these cytolysins mediate a connection to vascular tissue and/or blood components, such as plasma and cellular fibronectin. Therefore, we investigated the haemolytic activity of 72 aEPEC isolates and determined the correlation of this phenotype with the presence of genes encoding enterohaemolysins (Ehly) and cytolysin A (ClyA). In addition, the correlation between the expression of haemolysins and the ability of these secreted proteins to adhere to extracellular matrix (ECM) components was also assessed in this study. Our findings demonstrate that a subset of aEPEC presents haemolytic activity due to the expression of Ehlys and/or ClyA and that this activity is closely related to the ability of these isolates to bind to ECM components.

Human pregnane X receptor is expressed in breast carcinomas, potential heterodimers formation between hPXR and RXR-alpha.

BACKGROUND The human pregnane X receptor (hPXR) is an orphan nuclear receptor that induces transcription of response elements present in steroid-inducible cytochrome P-450 gene promoters. This activation requires the participation of retinoid X receptors (RXRs), needed partners of hPXR to form heterodimers. We have investigated the expression of hPXR and RXRs in normal, premalignant, and malignant breast tissues, in order to determine whether their expression profile in localized infiltrative breast cancer is associated with an increased risk of recurrent disease. METHODS Breast samples from 99 patients including benign breast diseases, in situ and infiltrative carcinomas were processed for immunohistochemistry and Western-blot analysis. RESULTS Cancer cells from patients that developed recurrent disease showed a high cytoplasmic location of both hPXR isoforms. Only the infiltrative carcinomas that relapsed before 48 months showed nuclear location of hPXR isoform 2. This location was associated with the nuclear immunoexpression of RXR-alpha. CONCLUSION Breast cancer cells can express both variants 1 and 2 of hPXR. Infiltrative carcinomas that recurred showed a nuclear location of both hPXR and RXR-alpha; therefore, the overexpression and the subcellular location changes of hPXR could be considered as a potential new prognostic indicator.

Probing the interactions of NK cell receptors with ligand expressed in trans and cis.

Certain receptors on natural killer (NK) cells, which are specific for MHC class I (MHC-I) molecules, do not only interact with ligand expressed on opposing cell membranes (in trans) but also interact with those on the same cell membrane (in cis). Cis interactions have been demonstrated for only a small number of cell surface receptors. However, this has not been tested systematically, raising the possibility that additional receptors may be able to bind ligand expressed in cis. Here we describe a number of approaches to evaluate trans and cis binding of the Ly49A NK cell receptor to its H-2D(d) ligand. These procedures should facilitate the investigation of cis/trans interactions of other receptor-ligand pairs and simplify the analysis of NK cell receptor variants.