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ABSTRACT: About sixty thousand new cases of Hepatitis C virus (HCV) infection are recorded in Brazil each year. These cases are currently treated with pegylated interferon (PEG-IFN) and ribavirin (RBV) with an overall success rate of 50%. New compounds for anti-HCV therapy targeted to the HCV NS3 protease are being developed and some already form the components of licensed therapies. Mapping NS3 protease resistance mutations to protease inhibitors or anti-viral drug candidates is important to direct anti-HCV drug treatment. METHODS: Sequence analysis of the HCV NS3 protease was conducted in a group of 68 chronically infected patients harboring the HCV genotype 1. The patients were sampled before, during and after a course of PEG-IFN-RBV treatment. RESULTS: Resistance mutations to the protease inhibitors, Boceprevir and Telaprevir were identified in HCV isolated from three patients (4.4%); the viral sequences contained at least one of the following mutations: V36L, T54S and V55A. In one sustained virological responder, the T54S mutation appeared during the course of PEG-IFN and RBV therapy. In contrast, V36L and V55A mutations were identified in virus isolated from one relapsing patient before, during, and after treatment, whereas the T54S mutation was identified in virus isolated from one non-responding patient, before and during the treatment course. CONCLUSIONS: The incidence and persistence of protease resistance mutations occurring in HCV from chronically infected patients in Brazil should be considered when using protease inhibitors to treat HCV disease. In addition, patients treated with the current therapy (PEG-IFN and RBV) that are relapsing or are non-responders should be considered candidates for protease inhibitor therapy.
Virology Journal 02/2013; 10(1):57. · 2.34 Impact Factor
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ABSTRACT: Single nucleotide polymorphisms (SNPs) in the interleukin (IL)28B locus have been associated with a sustained virological response (SVR) in interferon-ribavirin (IFN-RBV)-treated chronic hepatitis C virus (HCV)-infected patients in European and African populations. In this study, the genotype frequency of two IL28B SNPs (rs129679860 and rs8099917) in a cohort of chronic HCV-monoinfected patients in Brazil was evaluated and the SNP sufficient to predict the treatment response outcome was determined. A total of 66 naïve genotype-1 chronic HCV-infected patients were genotyped and the associated viral kinetics and SVR were assessed. The overall SVR was 38%. Both the viral kinetics and SVR were associated with rs129679860 genotypes (CC = 62% vs. CT = 33% vs. TT = 18%, p = 0.016). However, rs8099917 genotypes were only associated with SVR (TT = 53% vs. TG = 33% vs. GG = 18%; p = 0.032). In this population, the analysis of a single SNP, rs12979860, successfully predicts SVR in the IFN-RBV treatment of HCV.
Memórias do Instituto Oswaldo Cruz 11/2012; 107(7):888-92. · 2.15 Impact Factor
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ABSTRACT: BACKGROUND: Cytokines play an important role in the regulation of the immune response. In hepatitis C virus (HCV) infection, cytokine levels may influence the outcome of acute HCV infection. Polymorphisms in cytokine genes have been associated to different expression levels in response to infection. This study was carried out to investigate the association of several cytokine gene polymorphisms with disease outcome in HCV-infected patients. FINDINGS: Patients with chronic or spontaneously resolved HCV infection were included in a cross-sectional study. A comparative analysis was performed between the groups regarding frequency distribution of the following cytokines' gene polymorphisms: IL-10 (-1082 A/G; -819 T/C; -592 A/C), IL-4 (+33C/T), IFN-gamma (+874 T/A), TNF-alpha (-238 G/A and -308 G/A) and IL-28B (rs12979860 C/T and rs8099917 T/G). Results: Eighteen patients with spontaneous viral clearance and 161 with chronic HCV infection were included. In the comparative analysis, the GG genotype of the IL-10 polymorphism -1082A/G was more frequent in patients with spontaneous viral clearance when compared to patients with chronic HCV (41.2% vs 6.2%; p = 0.001). This association was also found for the CC genotype of the IL-4 polymorphism +33C/T (72.2% vs 36.7%; p = 0.017) and the CC and TT genotypes of the IL-28B polymorphisms rs 12979860 and rs 8099917 (88.9% vs 30.3%; p < 0.001 and 88.9% vs 49.6%; p = 0.002). The IL10 (A-1082 G) and IL-28B (Crs12979860T) gene polymorphisms showed odds ratios of 12.848 and 11.077, respectively, and thus may have a greater influence on HCV spontaneous viral clearance. The IFN-gamma (+874 T/A), TNF-alpha (-238 G/A and -308 G/A) polymorphisms did not show significant association with spontaneous viral clearance or chronicity. CONCLUSION: The G allele for IL-10 (-1082 A/G), the C allele for IL-4 (+3 C/T) and the C and T alleles for IL-28B (rs12979860 and rs8099917, respectively) are associated with spontaneous viral clearance in hepatitis C infection.
BMC Research Notes 09/2012; 5(1):508.
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ABSTRACT: We describe an association of two SNPs, rs3212345:C>T and rs3212346:G>A, located approximately 2.5 kb upstream of the melanocortin-1 receptor (MC1R) translation initiation codon, with pigmentation phenotype variation in a Southeast Brazilian miscegenated population.
One hundred thirty-eight genetically unrelated subjects, with multicolor phenotype, were selected from the southeast region of Brazil. Skin, hair and eye color, and tanning ability were rated. Genotypes for each SNP (rs3212345:C>T and rs3212346:G>A) were determined. A logistic regression analysis was performed with the additive model to determine which of the polymorphisms contributed to a specific phenotype.
We found that the rs3212345:C>T is associated with light skin, red hair, and poor tanning ability, while the rs3212346:G>A is associated with dark skin, black hair, and strong tanning ability. The presence of rs3212345-C and rs3212346-A alleles in human, chimpanzee, gorilla, orangutan, and marmoset genomes suggests that they are the ancestral alleles.
These data suggest that the rs3212345-T and rs3212346-G alleles may have contributed to lighter pigmentation phenotypes in modern humans. Genotyping for these SNPs may prove useful to the fields of molecular anthropology and forensic genetics. Am. J. Hum. Biol. 2012. © 2012 Wiley Periodicals, Inc.
American Journal of Human Biology 09/2012; 24(6):853-5. · 2.27 Impact Factor
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ABSTRACT: Induced pluripotent stem cells (iPSCs) were originally generated by forced ectopic expression of four transcription factors genes - OCT4, KLF4, SOX2 and c-MYC - in fibroblasts. However, the efficiency of iPSCs obtention is extremely low, and reprogramming takes about 20 days. We reasoned that adult cells showing basal expression of core embryonic stem (ES) cell regulator genes could be a better cell source for reprogramming. Menstrual blood-derived mesenchymal cells (MBMCs) are multipotent cells that show detectable levels of some of the core ES cells regulators. The aim of this study was to determine whether reprogramming efficiency could be increased by using MBMCs as a cell source to generate iPSCs. MBMCs were transduced with recombinant retroviruses expressing the coding regions of OCT4, SOX2 and KLF4 genes. Cells with high nucleus/cytoplasm ratio can be detected about 5 days posttransduction, and colonies of typical ES-like cells begun to appear after 7 days. At day 15, colonies were picked-up and expanded for characterization. Most of the clones were morphologically identical to ES cells and positive at the mRNA and protein levels for all pluripotency markers tested. The clones are capable of forming embryoid bodies and to differentiate in vitro into cells of the three germ cell layers. Our results show that the reprogramming was faster and with efficiency around 2 - 5%, even in the absence of ectopic expression of c-MYC. To date, this is the first study showing MBMCs as a cell source for nuclear reprogramming.
Cell Transplantation 07/2012; · 5.13 Impact Factor
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ABSTRACT: Glycosylated mouse cystatin C (mCysC), an endogenous inhibitor of cysteine cathepsin proteases (CP), has been suggested as a cofactor of β-FGF to induce the differentiation of mouse embryonic stem cells into neural progenitor cells (NPCs). To investigate the possible role of CP in neural differentiation, we treated embryoid bodies (EBs) with (i) E64, an inhibitor of papain-like CP and of calpains, (ii) an inhibitor of cathepsin L (iCatL), (iii) an inhibitor of calpains (iCalp), or (iv) cystatins, and their ability to differentiate into neural cells was assessed. We show that the inhibition of CP induces a significant increase in Pax6 expression in EBs, leading to an increase in the number of nestin-positive cells after 3 days. Fourteen days after E64 treatment, we observed increased numbers of β-III-tubulin-positive cells, showing greater percentage of immature neurons, and this feature persisted up to 24 days. At this point, we encountered higher numbers of neurons with inward Na(+) current compared with untreated EBs. Further, we show that mCysC and iCatL, but not unglycosylated egg white cystatin or iCalp, increased the numbers of NPCs. In contrast to E64 and iCatL, mCysC did not inhibit CP in EBs and its neural-inducing activity required β-FGF. We propose that the inhibition of CP induces the differentiation of mouse embryonic stem cells into NPCs and neurons through a mechanism that is distinct from CysC-induced neural differentiation.
Stem cells and development 03/2011; 20(11):1859-72. · 4.15 Impact Factor
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ABSTRACT: The long QT syndrome (LQTS) is an inherited arrhythmia syndrome with increased QT interval and risk of sudden death. Mutations in genes KCNQ1, KCNH2 and SCN5A account for 90% of cases with genotype determined, and genotyping is informative for genetic counseling and better disease management.
Molecular investigation and computational analysis of gene variants of KCNQ1, KCNH2 and SCN5A associated with LQTS, in families with the disease.
The coding regions of genes KCNQ1, KCNH2 and SCN5A in patients with LQTS and their family members were sequenced and analyzed using Geneious ProTM software.
Two families with clinical criteria for LQTS were investigated. The proband of Family A had QTC = 562 ms, Schwartz Score = 5.5. The genotyping identified the G1714A mutation in the KCNH2 gene. QTC = 521 ± 42 ms was observed in family members carrying the mutation against QTC = 391 ± 21 ms for non-carriers. The proband of Family B had QTc = 551 ms, Schwartz Score = 5.5. The genotyping identified the G1600T mutation, in the same gene. The analysis of family members revealed QTC = 497 ± 42 ms in mutation carriers, compared with QTC = 404 ± 29 ms in non-carriers.
Two gene variants previously associated with LQTS were found in two families clinically diagnosed with LQTS. The prolongation of the QT interval was observed in all family members carrying the mutations. A strategy was developed to identify variants of genes KCNQ1, KCNH2 and SCN5A, making it possible to train technical staff for future application to diagnosis routine.
Arquivos brasileiros de cardiologia 02/2011; 96(3):172-8. · 1.32 Impact Factor
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ABSTRACT: Gene regulation in trypanosomatids occurs mainly by post-transcriptional mechanisms modulating mRNA stability and translation. We have investigated heat shock protein (HSP) 70 gene regulation in Trypanosoma cruzi, the causal agent of Chagas' disease. The HSP70 mRNA's half-life increases after heat shock, and the stabilization is dependent on protein synthesis. In a cell-free RNA decay assay, a U-rich region in the 3' untranslated region (UTR) is a target for degradation, which is reduced when in the presence of protein extracts from heat shocked cells. In a transfected reporter gene assay, both the 5'- and 3'-UTRs confer temperature-dependent regulation. Both UTRs must be present to increase mRNA stability at 37 degrees C, indicating that the 5'- and 3'-UTRs act cooperatively to stabilize HSP70 mRNA during heat shock. We conclude that HSP70 5'- and 3'-UTRs regulate mRNA stability during heat shock in T. cruzi.
Experimental Parasitology 10/2010; 126(2):245-53. · 2.12 Impact Factor
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ABSTRACT: The illicit use of supraphysiological doses of androgenic steroids (AAS) has been suggested as a cause of arrhythmia in athletes. The objectives of the present study were to investigate the time-course and the cellular, ionic and molecular processes underlying ventricular repolarization in rats chronically treated with AAS. Male Wistar rats were treated weekly for 8 weeks with 10mg/kg of nandrolone decanoate (DECA n=21) or vehicle (control n=20). ECG was recorded weekly. Action potential (AP) and transient outward potassium current (I(to)) were recorded in rat hearts. Expression of KChIP2, Kv1.4, Kv4.2, and Kv4.3 was assessed by real-time PCR. Hematoxylin/eosin and Picrosirius red staining were used for histological analysis. QTc was greater in the DECA group. After DECA treatment the left, but not right, ventricle showed a longer AP duration than did the control. I(to) current densities were 47.5% lower in the left but not in the right ventricle after DECA. In the right ventricle the I(to) inactivation time-course was slower than in the control group. After DECA the left ventricle showed lower KChIP2 ( approximately 26%), Kv1.4 ( approximately 23%) and 4.3 ( approximately 70%) expression while the Kv 4.2 increased in 4 ( approximately 250%) and diminished in 3 ( approximately 30%) animals of this group. In the right ventricle the expression of I(to) subunits was similar between the treatment and control groups. DECA-treated hearts had 25% fewer nuclei and greater nuclei diameters in both ventricles. Our results strongly suggest that supraphysiological doses of AAS induce morphological remodeling in both ventricles. However, the electrical remodeling was mainly observed in the left ventricle.
Journal of Molecular and Cellular Cardiology 08/2010; 49(2):165-75. · 5.17 Impact Factor
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ABSTRACT: The bone marrow stromal cell line S17 has been used to study hematopoiesis in vitro. In this study, we demonstrate the presence of calcium and chloride currents in cultured S17 cells. Calcium currents were of low amplitude or barely detectable (50-100 pA). Hence to amplify the currents, we have used barium as a charge carrier. Barium currents were identified based on their distinct voltage-dependence, and sensitivity to dihydropyridines. S17 cells also exhibited a slowly activating outward current without inactivation, most commonly seen when the sodium of the extracellular solution was replaced either by TEA (TEA/Cs saline) or NMDG (NMDG saline), or by addition of amiloride to the extracellular solution. This current was abolished either by 500 microM SITS (4,4'-diisothiocyanatostilbene-2-2'-disulfonic acid) or 500 microM DPC (diphenylamine-2-carboxylic acid) a cystic fibrosis transmembrane conductance regulator (CFTR) chloride channel blocker, identifying it as a Cl(-) current. RT-PCR identified the presence of ENaC and CFTR transcripts. CFTR blockade reduced cell proliferation, suggesting that this channel plays a physiological role in regulation of S17 cell proliferation.
Journal of Cellular Physiology 04/2010; 223(1):244-51. · 3.87 Impact Factor
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ABSTRACT: The bone marrow stromal cell line S17 has been used to study hematopoiesis in vitro. In this study, we demonstrate the presence of calcium and chloride currents in cultured S17 cells. Calcium currents were of low amplitude or barely detectable (50–100 pA). Hence to amplify the currents, we have used barium as a charge carrier. Barium currents were identified based on their distinct voltage-dependence, and sensitivity to dihydropyridines. S17 cells also exhibited a slowly activating outward current without inactivation, most commonly seen when the sodium of the extracellular solution was replaced either by TEA (TEA/Cs saline) or NMDG (NMDG saline), or by addition of amiloride to the extracellular solution. This current was abolished either by 500 µM SITS (4,4′-diisothiocyanatostilbene-2-2′-disulfonic acid) or 500 µM DPC (diphenylamine-2-carboxylic acid) a cystic fibrosis transmembrane conductance regulator (CFTR) chloride channel blocker, identifying it as a Cl− current. RT-PCR identified the presence of ENaC and CFTR transcripts. CFTR blockade reduced cell proliferation, suggesting that this channel plays a physiological role in regulation of S17 cell proliferation. J. Cell. Physiol. 223: 244–251, 2010. © 2009 Wiley-Liss, Inc.
Journal of Cellular Physiology 01/2010; 223(1):244 - 251. · 3.87 Impact Factor
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Marcelo Bertalan,
Rodolpho Albano,
Vânia de Pádua,
Luc Rouws,
Cristian Rojas,
Adriana Hemerly,
Kátia Teixeira,
Stefan Schwab,
Jean Araujo,
André Oliveira, [......],
Paulo Bisch,
Shaila C Rössle,
Turán Urményi,
Alessandra Rael Pereira,
Rosane Silva, Edson Rondinelli,
Wanda von Krüger,
Orlando Martins,
José Ivo Baldani,
Paulo C G Ferreira
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ABSTRACT: Gluconacetobacter diazotrophicus Pal5 is an endophytic diazotrophic bacterium that lives in association with sugarcane plants. It has important biotechnological features such as nitrogen fixation, plant growth promotion, sugar metabolism pathways, secretion of organic acids, synthesis of auxin and the occurrence of bacteriocins.
Gluconacetobacter diazotrophicus Pal5 is the third diazotrophic endophytic bacterium to be completely sequenced. Its genome is composed of a 3.9 Mb chromosome and 2 plasmids of 16.6 and 38.8 kb, respectively. We annotated 3,938 coding sequences which reveal several characteristics related to the endophytic lifestyle such as nitrogen fixation, plant growth promotion, sugar metabolism, transport systems, synthesis of auxin and the occurrence of bacteriocins. Genomic analysis identified a core component of 894 genes shared with phylogenetically related bacteria. Gene clusters for gum-like polysaccharide biosynthesis, tad pilus, quorum sensing, for modulation of plant growth by indole acetic acid and mechanisms involved in tolerance to acidic conditions were identified and may be related to the sugarcane endophytic and plant-growth promoting traits of G. diazotrophicus. An accessory component of at least 851 genes distributed in genome islands was identified, and was most likely acquired by horizontal gene transfer. This portion of the genome has likely contributed to adaptation to the plant habitat.
The genome data offer an important resource of information that can be used to manipulate plant/bacterium interactions with the aim of improving sugarcane crop production and other biotechnological applications.
BMC Genomics 09/2009; 10:450. · 4.07 Impact Factor
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Marcelo Bertalan,
Rodolpho Albano,
de Pádua Vânia,
Luc Rouws,
Cristian Rojas,
Adriana Hemerly,
Kátia Teixeira,
Stefan Schwab,
Jean Araujo,
André Oliveira, [......],
Paulo Bisch,
Shaila Rössle,
Turán Ürményi,
Rael Pereira Alessandra,
Rosane Silva, Edson Rondinelli,
von Krüger Wanda,
Orlando Martins,
José Baldani,
Paulo Ferreira
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ABSTRACT: Abstract
Background
Gluconacetobacter diazotrophicus Pal5 is an endophytic diazotrophic bacterium that lives in association with sugarcane plants. It has important biotechnological features such as nitrogen fixation, plant growth promotion, sugar metabolism pathways, secretion of organic acids, synthesis of auxin and the occurrence of bacteriocins.
Results
Gluconacetobacter diazotrophicus Pal5 is the third diazotrophic endophytic bacterium to be completely sequenced. Its genome is composed of a 3.9 Mb chromosome and 2 plasmids of 16.6 and 38.8 kb, respectively. We annotated 3,938 coding sequences which reveal several characteristics related to the endophytic lifestyle such as nitrogen fixation, plant growth promotion, sugar metabolism, transport systems, synthesis of auxin and the occurrence of bacteriocins. Genomic analysis identified a core component of 894 genes shared with phylogenetically related bacteria. Gene clusters for gum -like polysaccharide biosynthesis, tad pilus, quorum sensing, for modulation of plant growth by indole acetic acid and mechanisms involved in tolerance to acidic conditions were identified and may be related to the sugarcane endophytic and plant-growth promoting traits of G. diazotrophicus . An accessory component of at least 851 genes distributed in genome islands was identified, and was most likely acquired by horizontal gene transfer. This portion of the genome has likely contributed to adaptation to the plant habitat.
Conclusion
The genome data offer an important resource of information that can be used to manipulate plant/bacterium interactions with the aim of improving sugarcane crop production and other biotechnological applications.
BMC Genomics. 01/2009;
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Marcelo A Carvalho,
Sylvia M Marsillac,
Rachel Karchin,
Siranoush Manoukian,
Scott Grist,
Ramona F Swaby,
Turan P Urmenyi, Edson Rondinelli,
Rosane Silva,
Luis Gayol,
Lisa Baumbach,
Rebecca Sutphen,
Jennifer L Pickard-Brzosowicz,
Katherine L Nathanson,
Andrej Sali,
David Goldgar,
Fergus J Couch,
Paolo Radice,
Alvaro N A Monteiro
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ABSTRACT: Germ line inactivating mutations in BRCA1 confer susceptibility for breast and ovarian cancer. However, the relevance of the many missense changes in the gene for which the effect on protein function is unknown remains unclear. Determination of which variants are causally associated with cancer is important for assessment of individual risk. We used a functional assay that measures the transactivation activity of BRCA1 in combination with analysis of protein modeling based on the structure of BRCA1 BRCT domains. In addition, the information generated was interpreted in light of genetic data. We determined the predicted cancer association of 22 BRCA1 variants and verified that the common polymorphism S1613G has no effect on BRCA1 function, even when combined with other rare variants. We estimated the specificity and sensitivity of the assay, and by meta-analysis of 47 variants, we show that variants with <45% of wild-type activity can be classified as deleterious whereas variants with >50% can be classified as neutral. In conclusion, we did functional and structure-based analyses on a large series of BRCA1 missense variants and defined a tentative threshold activity for the classification missense variants. By interpreting the validated functional data in light of additional clinical and structural evidence, we conclude that it is possible to classify all missense variants in the BRCA1 COOH-terminal region. These results bring functional assays for BRCA1 closer to clinical applicability.
Cancer Research 02/2007; 67(4):1494-501. · 7.86 Impact Factor
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ABSTRACT: DNA replication mechanisms are poorly understood in most of trypanosomatids, in particular the replication of the peculiar mitochondrial DNA, the kinetoplast DNA (kDNA). To contribute to the knowledge on the mechanism of kDNA replication in Trypanosoma cruzi, we have previously characterized the Universal Minicircle Sequence Binding Protein of this parasite (TcUMSBP), which was first called PDZ5 [E.R. Coelho, T.P. Urmenyi, J. Franco da Silveira, E. Rondinelli, R. Silva, Identification of PDZ5, a candidate universal minicircle sequence binding protein of Trypanosoma cruzi, Int. J. Parasitol. 33 (2003) 853-858]. In this work, we describe two highly polymorphic alleles of the TcUMSBP locus in the T. cruzi reference clone CL Brener and the differential expression pattern of these alleles. A 62 bp sequence in the TcUMSBP upstream intergenic region in one of its alleles affects the efficiency of polycistronic RNA processing and the polyadenylation sites, and therefore regulates the differential expression of TcUMSBP alleles of this locus.
Biochemical and Biophysical Research Communications 04/2006; 341(2):382-90. · 2.48 Impact Factor
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ABSTRACT: RAB proteins, which belong to the RAS superfamily, regulate exocytic and endocytic pathways of eukaryotic cells, controlling vesicle docking and fusion. Few RAB proteins have been identified in parasites. Molecular markers for cellular compartments are important to studies concerning about the protein traffic in Trypanosoma cruzi, the causal agent of Chagas disease. In this work, we describe the characterization of TcRABL4, the first RAB-like gene identified in T. cruzi (GenBank Accession No.: ), present as a single-copy gene. TcRABL4 contains all five consensus RAB motifs but lacks cysteine residues at the C terminus, which are essential to isoprenylation, an absolute prerequisite for membrane association of these proteins. TcRABL4 is a functional GTPase that is able to bind and hydrolyze GTP, and its gene is transcribed as a single 1.2 kb mRNA in epimastigotes. TcRABL4 appears to be differentially regulated in the three cell forms of the parasite, and the protein is not associated to membranes, unlike other RAB proteins. It is possible that TcRABL4 may be a member of a novel family of small GTPases.
Biochemical and Biophysical Research Communications 09/2005; 333(3):808-17. · 2.48 Impact Factor
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ABSTRACT: Heat shock protein (HSP) 10 is a member of the highly conserved group of molecular chaperons, which are necessary for efficient folding of many proteins in normal and stress conditions and have been implicated in several human diseases. We have characterized the HSP10 genes of Trypanosoma cruzi, the causative agent of Chagas' disease. After sequence analysis of clones obtained from the T. cruzi Genome Initiative, we show that the T. cruzi HSP10 coding region is 300 bp long, encoding a polypeptide of 100 amino acids with highest sequence identity (83%) to HSP10 of Trypanosoma brucei and lowest (28%) to HSP10 of Escherichia coli. The T. cruzi HSP10 genes are arranged in 3 tandemly repeated copies, which give rise to a major mRNA of 1.0 kb that remains unaltered during heat shock; a smaller mRNA species is induced at 37 degrees C by alternate polyadenylation. Finally, the presence of a conserved 5-amino acid residue deletion in trypanosomatid HSP10s led us to generate a molecular model of the T. cruzi HSP10 structure. The oligomeric assembly of this model shows some peculiar characteristics that may have functional significance.
Gene 05/2005; 349:135-42. · 2.34 Impact Factor
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ABSTRACT: RAB proteins are small GTPases involved in exocytic and endocytic pathways of eukaryotic cells, controlling vesicle docking and fusion. RABs show a remarkable specificity in subcellular localization, so they can be used as molecular markers for studying protein trafficking in Trypanosoma cruzi, the causal agent of Chagas' disease. RAB5 is a component of early endosomes. It has been identified in kinetoplastids such as Trypanosoma brucei and Leishmania donovani. In this work, we describe the characterization of the complete coding sequence of a RAB5 gene homologue in T. cruzi (TcRAB5, GenBank Accession No. AY730667). It is present as a single copy gene, located at chromosomal bands XIII and XIV. TcRAB5 shares the highest degrees of similarity (71%) and identity (63%) with Trypanosoma brucei rhodesiense RAB5a and contains all five characteristic RAB motifs. TcRAB5 is transcribed as a single 1.5kb mRNA in epimastigotes. Its transcript was also detected in the other two forms of the parasite, metacyclic trypomastigotes and spheromastigotes. The recombinant TcRAB5 protein was able to bind and hydrolyze GTP. The identification of proteins involved in T. cruzi endo- and exocytic pathways may generate cellular compartment markers, an invaluable tool to better understand the vesicular transport in this parasite.
Biochemical and Biophysical Research Communications 05/2005; 329(2):638-45. · 2.48 Impact Factor
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ABSTRACT: The identification and characterization of RNA binding proteins in Trypanosoma cruzi are particularly relevant as they play key roles in the regulatory mechanisms of gene expression. In this work, we have identified coding sequences for the proteins, named TcRRM1 and TcRRM2, in the EST database generated by the T. cruzi genomic initiative. TcRRM1 and TcRRM2 contain two RNA binding domains (RRM) and are very similar to two Trypanosoma brucei RNA binding proteins previously reported, Tbp34 and Tbp37, and to a not yet annotated ORF in Leishmania major genome project. The T. cruzi RRM genes are organized in tandem, alternating with copies of Tcp28, a gene of unknown function. However, TcRRM transcript accumulation is higher in the spheromastigote stage, while Tcp28 transcripts accumulate more in the trypomastigote stage suggesting developmental regulation.
Biochemical and Biophysical Research Communications 10/2004; 322(3):985-92. · 2.48 Impact Factor
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ABSTRACT: In mammalian cells, the Rab7 protein is a key element of late endocytic membrane traffic. Several results suggest that it is involved in the transport from early to late endosome or from late endosome to lysosome. We have previously characterized a Rab7 gene homologue (TcRAB7) in Trypanosoma cruzi. Now, using an affinity-purified antibody specific to TcRAB7 protein we have determined that it is localized at the Golgi apparatus of the parasite. Our results indicate that the T. cruzi Rab7 homologue may function in a different route than its counterparts in mammalian cells.
Biochemical and Biophysical Research Communications 09/2004; 321(2):397-402. · 2.48 Impact Factor
