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Sergio Verjovski-Almeida

Publications and source records attributed to Sergio Verjovski-Almeida.

At least 19 recordsLinked to original sources
PubMed2006

Characterization of Schistosoma mansoni ATPDase2 gene, a novel apyrase family member.

Schistosoma mansoni is a major causative agent of schistosomiasis, which constitutes a severe health problem in developing countries. We have previously described the SmATPDase1 gene, encoding a protein from the external surface of the parasites. In this work, we describe the cloning and characterization of SmATPDase2, a novel CD39-like ATP diphosphohydrolase gene in S. mansoni. In silico analysis of the protein encoded by SmATPDase2 predicts a single N-terminal transmembrane domain similar to that described for secreted human apyrase isoforms. Immuno-colocalization experiments detected both SmATPDase proteins at the S. mansoni adult worm tegument basal and apical membranes, but only SmATPDase2 in the tegument syncytium. SmATPDase2 but not SmATPDase1 protein was detected by Western blot in culture medium supernatants following incubation of adult worms in vitro, indicating that SmATPDase2 was secreted by the parasite to the medium. Taken together these data suggest a non-redundant role for SmATPDase2 in the parasite-host interplay.

Amino Acid Sequence↗
PubMed2006

SmTRC1, a novel Schistosoma mansoni DNA transposon, discloses new families of animal and fungi transposons belonging to the CACTA superfamily.

BACKGROUND: The CACTA (also called En/Spm) superfamily of DNA-only transposons contain the core sequence CACTA in their Terminal Inverted Repeats (TIRs) and so far have only been described in plants. Large transcriptome and genome sequence data have recently become publicly available for Schistosoma mansoni, a digenetic blood fluke that is a major causative agent of schistosomiasis in humans, and have provided a comprehensive repository for the discovery of novel genes and repetitive elements. Despite the extensive description of retroelements in S. mansoni, just a single DNA-only transposon belonging to the Merlin family has so far been reported in this organism. RESULTS: We describe a novel S. mansoni transposon named SmTRC1, for S. mansoni Transposon Related to CACTA 1, an element that shares several characteristics with plant CACTA transposons. Southern blotting indicates approximately 30-300 copies of SmTRC1 in the S. mansoni genome. Using genomic PCR followed by cloning and sequencing, we amplified and characterized a full-length and a truncated copy of this element. RT-PCR using S. mansoni mRNA followed by cloning and sequencing revealed several alternatively spliced transcripts of this transposon, resulting in distinct ORFs coding for different proteins. Interestingly, a survey of complete genomes from animals and fungi revealed several other novel TRC elements, indicating new families of DNA transposons belonging to the CACTA superfamily that have not previously been reported in these kingdoms. The first three bases in the S. mansoni TIR are CCC and they are identical to those in the TIRs of the insects Aedes aegypti and Tribolium castaneum, suggesting that animal TRCs may display a CCC core sequence. CONCLUSION: The DNA-only transposable element SmTRC1 from S. mansoni exhibits various characteristics, such as generation of multiple alternatively-spliced transcripts, the presence of terminal inverted repeats at the extremities of the elements flanked by direct repeats and the presence of a Transposase_21 domain, that suggest a distant relationship to CACTA transposons from Magnoliophyta. Several sequences from other Metazoa and Fungi code for proteins similar to those encoded by SmTRC1, suggesting that such elements have a common ancestry, and indicating inheritance through vertical transmission before separation of the Eumetazoa, Fungi and Plants.

Alternative Splicing↗
PubMed2006

Gender biased differential alternative splicing patterns of the transcriptional cofactor CA150 gene in Schistosoma mansoni.

The complex molecular systems involved in the process of sex-differentiation and fertility in Schistosoma mansoni have not yet been completely described. Using a 4608-element cDNA microarray, we have now determined 90 and 139 genes with significantly (q-value</=0.06) higher expression levels in adult males and females, respectively. Eight out of eleven (73%) selected transcripts had their differential expression levels validated by real-time RT-PCR. One of these transcripts was extended by RT-PCR and was shown to span the intronic region between exons 9 and 11 of the S. mansoni CA150 gene, a transcriptional cofactor known in humans to interact with both RNA polymerase II and the spliceosome complex. The longer transcript probably represents a novel isoform of S. mansoni CA150. Additionally, we obtained full-length sequences for three other isoforms of the SmCA150 gene, coding for proteins of different lengths and domain compositions. Semi-quantitative RT-PCR showed different expression ratios among these isoforms between male and female. Due to the role of CA150 in RNA transcription and processing, we hypothesize that these differential expression events may be important in the generation and maintenance of the different phenotypes between male and female.

Alternative Splicing↗
PubMed2006

Schistosoma mansoni TGF-beta receptor II: role in host ligand-induced regulation of a schistosome target gene.

Members of transforming growth factor-beta (TGF-beta) superfamily play pivotal roles in development in multicellular organisms. We report the functional characterization of the Schistosoma mansoni type II receptor (SmTbetaRII). Mining of the S. mansoni expressed sequence tag (EST) database identified an EST clone that shows homology to the kinase domain of type II receptors from different species. The amplified EST sequence was used as a probe to isolate a cDNA clone spanning the entire coding region of a type II serine/threonine kinase receptor. The interaction of SmTbetaRII with SmTbetaRI was elucidated and shown to be dependent on TGF-beta ligand binding. Furthermore, in the presence of human TGF-beta1, SmTbetaRII was able to activate SmTbetaRI, which in turn activated SmSmad2 and promoted its interaction with SmSmad4, proving the transfer of the signal from the receptor complex to the Smad proteins. Gynaecophoral canal protein (GCP), whose expression in male worms is limited to the gynaecophoric canal, was identified as a potential TGF-beta target gene in schistosomes. Knocking down the expression of SmTbetaRII using short interfering RNA molecules (siRNA) resulted in a concomitant reduction in the expression of GCP. These data provide evidence for the direct involvement of SmTbetaRII in mediating TGF-beta-induced activation of the TGF-beta target gene, SmGCP, within schistosome parasites. The results also provide additional evidence for a role for the TGF-beta signaling pathway in male-induced female reproductive development.

Activin Receptors, Type I↗
PubMed2006

Cloning the genes and DNA binding properties of High Mobility Group B1 (HMGB1) proteins from the human blood flukes Schistosoma mansoni and Schistosoma japonicum.

The parasitic helminth Schistosoma mansoni contains three HMGB proteins, HMGB1, HMGB2 and HMGB3, of primary amino acid sequences highly similar to vertebrate proteins. In this report we describe the characterization of the HMGB1 proteins and their genes from S. mansoni and Schistosoma japonicum. The deduced amino acid sequences of HMGB1 proteins from both schistosome species are identical, and comprise 176 residues. The proteins contain the two evolutionarily highly conserved HMG-box domains, A and B, exhibiting 60% similarity to mammalian HMGB1. Unlike the human HMGB1 which contains an unbroken run of 30 glutamic or aspartic residues, the SmHMGB1 or SjHMGB1 proteins possess unusually short acidic C-terminal tails (5 acidic residues interrupted by 2 serines). Southern hybridization and DNA sequencing revealed a single copy HMGB1 gene, composed of 3 exons and two introns, in S. mansoni. The exon/intron boundaries are identical to those of the human HMGB1 gene, with the exception that the second exon of the SmHMGB1 gene which is not split into two exons as in the human HMGB1 gene. RNA blot analysis revealed that the SmHMGB1 gene is constitutively expressed in similar levels both in male and female worms. The single-sized mRNA for SmHMGB1 is consistent with the size derived from the cDNA. Although DNA binding properties of SmHMGB1 (or SjHMGB1) protein seem to be similar to those previously reported with human HMGB1, i.e., preferential binding to supercoiled DNA over linear DNA, specific recognition of DNA four-way junctions, DNA-induced supercoiling in the presence of topoisomerase I, and DNA bending, we have observed two important differences relative to those observed with the human HMGB1: (i) the inability of the isolated SmHMGB1 domain A to bend DNA (as revealed by T4 ligase-mediated circularization assay), and (ii) higher DNA supercoiling and bending potential of the SmHMGB1 protein as compared to its human counterpart. The latter finding may indicate that the long acidic C-tail of human HMGB1 has much stronger repressive role on DNA bending or DNA supercoiling by topoisomerase I at physiological ionic strength than the short C-tail of the SmHMGB1 protein. Considering the important role of HMGB1 in DNA replication, transcription, recombination, and in particularly, the mediation of inflammation responses in mammalian cells, further studies on schistosome HMGB proteins may provide valuable information related to schistosomiasis, where inflammation plays a critical role in this disease.

Amino Acid Sequence↗
PubMed2006

Schistosomiasis--a century searching for chemotherapeutic drugs.

Schistosomiasis affects 200 million individuals in underdeveloped and developing regions and is a growing concern for travelers worldwide. There has been evidence of resistance to the praziquantel-based therapy and reports of acute-disease manifestation; therefore, other drugs affecting different stages of the schistosome parasites life cycle and alternative therapeutic regimens should be developed and become accessible. The present review results from a comprehensive search in the scientific literature for substances and compounds tested in the past centennial for schistosomiasis therapy. We gathered over 40 drugs providing information on therapeutic action in humans or animal model, toxicity, susceptible Schistosoma stages, species, etc. The drugs were grouped according to their known metabolic effects on the parasite, whether they are on membrane structure and function, carbohydrate metabolism, protein synthesis and function, or on nucleic acid metabolism. We discuss the current knowledge of drug-target interactions, their mechanism of action and possible therapy combinations. Furthermore, based in the literature and in our own experience with large-scale Schistosoma mansoni genome and transcriptome analyses, we put forward several recently described gene products that are promising target candidates for existing or new drugs.

Animals↗
PubMed2006

Evaluation of reference-based two-color methods for measurement of gene expression ratios using spotted cDNA microarrays.

BACKGROUND: Spotted cDNA microarrays generally employ co-hybridization of fluorescently-labeled RNA targets to produce gene expression ratios for subsequent analysis. Direct comparison of two RNA samples in the same microarray provides the highest level of accuracy; however, due to the number of combinatorial pair-wise comparisons, the direct method is impractical for studies including large number of individual samples (e.g., tumor classification studies). For such studies, indirect comparisons using a common reference standard have been the preferred method. Here we evaluated the precision and accuracy of reconstructed ratios from three indirect methods relative to ratios obtained from direct hybridizations, herein considered as the gold-standard. RESULTS: We performed hybridizations using a fixed amount of Cy3-labeled reference oligonucleotide (RefOligo) against distinct Cy5-labeled targets from prostate, breast and kidney tumor samples. Reconstructed ratios between all tissue pairs were derived from ratios between each tissue sample and RefOligo. Reconstructed ratios were compared to (i) ratios obtained in parallel from direct pair-wise hybridizations of tissue samples, and to (ii) reconstructed ratios derived from hybridization of each tissue against a reference RNA pool (RefPool). To evaluate the effect of the external references, reconstructed ratios were also calculated directly from intensity values of single-channel (One-Color) measurements derived from tissue sample data collected in the RefOligo experiments. We show that the average coefficient of variation of ratios between intra- and inter-slide replicates derived from RefOligo, RefPool and One-Color were similar and 2 to 4-fold higher than ratios obtained in direct hybridizations. Correlation coefficients calculated for all three tissue comparisons were also similar. In addition, the performance of all indirect methods in terms of their robustness to identify genes deemed as differentially expressed based on direct hybridizations, as well as false-positive and false-negative rates, were found to be comparable. CONCLUSION: RefOligo produces ratios as precise and accurate as ratios reconstructed from a RNA pool, thus representing a reliable alternative in reference-based hybridization experiments. In addition, One-Color measurements alone can reconstruct expression ratios without loss in precision or accuracy. We conclude that both methods are adequate options in large-scale projects where the amount of a common reference RNA pool is usually restrictive.

Adenocarcinoma↗
PubMed2006

The tegument surface membranes of the human blood parasite Schistosoma mansoni: a proteomic analysis after differential extraction.

The blood fluke Schistosoma mansoni can live for years in the hepatic portal system of its human host and so must possess very effective mechanisms of immune evasion. The key to understanding how these operate lies in defining the molecular organisation of the exposed parasite surface. The adult worm is covered by a syncytial tegument, bounded externally by a plasma membrane and overlain by a laminate secretion, the membranocalyx. In order to determine the protein composition of this surface, the membranes were detached using a freeze/thaw technique and enriched by sucrose density gradient centrifugation. The resulting preparation was sequentially extracted with three reagents of increasing solubilising power. The extracts were separated by 2-DE and their protein constituents were identified by MS/MS, yielding predominantly cytosolic, cytoskeletal and membrane-associated proteins, respectively. After extraction, the final pellet containing membrane-spanning proteins was processed by liquid chromatographic techniques before MS. Transporters for sugars, amino acids, ions and other solutes were found together with membrane enzymes and proteins concerned with membrane structure. The proteins identified were categorised by their function and putative location on the basis of their homology with annotated proteins in other organisms.

Animals↗
PubMed2005

Identification of 18 new transcribed retrotransposons in Schistosoma mansoni.

This work describes 18 new transcribed retrotransposons of the blood fluke Schistosoma mansoni. Among them, 9 were LTR, 8 non-LTR, and 1 Penelope-like element (PLE) retrotransposon. Sequences were generated by in silico reconstruction using S. mansoni ESTs and transcripts obtained by rapid amplification of cDNA ends, complemented in some cases by sequencing of genomic clones amplified by PCR. A novel element from the ancient R2/R4/CRE transposon group is described for the first time in S. mansoni. In addition, one non-LTR retrotransposon family displays long (40-450 bp) 3'-UTR with at least six different transcribed sequences among the copies, five LTR retrotransposons have abundantly transcribed incomplete copies lacking the sequence segment coding for the reverse transcriptase domain, and four non-LTR retrotransposons code for DNA-binding PHD domains that may give them a differential targeting. These results allow for a comprehensive description of the transcribed retrotransposon diversity of this complex human parasite.

Animals↗
PubMed2005

Large-scale transcriptome analyses reveal new genetic marker candidates of head, neck, and thyroid cancer.

A detailed genome mapping analysis of 213,636 expressed sequence tags (EST) derived from nontumor and tumor tissues of the oral cavity, larynx, pharynx, and thyroid was done. Transcripts matching known human genes were identified; potential new splice variants were flagged and subjected to manual curation, pointing to 788 putatively new alternative splicing isoforms, the majority (75%) being insertion events. A subset of 34 new splicing isoforms (5% of 788 events) was selected and 23 (68%) were confirmed by reverse transcription-PCR and DNA sequencing. Putative new genes were revealed, including six transcripts mapped to well-studied chromosomes such as 22, as well as transcripts that mapped to 253 intergenic regions. In addition, 2,251 noncoding intronic RNAs, eventually involved in transcriptional regulation, were found. A set of 250 candidate markers for loss of heterozygosis or gene amplification was selected by identifying transcripts that mapped to genomic regions previously known to be frequently amplified or deleted in head, neck, and thyroid tumors. Three of these markers were evaluated by quantitative reverse transcription-PCR in an independent set of individual samples. Along with detailed clinical data about tumor origin, the information reported here is now publicly available on a dedicated Web site as a resource for further biological investigation. This first in silico reconstruction of the head, neck, and thyroid transcriptomes points to a wealth of new candidate markers that can be used for future studies on the molecular basis of these tumors. Similar analysis is warranted for a number of other tumors for which large EST data sets are available.

Alternative Splicing↗
PubMed2005

Gene expression arrays in cancer research: methods and applications.

During the last 5 years, the number of papers describing data obtained by microarray technology increased exponentially with about 3000 papers in 2003. Undoubtedly, cancer is by far the disease that received most of the attention as far as the amount of data generated. As array technology is rather new and highly dependent on bioinformatics, mathematics and statistics, a clear understanding of the knowledge and information derived from array-based experiments is not widely appreciated. We shall review herein some of the issues related to the construction of DNA arrays, quantities and heterogeneity of probes and targets, the consequences of the physical characteristics of the probes, data extraction and data analysis as well as the applications of array technology. Our goal is to bring to the general audience, some of the basics of array technology and its possible application in oncology. By discussing some of the basic aspects of the methodology, we hope to stimulate criticism concerning the conclusions proposed by authors, especially in the light of the very low degree of reproducibility already proven when commercially available platforms were compared . Regardless of its pitfalls, it is unquestionable that array technology will have a great impact in the management of cancer and its applications will range from the discovery of new drug targets, new molecular tools for diagnosis and prognosis as well as for a tailored treatment that will take into account the molecular determinants of a given tumor. Hence, we shall also highlight some of the already available and promising applications of array technology on the day-to-day practice of oncology.

Cluster Analysis↗
PubMed2005

As antisense RNA gets intronic.

Recent work describing the transcriptional output of the human genome points to the existence of a significant number of non-coding RNA transcripts coming from intronic regions, with a fraction of these being oriented antisense relative to the protein-coding mRNA of the known gene. In this article, we survey the main findings of the large-scale expression analysis projects that led to the identification of antisense intronic messages and which demonstrate their ubiquitous expression in the human genome. We review the current knowledge on long, unspliced, intronic antisense transcripts, a new class of non-coding RNAs, recently described by our group to be correlated with the degree of tumor differentiation in prostate cancer, which we postulate is involved in the fine tuning of gene expression in eukaryotes. Possible mechanisms of antisense intronic transcript biogenesis and function in gene expression regulation are discussed, as is their involvement in human diseases. While there is still no conclusive evidence demonstrating a functional role for these long, intronic antisense messages, the far-reaching implications of their existence for the mechanisms regulating gene expression certainly warrant further experimentation.

Alternative Splicing↗
PubMed2005

Mapping transposon insertion sites by touchdown PCR and hybrid degenerate primers.

A novel mapping method based on touchdown PCR was developed for identifying a transposon insertion site in genomic DNA using a hybrid consensus-degenerate primer in combination with a specific primer that anneals to the transposon. The method was tested using Xanthomonas citri transposon mutants. PCR products contained adjacent DNA regions that belonged to both X. citri genomic DNA and the transposon. Products were directly sequenced from PCRs using only the specific primer. Different PCR conditions were tested, and the optimized reaction parameters that increased product yields and specificity are described. Best results were obtained with the HIB17 hybrid primer, which is a 25-mer oligonucleotide having degenerate bases at 6 different positions within the last 12 bases at the 3' end. An X. citri mutants library was produced by random transposition using the EZ::TN transposon, and we identified the insertion sites within the genome of 90 mutants. Insertions were found within both the chromosomal and the plasmid DNA in these X. citri mutants. Restriction mapping and Southern blot analysis confirmed the insertion sites for eight randomly chosen mutants. This method is a very useful tool for large-scale characterization of mutants in functional genomics studies.

Chromosome Mapping↗
PubMed2004

Antisense intronic non-coding RNA levels correlate to the degree of tumor differentiation in prostate cancer.

A large fraction of transcripts are expressed antisense to introns of known genes in the human genome. Here we show the construction and use of a cDNA microarray platform enriched in intronic transcripts to assess their biological relevance in pathological conditions. To validate the approach, prostate cancer was used as a model, and 27 patient tumor samples with Gleason scores ranging from 5 to 10 were analyzed. We find that a considerably higher fraction (6.6%, [23/346]) of intronic transcripts are significantly correlated (P< or =0.001) to the degree of prostate tumor differentiation (Gleason score) when compared to transcripts from unannotated genomic regions (1%, [6/539]) or from exons of known genes (2%, [27/1369]). Among the top twelve transcripts most correlated to tumor differentiation, six are antisense intronic messages as shown by orientation-specific RT-PCR or Northern blot analysis with strand-specific riboprobe. Orientation-specific real-time RT-PCR with six tumor samples, confirmed the correlation (P=0.024) between the low/high degrees of tumor differentiation and antisense intronic RASSF1 transcript levels. The need to use intron arrays to reveal the transcriptome profile of antisense intronic RNA in cancer has clearly emerged.

Cell Differentiation↗
PubMed2004

RASL11A, member of a novel small monomeric GTPase gene family, is down-regulated in prostate tumors.

We performed a genome-wide search for novel loci encoding for Ras-related proteins based on the genome mapping coordinates of the cancer-derived EST dataset at GenBank. Partial sequences from two novel human genes were identified and subsequently used for full length transcript cloning. RASL11A and ARL9 belong to two novel subfamilies coding for small GTPases that we found to be highly conserved among eukaryotes. The Arl9/Arl10 subfamily displays a conserved interswitch toggle that places it evolutionarily closer to the Arf family. Rasl11 proteins are more closely related to the Ras branch of GTPases. All orthologues newly identified here exhibit an Asn residue in place of the highly conserved Thr35 of the G domain, suggesting that the universal switch mechanism of small GTPases may be structurally different in this subfamily. We determined by Northern blot that RASL11A is transcribed in several human tissues and that it is down-regulated in prostate tumors as measured by quantitative real-time PCR. These results highlight a previously uncharacterized subfamily of Ras-related genes that may have a tumor suppressor role in prostate cancer.

ADP-Ribosylation Factors↗
PubMed2004

Characterization and immunolocalization of an NTP diphosphohydrolase of Trypanosoma cruzi.

An ecto-NTP diphosphohydrolase (NTPDase) activity, insensitive to inhibitors of ATPases and phosphatases, was characterized on the surface of live Trypanosoma cruzi intact parasites. The enzyme exhibits broad substrate specificity, typical of NTPDases, and a high hydrolysis rate for GTP. A 2282 bp message encoding a full-length NTPDase was cloned by RT-PCR using epimastigote mRNA. A single protein was immunoprecipitated from [(35)S]methionine-labeled parasites using antibodies against Toxoplasma gondii NTPase I. This antibody localized an NTPDase on the external surface of all forms of T. cruzi, as seen by confocal immuno-fluorescence microscopy. The NTPDase could be part of the parasite's purine salvage pathway. Additionally, trypomastigotes (infective form) presented a 2:1 ATP/ADP hydrolysis ratio, while epimastigotes (non-infective form) presented a 1:1 ratio, suggesting a possible role for the NTPDase in the parasite's virulence mechanisms.

Animals↗
PubMed2004

DNA microarray-based genome comparison of a pathogenic and a nonpathogenic strain of Xylella fastidiosa delineates genes important for bacterial virulence.

Xylella fastidiosa is a phytopathogenic bacterium that causes serious diseases in a wide range of economically important crops. Despite extensive comparative analyses of genome sequences of Xylella pathogenic strains from different plant hosts, nonpathogenic strains have not been studied. In this report, we show that X. fastidiosa strain J1a12, associated with citrus variegated chlorosis (CVC), is nonpathogenic when injected into citrus and tobacco plants. Furthermore, a DNA microarray-based comparison of J1a12 with 9a5c, a CVC strain that is highly pathogenic and had its genome completely sequenced, revealed that 14 coding sequences of strain 9a5c are absent or highly divergent in strain J1a12. Among them, we found an arginase and a fimbrial adhesin precursor of type III pilus, which were confirmed to be absent in the nonpathogenic strain by PCR and DNA sequencing. The absence of arginase can be correlated to the inability of J1a12 to multiply in host plants. This enzyme has been recently shown to act as a bacterial survival mechanism by down-regulating host nitric oxide production. The lack of the adhesin precursor gene is in accordance with the less aggregated phenotype observed for J1a12 cells growing in vitro. Thus, the absence of both genes can be associated with the failure of the J1a12 strain to establish and spread in citrus and tobacco plants. These results provide the first detailed comparison between a nonpathogenic strain and a pathogenic strain of X. fastidiosa, constituting an important step towards understanding the molecular basis of the disease.

Adhesins, Bacterial↗