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S Verjovski-Almeida

Publications and source records attributed to S Verjovski-Almeida.

At least 19 recordsLinked to original sources
PubMed2006

Auto-antibodies in prostate cancer: humoral immune response to antigenic determinants coded by the differentially expressed transcripts FLJ23438 and VAMP3.

BACKGROUND: Here we evaluate auto-antibody response against two potential antigenic determinants of genes highly expressed in low Gleason Score prostate cancer (PC) tumor samples, namely FLJ23438 and VAMP3. METHODS: RT-PCR assays were used to analyze mRNA expression profiles of FLJ23438 and VAMP3 transcripts. The auto-antibody response against FLJ23438 and VAMP3 recombinant proteins was tested by immunoblot assays using PC, benign prostate hyperplasia (BPH), healthy donors (HD), and other human cancers plasma samples. RESULTS: Our data showed that 37% (10/27) and 7.4% (2/27) of PC plasma samples presented auto-antibodies against FLJ23438 and VAMP3, respectively. Only 8.3% (1/12) of BPH plasma samples were reactive for both auto-antibodies, while none (0/12) of HD plasma samples tested were reactive. CONCLUSIONS: The prevalence of 37% of positive PC plasma samples for anti-FLJ23438 antibodies suggests that humoral immune response against this antigenic determinant could be a potential serum marker for this cancer.

Adenocarcinoma↗
PubMed2004

Genome features of Leptospira interrogans serovar Copenhageni.

We report novel features of the genome sequence of Leptospira interrogans serovar Copenhageni, a highly invasive spirochete. Leptospira species colonize a significant proportion of rodent populations worldwide and produce life-threatening infections in mammals. Genomic sequence analysis reveals the presence of a competent transport system with 13 families of genes encoding for major transporters including a three-member component efflux system compatible with the long-term survival of this organism. The leptospiral genome contains a broad array of genes encoding regulatory system, signal transduction and methyl-accepting chemotaxis proteins, reflecting the organism's ability to respond to diverse environmental stimuli. The identification of a complete set of genes encoding the enzymes for the cobalamin biosynthetic pathway and the novel coding genes related to lipopolysaccharide biosynthesis should bring new light to the study of Leptospira physiology. Genes related to toxins, lipoproteins and several surface-exposed proteins may facilitate a better understanding of the Leptospira pathogenesis and may serve as potential candidates for vaccine.

Animals↗
PubMed2004

Comparative genomics of two Leptospira interrogans serovars reveals novel insights into physiology and pathogenesis.

Leptospira species colonize a significant proportion of rodent populations worldwide and produce life-threatening infections in accidental hosts, including humans. Complete genome sequencing of Leptospira interrogans serovar Copenhageni and comparative analysis with the available Leptospira interrogans serovar Lai genome reveal that despite overall genetic similarity there are significant structural differences, including a large chromosomal inversion and extensive variation in the number and distribution of insertion sequence elements. Genome sequence analysis elucidates many of the novel aspects of leptospiral physiology relating to energy metabolism, oxygen tolerance, two-component signal transduction systems, and mechanisms of pathogenesis. A broad array of transcriptional regulation proteins and two new families of afimbrial adhesins which contribute to host tissue colonization in the early steps of infection were identified. Differences in genes involved in the biosynthesis of lipopolysaccharide O side chains between the Copenhageni and Lai serovars were identified, offering an important starting point for the elucidation of the organism's complex polysaccharide surface antigens. Differences in adhesins and in lipopolysaccharide might be associated with the adaptation of serovars Copenhageni and Lai to different animal hosts. Hundreds of genes encoding surface-exposed lipoproteins and transmembrane outer membrane proteins were identified as candidates for development of vaccines for the prevention of leptospirosis.

Animals↗
PubMed2001

The contribution of 700,000 ORF sequence tags to the definition of the human transcriptome.

Open reading frame expressed sequences tags (ORESTES) differ from conventional ESTs by providing sequence data from the central protein coding portion of transcripts. We generated a total of 696,745 ORESTES sequences from 24 human tissues and used a subset of the data that correspond to a set of 15,095 full-length mRNAs as a means of assessing the efficiency of the strategy and its potential contribution to the definition of the human transcriptome. We estimate that ORESTES sampled over 80% of all highly and moderately expressed, and between 40% and 50% of rarely expressed, human genes. In our most thoroughly sequenced tissue, the breast, the 130,000 ORESTES generated are derived from transcripts from an estimated 70% of all genes expressed in that tissue, with an equally efficient representation of both highly and poorly expressed genes. In this respect, we find that the capacity of the ORESTES strategy both for gene discovery and shotgun transcript sequence generation significantly exceeds that of conventional ESTs. The distribution of ORESTES is such that many human transcripts are now represented by a scaffold of partial sequences distributed along the length of each gene product. The experimental joining of the scaffold components, by reverse transcription-PCR, represents a direct route to transcript finishing that may represent a useful alternative to full-length cDNA cloning.

Expressed Sequence Tags↗
PubMed2001

Directed gap closure in large-scale sequencing projects.

A problem in many sequencing projects is the final closure of gaps left in the clone libraries, which serve as templates for sequencing, because of uncloned or unclonable genomic areas. By use of the Xylella fastidiosa genome as a test system, we present here an approach to generate, in a directed manner, sequence information from those gaps. We suggest using the complete clone library as a competitor against the genomic DNA of interest in a subtractive hybridization procedure similar to representational difference analysis (RDA). The resulting sequence information can be used to screen selectively other clone resources or serve directly for gap closure.

DNA, Bacterial↗
PubMed2000

Identification of human chromosome 22 transcribed sequences with ORF expressed sequence tags.

Transcribed sequences in the human genome can be identified with confidence only by alignment with sequences derived from cDNAs synthesized from naturally occurring mRNAs. We constructed a set of 250,000 cDNAs that represent partial expressed gene sequences and that are biased toward the central coding regions of the resulting transcripts. They are termed ORF expressed sequence tags (ORESTES). The 250,000 ORESTES were assembled into 81,429 contigs. Of these, 1, 181 (1.45%) were found to match sequences in chromosome 22 with at least one ORESTES contig for 162 (65.6%) of the 247 known genes, for 67 (44.6%) of the 150 related genes, and for 45 of the 148 (30.4%) EST-predicted genes on this chromosome. Using a set of stringent criteria to validate our sequences, we identified a further 219 previously unannotated transcribed sequences on chromosome 22. Of these, 171 were in fact also defined by EST or full length cDNA sequences available in GenBank but not utilized in the initial annotation of the first human chromosome sequence. Thus despite representing less than 15% of all expressed human sequences in the public databases at the time of the present analysis, ORESTES sequences defined 48 transcribed sequences on chromosome 22 not defined by other sequences. All of the transcribed sequences defined by ORESTES coincided with DNA regions predicted as encoding exons by genscan. (http://genes.mit.edu/GENSCAN.html).

Chromosomes, Human, Pair 22↗
PubMed2000

Requirement of the hinge domain for dimerization of Ca2+-ATPase large cytoplasmic portion expressed in bacteria.

The large cytoplasmic domain of rabbit sarcoplasmic reticulum Ca2+-ATPase was overexpressed in Escherichia coli as a 48 kDa fusion protein, designated p48, containing an N-terminal hexa-His tag. Purification conditions were optimized, thus conferring long-term stability to p48. Circular dichroism spectroscopy and the pattern of limited trypsinolysis confirmed the proper folding of the domain. p48 retained 0.5 +/- 0.1 mol of high affinity 2',3'-O-(2,4,6-trinitrophenyl)adenosine-5'-triphosphate (TNP-ATP) binding sites per mol of polypeptide chain with an apparent dissociation constant of about 8 microM. Size-exclusion FPLC using protein concentrations in the range 0.03 5 mg/ml showed that p48 was essentially monodisperse with apparent molecular mass and Stokes radius (Rs) values compatible with a dimer (100 kDa and 40 A, respectively). Analysis of p48 by small-angle X-ray scattering provided an independent second proof for a dimeric p48 particle with a radius of gyration (Rg) of 39 A, suggesting that the dimer was not spherical (Rs/Rg = 1.026). When digested by proteinase K, p48 was converted to a 30 kDa fragment, designated p30, which was very resistant to further proteolysis. p30 retained high affinity TNP-ATP binding (Kd = 8 microM) and eluted as a monomer (35 kDa) in size-exclusion FPLC. As opposed to p48, the p30 fragment did not react with monoclonal antibody A52 [Clarke et al., J. Biol. Chem. 264 (1989) 11246-11251] which recognizes region E657-R672 located upstream of the hinge domain of the Ca2+-ATPase. These results indicate a requirement of the hinge domain (670-728) region for self-association of the p48 large hydrophilic domain as a dimer. We propose that this behavior points to a possible role of the hinge domain in dimerization of sarcoplasmic reticulum Ca2+-ATPase in the native membrane.

Animals↗
PubMed2000

Shotgun sequencing of the human transcriptome with ORF expressed sequence tags.

Theoretical considerations predict that amplification of expressed gene transcripts by reverse transcription-PCR using arbitrarily chosen primers will result in the preferential amplification of the central portion of the transcript. Systematic, high-throughput sequencing of such products would result in an expressed sequence tag (EST) database consisting of central, generally coding regions of expressed genes. Such a database would add significant value to existing public EST databases, which consist mostly of sequences derived from the extremities of cDNAs, and facilitate the construction of contigs of transcript sequences. We tested our predictions, creating a database of 10,000 sequences from human breast tumors. The data confirmed the central distribution of the sequences, the significant normalization of the sequence population, the frequent extension of contigs composed of existing human ESTs, and the identification of a series of potentially important homologues of known genes. This approach should make a significant contribution to the early identification of important human genes, the deciphering of the draft human genome sequence currently being compiled, and the shotgun sequencing of the human transcriptome.

Animals↗
PubMed1999

N-terminal chimeric constructs improve the expression of sarcoplasmic reticulum Ca(2+)-ATPase in yeast.

Wild-type and chimeric constructs comprising rabbit sarcoplasmic reticulum (SR) Ca(2+)-ATPase and the N-terminal cytoplasmic portion of yeast plasma membrane H(+)-ATPase were expressed in yeast under control of a heat-shock regulated promoter. The wild-type ATPase was found predominantly in endoplasmic reticulum (ER) membranes. Addition of the first 88 residues of H(+)-ATPase to the Ca(2+)-ATPase N-terminal end promoted a marked shift in the localization of chimeric H(+)/Ca(2+)-ATPase which accumulated in a light membrane fraction associated with yeast smooth ER. Furthermore, there was a three-fold increase in the overall level of expression of chimeric H(+)/Ca(2+)-ATPase. Similar results were obtained for a chimeric Ca(2+)-ATPase containing a hexahistidine sequence added to its N-terminal end. Both H(+)/Ca(2+)-ATPase and 6xHis-Ca(2+)-ATPase were functional as demonstrated by their ability to form a phosphorylated intermediate and undergo fast turnover. Conversely, a replacement chimera in which the N-terminal end of SR Ca(2+)-ATPase was replaced by the corresponding segment of H(+)-ATPase was not stably expressed in yeast membranes. These results indicate that the N-terminal segment of Ca(2+)-ATPase plays an important role in enzyme assembly and contains structural determinants necessary for ER retention of the ATPase.

Animals↗
PubMed1998

Substitutions of aspartate 378 in the phosphorylation domain of the yeast PMA1 H+-ATPase disrupt protein folding and biogenesis.

There is strong evidence that Asp-378 of the yeast PMA1 ATPase plays an essential role in ATP hydrolysis by forming a covalent beta-aspartyl phosphate reaction intermediate. In this study, Asp-378 was replaced by Asn, Ser, and Glu, and the mutant ATPases were expressed in a temperature-sensitive secretion-deficient strain (sec6-4) that allowed their properties to be examined. Although all three mutant proteins were produced at nearly normal levels and remained stable for at least 2 h at 37 degrees C, they failed to travel to the vesicles that serve as immediate precursors of the plasma membrane; instead, they became arrested at an earlier step of the secretory pathway. A closer look at the mutant proteins revealed that they were firmly inserted into the bilayer and were not released by washing with high salt, urea, or sodium carbonate (pH 11), treatments commonly used to strip nonintegral proteins from membranes. However, all three mutant ATPases were extremely sensitive to digestion by trypsin, pointing to a marked abnormality in protein folding. Furthermore, in contrast to the wild-type enzyme, the mutant ATPases could not be protected against trypsinolysis by ligands such as MgATP, MgADP, or inorganic orthovanadate. Thus, Asp-378 functions in an unexpectedly complex way during the acquisition of a mature structure by the yeast PMA1 ATPase.

Amino Acid Substitution↗
PubMed1998

Divalent cation dependence and inhibition of Schistosoma mansoni ATP diphosphohydrolase by fluorosulfonylbenzoyl adenosine.

Hydrolysis of ATP or ADP catalyzed by the ATP diphosphohydrolase of Schistosoma mansoni tegument was measured in the presence of different cations. ATP diphosphohydrolase was stimulated by micromolar concentrations of either Ca2+ or Mg2+, Ca2+ producing threefold higher maximal activities than Mg2+. Kinetic studies indicated that Ca2+ and Mg2+ compete for the same binding site on the enzyme. The effect of covalent labeling of ATP diphosphohydrolase with the ATP analog fluorosulfonylbenzoyl adenosine (FSO2BzAdo) was studied. Schistosome tegument was passed through with Sephadex G-50 filtration centrifugation columns to remove endogenous nucleotides, and this was followed by labeling with FSO2BzAdo. Incubation of ATP diphosphohydrolase with 1 mM FSO2BzAdo for 1 h inhibited ATPase or ADPase activities by 60% and 50%, respectively. Addition of ATP together with FSO2BzAdo provided greater than 90% protection against FSO2BzAdo inactivation, indicating that FSO2BzAdo binds to an ATP-binding site on the ATP diphosphohydrolase. Furthermore, addition of FSO2BzAdo to a medium containing intact worms caused 30% and 50% inhibition of ATPase and ADPase activities, respectively, indicating that the ATP-binding site of diphosphohydrolase is accessible to FSO2BzAdo from the external surface of S. mansoni worms.

Adenosine↗
PubMed1996

Partial purification and immunohistochemical localization of ATP diphosphohydrolase from Schistosoma mansoni. Immunological cross-reactivities with potato apyrase and Toxoplasma gondii nucleoside triphosphate hydrolase.

ATP diphosphohydrolase from tegumental membranes of Schistosoma mansoni was solubilized with Triton X-100 plus deoxycholate and separated by preparative nondenaturing polyacrylamide gel electrophoresis. Two isoforms with ATP-hydrolytic activity were identified and excised from nondenaturing gels. For each of the active bands, two protein bands (63 and 55 kDa) were detected with Coomassie Blue staining, following sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Western blots developed with polyclonal anti-potato apyrase antibody revealed a single protein of 63 kDa, either with samples excised from active bands or with total S. mansoni tegument. Anti-potato apyrase antibody immobilized on Sepharose-Protein A depleted over 95% of ATPase and ADPase activities from detergent-solubilized tegument. Confocal laser scanning microscopy showed anti-potato apyrase antibody on the outer surface of S. mansoni tegument. A different antibody against a fusion protein derived from recently cloned Toxoplasma gondii nucleoside triphosphate hydrolase (Bermudes, D., Peck, K. R., Afifi, M. A., Beckers, C. J. M., and Joiner, K. A. (1994) J. Biol. Chem. 269, 29252-29260) revealed the same 63-kDa band in Western blots of S. mansoni tegument. Since anti-potato apyrase antibodies exhibited cross-reactivity with S. mansoni ATP diphosphohydrolase, we decided to gain further information on the primary structure of potato apyrase by sequencing the protein. Three novel peptides were obtained: amino-terminal sequence and two internal sequences from tryptic fragments. Eight sequences recently deposited in the data bank, including that of T. gondii nucleoside triphosphate hydrolase, have considerable homologies to potato apyrase suggesting a new family of nucleoside triphosphatases which contains a conserved motif (I/V)(V/M/I)(I/L/F/C)DAGS(S/T) near the amino-terminal. Antibody cross-reactivities in the present work suggest that conserved epitopes from S. mansoni ATP diphosphohydrolase are present in this family of nucleotide-splitting enzymes.

Acid Anhydride Hydrolases↗
PubMed1996

Heterologous expression of sarcoplasmic reticulum Ca(2+)-ATPase.

In recent years, expression of rabbit sarcoplasmic reticulum (SR) Ca(2+)-ATPase in heterologous systems has been a widely used strategy to study altered enzymes generated by site-directed mutagenesis. Various eukaryotic expression systems have been tested, all of them yielding comparable amounts of recombinant protein. However, the relatively low yield of recombinant protein obtained so far suggests that novel purification techniques will be required to allow further characterization of this enzyme based on direct ligand-binding measurements.

Animals↗
PubMed1993

Elimination of the hydroxyl groups in the ribose ring of ATP reduces its ability to phosphorylate the sarcoplasmic reticulum Ca(2+)-ATPase.

2'-Deoxyadenosine 5'-triphosphate, 3'-deoxyadenosine 5'-triphosphate, and 3'-amino-3'-deoxyadenosine 5'-triphosphate were substituted for ATP in the Ca2+ pumping cycle of the sarcoplasmic reticulum Ca(2+)-ATPase. The rate of phosphorylation of the enzyme decreased by more than an order of magnitude when either of the hydroxyl groups was eliminated from the ribose ring. This resulted in low rates of hydrolysis and low levels of phosphoenzyme intermediate. In addition, the Km(1) of hydrolysis and the K1/2 of phosphorylation of the derivatives modified in the 3' position were decreased by a factor of 5-10. Otherwise, the 3'-amino-3'-deoxyadenosine 5'-triphosphate was utilized in a manner equivalent to ATP. Because the observed rates of phosphoenzyme formation with the deoxynucleotides were lowered to the extent that they would be rate-limiting in the enzyme cycle, and the level of phosphoenzyme intermediate remained low when the enzyme was back-inhibited by high Ca2+ concentrations, it was concluded that the majority of the enzyme remained in a preliminary conformation, in which the phosphorylation reaction could not proceed although substrate and Ca2+ were bound. It was then proposed that, following Ca(2+)-induced changes in conformation, the hydroxyl groups are able to form hydrogen bonds with pertinent segments of the phosphorylation domain, helping to stabilize an enzyme-substrate complex, one function of which may be to provide the proper stereochemistry for phosphate transfer.

Adenosine Triphosphate↗
PubMed1993

Characterization and localization of an ATP-diphosphohydrolase on the external surface of the tegument of Schistosoma mansoni.

An ATP-diphosphohydrolase (EC 3.6.1.5) was identified in the tegumental fraction isolated from Schistosoma mansoni worms. Both ATP and ADP were hydrolyzed to AMP at similar rates by the enzyme. Other nucleotides were also degraded by the tegument enzyme, revealing a broad substrate specificity. Electrophoretic separation of tegumental proteins under non-denaturing conditions followed by addition of ATP or ADP as substrate revealed a single band of activity with similar mobility. In addition, similar heat-inactivation profiles were obtained for ATPase or ADPase activities, indicating that a single enzyme is responsible for degrading both nucleotides. The enzyme was not inhibited by vanadate, levamisole, tetramisole, ouabain or sodium azide. The ADPase activity was not affected by adenosine (5')-pentaphospho-(5')-adenosine (Ap5A) or by an excess of glucose and hexokinase used as an ATP-trapping system, thus excluding the presence of any significant adenylate kinase activity. The ATP-diphosphohydrolase displayed micromolar affinities for both Mg2+ and Ca2+, and the calcium-activated enzyme was inhibited by millimolar Mg2+. In intact live worms a calcium phosphate precipitate was formed on the outer tegumental surface upon incubation of the worms with either ATP or ADP, indicating the ectolocalization of this enzyme. In addition, ultrastructural histochemical localization of the enzyme was obtained. A distinct deposition of lead phosphate granules on the outer surface of the tegument was observed by electron microscopy, in the presence of either ATP or ADP as substrate. It is suggested that the ATP-diphosphohydrolase could regulate the concentration of purine nucleotides around the parasites and hence enable them to escape the host hemostasis by preventing ADP-induced platelet activation.

Adenosine Diphosphate↗
PubMed1992

Dissociation of F-actin induced by hydrostatic pressure.

F-actin purified from rabbit skeletal muscle undergoes reversible dissociation when subjected to hydrostatic pressures up to 240 MPa. Dissociation and reversibility were detected by the following procedures: fluorescence spectral changes observed under pressure, when either intrinsic tryptophan or pyrenyl emission of N-(1-pyrenyl)iodoacetamide-labeled actin were monitored; electron microscopy of samples fixed under pressure; size-exclusion HPLC of pressurized actin. The effect of pressure upon F-actin that had been polymerized in the presence of either Mg2+, Ca2+ or K+ was studied. The standard volume changes for the association of actin subunits, calculated from pressure/dissociation curves were 74 +/- 14 ml/mol for Mg-F-actin, 79 +/- 12 ml/mol for Ca-F-actin and 328 +/- 63 ml/mol for K-F-actin, indicating that actin subunits are packed differently in the polymer depending on which cation is present. All pressure/dissociation data could be fitted by a model for dissociation of a dimer, which suggests that in the F-actin filament there is a predominant intersubunit interaction interface, most likely the head-to-tail intrastrand interaction between two subunits which repeats itself along the polymer. A tenfold change in total protein concentration from 20 micrograms to 200 micrograms/ml Mg-F-actin did not cause a change in the pressure required for half-maximal dissociation. This indicates a heterogeneity of free energy of association among actin monomers in the Mg-F-actin polymer, suggesting that, in addition to the predominant intersubunit interaction, the disordered interactions in the filament significantly contribute to the heterogeneity of microenvironments in the interface between the subunits.

Actins↗
PubMed1989

Fluorescence decay of sarcoplasmic reticulum ATPase. Ligand binding and hydration effects.

Multifrequency phase-modulation lifetime data were acquired for sarcoplasmic reticulum Ca2+-ATPase. The intrinsic tryptophan fluorescence decay was complex and was fitted either with three exponentials or with bimodal Lorentzian distributions of lifetimes. Ca2+ binding to the high affinity sites in the ATPase produced an increase of 11% in the center of the main component of the bimodal distribution, shifting the lifetime from 4.04 to 4.50 ns. The effects of solvent on the ATPase were studied with the enzyme dissolved in reverse micelles of detergent bis-(2-ethylhexyl)sulfosuccinate in hexane. Increasing amounts of water up to a water/bis-(2-ethylhexyl)sulfosuccinate molar ratio of 4 produced marked changes in the fluorescence emission of the protein. Comparison of data obtained for micellar solutions of tryptophan or ATPase indicated that the tryptophan residues in the protein are protected from exposure to water. Correlation of water effects on emission intensity and lifetimes suggested that interaction with solvent may result in structural changes that cause a mixture of dynamic and static quenching of ATPase intrinsic fluorescence. Evidence for an effect of hydration on the structure of the active site was obtained by measurements of the fluorescence properties of fluorescein isothiocianate-labeled ATPase in reverse micelles.

Adenosine Triphosphatases↗