Publications by authors named "Aline M da Silva"

29 Publications

  • Page 1 of 1

Proteomic and Metabolomic Analyses of OMV-Enriched Fractions Reveal Association with Virulence Factors and Signaling Molecules of the DSF Family.

Phytopathology 2019 Aug 5;109(8):1344-1353. Epub 2019 Jul 5.

1Departamento de Bioquímica, Instituto de Química, Universidade de São Paulo, São Paulo, SP 05508-000, Brazil.

releases outer membrane vesicles (OMVs) known to play a role in the systemic dissemination of this pathogen. OMVs inhibit bacterial attachment to xylem wall and traffic lipases/esterases that act on the degradation of plant cell wall. Here, we extended the characterization of OMVs by identifying proteins and metabolites potentially associated with OMVs produced by Temecula1, a Pierce's disease strain, and by 9a5c and Fb7, two citrus variegated chlorosis strains. These results strengthen that one of the OMVs multiple functions is to carry determinants of virulence, such as lipases/esterases, adhesins, proteases, porins, and a pectin lyase-like protein. For the first time, we show that the two citrus variegated chlorosis strains produce diffusible signaling factor 2 (DSF2) and citrus variegated chlorosis-DSF (likewise, Temecula1) and most importantly, that these compounds of the DSF ( DSF) family are associated with OMV-enriched fractions. Altogether, our findings widen the potential functions of OMVs in intercellular signaling and host-pathogen interactions.
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http://dx.doi.org/10.1094/PHYTO-03-19-0083-RDOI Listing
August 2019

A ligand motif enables differential vascular targeting of endothelial junctions between brain and retina.

Proc Natl Acad Sci U S A 2019 02 22;116(6):2300-2305. Epub 2019 Jan 22.

Department of Biochemistry, Institute of Chemistry, University of São Paulo, São Paulo, SP 05508-000, Brazil;

Endothelial heterogeneity has important implications in health and disease. Molecular markers selectively expressed in the vasculature of different organs and tissues are currently being explored in targeted therapies with promising results in preclinical and clinical studies. Noteworthy is the role that combinatorial approaches such as phage display have had in identifying such markers by using phage as nanoparticles and surrogates for billions of different peptides, screening noninvasively the vascular lumen for binding sites. Here, we show that a new peptide motif that emerged from such combinatorial screening of the vasculature binds selectively to blood vessels in the brain in vivo but not to vessels in other organs. Peptides containing a conserved motif in which amino acids Phenylalanine-Arginine-Tryptophan (FRW) predominate could be visualized by transmission electron microscopy bound to the junctions between endothelial cells in all areas of the brain, including the optic nerve, but not in other barrier-containing tissues, such as intestines and testis. Remarkably, peptides containing the motif do not bind to vessels in the retina, implying an important molecular difference between these two vascular barriers. Furthermore, the peptide allows for in vivo imaging, demonstrating that new tools for studying and imaging the brain are likely to emerge from this motif.
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http://dx.doi.org/10.1073/pnas.1809483116DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6369793PMC
February 2019

MARVEL, a Tool for Prediction of Bacteriophage Sequences in Metagenomic Bins.

Front Genet 2018 7;9:304. Epub 2018 Aug 7.

Departamento de Bioquímica, Instituto de Química, Universidade de São Paulo, São Paulo, Brazil.

Here we present MARVEL, a tool for prediction of double-stranded DNA bacteriophage sequences in metagenomic bins. MARVEL uses a random forest machine learning approach. We trained the program on a dataset with 1,247 phage and 1,029 bacterial genomes, and tested it on a dataset with 335 bacterial and 177 phage genomes. We show that three simple genomic features extracted from contig sequences were sufficient to achieve a good performance in separating bacterial from phage sequences: gene density, strand shifts, and fraction of significant hits to a viral protein database. We compared the performance of MARVEL to that of VirSorter and VirFinder, two popular programs for predicting viral sequences. Our results show that all three programs have comparable specificity, but MARVEL achieves much better performance on the recall (sensitivity) measure. This means that MARVEL should be able to identify many more phage sequences in metagenomic bins than heretofore has been possible. In a simple test with real data, containing mostly bacterial sequences, MARVEL classified 58 out of 209 bins as phage genomes; other evidence suggests that 57 of these 58 bins are novel phage sequences. MARVEL is freely available at https://github.com/LaboratorioBioinformatica/MARVEL.
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http://dx.doi.org/10.3389/fgene.2018.00304DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6090037PMC
August 2018

A Tropical Composting Operation Unit at São Paulo Zoo as a Source of Bacterial Proteolytic Enzymes.

Appl Biochem Biotechnol 2019 Jan 23;187(1):282-297. Epub 2018 Jun 23.

Department of Biophysics, Escola Paulista de Medicina, Universidade Federal de São Paulo, Rua Três de Maio 100, São Paulo, SP, 04044-20, Brazil.

Composting operation systems are valuable sources of microorganisms and enzymes. This work reports the assessment of proteolytic enzymes from cultivable bacteria isolated from a composting facility of the São Paulo Zoo Park (SPZPF), São Paulo, Brazil. Three hundred bacterial isolates were obtained and identified based on 16S rRNA gene as belonging to 13 different genera. The most common genus among the isolates was Bacillus (67%); some of which show high proteolytic activity in their culture media. Biochemical assays of hydrolytic activities using FRET peptides as substrates allowed the characterization of a repertoire of serine proteases and metalloproteases with different molecular weights secreted by Bacillus strains isolated from composting. Furthermore, thermostable serine and metalloproteases were detected in the composting leachate, which might be of interest for industrial applications.
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http://dx.doi.org/10.1007/s12010-018-2810-7DOI Listing
January 2019

Genome-Centric Analysis of a Thermophilic and Cellulolytic Bacterial Consortium Derived from Composting.

Front Microbiol 2017 19;8:644. Epub 2017 Apr 19.

Departamento de Bioquímica, Instituto de Química, Universidade de São PauloSão Paulo, Brazil.

Microbial consortia selected from complex lignocellulolytic microbial communities are promising alternatives to deconstruct plant waste, since synergistic action of different enzymes is required for full degradation of plant biomass in biorefining applications. Culture enrichment also facilitates the study of interactions among consortium members, and can be a good source of novel microbial species. Here, we used a sample from a plant waste composting operation in the São Paulo Zoo (Brazil) as inoculum to obtain a thermophilic aerobic consortium enriched through multiple passages at 60°C in carboxymethylcellulose as sole carbon source. The microbial community composition of this consortium was investigated by shotgun metagenomics and genome-centric analysis. Six near-complete (over 90%) genomes were reconstructed. Similarity and phylogenetic analyses show that four of these six genomes are novel, with the following hypothesized identifications: a new species; the first genome (for which currently only 16S sequences are available) or else the first representative of a new family in the Bacillales order; the first representative of a new genus in the Paenibacillaceae family; and the first representative of a new deep-branching family in the Clostridia class. The reconstructed genomes from known species were identified as and . The metabolic potential of these recovered genomes based on COG and CAZy analyses show that these genomes encode several glycoside hydrolases (GHs) as well as other genes related to lignocellulose breakdown. The new species stands out for being the richest in diversity and abundance of GHs, possessing the greatest potential for biomass degradation among the six recovered genomes. We also investigated the presence and activity of the organisms corresponding to these genomes in the composting operation from which the consortium was built, using compost metagenome and metatranscriptome datasets generated in a previous study. We obtained strong evidence that five of the six recovered genomes are indeed present and active in that composting process. We have thus discovered three (perhaps four) new thermophillic bacterial species that add to the increasing repertoire of known lignocellulose degraders, whose biotechnological potential can now be investigated in further studies.
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http://dx.doi.org/10.3389/fmicb.2017.00644DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5395642PMC
April 2017

Xylella fastidiosa outer membrane vesicles modulate plant colonization by blocking attachment to surfaces.

Proc Natl Acad Sci U S A 2014 Sep 2;111(37):E3910-8. Epub 2014 Sep 2.

Department of Plant and Microbial Biology, University of California, Berkeley, CA 94720; and

Outer membrane vesicles (OMVs) of Gram-negative bacteria have been studied intensively in recent years, primarily in their role in delivering virulence factors and antigens during pathogenesis. However, the near ubiquity of their production suggests that they may play other roles, such as responding to envelope stress or trafficking various cargoes to prevent dilution or degradation by other bacterial species. Here we show that OMVs produced by Xylella fastidiosa, a xylem-colonizing plant pathogenic bacterium, block its interaction with various surfaces such as the walls of xylem vessels in host plants. The release of OMVs was suppressed by the diffusible signal factor-dependent quorum-sensing system, and a X. fastidiosa ΔrpfF mutant in which quorum signaling was disrupted was both much more virulent to plants and less adhesive to glass and plant surfaces than the WT strain. The higher virulence of the ΔrpfF mutant was associated with fivefold higher numbers of OMVs recovered from xylem sap of infected plants. The frequency of attachment of X. fastidiosa to xylem vessels was 20-fold lower in the presence of OMVs than in their absence. OMV production thus is a strategy used by X. fastidiosa cells to adjust attachment to surfaces in its transition from adhesive cells capable of insect transmission to an "exploratory" lifestyle for systemic spread within the plant host which would be hindered by attachment. OMV production may contribute to the movement of other bacteria in porous environments by similarly reducing their contact with environmental constituents.
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http://dx.doi.org/10.1073/pnas.1414944111DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4169949PMC
September 2014

Phenotype overlap in Xylella fastidiosa is controlled by the cyclic di-GMP phosphodiesterase Eal in response to antibiotic exposure and diffusible signal factor-mediated cell-cell signaling.

Appl Environ Microbiol 2013 Jun 29;79(11):3444-54. Epub 2013 Mar 29.

Department of Plant and Microbial Biology, University of California, Berkeley, Berkeley, CA, USA.

Eal is an EAL domain protein in Xylella fastidiosa homologous to one involved in resistance to tobramycin in Pseudomonas aeruginosa. EAL and HD-GYP domain proteins are implicated in the hydrolysis of the secondary messenger bis-(3'-5')-cyclic dimeric GMP (cyclic di-GMP). Cell density-dependent communication mediated by a Diffusible Signal Factor (DSF) also modulates cyclic di-GMP levels in X. fastidiosa, thereby controlling the expression of virulence genes and genes involved in insect transmission. The possible linkage of Eal to both extrinsic factors such as antibiotics and intrinsic factors such as quorum sensing, and whether both affect virulence, was thus addressed. Expression of eal was induced by subinhibitory concentrations of tobramycin, and an eal deletion mutant was more susceptible to this antibiotic than the wild-type strain and exhibited phenotypes similar to those of an rpfF deletion mutant blocked in DSF production, such as hypermotility, reduced biofilm formation, and hypervirulence to grape. Consistent with that, the rpfF mutant was more susceptible than the wild-type strain to tobramycin. Therefore, we propose that cell-cell communication and antibiotic stress can apparently lead to similar modulations of cyclic di-GMP in X. fastidiosa, resulting in similar phenotypes. However, the effect of cell density is dominant compared to that of antibiotic stress, since eal is suppressed by RpfF, which may prevent inappropriate behavioral changes in response to antibiotic stress when DSF accumulates.
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http://dx.doi.org/10.1128/AEM.03834-12DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3648042PMC
June 2013

Melatonin triggers PKA activation in the rodent malaria parasite Plasmodium chabaudi.

J Pineal Res 2011 Jan 22;50(1):64-70. Epub 2010 Oct 22.

Departamento de Biociências, Universidade Federal de São Paulo, Santos, SP, Brasil.

Calcium (Ca(2+) ) is a critical regulator of many aspects of the Plasmodium reproductive cycle. In particular, intra-erythrocyte Plasmodium parasites respond to circulating levels of the melatonin in a process mediated partly by intracellular Ca(2+) . Melatonin promotes the development and synchronicity of parasites, thereby enhancing their spread and worsening the clinical implications. The signalling mechanisms underlying the effects of melatonin are not fully established, although both Ca(2+) and cyclic AMP (cAMP) have been implicated. Furthermore, it is not clear whether different strains of Plasmodium use the same, or divergent, signals to control their development. The aim of this study was to explore the signalling mechanisms engaged by melatonin in P. chabaudi, a virulent rodent parasite. Using parasites at the throphozoite stage acutely isolated from mice erythrocytes, we demonstrate that melatonin triggers cAMP production and protein kinase A (PKA) activation. Interestingly, the stimulation of cAMP/PKA signalling by melatonin was dependent on elevation of Ca(2+) within the parasite, because buffering Ca(2+) changes using the chelator BAPTA prevented cAMP production in response to melatonin. Incubation with melatonin evoked robust Ca(2+) signals within the parasite, as did the application of a membrane-permeant analogue of cAMP. Our data suggest that P. chabaudi engages both Ca(2+) and cAMP signalling systems when stimulated by melatonin. Furthermore, there is positive feedback between these messengers, because Ca(2+) evokes cAMP elevation and vice versa. Melatonin more than doubled the observed extent of parasitemia, and the increase in cAMP concentration and PKA activation was essential for this effect. These data support the possibility to use melatonin antagonists or derivates in therapeutic approach.
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http://dx.doi.org/10.1111/j.1600-079X.2010.00810.xDOI Listing
January 2011

Development and validation of a Xanthomonas axonopodis pv. citri DNA microarray platform (XACarray) generated from the shotgun libraries previously used in the sequencing of this bacterial genome.

BMC Res Notes 2010 May 27;3:150. Epub 2010 May 27.

Departamento de Ciências Biológicas (DECBI), Instituto de Ciências Exatas e Biológicas, Universidade Federal de Ouro Preto, Campus Morro do Cruzeiro, Ouro Preto, MG, Brazil.

Background: From shotgun libraries used for the genomic sequencing of the phytopathogenic bacterium Xanthomonas axonopodis pv. citri (XAC), clones that were representative of the largest possible number of coding sequences (CDSs) were selected to create a DNA microarray platform on glass slides (XACarray). The creation of the XACarray allowed for the establishment of a tool that is capable of providing data for the analysis of global genome expression in this organism.

Findings: The inserts from the selected clones were amplified by PCR with the universal oligonucleotide primers M13R and M13F. The obtained products were purified and fixed in duplicate on glass slides specific for use in DNA microarrays. The number of spots on the microarray totaled 6,144 and included 768 positive controls and 624 negative controls per slide. Validation of the platform was performed through hybridization of total DNA probes from XAC labeled with different fluorophores, Cy3 and Cy5. In this validation assay, 86% of all PCR products fixed on the glass slides were confirmed to present a hybridization signal greater than twice the standard deviation of the deviation of the global median signal-to-noise ration.

Conclusions: Our validation of the XACArray platform using DNA-DNA hybridization revealed that it can be used to evaluate the expression of 2,365 individual CDSs from all major functional categories, which corresponds to 52.7% of the annotated CDSs of the XAC genome. As a proof of concept, we used this platform in a previously work to verify the absence of genomic regions that could not be detected by sequencing in related strains of Xanthomonas.
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http://dx.doi.org/10.1186/1756-0500-3-150DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2890508PMC
May 2010

Novel insights into the genomic basis of citrus canker based on the genome sequences of two strains of Xanthomonas fuscans subsp. aurantifolii.

BMC Genomics 2010 Apr 13;11:238. Epub 2010 Apr 13.

Departamento de Ciências Biológicas, Instituto de Ciências Exatas e Biológicas, Campus Morro do Cruzeiro, Universidade Federal de Ouro Preto, Ouro Preto, MG, Brazil.

Background: Citrus canker is a disease that has severe economic impact on the citrus industry worldwide. There are three types of canker, called A, B, and C. The three types have different phenotypes and affect different citrus species. The causative agent for type A is Xanthomonas citri subsp. citri, whose genome sequence was made available in 2002. Xanthomonas fuscans subsp. aurantifolii strain B causes canker B and Xanthomonas fuscans subsp. aurantifolii strain C causes canker C.

Results: We have sequenced the genomes of strains B and C to draft status. We have compared their genomic content to X. citri subsp. citri and to other Xanthomonas genomes, with special emphasis on type III secreted effector repertoires. In addition to pthA, already known to be present in all three citrus canker strains, two additional effector genes, xopE3 and xopAI, are also present in all three strains and are both located on the same putative genomic island. These two effector genes, along with one other effector-like gene in the same region, are thus good candidates for being pathogenicity factors on citrus. Numerous gene content differences also exist between the three cankers strains, which can be correlated with their different virulence and host range. Particular attention was placed on the analysis of genes involved in biofilm formation and quorum sensing, type IV secretion, flagellum synthesis and motility, lipopolysacharide synthesis, and on the gene xacPNP, which codes for a natriuretic protein.

Conclusion: We have uncovered numerous commonalities and differences in gene content between the genomes of the pathogenic agents causing citrus canker A, B, and C and other Xanthomonas genomes. Molecular genetics can now be employed to determine the role of these genes in plant-microbe interactions. The gained knowledge will be instrumental for improving citrus canker control.
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http://dx.doi.org/10.1186/1471-2164-11-238DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2883993PMC
April 2010

Effects of the antimicrobial peptide gomesin on the global gene expression profile, virulence and biofilm formation of Xylella fastidiosa.

FEMS Microbiol Lett 2010 May 11;306(2):152-9. Epub 2010 Mar 11.

Departamento de Bioquímica, Instituto de Química, Universidade de São Paulo, SP, Brazil.

In the xylem vessels of susceptible hosts, such as citrus trees, Xylella fastidiosa forms biofilm-like colonies that can block water transport, which appears to correlate to disease symptoms. Besides aiding host colonization, bacterial biofilms play an important role in resistance against antimicrobial agents, for instance antimicrobial peptides (AMPs). Here, we show that gomesin, a potent AMP from a tarantula spider, modulates X. fastidiosa gene expression profile upon 60 min of treatment with a sublethal concentration. DNA microarray hybridizations revealed that among the upregulated coding sequences, some are related to biofilm production. In addition, we show that the biofilm formed by gomesin-treated bacteria is thicker than that formed by nontreated cells or cells exposed to streptomycin. We have also observed that the treatment of X. fastidiosa with a sublethal concentration of gomesin before inoculation in tobacco plants correlates with a reduction in foliar symptoms, an effect possibly due to the trapping of bacterial cells to fewer xylem vessels, given the enhancement in biofilm production. These results warrant further investigation of how X. fastidiosa would respond to the AMPs produced by citrus endophytes and by the insect vector, leading to a better understanding of the mechanism of action of these molecules on bacterial virulence.
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http://dx.doi.org/10.1111/j.1574-6968.2010.01950.xDOI Listing
May 2010

Heterologous expression in Escherichia coli of Neurospora crassa neutral trehalase as an active enzyme.

Protein Expr Purif 2009 Jun 6;65(2):185-9. Epub 2008 Dec 6.

Departamento de Biologia, Faculdade de Filosofia Ciências e Letras, Universidade de São Paulo, Ribeirão Preto, SP, Brazil.

Neutral trehalase from Neurospora crassa was expressed in Escherichia coli as a polypeptide of approximately 84 kDa in agreement with the theoretical size calculated from the corresponding cDNA. The recombinant neutral trehalase, purified by affinity chromatography exhibited a specific activity of 80-150 mU/mg protein. Optima of pH and temperature were 7.0 and 30 degrees C, respectively. The enzyme was absolutely specific for trehalose, and was quite sensitive to incubation at 40 degrees C. The recombinant enzyme was totally dependent on calcium, and was inhibited by ATP, copper, silver, aluminium and cobalt. K(M) was 42 mM, and V(max) was 30.6 nmol of glucose/min. The recombinant protein was phosphorylated by cAMP-dependent protein kinase, but not significantly activated. Immunoblotting with polyclonal antiserum prepared against the recombinant protein showed that neutral trehalase protein levels increased during exponential phase of N. crassa growth and dropped at the stationary phase. This is the first report of a neutral trehalase produced in E. coli with similar biochemical properties described for fungi native neutral trehalases, including calcium-dependence.
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http://dx.doi.org/10.1016/j.pep.2008.11.010DOI Listing
June 2009

The iron stimulon of Xylella fastidiosa includes genes for type IV pilus and colicin V-like bacteriocins.

J Bacteriol 2008 Apr 25;190(7):2368-78. Epub 2008 Jan 25.

Departamento de Bioquímica, Instituto de Química, Universidade de São Paulo, Av. Prof. Lineu Prestes 748, 05508-000, São Paulo, SP, Brazil.

Xylella fastidiosa is the etiologic agent of a wide range of plant diseases, including citrus variegated chlorosis (CVC), a major threat to citrus industry. The genomes of several strains of this phytopathogen were completely sequenced, enabling large-scale functional studies. DNA microarrays representing 2,608 (91.6%) coding sequences (CDS) of X. fastidiosa CVC strain 9a5c were used to investigate transcript levels during growth with different iron availabilities. When treated with the iron chelator 2,2'-dipyridyl, 193 CDS were considered up-regulated and 216 were considered down-regulated. Upon incubation with 100 microM ferric pyrophosphate, 218 and 256 CDS were considered up- and down-regulated, respectively. Differential expression for a subset of 44 CDS was further evaluated by reverse transcription-quantitative PCR. Several CDS involved with regulatory functions, pathogenicity, and cell structure were modulated under both conditions assayed, suggesting that major changes in cell architecture and metabolism occur when X. fastidiosa cells are exposed to extreme variations in iron concentration. Interestingly, the modulated CDS include those related to colicin V-like bacteriocin synthesis and secretion and to functions of pili/fimbriae. We also investigated the contribution of the ferric uptake regulator Fur to the iron stimulon of X. fastidiosa. The promoter regions of the strain 9a5c genome were screened for putative Fur boxes, and candidates were analyzed by electrophoretic mobility shift assays. Taken together, our data support the hypothesis that Fur is not solely responsible for the modulation of the iron stimulon of X. fastidiosa, and they present novel evidence for iron regulation of pathogenicity determinants.
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http://dx.doi.org/10.1128/JB.01495-07DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2293194PMC
April 2008

The P450 oxidoreductase, RedA, controls development beyond the mound stage in Dictyostelium discoideum.

BMC Dev Biol 2008 Jan 24;8. Epub 2008 Jan 24.

Departamento de Bioquímica, Instituto de Química, Universidade de São Paulo, Av, Prof, Lineu Prestes 748, 05508-000, São Paulo, Brasil.

Background: NADPH-cytochrome-P450 oxidoreductase (CPR) is a ubiquitous enzyme that belongs to a family of diflavin oxidoreductases and is required for activity of the microsomal cytochrome-P450 monooxygenase system. CPR gene-disruption experiments have demonstrated that absence of this enzyme causes developmental defects both in mouse and insect.

Results: Annotation of the sequenced genome of D. discoideum revealed the presence of three genes (redA, redB and redC) that encode putative members of the diflavin oxidoreductase protein family. redA transcripts are present during growth and early development but then decline, reaching undetectable levels after the mound stage. redB transcripts are present in the same levels during growth and development while redC expression was detected only in vegetative growing cells. We isolated a mutant strain of Dictyostelium discoideum following restriction enzyme-mediated integration (REMI) mutagenesis in which redA was disrupted. This mutant develops only to the mound stage and accumulates a bright yellow pigment. The mound-arrest phenotype is cell-autonomous suggesting that the defect occurs within the cells rather than in intercellular signaling.

Conclusion: The developmental arrest due to disruption of redA implicates CPR in the metabolism of compounds that control cell differentiation.
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http://dx.doi.org/10.1186/1471-213X-8-8DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2257935PMC
January 2008

Genome mapping and expression analyses of human intronic noncoding RNAs reveal tissue-specific patterns and enrichment in genes related to regulation of transcription.

Genome Biol 2007 ;8(3):R43

Departamento de Bioquimica, Instituto de Quimica, Universidade de São Paulo, São Paulo, SP, Brazil.

Background: RNAs transcribed from intronic regions of genes are involved in a number of processes related to post-transcriptional control of gene expression. However, the complement of human genes in which introns are transcribed, and the number of intronic transcriptional units and their tissue expression patterns are not known.

Results: A survey of mRNA and EST public databases revealed more than 55,000 totally intronic noncoding (TIN) RNAs transcribed from the introns of 74% of all unique RefSeq genes. Guided by this information, we designed an oligoarray platform containing sense and antisense probes for each of 7,135 randomly selected TIN transcripts plus the corresponding protein-coding genes. We identified exonic and intronic tissue-specific expression signatures for human liver, prostate and kidney. The most highly expressed antisense TIN RNAs were transcribed from introns of protein-coding genes significantly enriched (p = 0.002 to 0.022) in the 'Regulation of transcription' Gene Ontology category. RNA polymerase II inhibition resulted in increased expression of a fraction of intronic RNAs in cell cultures, suggesting that other RNA polymerases may be involved in their biosynthesis. Members of a subset of intronic and protein-coding signatures transcribed from the same genomic loci have correlated expression patterns, suggesting that intronic RNAs regulate the abundance or the pattern of exon usage in protein-coding messages.

Conclusion: We have identified diverse intronic RNA expression patterns, pointing to distinct regulatory roles. This gene-oriented approach, using a combined intron-exon oligoarray, should permit further comparative analysis of intronic transcription under various physiological and pathological conditions, thus advancing current knowledge about the biological functions of these noncoding RNAs.
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http://dx.doi.org/10.1186/gb-2007-8-3-r43DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1868932PMC
June 2007

Identification and domain mapping of Dictyostelium discoideum type-1 protein phosphatase inhibitor-2.

Biochimie 2007 May 21;89(5):692-701. Epub 2007 Jan 21.

Departamento de Bioquímica, Instituto de Química, Universidade de São Paulo, Av. Prof. Lineu Prestes 748, 05508-000, São Paulo, Brazil.

The protein phosphatase type-1 catalytic subunit (PP1c) does not exist freely in the cell and its activity must be very strictly controlled. Several protein inhibitors of PP1c have been described including the classical mammalian inhibitor-1 (I-1) and inhibitor-2 (I-2). Association of these inhibitors with PP1c appears to involve multiple contacts and in the case of I-2 no less than five I-2 interaction subdomains have been proposed. In this report, we provide both in vitro and in vivo evidence that the Dictyostelium discoideum genome encodes a protein (DdI-2) that is an ortholog of mammalian I-2, being the first PP1c interacting protein characterized in this social amoeba. Despite the low overall sequence similarity of DdI-2 with other I-2 sequences and its long N-terminal extension, the five PP1c interaction motifs proposed for mammalian I-2 are reasonably conserved in the Dictyostelium ortholog. We demonstrate that DdI-2 interacts with and inhibits D. discoideum PP1c (DdPP1c), which we have previously characterized. Moreover, using yeast two-hybrid assays we show that a stable interaction of DdI-2 with DdPP1c requires multiple contacts.
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http://dx.doi.org/10.1016/j.biochi.2007.01.002DOI Listing
May 2007

Androgen responsive intronic non-coding RNAs.

BMC Biol 2007 Jan 30;5. Epub 2007 Jan 30.

Departamento de Bioquimica, Instituto de Quimica, Universidade de São Paulo, 05508-900 São Paulo, Brazil.

Background: Transcription of large numbers of non-coding RNAs originating from intronic regions of human genes has been recently reported, but mechanisms governing their biosynthesis and biological functions are largely unknown. In this work, we evaluated the existence of a common mechanism of transcription regulation shared by protein-coding mRNAs and intronic RNAs by measuring the effect of androgen on the transcriptional profile of a prostate cancer cell line.

Results: Using a custom-built cDNA microarray enriched in intronic transcribed sequences, we found 39 intronic non-coding RNAs for which levels were significantly regulated by androgen exposure. Orientation-specific reverse transcription-PCR indicated that 10 of the 13 were transcribed in the antisense direction. These transcripts are long (0.5-5 kb), unspliced and apparently do not code for proteins. Interestingly, we found that the relative levels of androgen-regulated intronic transcripts could be correlated with the levels of the corresponding protein-coding gene (asGAS6 and asDNAJC3) or with the alternative usage of exons (asKDELR2 and asITGA6) in the corresponding protein-coding transcripts. Binding of the androgen receptor to a putative regulatory region upstream from asMYO5A, an androgen-regulated antisense intronic transcript, was confirmed by chromatin immunoprecipitation.

Conclusion: Altogether, these results indicate that at least a fraction of naturally transcribed intronic non-coding RNAs may be regulated by common physiological signals such as hormones, and further corroborate the notion that the intronic complement of the transcriptome play functional roles in the human gene-expression program.
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http://dx.doi.org/10.1186/1741-7007-5-4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1800835PMC
January 2007

Cyclic AMP and calcium interplay as second messengers in melatonin-dependent regulation of Plasmodium falciparum cell cycle.

J Cell Biol 2005 Aug;170(4):551-7

Departamento de Parasitologia, Instituto de Ciências Biomédicas, Universidade de São Paulo, São Paulo, SP, Brazil.

The host hormone melatonin increases cytoplasmic Ca(2+) concentration and synchronizes Plasmodium cell cycle (Hotta, C.T., M.L. Gazarini, F.H. Beraldo, F.P. Varotti, C. Lopes, R.P. Markus, T. Pozzan, and C.R. Garcia. 2000. Nat. Cell Biol. 2:466-468). Here we show that in Plasmodium falciparum melatonin induces an increase in cyclic AMP (cAMP) levels and cAMP-dependent protein kinase (PKA) activity (40 and 50%, respectively). When red blood cells infected with P. falciparum are treated with cAMP analogue adenosine 3',5'-cyclic monophosphate N6-benzoyl/PKA activator (6-Bz-cAMP) there is an alteration of the parasite cell cycle. This effect appears to depend on activation of PKA (abolished by the PKA inhibitors adenosine 3',5'-cyclic monophosphorothioate/8 Bromo Rp isomer, PKI [cell permeable peptide], and H89). An unexpected cross talk was found to exist between the cAMP and the Ca(2+)-dependent signaling pathways. The increases in cAMP by melatonin are inhibited by blocker of phospholipase C U73122, and addition of 6-Bz-cAMP increases cytosolic Ca(2+) concentration, through PKA activation. These findings suggest that in Plasmodium a highly complex interplay exists between the Ca(2+) and cAMP signaling pathways, but also that the control of the parasite cell cycle by melatonin requires the activation of both second messenger controlled pathways.
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http://dx.doi.org/10.1083/jcb.200505117DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2171486PMC
August 2005

Whole-genome expression profiling of Xylella fastidiosa in response to growth on glucose.

OMICS 2005 ;9(1):77-90

Departamento de Bioquímica, Instituto de Química, Universidade de São Paulo, São Paulo, Brasil.

Xylella fastidiosa is the etiologic agent of diseases in a wide range of economically important crops including citrus variegated chlorosis, a major threat to the Brazilian citrus industry. The genomes of several strains of this phytopathogen have been completely sequenced enabling large-scale functional studies. In this work we used whole-genome DNA microarrays to investigate the transcription profile of X. fastidiosa grown in defined media with different glucose concentrations. Our analysis revealed that while transcripts related to fastidian gum production were unaffected, colicin-V-like and fimbria precursors were induced in high glucose medium. Based on these results, we suggest a model for colicin-defense mechanism in X. fastidiosa.
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http://dx.doi.org/10.1089/omi.2005.9.77DOI Listing
September 2005

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

Cancer Res 2005 Mar;65(5):1693-9

Departamento de Bioquímica, Faculdade de Medicina, Universidade de São Paulo, Brazil.

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.
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http://dx.doi.org/10.1158/0008-5472.CAN-04-3506DOI Listing
March 2005

Polyductin, the PKHD1 gene product, comprises isoforms expressed in plasma membrane, primary cilium, and cytoplasm.

Kidney Int 2004 Oct;66(4):1345-55

Department of Medicine, University of São Paulo School of Medicine, São Paulo, Brazil.

Background: PKHD1, the autosomal-recessive polycystic kidney disease (ARPKD) gene, encodes multiple alternatively spliced transcripts predicted to generate membrane-bound and secreted proteins. The longest open reading frame encodes polyductin (fibrocystin), a putative 4074 amino acid protein with a single transmembrane domain and an intracellular C-terminus.

Methods: To characterize the PKHD1 products and their expression profile, we raised polyclonal antibodies against different portions of polyductin and analyzed different organs using various methods.

Results: Western blot analyses demonstrated specific bands of >440 kD in human adult kidney, liver, and pancreas and approximately 230 kD in kidney and liver, predominantly observed in membrane fractions. The >440-kD putative membrane protein was immunoprecipitated from kidney and subsequently detected by Western blotting using two distinct antisera. An additional product of approximately 140 kD was specifically recognized by affinity-purified antisera predominantly in soluble fractions. Immunohistochemistry studies revealed specific staining in cortical and medullary collecting ducts and thick ascending limbs of Henle (TALH). Serial sections were stained with antibodies against aquaporin-2 and Tamm-Horsfall protein to confirm the nephron segment localization. Positive staining was also detected in biliary and pancreatic duct epithelia. Analyses of mouse developing tissues showed specific staining in the ureteric bud branches, intra- and extrahepatic biliary ducts, pancreatic ducts, and salivary glands. Immunofluorescence studies in inner medullary collecting duct cultured cells and immunoelectron microscopy analysis of medullary collecting ducts demonstrated that the protein localizes to the primary cilium. Positive signal was also detected in the apical membrane and in cytoplasm.

Conclusion: The results indicate that polyductin is part of the group of polycystic kidney disease (PKD)-related proteins expressed in primary apical cilia. Our data also suggest that, in addition to its likely involvement in cilia function, polyductin probably serves in other subcellular functional roles. The detection of three different products using two antisera, with evidence for distinct subcellular localizations, suggests that PKHD1 encodes membrane-bound and soluble isoforms.
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http://dx.doi.org/10.1111/j.1523-1755.2004.00844.xDOI Listing
October 2004

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

J Bacteriol 2004 Aug;186(16):5442-9

Departamento de Bioquímica, Instituto de Química, Universidade de São Paulo, SP, Brazil.

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.
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http://dx.doi.org/10.1128/JB.186.16.5442-5449.2004DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC490883PMC
August 2004

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

Oncogene 2004 Aug;23(39):6684-92

Departamento de Bioquímica, Instituto de Química, Universidade de São Paulo, 05508-900 São Paulo, SP, Brasil.

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.
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http://dx.doi.org/10.1038/sj.onc.1207880DOI Listing
August 2004

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

Biochem Biophys Res Commun 2004 Apr;316(3):618-27

Departamento de Bioquímica, Instituto de Química, Universidade de São Paulo, 05508-900 São Paulo, SP, Brazil.

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.
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http://dx.doi.org/10.1016/j.bbrc.2004.02.091DOI Listing
April 2004

Analysis and functional annotation of an expressed sequence tag collection for tropical crop sugarcane.

Genome Res 2003 Dec 12;13(12):2725-35. Epub 2003 Nov 12.

Centro de Biologia Molecular e Engenharia Genética, Instituto da Computação, Universidade Estadual de Campinas, 13083-970 Campinas-SP, Brazil.

To contribute to our understanding of the genome complexity of sugarcane, we undertook a large-scale expressed sequence tag (EST) program. More than 260,000 cDNA clones were partially sequenced from 26 standard cDNA libraries generated from different sugarcane tissues. After the processing of the sequences, 237,954 high-quality ESTs were identified. These ESTs were assembled into 43,141 putative transcripts. Of the assembled sequences, 35.6% presented no matches with existing sequences in public databases. A global analysis of the whole SUCEST data set indicated that 14,409 assembled sequences (33% of the total) contained at least one cDNA clone with a full-length insert. Annotation of the 43,141 assembled sequences associated almost 50% of the putative identified sugarcane genes with protein metabolism, cellular communication/signal transduction, bioenergetics, and stress responses. Inspection of the translated assembled sequences for conserved protein domains revealed 40,821 amino acid sequences with 1415 Pfam domains. Reassembling the consensus sequences of the 43,141 transcripts revealed a 22% redundancy in the first assembling. This indicated that possibly 33,620 unique genes had been identified and indicated that >90% of the sugarcane expressed genes were tagged.
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http://dx.doi.org/10.1101/gr.1532103DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC403815PMC
December 2003

The generation and utilization of a cancer-oriented representation of the human transcriptome by using expressed sequence tags.

Authors:
Helena Brentani Otávia L Caballero Anamaria A Camargo Aline M da Silva Wilson Araújo da Silva Emmanuel Dias Neto Marco Grivet Arthur Gruber Pedro Edson Moreira Guimaraes Winston Hide Christian Iseli C Victor Jongeneel Janet Kelso Maria Aparecida Nagai Elida Paula Benquique Ojopi Elisson C Osorio Eduardo M R Reis Gregory J Riggins Andrew John George Simpson Sandro de Souza Brian J Stevenson Robert L Strausberg Eloiza H Tajara Sergio Verjovski-Almeida Marcio Luis Acencio Mário Henrique Bengtson Fabiana Bettoni Walter F Bodmer Marcelo R S Briones Luiz Paulo Camargo Webster Cavenee Janete M Cerutti Luis Eduardo Coelho Andrade Paulo César Costa dos Santos Maria Cristina Ramos Costa Israel Tojal da Silva Marcos Roberto H Estécio Karine Sa Ferreira Frank B Furnari Milton Faria Pedro A F Galante Gustavo S Guimaraes Adriano Jesus Holanda Edna Teruko Kimura Maarten R Leerkes Xin Lu Rui M B Maciel Elizabeth A L Martins Katlin Brauer Massirer Analy S A Melo Carlos Alberto Mestriner Elisabete Cristina Miracca Leandro Lorenco Miranda Francisco G Nobrega Paulo S Oliveira Apua C M Paquola José Rodrigo C Pandolfi Maria Ines de Moura Campos Pardini Fabio Passetti John Quackenbush Beatriz Schnabel Mari Cleide Sogayar Jorge E Souza Sandro R Valentini Andre C Zaiats Elisabete Jorge Amaral Liliane A T Arnaldi Amelia Goes de Araújo Simone Aparecida de Bessa David C Bicknell Maria Eugenia Ribeiro de Camaro Dirce Maria Carraro Helaine Carrer Alex F Carvalho Christian Colin Fernando Costa Cyntia Curcio Ismael Dale Cotrim Guerreiro da Silva Neusa Pereira da Silva Márcia Dellamano Hamza El-Dorry Enilza Maria Espreafico Ari José Scattone Ferreira Cristiane Ayres Ferreira Maria Angela H Z Fortes Angelita Habr Gama Daniel Giannella-Neto Maria Lúcia C C Giannella Ricardo R Giorgi Gustavo Henrique Goldman Maria Helena S Goldman Christine Hackel Paulo Lee Ho Elza Myiuki Kimura Luiz Paulo Kowalski Jose E Krieger Luciana C C Leite Ademar Lopes Ana Mercedes S C Luna Alan Mackay Suely Kazue Nagahashi Mari Adriana Aparecida Marques Waleska K Martins André Montagnini Mario Mourão Neto Ana Lucia T O Nascimento A Munro Neville Marina P Nobrega Mike J O'Hare Audrey Yumi Otsuka Anna Izabel Ruas de Melo Maria Luisa Paco-Larson Gonçalo Guimarães Pereira Neusa Pereira da Silva Joao Bosco Pesquero Juliana Gilbert Pessoa Paula Rahal Claudia Aparecida Rainho Vanderlei Rodrigues Silvia Regina Rogatto Camila Malta Romano Janaina Gusmao Romeiro Benedito Mauro Rossi Monica Rusticci Renata Guerra de Sá Simone Cristina Sant' Anna Miriam L Sarmazo Teresa Cristina de Lima E Silva Fernando Augusto Soares Maria de Fátima Sonati Josane de Freitas Sousa Diana Queiroz Valéria Valente André Luiz Vettore Fabiola Elizabeth Villanova Marco Antonio Zago Heloisa Zalcberg

Proc Natl Acad Sci U S A 2003 Nov 30;100(23):13418-23. Epub 2003 Oct 30.

Laboratorio de Genética Molecular do Cancer, Departmento de Radiologia, Universidade de São Paulo, Travessa da Rua Dr. Ovídeo Pires de Campos S/N, 4deg, Brazil.

Whereas genome sequencing defines the genetic potential of an organism, transcript sequencing defines the utilization of this potential and links the genome with most areas of biology. To exploit the information within the human genome in the fight against cancer, we have deposited some two million expressed sequence tags (ESTs) from human tumors and their corresponding normal tissues in the public databases. The data currently define approximately 23,500 genes, of which only approximately 1,250 are still represented only by ESTs. Examination of the EST coverage of known cancer-related (CR) genes reveals that <1% do not have corresponding ESTs, indicating that the representation of genes associated with commonly studied tumors is high. The careful recording of the origin of all ESTs we have produced has enabled detailed definition of where the genes they represent are expressed in the human body. More than 100,000 ESTs are available for seven tissues, indicating a surprising variability of gene usage that has led to the discovery of a significant number of genes with restricted expression, and that may thus be therapeutically useful. The ESTs also reveal novel nonsynonymous germline variants (although the one-pass nature of the data necessitates careful validation) and many alternatively spliced transcripts. Although widely exploited by the scientific community, vindicating our totally open source policy, the EST data generated still provide extensive information that remains to be systematically explored, and that may further facilitate progress toward both the understanding and treatment of human cancers.
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http://dx.doi.org/10.1073/pnas.1233632100DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC263829PMC
November 2003

ESTWeb: bioinformatics services for EST sequencing projects.

Bioinformatics 2003 Aug;19(12):1587-8

Departamento de Bioquímica, Instituto de Química, Universidade de São Paulo, 05508-900, São Paulo, SP, Brazil.

ESTWeb is an internet based software package designed for uniform data processing and storage for large-scale EST sequencing projects. The package provides for: (a) reception of sequencing chromatograms; (b) sequence processing such as base-calling, vector screening, comparison with public databases; (c) storage of data and analysis in a relational database, (d) generation of a graphical report of individual sequence quality; and (e) issuing of reports with statistics of productivity and redundancy. The software facilitates real-time monitoring and evaluation of EST sequence acquisition progress along an EST sequencing project.
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http://dx.doi.org/10.1093/bioinformatics/btg196DOI Listing
August 2003

Zerg: a very fast BLAST parser library.

Bioinformatics 2003 May;19(8):1035-6

Departamento de Bioquímica, Instituto de Química, Universidade de São Paulo 05508-900, São Paulo, SP, Brazil.

Summary: Zerg is a library of sub-routines that parses the output from all NCBI BLAST programs (Blastn, Blastp, Blastx, Tblastn and Tblastx) and returns the attributes of a BLAST report to the user. It is optimized for speed, being especially useful for large-scale genomic analysis. Benchmark tests show that Zerg is over two orders of magnitude faster than some widely used BLAST parsers.

Availability: http://bioinfo.iq.usp.br/zerg
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http://dx.doi.org/10.1093/bioinformatics/btg122DOI Listing
May 2003

Dictyostelium discoideum protein phosphatase-1 catalytic subunit exhibits distinct biochemical properties.

Biochem J 2003 Aug;373(Pt 3):703-11

Departamento de Bioquímica, Instituto de Química, Universidade de São Paulo, Avenida Professor Lineu Prestes 748, 05508-900, São Paulo, SP, Brazil.

Protein phosphatase-1 (PP1) is expressed ubiquitously and is involved in many eukaryotic cellular functions, although PP1 enzyme activity could not be detected in the social amoeba Dictyostelium discoideum cell extracts. In the present paper, we show that D. discoideum has a single copy gene that codes for the catalytic subunit of PP1 (DdPP1c). DdPP1c is expressed throughout the D. discoideum life cycle with constant levels of mRNA, and its protein and amino acid sequence show a mean identity of 80% with other PP1c enzymes. However, it has a distinctive difference: the substitution of a phenylalanine residue (Phe(269) in the DdPP1c) for a highly conserved cysteine residue (Cys(273) in rabbit PP1c) in a region that was shown to have a critical role in the interaction of rabbit PP1c with toxin inhibitors. Wild-type DdPP1c and an engineered mutant form in which Phe(269) was replaced by a cysteine residue were expressed in Escherichia coli. Both recombinant activities were similarly inhibited by okadaic acid, tautomycin and microcystin. However, the Phe(269)-->Cys mutation resulted in a large increase in enzyme activity towards phosphorylase a and a higher sensitivity to calyculin A. These results, together with the molecular modelling of DdPP1c structure, indicate that the Phe(269) residue, which occurs naturally in D. discoideum, confers distinct biochemical properties on this enzyme.
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http://dx.doi.org/10.1042/BJ20021964DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1223547PMC
August 2003