[Show abstract][Hide abstract] ABSTRACT: The development and progression of cancer depend on its genetic characteristics as well as on the interactions with its microenvironment. Understanding these interactions may contribute to diagnostic and prognostic evaluations and to the development of new cancer therapies. Aiming to investigate potential mechanisms by which the tumor microenvironment might contribute to a cancer phenotype, we evaluated soluble paracrine factors produced by stromal and neoplastic cells which may influence proliferation and gene and protein expression.
The study was carried out on the epithelial cancer cell line (Hep-2) and fibroblasts isolated from a primary oral cancer. We combined a conditioned-medium technique with subtraction hybridization approach, quantitative PCR and proteomics, in order to evaluate gene and protein expression influenced by soluble paracrine factors produced by stromal and neoplastic cells.
We observed that conditioned medium from fibroblast cultures (FCM) inhibited proliferation and induced apoptosis in Hep-2 cells. In neoplastic cells, 41 genes and 5 proteins exhibited changes in expression levels in response to FCM and, in fibroblasts, 17 genes and 2 proteins showed down-regulation in response to conditioned medium from Hep-2 cells (HCM). Nine genes were selected and the expression results of 6 down-regulated genes (ARID4A, CALR, GNB2L1, RNF10, SQSTM1, USP9X) were validated by real time PCR.
A significant and common denominator in the results was the potential induction of signaling changes associated with immune or inflammatory response in the absence of a specific protein.
BMC Medical Genomics 01/2010; 3:14. · 3.91 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Annexin A1 (ANXA1) is a soluble cytoplasmic protein, moving to membranes when calcium levels are elevated. ANXA1 has also been shown to move to the nucleus or outside the cells, depending on tyrosine-kinase signalling, thus interfering in cytoskeletal organization and cell differentiation, mostly in inflammatory and neoplastic processes. The aim was to investigate subcellular patterns of immunohistochemical expression of ANXA1 in neoplastic and non-neoplastic samples from patients with laryngeal squamous cell carcinomas (LSCC), to elucidate the role of ANXA1 in laryngeal carcinogenesis.
Serial analysis of gene expression experiments detected reduced expression of ANXA1 gene in LSCC compared with the corresponding non-neoplastic margins. Quantitative polymerase chain reaction confirmed ANXA1 low expression in 15 LSCC and eight matched normal samples. Thus, we investigated subcellular patterns of immunohistochemical expression of ANXA1 in 241 paraffin-embedded samples from 95 patients with LSCC. The results showed ANXA1 down-regulation in dysplastic, tumourous and metastatic lesions and provided evidence for the progressive migration of ANXA1 from the nucleus towards the membrane during laryngeal tumorigenesis.
ANXA1 dysregulation was observed early in laryngeal carcinogenesis, in intra-epithelial neoplasms; it was not found related to prognostic parameters, such as nodal metastases.
[Show abstract][Hide abstract] ABSTRACT: Denture stomatitis is a common lesion that affects denture wearers. Its multifactorial etiology seems to depend on a complex and poorly characterized biofilm. The purpose of this study was to assess the composition of the microbial biofilm obtained from complete denture wearers with and without denture stomatitis using culture-independent methods.
Samples were collected from healthy denture wearers and from patients with denture stomatitis. Libraries comprising about 600 cloned 16S ribosomal DNA (rDNA) bacterial sequences and 192 cloned eukaryotic internal transcribed spacer (ITS) region sequences, obtained by polymerase chain reactions, were analyzed.
The partial 16S rDNA sequences revealed a total of 82 bacterial species identified in healthy subjects and patients with denture stomatitis. Twenty-seven bacterial species were detected in both biofilms, 29 species were exclusively present in patients with denture stomatitis, and 26 were found only in healthy subjects. Analysis of the ITS region revealed the presence of Candida sp. in both biofilms.
The results revealed the extent of the microbial flora, suggesting the existence of distinct biofilms in healthy subjects and in patients with denture stomatitis.
Oral Microbiology and Immunology 11/2008; 23(5):419-24. · 2.81 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: BACKGROUND: Head and neck squamous cell carcinoma (HNSCC) is one of the most common malignancies in humans. The average 5-year survival rate is one of the lowest among aggressive cancers, showing no significant improvement in recent years. When detected early, HNSCC has a good prognosis, but most patients present metastatic disease at the time of diagnosis, which significantly reduces survival rate. Despite extensive research, no molecular markers are currently available for diagnostic or prognostic purposes. METHODS: Aiming to identify differentially-expressed genes involved in laryngeal squamous cell carcinoma (LSCC) development and progression, we generated individual Serial Analysis of Gene Expression (SAGE) libraries from a metastatic and non-metastatic larynx carcinoma, as well as from a normal larynx mucosa sample. Approximately 54,000 unique tags were sequenced in three libraries. RESULTS: Statistical data analysis identified a subset of 1,216 differentially expressed tags between tumor and normal libraries, and 894 differentially expressed tags between metastatic and non-metastatic carcinomas. Three genes displaying differential regulation, one down-regulated (KRT31) and two up-regulated (BST2, MFAP2), as well as one with a non-significant differential expression pattern (GNA15) in our SAGE data were selected for real-time polymerase chain reaction (PCR) in a set of HNSCC samples. Consistent with our statistical analysis, quantitative PCR confirmed the upregulation of BST2 and MFAP2 and the downregulation of KRT31 when samples of HNSCC were compared to tumor-free surgical margins. As expected, GNA15 presented a non-significant differential expression pattern when tumor samples were compared to normal tissues. CONCLUSION: To the best of our knowledge, this is the first study reporting SAGE data in head and neck squamous cell tumors. Statistical analysis was effective in identifying differentially expressed genes reportedly involved in cancer development. The differential expression of a subset of genes was confirmed in additional larynx carcinoma samples and in carcinomas from a distinct head and neck subsite. This result suggests the existence of potential common biomarkers for prognosis and targeted-therapy development in this heterogeneous type of tumor.
BMC Medical Genomics 01/2008; 1:56. · 3.91 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Recurrent aphthous ulcers are common lesions of the oral mucosa of which the etiology is unknown. This study aimed to estimate the bacterial diversity in the lesions and in control mucosa in pooled samples using a culture-independent molecular approach.
Samples were collected from ten healthy individuals and ten individuals with a clinical history of recurrent aphthous ulcers. After DNA extraction, the 16S ribosomal RNA bacterial gene was amplified by polymerase chain reaction with universal primers; amplicons were cloned, sequenced and matched to the GenBank database.
A total of 535 clones were analyzed, defining 95 bacterial species. We identified 62 putative novel phylotypes. In recurrent aphthous ulcer lesions 57 phylotypes were detected, of which 11 were known species. Control samples had 38 phylotypes, five of which were already known. Only three species or phylotypes were abundant and common to both groups (Gemella haemolysans, Streptococcus mitis strain 209 and Streptococcus pneumoniae R6). One genus was found only in recurrent aphthous ulcer samples (Prevotella) corresponding to 16% of all lesion-derived clones.
The microbiota found in recurrent aphthous ulcers and in the control groups diverged markedly and the rich variety of genera found can provide a new starting point for individual qualitative and quantitative analyses of bacteria associated with this oral condition.
Oral Microbiology and Immunology 09/2007; 22(4):225-31. · 2.81 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Heme A is a prosthetic group of all eukaryotic and some prokaryotic cytochrome oxidases. This heme differs from heme B (protoheme) at two carbon positions of the porphyrin ring. The synthesis of heme A begins with farnesylation of the vinyl group at carbon C-2 of heme B. The heme O product of this reaction is then converted to heme A by a further oxidation of a methyl to a formyl group on C-8. In a previous study (Barros, M. H., Carlson, C. G., Glerum, D. M., and Tzagoloff, A. (2001) FEBS Lett. 492, 133-138) we proposed that the formyl group is formed by an initial hydroxylation of the C-8 methyl by a three-component monooxygenase consisting of Cox15p, ferredoxin, and ferredoxin reductase. In the present study three lines of evidence confirm a requirement of ferredoxin in heme A synthesis. 1) Temperature-conditional yah1 mutants grown under restrictive conditions display a decrease in heme A relative to heme B. 2) The incorporation of radioactive delta-aminolevulinic acid into heme A is reduced in yah1 ts but not in the wild type after the shift to the restrictive temperature; and 3) the overexpression of Cox15p in cytochrome oxidase mutants that accumulate heme O leads to an increased mitochondrial concentration of heme A. The increase in heme A is greater in mutants that overexpress Cox15p and ferredoxin. These results are consistent with a requirement of ferredoxin and indirectly of ferredoxin reductase in hydroxylation of heme O.
Journal of Biological Chemistry 04/2002; 277(12):9997-10002. · 4.65 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: 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.
Proceedings of the National Academy of Sciences 11/2001; 98(21):12103-8. · 9.81 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: 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).
Proceedings of the National Academy of Sciences 12/2000; 97(23):12690-3. · 9.81 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Xylella fastidiosa is a fastidious, xylem-limited bacterium that causes a range of economically important plant diseases. Here we report the complete genome sequence of X. fastidiosa clone 9a5c, which causes citrus variegated chlorosis--a serious disease of orange trees. The genome comprises a 52.7% GC-rich 2,679,305-base-pair (bp) circular chromosome and two plasmids of 51,158 bp and 1,285 bp. We can assign putative functions to 47% of the 2,904 predicted coding regions. Efficient metabolic functions are predicted, with sugars as the principal energy and carbon source, supporting existence in the nutrient-poor xylem sap. The mechanisms associated with pathogenicity and virulence involve toxins, antibiotics and ion sequestration systems, as well as bacterium-bacterium and bacterium-host interactions mediated by a range of proteins. Orthologues of some of these proteins have only been identified in animal and human pathogens; their presence in X. fastidiosa indicates that the molecular basis for bacterial pathogenicity is both conserved and independent of host. At least 83 genes are bacteriophage-derived and include virulence-associated genes from other bacteria, providing direct evidence of phage-mediated horizontal gene transfer.
[Show abstract][Hide abstract] ABSTRACT: Nuclear mutants of Saccharomyces cerevisiae assigned to complementation group G34 are respiratory-deficient and lack cytochrome oxidase activity and the characteristic spectral peaks of cytochromes a and a(3). The corresponding gene was cloned by complementation, sequenced, and identified as reading frame YGR062C on chromosome VII. This gene was named COX18. The COX18 gene product is a polypeptide of 316 amino acids with a putative amino-terminal mitochondrial targeting sequence and predicted transmembrane domains. Respiratory chain carriers other than cytochromes a and a(3) and the ATPase complex are present at near wild-type levels in cox18 mutants, indicating that the mutations specifically affect cytochrome oxidase. The synthesis of Cox1p and Cox3p in mutant mitochondria is normal whereas Cox2p is barely detected among labeled mitochondrial polypeptides. Transcription of COX2 does not require COX18 function, and a chimeric COX3-COX2 mRNA did not suppress the respiratory defect in the null mutant, indicating that the mutation does not impair transcription or translation of the mRNA. Western analysis of cytochrome oxidase subunits shows that inactivation of the COX18 gene greatly reduces the steady state amounts of subunit 2 and results in variable decreases in other subunits of cytochrome oxidase. A gene fusion expressing a biotinylated form of Cox18p complements cox18 mutants. Biotinylated Cox18p is a mitochondrial integral membrane protein. These results indicate Cox18p to be a new member of a group of mitochondrial proteins that function at a late stage of the cytochrome oxidase assembly pathway.
Journal of Biological Chemistry 06/2000; 275(20):14898-902. · 4.65 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Xylella fastidiosa is a fastidious, xylem-limited bacterium that causes a range of economically important plant diseases. Here we report the complete genome sequence of X. fastidiosa clone 9a5c, which causes citrus variegated chlorosis—a serious disease of orange trees. The genome comprises a 52.7% GC-rich 2,679,305-base-pair (bp) circular chromosome and two plasmids of 51,158 bp and 1,285 bp. We can assign putative functions to 47% of the 2,904 predicted coding regions. Efficient metabolic functions are predicted, with sugars as the principal energy and carbon source, supporting existence in the nutrient-poor xylem sap. The mechanisms associated with pathogenicity and virulence involve toxins, antibiotics and ion sequestration systems, as well as bacterium–bacterium and bacterium–host interactions mediated by a range of proteins. Orthologues of some of these proteins have only been identified in animal and human pathogens; their presence in X. fastidiosa indicates that the molecular basis for bacterial pathogenicity is both conserved and independent of host. At least 83 genes are bacteriophage-derived and include virulence-associated genes from other bacteria, providing direct evidence of phage-mediated horizontal gene transfer.
[Show abstract][Hide abstract] ABSTRACT: Here we describe the identification of a yeast gene (YAH1) with significant homology to a mammalian enzyme, adrenodoxin, encoded in open reading frame (ORF) YPL252C. Adrenodoxin is the second electron carrier that participates in a mitochondrial electron transfer chain that, in mammals, catalyses the conversion of cholesterol into pregnenolone, the first step in the synthesis of all steroid hormones. The inactivation of the yeast gene's chromosomal copy reveals that it performs an essential function. We show that the protein is targeted to the mitochondrial matrix and describe attempts to complement the yeast knockout with the human adrenodoxin gene (FDX1) and with chimerical proteins constructed with the fusion of the yeast and the human gene. The previous identification of a homolog of the first mammalian enzyme in yeast, ARH1, also shown to be essential (Manzella, L., Barros, M.H., Nobrega, F.G., 1998. Yeast 14, 839-846), strongly suggests that there is a novel electron transfer chain, unlinked to respiration, and of essential function in mitochondria.
[Show abstract][Hide abstract] ABSTRACT: A yeast gene was found in which the derived protein sequence has similarity to human and bovine adrenodoxin reductase (Nobrega, F. G., Nobrega, M. P. and Tzagoloff, A. (1992). EMBO J. 11, 3821-3829; Lacour, T. and Dumas, B. (1996). Gene 174, 289 292), an enzyme in the mitochondrial electron transfer chain that catalyses in mammals the conversion of cholesterol into pregnenolone, the first step in the synthesis of all steroid hormones. It was named ARH1 (Adrenodoxin Reductase Homologue 1) and here we show that it is essential. Rescue was possible by the yeast gene, but failed with the human gene. Supplementation was tried without success with various sterols, ruling out its involvement in the biosynthesis of ergosterol. Immunodetection with a specific polyclonal antibody located the gene product in the mitochondrial fraction. Consequently ARH1p joins the small group of gene products that affect essential functions carried out by the organelle and not linked to oxidative phosphorylation.
[Show abstract][Hide abstract] ABSTRACT: The mutants of Saccharomyces cerevisiae assigned to complementation group G199 are deficient in mitochondrial respiration and lack a functional cytochrome oxidase complex. Recombinant plasmids capable of restoring respiration were cloned by transformation of mutants of this group with a yeast genomic library. Sequencing indicated that a 2.1-kb subclone encompasses the very end (last 11 amino acids) of the PET111 gene, the COX7 gene and a new gene (YMR255W) of unknown function that potentially codes for a polypeptide of 188 amino acids (about 21.5 kDa) without significant homology to any known protein. We have shown that the respiratory defect corresponding to group G199 is complemented by plasmids carrying only the COX7 gene. The gene YMR255W was inactivated by one-step gene replacement and the disrupted strain was viable and unaffected in its ability to grow in a variety of different test media such as minimal or complete media using eight distinct carbon sources at three pH values and temperatures. Inactivation of this gene also did not affect mating or sporulation.
Brazilian Journal of Medical and Biological Research 04/1998; 31(3):355-63. · 1.14 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The nucleotide changes present in a group of five cytochrome b mit- mutants were analyzed at the sequence level. Two single-base changes were found: one (M10-152) generated a nonsense codon in the first exon while the other (M8-181) created a missense substitution in the second exon. The other mutants all have multiple (three) substitutions that either resulted in a missense mutation in a coding region (M17-162) or else changed nucleotides in the last intron of the gene, so blocking its excision (M6-200 and M8-53). The synthesis of mitochondrial polypeptides and the steady state concentration of the complex-III subunits were examined. The Rieske protein and the core-4 and core-5 subunits were much reduced in all mutants. Consequently the overall stability of complex III is very sensitive even to amino-acid substitutions in the cytochrome b protein. Mutant M8-53 provides direct evidence for the proposed role of the P9.1 stem in the core structure of the group-I type last intron of this gene.
Current Genetics 09/1996; 30(3):200-5. · 2.41 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Respiratory deficient pet mutants of Saccharomyces cerevisiae assigned to complementation group G2 define a new gene, named BCS1, whose product is shown to be necessary for the expression of functional ubiquinol-cytochrome c reductase (bc1) complex. Immunological assays indicate a gross reduction in the Rieske iron-sulfur subunit in bcs1 mutants, while other subunits of the ubiquinol-cytochrome c reductase complex are present at concentrations comparable to the wild type. Transformation of bcs1 mutants with the iron-sulfur protein gene on a multicopy plasmid led to elevated mitochondrial concentrations of Rieske protein, but did not correct the enzymatic defect, indicating that BCS1 is involved either in forming the active site iron-sulfur cluster or providing a chaperone-like function in assembling the Rieske protein with the other subunits of the complex. Both postulated functions are consistent with the localization of BCS1 in mitochondria. To facilitate further studies on this novel protein, BCS1 was cloned by transformation of a bcs1 mutant and its structure determined. The primary structure of the encoded BCS1 protein bears similarity to a group of proteins that have been implicated in intracellular protein sorting, membrane fusion and regulation of transcription. The region of BCS1 homologous to this diverse group of proteins is approximately 200 amino acids long and includes several signature sequences commonly found in ATPases and nucleotide binding proteins.
The EMBO Journal 12/1992; 11(11):3821-9. · 9.82 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The insert-containing, non-canonical ori 6 region of yeast mitochondrial DNA of Saccharomyces cerevisiae was dissected into 15 different segments that were ligated to the integrative yeast vector YIp5. Six recombinant plasmids exhibited replicative ability in yeast and carried consensus sequences similar to the previously described 11 bp motifs active as autonomous replication sequences (ARS). In addition, all active constructions carry one or more of the characteristic GC-rich domains A, B or C present in the ori 6 region, thus confirming and expanding the study of Blanc (Gene 30 (1984) 47-61) with the canonical ori 5. Also a new transcriptional origin is activated in the ori 6 region, apparently circumventing a disruption by insertion of a GC-rich sequence that, in this ori, removes the mitochondrial promoter usually present next to the C element. The ARS-positive constructions correspond to the retained segments of spontaneous well-characterized suppressive or neutral petite genomes that contain segments of the ori sequence.
[Show abstract][Hide abstract] ABSTRACT: Respiratory-defective mutants of Saccharomyces cerevisiae assigned to pet complementation group G19 lack cytochrome oxidase activity and cytochromes a and a3. The enzyme deficiency is caused by recessive mutations in the nuclear gene COX10. Analyses of cytochrome oxidase subunits suggest that the product of COX10 provides an essential function at a posttranslational stage of enzyme assembly. The wild type COX10 gene has been cloned by transformation of a mutant from complementation group G19 with a yeast genomic library. Based on the nucleotide sequence of COX10, the primary translation product has an Mr of 52,000. The amino-terminal 190 residues constitute a hydrophilic domain while the carboxyl-terminal region is hydrophobic and has nine potential membrane-spanning segments. The sequence of the carboxyl-terminal hydrophobic region is homologous to an unidentified protein encoded by a reading frame (ORF1) located in one of the cytochrome oxidase operons of Paracoccus denitrificans. The two proteins share 24% identical residues and exhibit very similar hydrophobicity profiles. The bacterial homolog, however, lacks the hydrophilic amino-terminal region of the yeast protein.
Journal of Biological Chemistry 09/1990; 265(24):14220-6. · 4.65 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Seven MboI fragments spanning the mitochondrial apocytochrome b gene in Saccharomyces cerevisiae strain D273-10B were cloned in the BamHI site of the integrative yeast vector YIp5 and the capacity for autonomous replication was subsequently assayed in yeast. The positive correlation found between the ars-like activity in four fragments and the presence of regions common to multiple ethidium bromide-induced petite (rho-) genomes suggests that the mitochondrial sequences possibly active as origins of replication in low-complexity neutral or weakly suppressive rho- mutants could be functionally related to the yeast nuclear replicator 11 nucleotide motif defined by Broach et al. (1983).
Current Genetics 02/1987; 12(8):583-9. · 2.41 Impact Factor