Annexin A1 subcellular expression in laryngeal squamous cell carcinoma.

AIMS: 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. METHODS AND RESULTS: 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. CONCLUSIONS: ANXA1 dysregulation was observed early in laryngeal carcinogenesis, in intra-epithelial neoplasms; it was not found related to prognostic parameters, such as nodal metastases.

Biofilm microbial communities of denture stomatitis.

INTRODUCTION: 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. 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. RESULTS: 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. CONCLUSION: The results revealed the extent of the microbial flora, suggesting the existence of distinct biofilms in healthy subjects and in patients with denture stomatitis.

YAH1 of Saccharomyces cerevisiae: a new essential gene that codes for a protein homologous to human adrenodoxin.

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.

Nucleotide substitutions in a yeast mitochondria cis-acting mutant located in the last intron of the apocytochrome b gene.

The region of mitochondrial DNA corresponding to the intron mutant M6-200 in Saccharomyces cerevisiae D273-10B has been isolated, and the nucleotide sequence of a 519 bp RsaI fragment has been determined. Three nucleotide substitutions were found at nucleotides +2650 (G----T), +2668 (G----A) and +2798 (A----G), all within the genetically defined location in the gene. Particular significance can be attributed to the first two changes (+2650 and +2668), that can be genetically isolated from the third substitution and, in addition, alter conserved sequence features detected in a study [(1982) Biochimie 64, 867-881] of fungal mitochondrial introns.

Revision of the nucleotide sequence at the last intron of the mitochondrial apocytochrome b gene in Saccharomyces cerevisiae.

The nucleotide sequence around a G + C-rich cluster in the last intron of the mitochondrial apocytochrome b gene (F.G. Nóbrega and A. Tzagoloff, Journal of Biological Chemistry, 255: 9828-9837, 1980) was revised when restriction digests failed to show a predicted Rsa I site at position +2769. The corrected sequence has four additional nucleotides, one Hae III and two Hpa II sites. Previously undetected typographical errors were also found in the A + T-rich sequence.

Study of a region on yeast chromosome XIII that complements pet G199 mutants (COX7) and carries a new non-essential gene.

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.

Genomics and proteomics approaches to the study of cancer-stroma interactions.

BACKGROUND: 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. METHODS: 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. RESULTS: 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. CONCLUSIONS: 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.

Bacterial diversity in aphthous ulcers.

INTRODUCTION: 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. METHODS: 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. RESULTS: 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. CONCLUSION: 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.

Mapping and sequencing of the wild-type and mutant (G116-40) alleles of the tyrosyl-tRNA mitochondrial gene in Saccharomyces cerevisiae.

The Saccharomyces cerevisiae syn- mitochondrial mutant G116-40 isolated by Berlani et al. (Berlani, R. E., Pentella, C., Macino, G., and Tzagoloff, A. (1980) J. Bacteriol. 141, 1086-1097) is shown to have a mutation in the tyrosyl-tRNA gene by genetic data combined with restriction analysis and DNA sequencing of the appropriate rho- mitochondrial DNAs derived from wild-type and mutant strains. The new region sequenced spans 685 base pairs located between 9.5 and 10.4 map units, the gene being located at 10.0 units. The tRNA structure, as deduced from the DNA sequence, is in agreement with the data derived from sequencing the purified tyrosyl-tRNA reported by Sibler et al. (Sibler, A., Dirheimer, G., and Martin, R.P. (1983) FEBS Lett. 152, 153-156). No in vitro tyrosyl-tRNA aminoacylation could be detected using mitochondrial RNA from the mutant. S1 nuclease mapping experiments showed that the mutant produces a transcript that is identical to the wild-type at its 5'-end. The same analysis carried out with the mitochondrial RNA from a rho- strain with the tyrosyl-tRNA region of mitochondrial DNA reveals a 5'-end shorter by about 3 nucleotides. The mutant gene has a single substitution (C----T) at the penultimate nucleotide near the 3'-end of the molecule creating an acceptor stem that lacks the two terminal Watson-Crick base pairs.

Mapping of the ARS-like activity and transcription initiation sites in the non-canonical yeast mitochondrial ori 6 region.

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.

Assembly of the mitochondrial membrane system. Complete restriction map of the cytochrome b region of mitochondrial DNA in Saccharomyces cerevisiae D273-10B.

The cytoplasmic petite (rho-) mutant DS400/A12 has been obtained from the wild type strain of Saccharomyces cerevisiae D273-10B/A21. The DS400/A12 clone has a mitochondrial genome with a 7.6-kilobase pair, tandemly repeated segment of DNA. Genetic tests indicate that DS400/A12 contains all the cob1 and cob2 markers of the cytochrome b gene. The gene has been further dissected by mutagenesis of DS400/A12 and selection of secondary rho- clones with simpler genotypes. Restriction analysis of the mtDNAs of the rho- clones was used to construct the complete restriction map of the cytochrome b region and to map the mutations within narrowly defined physical limits. The cytochrome b mutants scatter over a maximal distance of 3.3 kilobase pairs. All the mutations assigned previously to the cob2 locus are found between 71.6 and 73.2 units. The cob1 mutations are located between 74.6 and 76.3 units. The estimated distance between the two loci is at least 1 kilobase pair.

Assembly of the mitochondrial membrane system. DNA sequence and organization of the cytochrome b gene in Saccharomyces cerevisiae D273-10B.

The mitochondrial genomes of cytoplasmic "petite" (rho-) mutants of Saccharomyces cerevisiae have been used to sequence the cytochrome b gene. A continuous sequence of 6.2 kilobase pairs has been obtained from 71.4 to 80.2 units of the wild type map. This region contains all the cytochrome b mutations previously assigned to the cob1 and cob2 genetic loci. Analysis of the DNA sequence has revealed that in the strain D273-10B, the cytochrome b gene is composed of three exons. The longest exon (b1) codes for the first 252 to 253 amino acids from the NH2-terminal end of the protein. The next two exons (b2 and b3) code for 16 to 18 and 115 to 116 amino acids, respectively. The complete cytochrome b polypeptide chain consists of 385 amino acids. Based on the amino acid composition, the yeast protein has a molecular weight of 44,000. The three exon regions of the cytochrome b gene are separated by two introns. The intron between b1 and b2 is 1414 nucleotides long and contains a reading frame that is continuous with the reading frame of exon b1. This intron sequence is potentially capable of coding for another protein of 384 amino acid residues. The second intron is 733 nucleotides long. This sequence is rich in A + T and includes a G + C cluster that may be involved in processing of the cytochrome b messenger. The organization of the cytochrome b region in S. cerevisiae D273-10B is somewhat less complex than has been reported for other yeast strains i which exon b1 appears to be further fragmented into three smaller exons.

Proteolytic cleavage of horse liver cytochrome b5. Primary structure of the heme-containing moiety.

The amino acid sequence of the NH2-terminal segment of horse cytochrome b5, containing the heme binding site, has been determined. A fragment, representing residues 7 through 90, was obtained by tryptic cleavage of native cytochrome b5. Chymotryptic cleavage of native cytochrome b5 yields a peptide containing residues 1 through 98. Contrary to native cytochrome b5, neither derivative showed binding to horse liver microsomal vesicles. The complete primary structure of the polar moiety has been deducted from automated and manual sequence analysis of peptides obtained from tryptic and chymotryptic digests of native cytochrome and apocytochrome preparations. Glutamyl residues at positions 41, 42, 47, and 48 appear to be replaced by aspartyl residues in some molecules. Such microheterogeneity is not observed at glutamyl residues at other positions. The native cytochrome b5 preparation contains a blocked NH2-terminal residue.

ARS activity along the yeast mitochondrial apocytochrome b region: correlation with the location of petite genomes and consensus sequences.

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).

COX10 codes for a protein homologous to the ORF1 product of Paracoccus denitrificans and is required for the synthesis of yeast cytochrome oxidase.

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.

A rapid method for detecting specific RNA transcripts by hybridization to DNA probes in solution.

A method is described for detecting specific transcripts in crude mixtures of RNA. The method employs hybridization of single-stranded or double-stranded radioactive DNA probes in solution, followed by electrophoretic separation of the hybrid and probe on agarose and visualization by radioautography. The procedure offers the advantages of decreased preparation time and increased sensitivity over currently used methods.

BCS1, a novel gene required for the expression of functional Rieske iron-sulfur protein in Saccharomyces cerevisiae.

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.

ARH1 of Saccharomyces cerevisiae: a new essential gene that codes for a protein homologous to the human adrenodoxin reductase.

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.