A PCR-RFLP assay for gender assignment in the three-toed sloths (Bradypus, Pilosa, Bradypodidae)

The three-toed sloths (Bradypus) are slow-moving arboreal neotropical mammals. Understanding demographic variables (such as sex ratio) of populations is a key for conservation purposes. Nevertheless, gender assignment of Bradypus is particularly challenging because of the lack of sexual dimorphism in infants and in adults, particularly B. torquatus, the most endangered of the three-toed sloths, in which sex is attributed by visual observation of the reproductively active males. Here, we standardized a method for sexing Bradypus individuals using PCR-RFLP of sex-linked genes ZFX/ZFY. This assay was validated with known-gender animals and proved accurate to assign gender on three Bradypus species.

The R2 mobile element of Rhynchosciara americana: Molecular, cytological and dynamic aspects

Ribosomal RNA genes are encoded by large units clustered (18S, 5S, and 28S) in the nucleolar organizer region in several organisms. Sometimes additional insertions are present in the coding region for the 28S rDNA. These insertions are specific non-long terminal repeat retrotransposons that have very restricted integration targets within the genome. The retrotransposon present in the genome of Rhynchosciara americana, RaR2, was isolated by the screening of a genomic library. Sequence analysis showed the presence of conserved regions, such as a reverse transcriptase domain and a zinc finger motif in the amino terminal region. The insertion site was highly conserved in R. americana and a phylogenetic analysis showed that this element belongs to the R2 clade. The chromosomal localization confirmed that the RaR2 mobile element was inserted into a specific site in the rDNA gene. The expression level of RaR2 in salivary glands during larval development was determined by quantitative RT-PCR, and the increase of relative expression in the 3P of the fourth instar larval could be related to intense gene activity characteristic of this stage. 5`-Truncated elements were identified in different DNA samples. Additionally, in three other Rhynchosciara species, the R2 element was present as a full-length element.

Gene expression profiling reveals molecular marker candidates of laryngeal squamous cell carcinoma

Laryngeal squamous cell carcinoma is very common in head and neck cancer, with high mortality rates and poor prognosis. In this study, we compared expression profiles of clinical samples from 13 larynx tumors and 10 non-neoplastic larynx tissues using a custom-built cDNA microarray containing 331 probes for 284 genes previously identified by informatics analysis of EST databases as markers of head and neck tumors. Thirty-five genes showed statistically significant differences (SNR >= 11.01, p <= 0.001) in the expression between tumor and non-tumor larynx tissue samples. Functional annotation indicated that these genes are involved in cellular processes relevant to the cancer phenotype, such as apoptosis, cell cycle, DNA repair, proteolysis, protease inhibition, signal transduction and transcriptional regulation. Six of the identified transcripts map to intronic regions of protein-coding genes and may comprise non-annotated exons or as yet uncharacterized long ncRNAs with a regulatory role in the gene expression program of larynx tissue. The differential expression of 10 of these genes (ADCY6, AES, AL2SCR3, CRR9, CSTB, DUSP1, MAP3K5, PLAT, UBL1 and ZNF706) was independently confirmed by quantitative real-time RT-PCR. Among these, the CSTB gene product has cysteine protease inhibitor activity that has been associated with an antimetastatic function. Interestingly, CSTB showed a low expression in the tumor samples analyzed (p<0.0001). The set of genes identified here contribute to a better understanding of the molecular basis of larynx cancer, and provide candidate markers for improving diagnosis, prognosis and treatment of this carcinoma.

Overexpression of Nrp/b (nuclear restrict protein in brain) suppresses the malignant phenotype in the C6/ST1 glioma cell line

Upon searching for glucocorticoid-regulated cDNA sequences associated with the transformed to normal phenotypic reversion of C6/ST1 rat glioma cells, we identified Nrp/b (nuclear restrict protein in brain) as a novel rat gene. Here we report on the identification and functional characterization of the complete sequence encoding the rat NRP/B protein. The cloned cDNA presented a 1767 nucleotides open-reading frame encoding a 589 aminoacids residues sequence containing a BTB/POZ (broad complex Tramtrack bric-a-brac/Pox virus and zinc finger) domain in its N-terminal region and kelch motifs in its C-terminal region. Sequence analysis indicates that the rat Nrp/b displays a high level of identity with the equivalent gene orthologs from other organisms. Among rat tissues, Nrp/b expression is more pronounced in brain tissue. We show that overexpression of the Nrp/b cDNA in C6/ST1 cells suppresses anchorage independence in vitro and tumorigenicity in vivo, altering their malignant nature towards a more benign phenotype. Therefore, Nrp/b may be postulated as a novel tumor suppressorgene, with possible relevance for glioblastoma therapy. (C) 2009 Elsevier Ltd. All rights reserved.

Análise do perfil de expressão dos genes da cana-de-açúcar envolvidos na interação com Leifsonia xyli subsp: xyli

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

An interaction between two RNA binding proteins, Nab2 and Pub1, links mRNA Processing/Export and mRNA stability

mRNA stability is modulated by elements in the mRNA transcript and their cognate RNA binding proteins. Poly(U) binding protein 1 (Pub1) is a cytoplasmic Saccharomyces cerevisiae mRNA binding protein that stabilizes transcripts containing AU-rich elements (AREs) or stabilizer elements (STEs). In a yeast two-hybrid screen, we identified nuclear poly(A) binding protein 2 (Nab2) as being a Pub1-interacting protein. Nab2 is an essential nucleocytoplasmic shuttling mRNA binding protein that regulates poly(A) tail length and mRNA export. The interaction between Pub1 and Nab2 was confirmed by copurification and in vitro binding assays. The interaction is mediated by the Nab2 zinc finger domain. Analysis of the functional link between these proteins reveals that Nab2, like Pub1, can modulate the stability of specific mRNA transcripts. The half-life of the RPS16B transcript, an ARE-like sequence-containing Pub1 target, is decreased in both nab2-1 and nab2-67 mutants. In contrast, GCN4, an STE-containing Pub1 target, is not affected. Similar results were obtained for other ARE- and STE-containing Pub1 target transcripts. Further analysis reveals that the ARE-like sequence is necessary for Nab2-mediated transcript stabilization. These results suggest that Nab2 functions together with Pub1 to modulate mRNA stability and strengthen a model where nuclear events are coupled to the control of mRNA turnover in the cytoplasm.

Mudanças dimensionais dos arcos dentários em crianças entre 3 e 6 anos de idade

Para o presente trabalho, 235 crianças pertencentes às creches da Prefeitura Municipal de Araraquara foram avaliadas e moldadas com um dispositivo confeccionado com cera utilidade e palito abaixador de língua na forma dos arcos dentários. No intervalo de um ano as mesmas crianças foram remoldadas afim de se verificar se houveram ou não mudanças nas dimensões do arco dentário decíduo. A partir da obtenção dos modelos em gesso, foram realizadas medições por meio de um dispositivo digitalizador tridimensional denominado MicroScribe-3DX nos instantes inicial (primeira moldagem) e final (moldagem após um ano). Foram avaliadas medidas referentes às distâncias inter-segundos molares, interprimeiros molares, intercaninos, perímetro, comprimento de arco e espaços primatas. Consideraram-se ainda dimorfismo sexual, tipo de arco e influência de hábitos. Concluiu-se que as dimensões transversais sofreram aumento significativo na dentadura decídua, enquanto que o perímetro, o comprimento e os espaços primatas permaneceram constantes. As distâncias intermolares apresentaram dimorfismo sexual, com dimensões maiores no gênero feminino. Com relação às medidas de perímetro, comprimento e espaços primatas não ocorreram diferenças significantes em relação ao gênero. As dimensões de comprimento nos arcos não diferem nos arcos decíduos Tipo I e Tipo II de Baume, enquanto que o diâmetro dos arcos Tipo I de Baume, são maiores do que os do Tipo II. Os hábitos de sucção de dedo e chupeta não provocaram alterações nas dimensões dos arcos decíduos no período observado, de um ano.

Amino acid sequence and crystal structure of BaP1, a metalloproteinase from Bothrops asper snake venom that exerts multiple tissue-damaging activities

BaP1 is a 22.7-kD P-I-type zinc-dependent metalloproteinase isolated from the venom of the snake Bothrops asper, a medically relevant species in Central America. This enzyme exerts multiple tissue-damaging activities, including hemorrhage, myonecrosis, dermonecrosis, blistering, and edema. BaP1 is a single chain of 202 amino acids that shows highest sequence identity with metalloproteinases isolated front the venoms of snakes of the subfamily Crotalinae. It has six Cys residues involved in three disulfide bridges (Cys 117-Cys 197, Cys 159-Cys 181, Cys 157-Cys 164). It has the consensus sequence H(142)E(143)XXH(146)XXGXXH(152), as well as the sequence C164I165M166, which characterize the metzincin superfamily of metalloproteinases. The active-site cleft separates a major subdomain (residues 1-152), comprising four a-helices and a five-stranded beta-sheet, from the minor subdomain, which is formed by a single a-helix and several loops. The catalytic zinc ion is coordinated by the N-epsilon2 nitrogen atoms of His 142, His 146, and His 152, in addition to a solvent water molecule, which in turn is bound to Glu 143. Several conserved residues contribute to the formation of the hydrophobic pocket, and Met 166 serves as a hydrophobic base for the active-site groups. Sequence and structural comparisons of hemorrhagic and nonhemorrhagic P-I metalloproteinases from snake venoms revealed differences in several regions. In particular, the loop comprising residues 153 to 176 has marked structural differences between metalloproteinases with very different hemorrhagic activities. Because this region lies in close proximity to the active-site microenvironment, it may influence the interaction of these enzymes with physiologically relevant substrates in the extracellular matrix.

The contribution of transposable elements to Bos taurus gene structure

In an effort to identify the contribution of TEs to bovine genome evolution, the abundance, distribution and insertional orientation of TEs were examined in all bovine nuclear genes identified in sequence build 2.1 (released October 11, 2005). Exons, introns and promoter segments (3 kb upstream the transcription initiation sites) were screened with the RepeatMasker program. Most of the genes analyzed contained TE insertions, with an average of 18 insertions/gene. The majority of TE insertions identified were classified as retrotransposons and the remainder classified as DNA transposons. TEs were inserted into exons and promoter segments infrequently, while insertion into intron sequences was strikingly more abundant. The contribution of TEs to exon sequence is of great interest because TE insertions can directly influence the phenotype by altering protein sequences. We report six cases where the entire exon sequences of bovine genes are apparently derived from TEs and one of them, the insertion of Charlie into a bovine transcript similar to the zinc finger 452 gene is analyzed in detail. The great similarity of the TE-cassette sequence to the ZNF452 protein and phylogenetic relationship strongly suggests the occurrence of Charlie 10 DNA exaptation in the mammalian zinc finger 452 gene. (c) 2006 Published by Elsevier B.V.

Expression, purification and molecular analysis of the human ZNF706 protein

Background: The ZNF706 gene encodes a protein that belongs to the zinc finger family of proteins and was found to be highly expressed in laryngeal cancer, making the structure and function of ZNF706 worthy of investigation. In this study, we expressed and purified recombinant human ZNF706 that was suitable for structural analysis in Escherichia coli BL21(DH3). Findings. ZNF706 mRNA was extracted from a larynx tissue sample, and cDNA was ligated into a cloning vector using the TOPO method. ZNF706 protein was expressed according to the E. coli expression system procedures and was purified using a nickel-affinity column. The structural qualities of recombinant ZNF706 and quantification alpha, beta sheet, and other structures were obtained by spectroscopy of circular dichroism. ZNF706's structural modeling showed that it is composed of α-helices (28.3%), β-strands (19.4%), and turns (20.9%), in agreement with the spectral data from the dichroism analysis. Conclusions: We used circular dichroism and molecular modeling to examine the structure of ZNF706. The results suggest that human recombinant ZNF706 keeps its secondary structures and is appropriate for functional and structural studies. The method of expressing ZNF706 protein used in this study can be used to direct various functional and structural studies that will contribute to the understanding of its function as well as its relationship with other biological molecules and its putative role in carcinogenesis. © 2013 Colombo et al.; licensee BioMed Central Ltd.

Mudanças dimensionais dos arcos dentários em crianças entre 3 e 6 anos de idade

Pós-graduação em Odontologia - FOAR

Análise funcional e estrutural da proteína Pub 1 de Saccharomyces cerevisiae

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

O gene PHF21B como candidato a predisposição familia em familial em cânceres de cabeça e pescoço

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Expressão gênica diferencial por cDNA-AFLP em raízes de cana-de-açúcar submetida ao déficit hídrico

Pós-graduação em Agronomia (Genética e Melhoramento de Plantas) - FCAV