RESUMO
OBJECTIVE: We investigated early biological events initiated by Porphyromonas gingivalis infection of human osteoblasts, focusing on tyrosine-phosphorylation and the expression of key components in focal adhesion and cell signalling. DESIGN: Human primary osteoblasts were challenged for 1h with Porphyromonas gingivalis. Tyrosine-phosphorylation of paxillin and focal adhesion kinase (FAK) was examined by Western blotting. Changes in alpha3- and beta1-integrin mRNA expression were quantified by RT-PCR. RESULTS: Tyrosine-phosphorylation of paxillin was proportional to the size of the Porphyromonas gingivalis inoculum. FAK, a potential kinase for paxillin, was not activated. The amount of alpha3- and beta1-integrins, determined by Western blotting, did not vary significantly, while the corresponding mRNA levels fell significantly when a large bacterial inoculum was used. CONCLUSIONS: These results indicate that Porphyromonas gingivalis infection of osteoblasts in vitro triggers tyrosine-phosphorylation of paxillin but not FAK and modify alpha3- and beta1-integrin mRNA expression. This infection thus appears to have different effects on components with essential roles in focal adhesion (paxillin) and cell signalling (FAK and integrins).
Assuntos
Infecções por Bacteroidaceae/metabolismo , Integrinas/metabolismo , Osteoblastos/microbiologia , Paxilina/metabolismo , Porphyromonas gingivalis/metabolismo , Tirosina/metabolismo , Aderência Bacteriana , Western Blotting , Células Cultivadas , Proteína-Tirosina Quinases de Adesão Focal/metabolismo , Humanos , Integrina alfa3/genética , Integrina beta1/genética , Osteoblastos/patologia , Fosforilação , RNA Mensageiro/análise , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
We mined the zebrafish genomic sequence database and identified contigs containing segments of several dopamine receptor genes. By using a polymerase chain reaction amplification strategy, we generated full-length cDNAs encoding a single dopamine D3 receptor and three distinct D2 receptor subtypes. Zebrafish dopamine receptor genes were mapped by using the T51 radiation hybrid panel. The D3 receptor gene (drd3) mapped to linkage group (LG) 24. The three D2 receptor genes were localized to LG 15 (drd2a), LG 16, (drd2b), and LG 5 (drd2c). With the exception of the drd2b gene, each of these map positions was syntenic with regions of human chromosomes containing orthologs of the zebrafish dopamine receptor genes. Whole-mount in situ hybridization was used to investigate expression of the D2 and D3 receptor genes. Expression of the drd3 gene was first detected at mid-somitogenesis and was particularly prominent in somites. Thereafter, the drd3 gene was expressed diffusely throughout the brain and spinal cord. The three D2 receptor genes were expressed throughout the central nervous system (CNS) in distinct but overlapping patterns. In early embryos, the drd2a gene was expressed exclusively in the epiphysis, whereas the drd2c gene was localized to the notochord. After 24 hpf, the drd2a, drd2b, and drd2c genes were differentially expressed throughout the CNS. The identification of dopamine receptor genes in zebrafish should allow us to use the power of zebrafish genetics to analyze the functional properties of this important class of neurotransmitter receptors.
Assuntos
Evolução Molecular , Receptores de Dopamina D2/genética , Receptores de Dopamina D2/metabolismo , Peixe-Zebra/genética , Sequência de Aminoácidos , Animais , Sistema Nervoso Central/metabolismo , Mapeamento Cromossômico , Sequência Conservada , Embrião não Mamífero , Éxons , Ligação Genética , Humanos , Íntrons , Dados de Sequência Molecular , Filogenia , Estrutura Terciária de Proteína , Receptores de Dopamina D2/química , Receptores de Dopamina D3 , Homologia de Sequência de Aminoácidos , Somitos/metabolismo , Sintenia , Distribuição TecidualRESUMO
Estrogen-receptor related receptors (ERRalpha, beta and gamma) are so-called orphan (i.e. for which no natural ligand has been identified to date) nuclear receptors that are closely related to the estrogen receptors. To gain insights into the possible functions of ERRs during early development, we have cloned their homologs in the zebrafish. Five err cDNA types were recovered in a PCR screen. Transient transfection experiments indicated that zebrafish ERRalpha, ERRgamma and ERRdelta display transcriptional activities that are identical to those of their mammalian counterparts. The expression patterns of err genes were determined during zebrafish development. Tissues such as the hindbrain or the pronephric tubes express several errs whereas others, such as the presumptive mesoderm at the level of the margin, specifically express a single err. Our analysis also points to tissues in which err expression is conserved through evolution, such as slow muscles that specifically express erralpha, suggesting important functions in these tissues.
Assuntos
Clonagem Molecular , Regulação da Expressão Gênica no Desenvolvimento , Receptores de Estrogênio/genética , Peixe-Zebra/genética , Sequência de Aminoácidos , Animais , Dados de Sequência Molecular , Filogenia , Receptores Citoplasmáticos e Nucleares , Peixe-Zebra/metabolismoRESUMO
Selenium is important for embryogenesis in vertebrates but little is known about the expression patterns and biological functions of most selenoprotein genes. Taking advantage of the zebrafish model, systematic analysis of selenoprotein gene expression was performed by in situ hybridization on whole-mount embryos at different developmental stages. Twenty-one selenoprotein mRNAs were analyzed and all of them exhibited expression patterns restricted to specific tissues. Moreover, we demonstrated that highly similar selenoprotein paralogs were expressed within distinct territories. Therefore, tissue- and development-specific expression patterns provided new information for selenoproteins of unknown function.
