RESUMO
BACKGROUND: Salicylic acid (SA) is a major plant hormone that mediates the defence pathway against pathogens. SA accumulates in highly variable amounts depending on the plant-pathogen system, and several enzyme activities participate in the restoration of its levels. Gentisic acid (GA) is the product of the 5-hydroxylation of SA, which is catalysed by S5H, an enzyme activity regarded as a major player in SA homeostasis. GA accumulates at high levels in tomato plants infected by Citrus Exocortis Viroid (CEVd), and to a lesser extend upon Pseudomonas syringae DC3000 pv. tomato (Pst) infection. RESULTS: We have studied the induction of tomato SlS5H gene by different pathogens, and its expression correlates with the accumulation of GA. Transient over-expression of SlS5H in Nicotiana benthamiana confirmed that SA is processed by SlS5H in vivo. SlS5H-silenced tomato plants were generated, displaying a smaller size and early senescence, together with hypersusceptibility to the necrotrophic fungus Botrytis cinerea. In contrast, these transgenic lines exhibited an increased defence response and resistance to both CEVd and Pst infections. Alternative SA processing appears to occur for each specific pathogenic interaction to cope with SA levels. In SlS5H-silenced plants infected with CEVd, glycosylated SA was the most discriminant metabolite found. Instead, in Pst-infected transgenic plants, SA appeared to be rerouted to other phenolics such as feruloyldopamine, feruloylquinic acid, feruloylgalactarate and 2-hydroxyglutarate. CONCLUSION: Using SlS5H-silenced plants as a tool to unbalance SA levels, we have studied the re-routing of SA upon CEVd and Pst infections and found that, despite the common origin and role for SA in plant pathogenesis, there appear to be different pathogen-specific, alternate homeostasis pathways.
Assuntos
Solanum lycopersicum , Solanum lycopersicum/genética , Ácido Salicílico , Gentisatos , Pseudomonas syringaeRESUMO
BACKGROUND AND OBJECTIVE: Cells with osteoprogenitor potential are present within periodontal tissues during development and in postnatal life. To identify an osteoprogenitor population, this study utilized a transgenic model in which an alpha-smooth muscle actin (alphaSMA) promoter directed green fluorescent protein (GFP) expression. MATERIAL AND METHODS: Observation of GFP expression was complemented with analysis of osteogenic differentiation by determining the expression of RNA of bone markers, by histochemical staining for alkaline phosphatase and by the detection of mineralized nodules using xylenol orange. Flow cytometry was utilized to determine the proliferative potential and cell-surface phenotype of cultured alphaSMA-positive cells. RESULTS: alphaSMA-GFP expression was detected within the dental follicle and in the apical region of the root (i.e. areas rich in vascularization) but not in mature bone. alphaSMA-GFP expression was observed during the early stages of primary cultures derived from the dental follicle and periodontal ligament and was diminished in areas undergoing mineralization. Intense alkaline phosphatase activity and the presence of mineralized nodules was observed 2 wk after osteogenic induction. Consequently, the expression of bone sialoprotein, osteocalcin and dentin matrix protein-1 was increased. Flow cytometry revealed that in vitro expansion enriched for an alphaSMA-GFP-positive population in which 55-65% of cells expressed the cell-surface markers Thy1(+) and Sca1(+). The alphaSMA-GFP-positive population exhibited high proliferative and osteogenic potentials when compared with an alphaSMA-GFP-negative population. CONCLUSION: Our data indicate that the alphaSMA promoter can be used to identify a population of osteoprogenitor cells residing within the dental follicle and periodontal ligament that can differentiate into mature osteoblasts.
Assuntos
Actinas/análise , Processo Alveolar/citologia , Periodonto/citologia , Células-Tronco/citologia , Fosfatase Alcalina/análise , Animais , Antígenos Ly/análise , Biomarcadores/análise , Calcificação Fisiológica/fisiologia , Diferenciação Celular/fisiologia , Proliferação de Células , Saco Dentário/citologia , Proteínas da Matriz Extracelular/análise , Proteínas de Fluorescência Verde , Sialoproteína de Ligação à Integrina , Substâncias Luminescentes , Proteínas de Membrana/análise , Camundongos , Camundongos Transgênicos , Odontoblastos/citologia , Osteocalcina/análise , Osteócitos/citologia , Osteogênese/fisiologia , Ligamento Periodontal/citologia , Fenótipo , Fosfoproteínas/análise , RNA/análise , Sialoglicoproteínas/análise , Antígenos Thy-1/análise , Ápice Dentário/citologiaAssuntos
Desenvolvimento Ósseo/genética , Linhagem da Célula/genética , Fibroblastos/metabolismo , Mioblastos/metabolismo , Pericitos/metabolismo , Células-Tronco/metabolismo , Actinas/genética , Actinas/metabolismo , Animais , Osso e Ossos/citologia , Osso e Ossos/metabolismo , Diferenciação Celular/genética , Separação Celular/métodos , Células Cultivadas , Fibroblastos/citologia , Regulação da Expressão Gênica no Desenvolvimento/genética , Genes Reporter/genética , Proteínas de Fluorescência Verde/genética , Mesoderma/citologia , Mesoderma/embriologia , Mesoderma/metabolismo , Camundongos , Camundongos Transgênicos , Mioblastos/citologia , Osteoblastos/citologia , Osteoblastos/metabolismo , Pericitos/citologia , Fenótipo , Regiões Promotoras Genéticas/genética , Células Estromais/citologia , Células Estromais/metabolismoRESUMO
Retinoic acid is a potent inducer of tissue-non-specific alkaline phosphatase (TNSALP) expression in various osteoblastic and fibroblastic cells, and may be involved in morphogenesis, cellular growth and differentiation. This study investigates the effects of retinoic acid on alkaline phosphatase activity and TNSALP gene expression in human dental pulp cells. Cultured cells were treated with various concentrations of retinoic acid (0, 10(-7), 10(- 6), 10 (-5) M) in 0.5% bovine serum albumin without serum. Alkaline phosphatase activity was determined by the rate of p-nitrophenyl phosphate hydrolysis and was also assayed in the presence of various inhibitors and under thermal inactivation. A set of specific oligonucleotide primers was selected, based on the nucleotide sequences of two human TNSALP mRNA (bone and liver) types, and reverse transcription-polymerase chain reaction (RT-PCR) performed. Inhibitory and thermal inactivation experiments revealed that the elevated alkaline phosphatase activity had properties of the TNSALP type. RT-PCR showed that retinoic acid enhanced the expression of bone-type TNSALP mRNA in pulp cells. However, the liver-type TNSALP mRNA was not detected. These findings suggest that the high alkaline phosphatase activity of retinoic acid-treated dental pulp cells is associated with increased transcription of the bone-type mRNA of the TNSALP gene and not with liver-type.
Assuntos
Fosfatase Alcalina/efeitos dos fármacos , Fosfatase Alcalina/genética , Polpa Dentária/efeitos dos fármacos , Polpa Dentária/enzimologia , Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , RNA Mensageiro/efeitos dos fármacos , RNA Mensageiro/genética , Tretinoína/farmacologia , Fosfatase Alcalina/análise , Fosfatase Alcalina/biossíntese , Análise de Variância , Sequência de Bases , Células Cultivadas , Primers do DNA , Polpa Dentária/citologia , Relação Dose-Resposta a Droga , Indução Enzimática/efeitos dos fármacos , Regulação Enzimológica da Expressão Gênica/fisiologia , Humanos , Dados de Sequência Molecular , RNA Mensageiro/metabolismo , Valores de Referência , Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodos , Reação em Cadeia da Polimerase Via Transcriptase Reversa/estatística & dados numéricos , Especificidade por Substrato/efeitos dos fármacos , Fatores de TempoRESUMO
Alkaline phosphatase (ALP) in human periodontal ligament (HPDL) cells is classified as a tissue-non-specific alkaline phosphatase (TNSALP) by its enzymatic and immunological properties. Since retinoic acid (RA) has been shown as a potent inducer of TNSALP expression in various osteoblastic and fibroblastic cells, we investigated the effects of RA on the level of ALP activity and expression of TNSALP mRNAs in HPDL cells. Cultured cells were treated with desired RA concentrations (0, 10(-7), 10(-6), 10(-5) M) in medium containing 1% bovine serum albumin without serum. ALP activity was determined by the rate of hydrolysis of p-nitrophenyl phosphate and was also assayed in the presence of specific inhibitors. In order to identify the TNSALP mRNA type expressed by HPDL, a set of oligonucleotide primers corresponding to 2 types of human TNSALP mRNA (i.e. bone-type and liver-type) were designed, and mRNA isolated from HPDL was amplified by means of reverse transcription-polymerase chain reaction (RT-PCR). After treatment with RA (10(-6) M) for 4 d, there was a significant increase in the ALP activity of HPDL cells. The use of inhibitors and thermal inactivation experiments showed that the increased ALP activity had properties of the TNSALP type. RT-PCR analysis revealed that bone-type mRNA was highly stimulated in HPDL cells by RA treatment, but the expression of liver-type mRNA was not detected. These results indicated that the upregulation of ALP activity in HPDL cells by RA was due to the increased transcription of bone-type mRNA of the TNSALP gene.
