RESUMO
Telomerase is essential for the maintenance of telomeres, structures located at the ends of linear eukaryotic chromosomes that are crucial for genomic stability. Telomerase has been frequently explored in mammals because of its activity in many types of cancers, but knowledge in plants is rather sketchy despite plants representing useful models due to peculiarities in their telomeres and telomerase biology. We studied in planta complementation of telomerase in Arabidopsis thaliana mutant plants with disrupted expression of the gene encoding the telomerase protein subunit (AtTERT) and significantly shortened telomeres. We found that the upstream region of AtTERT, previously identified as a putative minimal promoter, was essential for reconstitution of telomerase function, as demonstrated by the full or partial recovery of the telomere phenotype in mutants. In contrast, transformation by the full length AtTERT gene construct resulted in more progressive telomere shortening in mutants and even in wild type plants, despite the high level of AtTERT transcript and telomerase activity detected by in vitro assay. Thus, the telomerase protein subunit putative promoter is essential for in planta telomerase reconstitution and restoration of its catalytical activity. Contributions from other factors, including those tissue-specific, for proper telomerase function are discussed.
Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Telomerase/metabolismo , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Regiões Promotoras Genéticas/genética , Telomerase/genéticaRESUMO
Exposure to ultraviolet (UV) irradiation has detrimental effects on skin accompanied by the increased metabolism of hyaluronan (HA), a linear polysaccharide important for the normal physiological functions of skin. In this study, the modulation of human keratinocyte response to UVB irradiation by HA (970 kDa) was investigated. Immortalized human keratinocytes (HaCaT) were irradiated by a single dose of UVB and immediately treated with HA for 6 and 24 h. The irradiation induced a significant decrease in the gene expression of CD44 and toll-like receptor 2 6 h after irradiation. The expressions of other HA receptors, including toll-like receptor 4 and the receptor for HA-mediated motility, were not detected in either the control or UVB-irradiated or HA-treated HaCaT cells. UVB irradiation induced a significant decrease in the gene expression of HA synthase-2 and hyaluronidase-2 6 h after irradiation. The expressions of HA synthase-3 and hyaluronidase-3 were not significantly modulated by UV irradiation. Interestingly, HA treatment did not significantly modulate any of these effects. In contrast, HA significantly suppressed UVB-induced pro-inflammatory cytokine release including interleukin-6 and interleukin-8. Similarly, HA treatment reduced the UVB-mediated production of transforming growth factor ß1. HA treatment also significantly reduced the UV irradiation-mediated release of soluble CD44 into the media. Finally, HA partially, but significantly, suppressed the UVB-induced decrease in cell viability. Data indicate that HA had significant protective effects for HaCaT cells against UVB irradiation.
Assuntos
Ácido Hialurônico/farmacologia , Queratinócitos/efeitos dos fármacos , Pele/efeitos dos fármacos , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Expressão Gênica , Glucuronosiltransferase/biossíntese , Glucuronosiltransferase/genética , Humanos , Receptores de Hialuronatos/biossíntese , Receptores de Hialuronatos/genética , Hialuronan Sintases , Ácido Hialurônico/metabolismo , Hialuronoglucosaminidase/biossíntese , Hialuronoglucosaminidase/genética , Interleucina-6/biossíntese , Interleucina-8/biossíntese , Queratinócitos/metabolismo , Queratinócitos/efeitos da radiação , Pele/metabolismo , Pele/efeitos da radiação , Receptor 2 Toll-Like/biossíntese , Receptor 2 Toll-Like/genética , Fator de Crescimento Transformador beta/biossíntese , Raios UltravioletaRESUMO
Reactive oxygen and nitrogen species are among the crucial mediators in the development of the pathological inflammatory process in the lungs and contribute to the damage of lung epithelium. The aim of the present study was to evaluate the potential of selected antioxidants or inhibitors of NADPH oxidase (glutathione, N-acetyl cysteine, trolox, apocynin, and diphenyleneiodonium chloride) to modulate nitric oxide (NO) production and inducible nitric oxide synthase (iNOS) expression by mouse macrophages induced by lipopolysaccharide (LPS) in vitro and to evaluate the potential of apocynin to modulate the course of LPS-induced lung inflammation in vivo. All the tested drugs revealed inhibitory effects on LPS-induced NO production and iNOS expression in RAW 264.7 macrophages. Further, apocynin significantly inhibited activation of nuclear factor kappa B induced by LPS. Ex vivo, diphenyleneiodonium chloride and apocynin significantly reduced ROS production by inflammatory cells isolated from bronchoalveolar lavage fluid. In contrast, in vivo intranasal application of apocynin did not exert any significant effect on the course of lung inflammation in mice induced by LPS that was evaluated based on the accumulation of cells, interleukine-6, interleukine-12, RANTES, tumor necrosis factor-alpha, and protein concentration in bronchoalveolar lavage fluid and expression of iNOS in lung tissue. Only effected were the levels of nitrites 36 h after induction of lung inflammation that were reduced in the apocynin-treated group. In conclusion, our data suggest that the inhibitors of NADPH oxidase possess inhibitory potential against LPS-induced NO production by mouse macrophages; however, apocynin failed to reduce LPS-induced lung inflammation in mice.
