RESUMO
Ferritin is a storage protein that plays a key role in iron metabolism. In this study, we report on the sequence characterization of a ferritin-coding cDNA in Eisenia andrei earthworms isolated by RT-PCR using degenerated primers, and we suggest the presence of a putative IRE in the 5'-UTR of ferritin mRNA. The obtained ferritin sequence was compared with those of other animals showing sequence and structure homology in consensus sites, including the iron-responsive element (IRE) and ferroxidase centers. Despite the sequence homology in the E. andrei mRNA of ferritin with the sequences of other animals in consensus IRE sites, the presented cytosine in the IRE of E. andrei ferritin in the expected position does not form a conventional bulge. The presence of ferritin in the coelomic fluid of E. andrei was proven by iron staining assay. Moreover, aconitase activity in the coelomic fluid was assessed by aconitase assay, suggesting the presence of an iron regulatory protein. Quantitative analysis revealed changes in the gene expression levels of ferritin in coelomocytes in response to bacterial challenge, reaching the maximum level 8h after the stimulation with both Gram-positive and Gram-negative bacteria.
Assuntos
Ferritinas/química , Ferritinas/genética , Oligoquetos/genética , Aconitato Hidratase/análise , Aconitato Hidratase/genética , Aconitato Hidratase/metabolismo , Sequência de Aminoácidos , Animais , Bacillus subtilis/metabolismo , Sequência de Bases , Ceruloplasmina/genética , Clonagem Molecular , Primers do DNA , DNA Complementar/biossíntese , DNA Complementar/genética , DNA Complementar/isolamento & purificação , Escherichia coli K12/metabolismo , Regulação da Expressão Gênica , Dados de Sequência Molecular , Conformação de Ácido Nucleico , Oligoquetos/metabolismo , RNA/genética , RNA/isolamento & purificação , RNA Mensageiro/química , RNA Mensageiro/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Análise de Sequência de DNARESUMO
This study examined the time-dependent effects of a cell permeable SOD mimetic, MnTMPyP, on mitochondrial function in renal ischemia-reperfusion injury (IRI). Male SD rats were subject to either sham operation or bilateral renal ischemia for 45 min followed by reperfusion for 1, 4 or 24 h. A sub-set of animals was treated with either saline vehicle or 5 mg/Kg of MnTMPyP (i.p.). EPR measurements showed that at 1-h reperfusion MnTMPyP prevented a decrease in aconitase activity (p < 0.05) and attenuated the increase in the high spin heme at g = 6 and oxidation of 4Fe4S to 3Fe4S signal at g = 2.015 (p < 0.01). MnTMPyP was effective in preventing loss of mitochondrial complexes and prevented the loss of cytochrome c and Smac/Diablo from mitochondria early in reperfusion. Following 24 h of reperfusion MnTMPyP was effective in attenuating caspase-3 and blocking apoptosis (p < 0.05). In conclusion, MnTMPyP has biphasic effects in renal IRI, inhibiting mitochondrial dysfunction at the early phases of reperfusion and prevention of apoptosis following longer durations of reperfusion.