RESUMO
BACKGROUND & AIMS: RAC1 is a guanosine triphosphatase that has an evolutionarily conserved role in coordinating immune defenses, from plants to mammals. Chronic inflammatory bowel diseases are associated with dysregulation of immune defenses. We studied the role of RAC1 in inflammatory bowel diseases using human genetic and functional studies and animal models of colitis. METHODS: We used a candidate gene approach to HapMap-Tag single nucleotide polymorphisms in a discovery cohort; findings were confirmed in 2 additional cohorts. RAC1 messenger RNA expression was examined from peripheral blood cells of patients. Colitis was induced in mice with conditional disruption of Rac1 in phagocytes by administration of dextran sulfate sodium. RESULTS: We observed a genetic association between RAC1 with ulcerative colitis in a discovery cohort, 2 independent replication cohorts, and in combined analysis for the single nucleotide polymorphisms rs10951982 (P(combined UC) = 3.3 × 10(-8), odds ratio = 1.43 [95% confidence interval: 1.26-1.63]) and rs4720672 (P(combined UC) = 4.7 × 10(-6), odds ratio = 1.36 [95% confidence interval: 1.19-1.58]). Patients with inflammatory bowel disease who had the rs10951982 risk allele had increased expression of RAC1 compared to those without this allele. Conditional disruption of Rac1 in macrophage and neutrophils of mice protected against dextran sulfate sodium-induced colitis. CONCLUSIONS: Human studies and knockout mice demonstrated a role for the guanosine triphosphatase RAC1 in the development of ulcerative colitis; increased expression of RAC1 was associated with susceptibility to colitis.
Assuntos
Colite Ulcerativa/genética , Colite Ulcerativa/metabolismo , Doença de Crohn/genética , Doença de Crohn/metabolismo , RNA Mensageiro/sangue , Proteínas rac1 de Ligação ao GTP/genética , Proteínas rac1 de Ligação ao GTP/metabolismo , Alelos , Animais , Estudos de Coortes , Colite Ulcerativa/induzido quimicamente , Colite Ulcerativa/patologia , Sulfato de Dextrana , Estudos de Associação Genética , Humanos , Interleucina-1beta/metabolismo , Camundongos , Camundongos Knockout , Razão de Chances , Peroxidase/metabolismo , Polimorfismo de Nucleotídeo Único , Estatísticas não Paramétricas , População Branca/genéticaRESUMO
In mammals, aging is linked to a decline in the activity of citrate synthase (CS; E.C. 2.3.3.1), the first enzyme of the citric acid cycle. We used 2,2'-azobis(2-amidinopropane) dihydrochloride (AAPH), a water-soluble generator of peroxyl and alkoxyl radicals, to investigate the susceptibility of CS to oxidative damage. Treatment of isolated mitochondria with AAPH for 8-24 h led to CS inactivation; however, the activity of aconitase, a mitochondrial enzyme routinely used as an oxidative stress marker, was unaffected. In addition to enzyme inactivation, AAPH treatment of purified CS resulted in dityrosine formation, increased protein surface hydrophobicity, and loss of tryptophan fluorescence. Propyl gallate, 1,8-naphthalenediol, 2,3-naphthalenediol, ascorbic acid, glutathione, and oxaloacetate protected CS from AAPH-mediated inactivation, with IC(50) values of 9, 14, 34, 37, 150, and 160 muM, respectively. Surprisingly, the antioxidant epigallocatechin gallate offered no protection against AAPH, but instead caused CS inactivation. Our results suggest that the current practice of using the enzymatic activity of CS as an index of mitochondrial abundance and the use of aconitase activity as an oxidative stress marker may be inappropriate, especially in oxidative stress-related studies, during which alkyl peroxyl and alkoxyl radicals can be generated.
Assuntos
Antioxidantes/farmacologia , Citrato (si)-Sintase/metabolismo , Oxidantes/farmacologia , Estresse Oxidativo/efeitos dos fármacos , Peróxidos/farmacologia , Aconitato Hidratase/antagonistas & inibidores , Aconitato Hidratase/metabolismo , Amidinas/farmacologia , Amidinas/toxicidade , Ácido Ascórbico/farmacologia , Catequina/análogos & derivados , Catequina/farmacologia , Catequina/toxicidade , Citrato (si)-Sintase/antagonistas & inibidores , Ativação Enzimática , Glutationa/farmacologia , Concentração Inibidora 50 , Mitocôndrias/enzimologia , Naftóis/farmacologia , Ácido Oxaloacético/farmacologia , Oxidantes/toxicidade , Oxirredução/efeitos dos fármacos , Estresse Oxidativo/fisiologia , Peróxidos/toxicidade , Galato de Propila/farmacologiaRESUMO
We present a case of a high cardiac output (CO) arteriovenous fistula (AVF) with pulmonary hypertension (PH) post-double lung transplant presenting for AVF occlusion. The patient presented with a CO of 9.83 L/min, pulmonary artery pressures of 64/16, inferior vena cava dilatation and an AVF between the left common iliac artery and vein. Given her anaesthetic considerations, we elected to proceed with local anaesthesia and sedation. Trial balloon occlusion resulted in an increase in blood pressure and a headache that resolved with balloon deflation. Successful final occlusion with an endovascular stent was completed without adverse events. PH is a complex pathophysiology with the potential for catastrophic decompensation. Anaesthesiologists must consider a patient's comorbidities and the procedure to safely administer anaesthesia without complications.
Assuntos
Fístula Arteriovenosa/complicações , Insuficiência Cardíaca/etiologia , Transplante de Pulmão/efeitos adversos , Injúria Renal Aguda/etiologia , Injúria Renal Aguda/terapia , Adulto , Angiografia , Fístula Arteriovenosa/terapia , Oclusão com Balão/métodos , Débito Cardíaco/fisiologia , Ecocardiografia , Feminino , Insuficiência Cardíaca/diagnóstico por imagem , Insuficiência Cardíaca/fisiopatologia , Humanos , Hipertensão Pulmonar/complicações , Artéria Ilíaca/diagnóstico por imagem , Artéria Ilíaca/fisiopatologia , Diálise Renal , Stents , Resultado do TratamentoRESUMO
BACKGROUND: Nrf1 (nuclear factor-erythroid 2 p45 subunit-related factor 1) is a transcription factor mediating cellular responses to xenobiotic and pro-oxidant stress. Nrf1 regulates the transcription of many stress-related genes through the electrophile response elements (EpREs) located in their promoter regions. Despite its potential importance in human health, the mechanisms controlling Nrf1 have not been addressed fully. PRINCIPAL FINDINGS: We found that proteasomal inhibitors MG-132 and clasto-lactacystin-ß-lactone stabilized the protein expression of full-length Nrf1 in both COS7 and WFF2002 cells. Concomitantly, proteasomal inhibition decreased the expression of a smaller, N-terminal Nrf1 fragment, with an approximate molecular weight of 23 kDa. The EpRE-luciferase reporter assays revealed that proteasomal inhibition markedly inhibited the Nrf1 transactivational activity. These results support earlier hypotheses that the 26 S proteasome processes Nrf1 into its active form by removing its inhibitory N-terminal domain anchoring Nrf1 to the endoplasmic reticulum. Immunoprecipitation demonstrated that Nrf1 is ubiquitinated and that proteasomal inhibition increased the degree of Nrf1 ubiquitination. Furthermore, Nrf1 protein had a half-life of approximately 5 hours in COS7 cells. In contrast, hypoxia (1% O(2)) significantly increased the luciferase reporter activity of exogenous Nrf1 protein, while decreasing the protein expression of p65, a shorter form of Nrf1, known to act as a repressor of EpRE-controlled gene expression. Finally, the protein phosphatase inhibitor okadaic acid activated Nrf1 reporter activity, while the latter was repressed by the PKC inhibitor staurosporine. CONCLUSIONS: Collectively, our data suggests that Nrf1 is controlled by several post-translational mechanisms, including ubiquitination, proteolytic processing and proteasomal-mediated degradation as well as by its phosphorylation status.