RESUMO
BACKGROUND: Patients with Crohn's disease have defects in intestinal epithelial permeability that are inadequately explained by known inflammatory bowel disease (IBD) susceptibility genes. E-cadherin (CDH1) plays a vital role in maintaining the integrity of the intestinal barrier and its cellular localisation is disrupted in patients with Crohn's disease. AIM: To determine if polymorphisms in the CDH1 gene are associated with Crohn's disease and to determine the function associated with these polymorphisms. METHODS: The hypothesis was tested using a candidate gene approach using 20 Tag SNPs derived from the HapMap and Crohn's disease trios. Functional studies were carried out using HapMap cell lines and polarised epithelial cell lines (MDCK-1 and Caco2). RESULTS: Here we show that CDH1 is associated with Crohn's disease in 327 trios (rs10431923 excess transmission of "TT" genotype; p = 0.0020) and is replicated in the Wellcome Trust Case Control Consortium CD data set (TT risk allele; OR 1.2, p = 0.005). Patients with the Crohn's disease risk haplotype (rs12597188, rs10431923 and rs9935563; GTC allelic frequency 21%; p = 0.000016) exhibited increased E-cadherin cytoplasmic accumulation in their intestinal epithelium which may be explained by the presence of a novel truncated form of E-cadherin. Accordingly, expression of this truncated E-cadherin in cultured polarised epithelial cells resulted in abnormal intracellular accumulation and impaired plasma membrane localisation of both E-cadherin and beta-catenin. CONCLUSION: The mis-localisation of E-cadherin and beta-catenin may explain the increased permeability seen in some patients with Crohn's disease. Thus, the polymorphisms identified in CDH1 are important for understanding the pathogenesis of Crohn's disease and point to a defect in barrier defence.
Assuntos
Caderinas/genética , Doença de Crohn/genética , Citoplasma/metabolismo , Polimorfismo de Nucleotídeo Único , Adolescente , Caderinas/metabolismo , Linhagem Celular , Criança , Doença de Crohn/metabolismo , Doença de Crohn/patologia , Células Epiteliais/metabolismo , Feminino , Predisposição Genética para Doença , Humanos , Mucosa Intestinal/metabolismo , Desequilíbrio de Ligação , Masculino , Microscopia ConfocalRESUMO
BACKGROUND AND AIMS: Incidence of paediatric inflammatory bowel disease [IBD] in Canada is among the highest worldwide, and age of onset may be decreasing. In a multicentre nationwide inception cohort study, we examined variation in phenotype of IBD throughout the paediatric age spectrum. METHODS: Children aged ≥2 years [y] and <17y [A1 age at diagnosis], with new onset IBD, were systematically evaluated at sites of the Canadian Children IBD Network. Prospectively recorded phenotypic data were compared between age groups. RESULTS: Among 1092 children (70% Caucasian; 64% Crohn's disease [CD], 36% ulcerative colitis/inflammatory bowel disease unclassified [UC/IBD-U]; median age 13 y, interquartile range [IQR] 11-15 y), 210 [19%] were diagnosed before the age of age 10 y [Paris A1a] and 43 [4%] before age 6 y (very-early-onset [VEO-IBD]). CD was less common in younger children [42%, 56%, 66%, respectively, of VEO-IBD, A1a; A1b]. Colon-only IBD [UC/IBDU or CD-colon] was present in 81% of VEO-IBD and 65% of A1a; ileal disease increased progressively, reaching plateau at age 10 y. CD location was ileocolonic [L3] in 53% overall. Ileitis [L1] increased with age [6% of VEO-IBD; 13% of A1a; 21% of A1b], as did stricturing/penetrating CD [4% of A1a; 11% of A1b]. At all ages UC was extensive [E3/E4] in >85%, and disease activity moderate to severe according to Physician's Global Assessment [PGA] and weighted Paediatric Crohn's Disease Activity Index/Paediatric Ulcerative Colitis Activity Index [wPCDAI/PUCAI] in >70%. Heights were modestly reduced in CD [mean height z score -0.30 ± 1.23], but normal in UC/IBD-U. CONCLUSIONS: Paris classification of age at diagnosis is supported by age-related increases in ileal disease until age 10 years. Other phenotypic features, including severity, are similar across all ages. Linear growth is less impaired in CD than in historical cohorts, reflecting earlier diagnosis.
Assuntos
Colite Ulcerativa , Doença de Crohn , Idade de Início , Variação Biológica da População , Canadá/epidemiologia , Criança , Estudos de Coortes , Colite Ulcerativa/diagnóstico , Colite Ulcerativa/epidemiologia , Colite Ulcerativa/fisiopatologia , Doença de Crohn/diagnóstico , Doença de Crohn/epidemiologia , Doença de Crohn/fisiopatologia , Progressão da Doença , Feminino , Humanos , Incidência , Masculino , Estudos Prospectivos , Índice de Gravidade de DoençaRESUMO
Genetic defects that affect intestinal epithelial barrier function can present with very early-onset inflammatory bowel disease (VEOIBD). Using whole-genome sequencing, a novel hemizygous defect in NOX1 encoding NAPDH oxidase 1 was identified in a patient with ulcerative colitis-like VEOIBD. Exome screening of 1,878 pediatric patients identified further seven male inflammatory bowel disease (IBD) patients with rare NOX1 mutations. Loss-of-function was validated in p.N122H and p.T497A, and to a lesser degree in p.Y470H, p.R287Q, p.I67M, p.Q293R as well as the previously described p.P330S, and the common NOX1 SNP p.D360N (rs34688635) variant. The missense mutation p.N122H abrogated reactive oxygen species (ROS) production in cell lines, ex vivo colonic explants, and patient-derived colonic organoid cultures. Within colonic crypts, NOX1 constitutively generates a high level of ROS in the crypt lumen. Analysis of 9,513 controls and 11,140 IBD patients of non-Jewish European ancestry did not reveal an association between p.D360N and IBD. Our data suggest that loss-of-function variants in NOX1 do not cause a Mendelian disorder of high penetrance but are a context-specific modifier. Our results implicate that variants in NOX1 change brush border ROS within colonic crypts at the interface between the epithelium and luminal microbes.
Assuntos
Colo/fisiologia , Genes Modificadores/genética , Genótipo , Doenças Inflamatórias Intestinais/genética , NADPH Oxidase 1/genética , Animais , Criança , Pré-Escolar , Estudos de Associação Genética , Predisposição Genética para Doença , Genoma , Sequenciamento de Nucleotídeos em Larga Escala , Interações Hospedeiro-Patógeno , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Mutação de Sentido Incorreto/genética , Polimorfismo de Nucleotídeo Único , Espécies Reativas de Oxigênio/metabolismoRESUMO
Hexokinase from larvae of the freeze-avoiding goldenrod gall moth, Epiblema scudderiana, was purified 20-fold using chromatography on DE52 Sephadex, phosphocellulose, and blue dextran. Final specific activity was 75.8 U/mg and SDS-PAGE gave a molecular weight of 94,000 for the monomer. Arrhenius plot showed a break at 16 degrees or 12 degrees C in the absence vs. presence of 10% v/v glycerol, indicating a conformational change in the enzyme at lower temperatures but suggesting a stabilizing effect of glycerol. Comparison of hexokinase kinetic properties at 22 degrees and 4 degrees C showed higher affinity for both glucose and ATP (Km values were 45-50% lower), as well as for the cofactor Mg(2+), at the lower temperature. Furthermore, product inhibition by glucose-6-phosphate and ADP was reduced at 4 degrees C. Glucose levels rise in E. scudderiana as an apparent by-product of high rates of glycogenolysis during glycerol synthesis. The temperature-dependent properties of hexokinase would facilitate the recycling of this glucose back into the pathway of glycerol synthesis and could help to achieve the near stoichiometric conversion of glycogen to glycerol that is seen during cold hardening. Arch.
Assuntos
Glicerol/metabolismo , Hexoquinase/fisiologia , Mariposas/enzimologia , Animais , Congelamento , Hexoquinase/isolamento & purificação , Hexoquinase/metabolismo , Cinética , Peso Molecular , Estações do AnoRESUMO
The adipocyte-enhancer binding protein (AEBP) 1 is a novel transcriptional repressor with carboxypeptidase (CP) activity. AEBP1 binds to a regulatory sequence (termed adipocyte enhancer 1, AE-1) located in the proximal promoter region of the adipose P2 (aP2) gene, which encodes the adipocyte fatty-acid binding protein. Sequence comparisons and kinetic studies using known carboxypeptidase substrates, activators and inhibitors have characterized AEBP1 as a member of the regulatory B-like CP family. Significantly, the inherent CP activity of AEBP1 is stimulated by the AE-1 sequence. Our results indicate that AEBP1 is activated by a novel mechanism, wherby the direct binding of DNA enhances its protease activity. These results represent the first demonstration of DNA-mediated regulation of CP activity.
Assuntos
Carboxipeptidases/metabolismo , DNA/metabolismo , Proteínas de Membrana/agonistas , Proteínas de Membrana/metabolismo , Proteínas Repressoras/agonistas , Proteínas Repressoras/metabolismo , Sítios de Ligação , Carboxipeptidase B , Carboxipeptidases/antagonistas & inibidores , Domínio Catalítico , DNA/farmacologia , Proteínas de Ligação a DNA/agonistas , Proteínas de Ligação a DNA/antagonistas & inibidores , Proteínas de Ligação a DNA/metabolismo , Elementos Facilitadores Genéticos/genética , Ativação Enzimática/efeitos dos fármacos , Concentração Inibidora 50 , Peptídeos e Proteínas de Sinalização Intercelular , Cinética , Proteínas de Membrana/antagonistas & inibidores , Regiões Promotoras Genéticas/genética , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/metabolismo , Proteínas Repressoras/antagonistas & inibidores , Especificidade por Substrato , Zinco/metabolismoRESUMO
Mitogen-activated protein kinase (MAPK) is required for cell growth and cell differentiation. In adipogenesis, MAPK activation opposes the differentiation process. The regulatory mechanisms or the cellular factors that regulate the switch between growth and differentiation in the adipogenic lineage have been largely unelucidated. We show here that AEBP1, a transcriptional repressor that is down-regulated during adipogenesis, complexes and protects MAPK from its specific phosphatase in mammalian cells. We further show evidence that the modulation of MAPK activation by AEBP1 is a biologically relevant process in adipogenesis. Our results suggest that modulation of MAPK activation by the protective effect of AEBP1 may constitute a critical part in the determination between cell growth and differentiation in the adipogenic lineage. The proposed mode of action by which a transcription factor regulates MAPK activation is novel.