RESUMO
OBJECTIVE: Clinical severity of alternating hemiplegia of childhood (AHC) is extremely variable. To investigate genotype-phenotype correlations in AHC, we analyzed the clinical information and ATP1A3 mutations in patients with AHC. METHODS: Thirty-five Japanese patients who were clinically diagnosed with AHC participated in this study. ATP1A3 mutations were analyzed using Sanger sequencing. Detailed clinical information was collected from family members of patients with AHC and clinicians responsible for their care. RESULTS: Gene analysis revealed 33 patients with de novo heterozygous missense mutations of ATP1A3: Glu815Lys in 12 cases (36%), Asp801Asn in 10 cases (30%), and other missense mutations in 11 cases. Clinical information was compared among the Glu815Lys, Asp801Asn, and other mutation groups. Statistical analysis revealed significant differences in the history of neonatal onset, gross motor level, status epilepticus, and respiratory paralysis in the Glu815Lys group compared with the other groups. In addition, 8 patients who did not receive flunarizine had severe motor deteriorations. CONCLUSIONS: The Glu815Lys genotype appears to be associated with the most severe AHC phenotype. Although AHC is not generally seen as a progressive disorder, it should be considered a disorder that deteriorates abruptly or in a stepwise fashion, particularly in patients with the Glu815Lys mutation.
Assuntos
Hemiplegia/genética , Transtornos das Habilidades Motoras/genética , Paralisia Respiratória/genética , ATPase Trocadora de Sódio-Potássio/genética , Estado Epiléptico/genética , Adolescente , Adulto , Criança , Pré-Escolar , Feminino , Estudos de Associação Genética , Hemiplegia/complicações , Hemiplegia/fisiopatologia , Heterozigoto , Humanos , Lactente , Masculino , Transtornos das Habilidades Motoras/etiologia , Transtornos das Habilidades Motoras/fisiopatologia , Mutação de Sentido Incorreto/genética , Paralisia Respiratória/etiologia , Paralisia Respiratória/fisiopatologia , Índice de Gravidade de Doença , Estado Epiléptico/etiologia , Estado Epiléptico/fisiopatologia , Adulto JovemRESUMO
BACKGROUND: Collectrin, a homologue of angiotensin converting enzyme 2, is expressed in pancreatic beta cells and renal proximal tubular and collecting duct cells under the control of hepatocyte nuclear factors-1alpha and -1beta. Because collectrin interacts with the soluble N-ethylmaleiamide-sensitive factor attachment protein receptor (SNARE) complexes, we investigated whether collectrin is involved in sodium handling in hypertension by vesicle trafficking of apical membrane proteins. METHODS AND RESULTS: Collectrin physically interacts with the SNARE complex: snapin, synaptosomal-associated protein 23 kDa, syntaxin-4, and vesicle-associated membrane protein-2 in mIMCD-3 cells. siRNA knockdown of collectrin resulted in a reduction in membrane-associated aquaporin-2, alpha-epithelial Na+ channel, and H+-ATPase. Collectrin and SNARE proteins were abundantly expressed in collecting ducts of Wistar-Kyoto rats. Wistar-Kyoto rats and spontaneously hypertensive rats 7 weeks of age were subjected to normal-salt (1% NaCl) and high-salt (8% NaCl) chow for 10 weeks. High-salt chow prominently elevated blood pressure, oral intake, and urinary excretion of NaCl and water in both groups. Although urinary excretion of aldosterone was significantly suppressed in both groups, collectrin expression was upregulated and associated with the maintenance of aquaporin-2, alpha-epithelial Na+ channel, and H+-ATPase in membrane fractions. Collectrin promoter activities and mRNA and protein expressions were upregulated and ubiquitinated collectrin was reduced by high NaCl (175 to 225 mmol/L) and not altered by 1 micromol/L aldosterone in mIMCD-3 cells. CONCLUSIONS: Upregulation of collectrin by high NaCl independent of aldosterone functionally links to the trafficking of apical membrane proteins via the SNARE complex, and collectrin may be responsible for the sodium retention in salt-sensitive hypertension.
Assuntos
Colectinas/biossíntese , Hipertensão/induzido quimicamente , Complexos Multiproteicos/metabolismo , Proteínas SNARE/metabolismo , Cloreto de Sódio/toxicidade , Regulação para Cima/efeitos dos fármacos , Aldosterona/urina , Animais , Aquaporina 2/metabolismo , Pressão Sanguínea/efeitos dos fármacos , Linhagem Celular , Canais Epiteliais de Sódio , Fator 1-alfa Nuclear de Hepatócito/metabolismo , Fator 1-beta Nuclear de Hepatócito/metabolismo , Hipertensão/metabolismo , Células Secretoras de Insulina/metabolismo , Túbulos Renais Coletores/metabolismo , Túbulos Renais Proximais/metabolismo , ATPases Translocadoras de Prótons/metabolismo , Proteínas Qa-SNARE/metabolismo , RNA Interferente Pequeno/farmacologia , Ratos , Ratos Endogâmicos SHR , Ratos Endogâmicos WKY , Proteína 2 Associada à Membrana da Vesícula/metabolismoRESUMO
Dialysis related amyloidosis (DRA) is a progressive and serious complication in patients under long-term hemodialysis and mainly leads to osteo-articular diseases. Although beta(2)-microglobulin (beta2-m) is the major structural component of beta2-m amyloid fibrils, the initiation of amyloid formation is not clearly understood. Here, we have identified procollagen C-proteinase enhancer-1 (PCPE-1) as a new interacting protein with beta2-m by screening a human synovium cDNA library. The interaction of beta2-m with full-length PCPE-1 was confirmed by immunoprecipitation, solid-phase binding and pull-down assays. By yeast two-hybrid analysis and pull-down assay, beta2-m appeared to interact with PCPE-1 via the NTR (netrin-like) domain and not via the CUB (C1r/C1s, Uegf and BMP-1) domain region. In synovial tissues derived from hemodialysis patients with DRA, beta2-m co-localized and formed a complex with PCPE-1. beta2-m did not alter the basal activity of bone morphogenetic protein-1/procollagen C-proteinase (BMP-1/PCP) nor BMP-1/PCP activity enhanced by PCPE-1. PCPE-1 did not stimulate beta2-m amyloid fibril formation from monomeric beta2-m in vitro under acidic and neutral conditions as revealed by thioflavin T fluorescence spectroscopy and electron microscopy. Since PCPE-1 is abundantly expressed in connective tissues rich in type I collagen, it may be involved in the initial accumulation of beta2-m in selected tissues such as tendon, synovium and bone. Furthermore, since such preferential deposition of beta2-m may be linked to subsequent beta2-m amyloid fibril formation, the disruption of the interaction between beta2-m and PCPE-1 may prevent beta2-m amyloid fibril formation and therefore PCPE-1 could be a new target for the treatment of DRA.
Assuntos
Amiloide/química , Proteínas da Matriz Extracelular/metabolismo , Glicoproteínas/metabolismo , Microglobulina beta-2/química , Sequência de Aminoácidos , Proteína Morfogenética Óssea 1 , Proteínas Morfogenéticas Ósseas/química , Relação Dose-Resposta a Droga , Elementos Facilitadores Genéticos , Biblioteca Gênica , Humanos , Metaloendopeptidases/química , Dados de Sequência Molecular , Ligação Proteica , Estrutura Terciária de Proteína , Proteínas Recombinantes/química , Técnicas do Sistema de Duplo-HíbridoRESUMO
Collectrin, a homologue of angiotensin converting enzyme 2 (ACE2), is a type I transmembrane protein, and we originally reported its localization to the cytoplasm and apical membrane of collecting duct cells. Recently, two independent studies of targeted disruption of collectrin in mice resulted in severe and general defects in renal amino acid uptake. Collectrin has been reported to be under the transcriptional regulation by HNF-1alpha, which is exclusively expressed in proximal tubules and localized at the luminal side of brush border membranes. The deficiency of collectrin was associated with reduction of multiple amino acid transporters on luminal membranes. In the current study, we describe that collectrin is a target of HNF-1beta and heavily expressed in the primary cilium of renal collecting duct cells. Collectrin is also localized in the vesicles near the peri-basal body region and binds to gamma-actin-myosin II-A, SNARE, and polycystin-2-polaris complexes, and all of these are involved in intracellular and ciliary movement of vesicles and membrane proteins. Treatment of mIMCD3 cells with collectrin siRNA resulted in defective cilium formation, increased cell proliferation and apoptosis, and disappearance of polycystin-2 in the primary cilium. Suppression of collectrin mRNA in metanephric culture resulted in the formation of multiple longitudinal cysts in ureteric bud branches. Taken together, the cystic change and formation of defective cilium with the interference in the collectrin functions would suggest that it is necessary for recycling of the primary cilia-specific membrane proteins, the maintenance of the primary cilia and cell polarity of collecting duct cells. The transcriptional hierarchy between HNF-1beta and PKD (polycystic kidney disease) genes expressed in the primary cilia of collecting duct cells has been suggested, and collectrin is one of such HNF-1beta regulated genes.
Assuntos
Polaridade Celular , Cílios/fisiologia , Fator 1-beta Nuclear de Hepatócito/fisiologia , Túbulos Renais Coletores/citologia , Glicoproteínas de Membrana/fisiologia , Animais , Sequência de Bases , Linhagem Celular , Primers do DNA , Ensaio de Desvio de Mobilidade Eletroforética , Imunoprecipitação , Camundongos , Camundongos Transgênicos , RNA Interferente PequenoRESUMO
Thiazolidinediones are ligands for peroxisome proliferator-activated receptor (PPAR)-gamma, widely used as insulin sensitizer in type 2 diabetic patients and implicated in apoptosis, cell proliferation, and cell cycle regulation. Here, the effect of thiazolidinediones on G1-phase cell cycle arrest, the hallmark in diabetic nephropathy, was investigated. Eight-week-old male Otsuka Long-Evans Tokushima fatty rats were treated with pioglitazone (1 mg x kg body wt(-1) x day(-1)) until 50 weeks of age and compared with insulin treatment. Although similar HbA(1c) levels were observed in both groups, pioglitazone significantly inhibited glomerular hypertrophy and mesangial matrix expansion and reduced urinary albumin excretion compared with the insulin-treated group. In addition, pioglitazone significantly reduced the number of glomerular p27(Kip1)-positive cells. Because prominent expression of PPAR-gamma was observed in podocytes in glomeruli and cultured cells, conditionally immortalized mouse podocyte cells were cultured under 5.5 and 25 mmol/l D-glucose supplemented with pioglitazone. Pioglitazone inhibited cell hypertrophy revealed by [(3)H]thymidine and [(3)H]proline incorporation, and pioglitazone reversed high glucose-induced G1-phase cell cycle arrest, i.e., an increase in G0/G1 phase and decrease in S and G2 phases. Pioglitazone suppressed high glucose-induced phosphorylation of p44/42 mitogen-activated protein kinase and reduced Bcl-2 and p27(Kip1) protein levels. Besides glucose-lowering action, pioglitazone ameliorates diabetic nephropathy via cell cycle-dependent mechanisms.
Assuntos
Ciclo Celular/efeitos dos fármacos , Nefropatias Diabéticas/tratamento farmacológico , Tiazolidinedionas/farmacologia , Albuminúria/tratamento farmacológico , Animais , Western Blotting , Peso Corporal/efeitos dos fármacos , Ciclo Celular/fisiologia , Linhagem Celular , Colágeno Tipo IV/metabolismo , Inibidor de Quinase Dependente de Ciclina p27/metabolismo , Nefropatias Diabéticas/metabolismo , Nefropatias Diabéticas/fisiopatologia , Glucose/farmacologia , Hipoglicemiantes/farmacologia , Imuno-Histoquímica , Insulina/farmacologia , Metabolismo dos Lipídeos/efeitos dos fármacos , Macrófagos/efeitos dos fármacos , Macrófagos/metabolismo , Macrófagos/patologia , Masculino , Camundongos , PPAR gama/metabolismo , Fosforilação/efeitos dos fármacos , Pioglitazona , Podócitos/citologia , Podócitos/efeitos dos fármacos , Podócitos/metabolismo , Ratos , Ratos Long-Evans , Fator de Crescimento Transformador beta1/metabolismoRESUMO
Hyperglycemia-induced overproduction of mitochondrial reactive oxygen species has emerged as a major player in diabetic vascular complications. Mammalian translocase of inner mitochondrial membrane 44 (TIM44) was identified by upregulation in diabetic mouse kidneys. TIM44 functions as a membrane anchor of mitochondrial heat-shock protein 70 (mtHsp70) to TIM23 complex and is involved in the import of mitochondria-targeted preproteins into mitochondrial matrix. The process is dependent on inner membrane potential and ATP hydrolysis on ATPase domain of mitochondrial heat-shock protein 70. Hemagglutination virus of Japan-envelope vector that carries pcDNA3.1 plasmid that contains the full-length cDNA of TIM44 and control plasmid were injected weekly into the tail vein of uninephrectomized streptozotocin-induced diabetic CD-1 mice. The gene delivery alleviated proteinuria and renal hypertrophy at 8 wk after the injection, inhibited renal cell proliferation and apoptosis, and suppressed superoxide production. In vitro experiments, using human proximal tubular (HK2) cells, revealed that the gene delivery of TIM44 reversed high glucose-induced metabolic and cellular abnormalities such as enhanced reactive oxygen species production, increased ATP contents, alterations in inner membrane potential, increased cell proliferation, and apoptosis. Transfection with siRNA and expressing vector of TIM44 revealed that TIM44 facilitates import of antioxidative enzymes such as superoxide dismutase and glutathione peroxidase into mitochondria. The gene delivery of TIM44 therefore seems to be beneficial for the maintenance of mitochondrial function and is a novel therapeutic approach for diabetic nephropathy.
Assuntos
Proteínas de Transporte/genética , Nefropatias Diabéticas/terapia , Terapia Genética/métodos , Proteínas de Membrana/genética , Proteínas Mitocondriais/genética , Trifosfato de Adenosina/biossíntese , Animais , Apoptose , Proteínas de Transporte/metabolismo , Linhagem Celular , Proliferação de Células , Nefropatias Diabéticas/metabolismo , Nefropatias Diabéticas/patologia , Glutationa Redutase/metabolismo , Humanos , Rim/metabolismo , Rim/patologia , Masculino , Potenciais da Membrana , Proteínas de Membrana/metabolismo , Camundongos , Mitocôndrias/metabolismo , Proteínas de Transporte da Membrana Mitocondrial , Proteínas do Complexo de Importação de Proteína Precursora Mitocondrial , Proteínas Mitocondriais/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Superóxido Dismutase/metabolismo , Superóxidos/metabolismoRESUMO
Methyl-CpG binding protein 2 gene (MECP2), the gene implicated in Rett syndrome, was also reported to be involved in mental retardation and autism. MECP2, MBD1, MBD2, MBD3, and MBD4 comprise a nuclear protein family sharing the methyl-CpG binding domain (MBD) and are related to transcriptional repression. In 65 Japanese autistic patients, all the exons of each gene were screened for mutations by DHPLC, and the results were confirmed by direct sequencing. An R269C mutation that resulted in the addition of cysteine near a cysteine rich region was found in the MBD1 gene in one patient. This mutation was also detected in the patient's father with some phenotypes of autism and his normal sister, but not in 151 controls. Two repeat length polymorphisms, (GGGGCC)2 to 3 and (GGC)4 to 5, were detected in MBD2, and several polymorphisms were detected in each gene. Although our findings could not confirm that the genes of this family are responsible for the etiology in the majority of autistic patients, the R269C mutation in the MBD1 gene may relate to autism. The potential association of the high-polymorphic gene variants with autism needs to be studied further. Furthermore, these polymorphisms are useful for linkage analysis.
Assuntos
Transtorno Autístico/genética , Proteínas de Ligação a DNA/genética , Sequência de Aminoácidos , Animais , Sequência de Bases , Criança , Pré-Escolar , Ilhas de CpG/genética , Análise Mutacional de DNA , Primers do DNA , Feminino , Humanos , Masculino , Camundongos , Dados de Sequência Molecular , Homologia de SequênciaRESUMO
BACKGROUND: Since adrenomedullin (AM) elicits vasodilatation by binding to specific AM receptors consisted of calcitonin-receptor-like receptor (CRLR)/receptor-activity-modifying protein 2 (RAMP2) or CRLR/receptor-activity-modifying protein 3 (RAMP3) on endothelial cells and stimulating nitric oxide production, AM possibly involves in glomerular capillary dilatation in early phase of diabetic nephropathy. METHODS: Streptozotocin (STZ)-induced diabetic Sprague-Dawley rats at 4 weeks after the injection were employed for expression studies of AM, RAPM2, and RAMP3. The measurement of AM peptide levels in kidney tissue, plasma, and urine was performed. Human aortic endothelial cells (HAEC) were used to investigate functional link between glucose-induced AM production and nitric oxide release. RESULTS: STZ rats showed glomerular hypertrophy and increased urinary NO2- and NO3- excretion. By Northern blot analyses, AM and RAPM2 mRNAs significantly increased in the kidneys of STZ rats, while RAMP3 mRNA was not altered. In STZ rats, AM peptide was actively secreted into urine (1280 +/- 360 fmol/day vs. control 110 +/- 36 fmol/day). AM peptide was mainly detected on cortical and medullary collecting duct cells in control rat kidneys and AM peptide and mRNA were up-regulated on afferent arterioles and glomeruli of STZ rats. RAMP2 expression was detected on afferent arterioles and not in glomeruli in control rats and it was up-regulated on glomerular endothelial cells in STZ rats. In HAEC culture, d-glucose stimulated AM and nitric oxide production and they were suppressed by addition of AM antisense oligodeoxynucleotides. CONCLUSION: Up-regulated expression of AM and RAMP2 in afferent arterioles and glomeruli may be related to selective dilatation of glomerular capillary in acute phase of type 1 diabetes.