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1.
Nature ; 471(7338): 325-30, 2011 Mar 17.
Artigo em Inglês | MEDLINE | ID: mdl-21297615

RESUMO

Geographic atrophy (GA), an untreatable advanced form of age-related macular degeneration, results from retinal pigmented epithelium (RPE) cell degeneration. Here we show that the microRNA (miRNA)-processing enzyme DICER1 is reduced in the RPE of humans with GA, and that conditional ablation of Dicer1, but not seven other miRNA-processing enzymes, induces RPE degeneration in mice. DICER1 knockdown induces accumulation of Alu RNA in human RPE cells and Alu-like B1 and B2 RNAs in mouse RPE. Alu RNA is increased in the RPE of humans with GA, and this pathogenic RNA induces human RPE cytotoxicity and RPE degeneration in mice. Antisense oligonucleotides targeting Alu/B1/B2 RNAs prevent DICER1 depletion-induced RPE degeneration despite global miRNA downregulation. DICER1 degrades Alu RNA, and this digested Alu RNA cannot induce RPE degeneration in mice. These findings reveal a miRNA-independent cell survival function for DICER1 involving retrotransposon transcript degradation, show that Alu RNA can directly cause human pathology, and identify new targets for a major cause of blindness.


Assuntos
Elementos Alu/genética , RNA Helicases DEAD-box/deficiência , Degeneração Macular/genética , Degeneração Macular/patologia , RNA/genética , RNA/metabolismo , Ribonuclease III/deficiência , Animais , Morte Celular , Sobrevivência Celular , Células Cultivadas , RNA Helicases DEAD-box/genética , RNA Helicases DEAD-box/metabolismo , Técnicas de Silenciamento de Genes , Humanos , Camundongos , MicroRNAs/metabolismo , Dados de Sequência Molecular , Oligonucleotídeos Antissenso , Fenótipo , Epitélio Pigmentado da Retina/enzimologia , Epitélio Pigmentado da Retina/metabolismo , Epitélio Pigmentado da Retina/patologia , Ribonuclease III/genética , Ribonuclease III/metabolismo
2.
Arterioscler Thromb Vasc Biol ; 35(5): 1156-65, 2015 May.
Artigo em Inglês | MEDLINE | ID: mdl-25745063

RESUMO

OBJECTIVE: Rupture of abdominal aortic aneurysm (AAA), a major cause of death in the aged population, is characterized by vascular inflammation and matrix degradation. Serum amyloid A (SAA), an acute-phase reactant linked to inflammation and matrix metalloproteinase induction, correlates with aortic dimensions before aneurysm formation in humans. We investigated whether SAA deficiency in mice affects AAA formation during angiotensin II (Ang II) infusion. APPROACH AND RESULTS: Plasma SAA increased ≈60-fold in apoE(-/-) mice 24 hours after intraperitoneal Ang II injection (100 µg/kg; n=4) and ≈15-fold after chronic 28-day Ang II infusion (1000 ng/kg per minute; n=9). AAA incidence and severity after 28-day Ang II infusion was significantly reduced in apoE(-/-) mice lacking both acute-phase SAA isoforms (SAAKO; n=20) compared with apoE(-/-) mice (SAAWT; n=20) as assessed by in vivo ultrasound and ex vivo morphometric analyses, despite a significant increase in systolic blood pressure in SAAKO mice compared with SAAWT mice after Ang II infusion. Atherosclerotic lesion area of the aortic arch was similar in SAAKO and SAAWT mice after 28-day Ang II infusion. Immunostaining detected SAA in AAA tissues of Ang II-infused SAAWT mice that colocalized with macrophages, elastin breaks, and enhanced matrix metalloproteinase activity. Matrix metalloproteinase-2 activity was significantly lower in aortas of SAAKO mice compared with SAAWT mice after 10-day Ang II infusion. CONCLUSIONS: Lack of endogenous acute-phase SAA protects against experimental AAA through a mechanism that may involve reduced matrix metalloproteinase-2 activity.


Assuntos
Angiotensina II/farmacologia , Aneurisma da Aorta Abdominal/prevenção & controle , Apolipoproteínas E/deficiência , Metaloproteinase 2 da Matriz/metabolismo , Proteína Amiloide A Sérica/deficiência , Animais , Aneurisma da Aorta Abdominal/patologia , Biomarcadores/sangue , Modelos Animais de Doenças , Elastina/metabolismo , Macrófagos/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Distribuição Aleatória , Sensibilidade e Especificidade , Proteína Amiloide A Sérica/metabolismo
3.
Nature ; 443(7114): 993-7, 2006 Oct 26.
Artigo em Inglês | MEDLINE | ID: mdl-17051153

RESUMO

Corneal avascularity-the absence of blood vessels in the cornea-is required for optical clarity and optimal vision, and has led to the cornea being widely used for validating pro- and anti-angiogenic therapeutic strategies for many disorders. But the molecular underpinnings of the avascular phenotype have until now remained obscure and are all the more remarkable given the presence in the cornea of vascular endothelial growth factor (VEGF)-A, a potent stimulator of angiogenesis, and the proximity of the cornea to vascularized tissues. Here we show that the cornea expresses soluble VEGF receptor-1 (sVEGFR-1; also known as sflt-1) and that suppression of this endogenous VEGF-A trap by neutralizing antibodies, RNA interference or Cre-lox-mediated gene disruption abolishes corneal avascularity in mice. The spontaneously vascularized corneas of corn1 and Pax6+/- mice and Pax6+/- patients with aniridia are deficient in sflt-1, and recombinant sflt-1 administration restores corneal avascularity in corn1 and Pax6+/- mice. Manatees, the only known creatures uniformly to have vascularized corneas, do not express sflt-1, whereas the avascular corneas of dugongs, also members of the order Sirenia, elephants, the closest extant terrestrial phylogenetic relatives of manatees, and other marine mammals (dolphins and whales) contain sflt-1, indicating that it has a crucial, evolutionarily conserved role. The recognition that sflt-1 is essential for preserving the avascular ambit of the cornea can rationally guide its use as a platform for angiogenic modulators, supports its use in treating neovascular diseases, and might provide insight into the immunological privilege of the cornea.


Assuntos
Córnea/irrigação sanguínea , Córnea/metabolismo , Receptor 1 de Fatores de Crescimento do Endotélio Vascular/metabolismo , Animais , Deleção de Genes , Camundongos , Neovascularização Fisiológica , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Solubilidade , Trichechus , Fator A de Crescimento do Endotélio Vascular/metabolismo , Receptor 1 de Fatores de Crescimento do Endotélio Vascular/deficiência , Receptor 1 de Fatores de Crescimento do Endotélio Vascular/genética
4.
BMC Gastroenterol ; 10: 133, 2010 Nov 10.
Artigo em Inglês | MEDLINE | ID: mdl-21067563

RESUMO

BACKGROUND: Serum Amyloid A (SAA) is a major acute phase protein of unknown function. SAA is mostly expressed in the liver, but also in other tissues including the intestinal epithelium. SAA reportedly has anti-bacterial effects, and because inflammatory bowel diseases (IBD) result from a breakdown in homeostatic interactions between intestinal epithelia and bacteria, we hypothesized that SAA is protective during experimental colitis. METHODS: Intestinal SAA expression was measured in mouse and human samples. Dextran sodium sulfate (DSS) colitis was induced in SAA 1/2 double knockout (DKO) mice and in wildtype controls. Anti-bacterial effects of SAA1/2 were tested in intestinal epithelial cell lines transduced with adenoviral vectors encoding the CE/J SAA isoform or control vectors prior to exposure to live Escherichia coli. RESULTS: Significant levels of SAA1/SAA2 RNA and SAA protein were detected by in situ hybridization and immunohistochemistry in mouse colonic epithelium. SAA3 expression was weaker, but similarly distributed. SAA1/2 RNA was present in the ileum and colon of conventional mice and in the colon of germfree mice. Expression of SAA3 was strongly regulated by bacterial lipopolysaccharides in cultured epithelial cell lines, whereas SAA1/2 expression was constitutive and not LPS inducible. Overexpression of SAA1/2 in cultured epithelial cell lines reduced the viability of co-cultured E. coli. This might partially explain the observed increase in susceptibility of DKO mice to DSS colitis. SAA1/2 expression was increased in colon samples obtained from Crohn's Disease patients compared to controls. CONCLUSIONS: Intestinal epithelial SAA displays bactericidal properties in vitro and could play a protective role in experimental mouse colitis. Altered expression of SAA in intestinal biopsies from Crohn's Disease patients suggests that SAA is involved in the disease process..


Assuntos
Bactérias/crescimento & desenvolvimento , Colite/genética , DNA/genética , Regulação da Expressão Gênica , Proteína Amiloide A Sérica/genética , Animais , Bactérias/efeitos dos fármacos , Biópsia , Linhagem Celular , Colite/microbiologia , Colite/patologia , Modelos Animais de Doenças , Progressão da Doença , Humanos , Immunoblotting , Hibridização In Situ , Mucosa Intestinal/metabolismo , Mucosa Intestinal/patologia , Camundongos , Camundongos Endogâmicos C57BL , Reação em Cadeia da Polimerase , Proteína Amiloide A Sérica/biossíntese
5.
J Clin Invest ; 116(2): 422-9, 2006 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-16453023

RESUMO

VEGF-A promotes angiogenesis in many tissues. Here we report that choroidal neovascularization (CNV) incited by injury was increased by excess VEGF-A before injury but was suppressed by VEGF-A after injury. This unorthodox antiangiogenic effect was mediated via VEGFR-1 activation and VEGFR-2 deactivation, the latter via Src homology domain 2-containing (SH2-containing) tyrosine phosphatase-1 (SHP-1). The VEGFR-1-specific ligand placental growth factor-1 (PlGF-1), but not VEGF-E, which selectively binds VEGFR-2, mimicked these responses. Excess VEGF-A increased CNV before injury because VEGFR-1 activation was silenced by secreted protein, acidic and rich in cysteine (SPARC). The transient decline of SPARC after injury revealed a temporal window in which VEGF-A signaling was routed principally through VEGFR-1. These observations indicate that therapeutic design of VEGF-A inhibition should include consideration of the level and activity of SPARC.


Assuntos
Inibidores da Angiogênese/metabolismo , Neovascularização de Coroide , Osteonectina/metabolismo , Fator A de Crescimento do Endotélio Vascular/metabolismo , Receptor 1 de Fatores de Crescimento do Endotélio Vascular/metabolismo , Animais , Olho/anatomia & histologia , Olho/metabolismo , Olho/patologia , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/genética , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Camundongos , Camundongos Knockout , Osteonectina/genética , Fator de Crescimento Placentário , Proteínas da Gravidez/metabolismo , Proteína Fosfatase 1 , Proteína Tirosina Fosfatase não Receptora Tipo 6 , Proteínas Tirosina Fosfatases/genética , Proteínas Tirosina Fosfatases/metabolismo , Transdução de Sinais/fisiologia , Receptor 1 de Fatores de Crescimento do Endotélio Vascular/genética , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/genética , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/metabolismo
6.
J Neurosci ; 25(42): 9591-601, 2005 Oct 19.
Artigo em Inglês | MEDLINE | ID: mdl-16237164

RESUMO

The opioid-like neuropeptide nociceptin/orphanin FQ (N/OFQ) and its receptor (NOP) are expressed in the substantia nigra (SN), a brain area containing dopamine neurons that degenerate in Parkinson's disease. Endogenous N/OFQ facilitates nigral glutamate release and inhibits nigrostriatal dopamine transmission and motor behavior. Here, we present evidence suggesting that endogenous N/OFQ may contribute to Parkinson's disease. Pharmacological blockade of the SN N/OFQ-NOP receptor system attenuated parkinsonian-like akinesia/hypokinesia in 6-hydroxydopamine hemilesioned or haloperidol-treated rats, whereas deletion of the NOP receptor gene conferred mice partial protection from haloperidol-induced motor depression. The antiparkinsonian action of NOP receptor antagonists was associated with reduction of glutamate release in the SN. In 6-hydroxydopamine hemilesioned rats, enhancement of N/OFQ expression and release was detected in the lesioned compared with the unlesioned SN, indicating that parkinsonism may be associated with overactivation of the N/OFQ-NOP receptor system in the SN. Finally, deletion of the N/OFQ gene conferred mice partial protection against 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine-induced loss of SN dopamine neurons. Based on these data, we propose that NOP receptor antagonists may represent a novel approach for combined (symptomatic and neuroprotective) therapy of Parkinson's disease.


Assuntos
Degeneração Neural/fisiopatologia , Peptídeos Opioides/antagonistas & inibidores , Peptídeos Opioides/fisiologia , Doença de Parkinson/fisiopatologia , Transmissão Sináptica/fisiologia , 1-Metil-4-Fenil-1,2,3,6-Tetra-Hidropiridina/farmacologia , Animais , Levodopa/farmacologia , Levodopa/uso terapêutico , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Degeneração Neural/tratamento farmacológico , Doença de Parkinson/tratamento farmacológico , Ratos , Ratos Sprague-Dawley , Transmissão Sináptica/efeitos dos fármacos , Nociceptina
7.
J Comp Neurol ; 494(5): 815-33, 2006 Feb 10.
Artigo em Inglês | MEDLINE | ID: mdl-16374808

RESUMO

Ts65Dn, a mouse model of Down syndrome (DS), demonstrates abnormal hippocampal synaptic plasticity and behavioral abnormalities related to spatial learning and memory. The molecular mechanisms leading to these impairments have not been identified. In this study, we focused on the G-protein-activated inwardly rectifying potassium channel 2 (GIRK2) gene that is highly expressed in the hippocampus region. We studied the expression pattern of GIRK subunits in Ts65Dn and found that GIRK2 was overexpressed in all analyzed Ts65Dn brain regions. Interestingly, elevated levels of GIRK2 protein in the Ts65Dn hippocampus and frontal cortex correlated with elevated levels of GIRK1 protein. This suggests that heteromeric GIRK1-GIRK2 channels are overexpressed in Ts65Dn hippocampus and frontal cortex, which could impair excitatory input and modulate spike frequency and synaptic kinetics in the affected regions. All GIRK2 splicing isoforms examined were expressed at higher levels in the Ts65Dn in comparison to the diploid hippocampus. The pattern of GIRK2 expression in the Ts65Dn mouse brain revealed by in situ hybridization and immunohistochemistry was similar to that previously reported in the rodent brain. However, in the Ts65Dn mouse a strong immunofluorescent staining of GIRK2 was detected in the lacunosum molecular layer of the CA3 area of the hippocampus. In addition, tyrosine hydroxylase containing dopaminergic neurons that coexpress GIRK2 were more numerous in the substantia nigra compacta and ventral tegmental area in the Ts65Dn compared to diploid controls. In summary, the regional localization and the increased brain levels coupled with known function of the GIRK channel may suggest an important contribution of GIRK2 containing channels to Ts65Dn and thus to DS neurophysiological phenotypes.


Assuntos
Síndrome de Down/metabolismo , Lobo Frontal/metabolismo , Canais de Potássio Corretores do Fluxo de Internalização Acoplados a Proteínas G/metabolismo , Hipocampo/metabolismo , Substância Negra/metabolismo , Animais , Modelos Animais de Doenças , Síndrome de Down/genética , Canais de Potássio Corretores do Fluxo de Internalização Acoplados a Proteínas G/genética , Regulação da Expressão Gênica , Imuno-Histoquímica , Hibridização In Situ , Masculino , Camundongos , Camundongos Endogâmicos C3H , Camundongos Endogâmicos C57BL , Camundongos Mutantes , Plasticidade Neuronal/genética , Plasticidade Neuronal/fisiologia , Subunidades Proteicas/genética , Subunidades Proteicas/metabolismo , RNA Mensageiro/análise , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Estatísticas não Paramétricas
8.
FASEB J ; 17(11): 1502-4, 2003 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-12824283

RESUMO

Lactoperoxidase (LPO) is an enzyme with antimicrobial properties present in saliva, milk, tears, and airway secretions. Although the formation of microbicidal oxidants by LPO has been recognized for some time, the source of hydrogen peroxide (H2O2) for LPO-catalyzed reactions remains unknown. Reactive oxygen species produced by the phagocyte NADPH oxidase (phox) play a critical role in host defense against pathogens; however, analogous oxidant-generating systems in other tissues have not been associated with antimicrobial activity. Several homologues of gp91phox, the catalytic core of this enzyme, were described recently; dual oxidase (Duox)1/thyroid oxidase 1 and Duox2/thyroid oxidase 2 were identified in the thyroid gland and characterized as H2O2 donors for thyroxin biosynthesis. We examined Duox1 and Duox2 expression in secretory glands and on mucosal surfaces and give evidence for their presence and activity in salivary glands, rectum, trachea, and bronchium. Epithelial cells in salivary excretory ducts and rectal glands express Duox2, whereas tracheal and bronchial epithelial cells express Duox1. Furthermore, we detected Duox1-dependent H2O2 release by cultured human bronchial epithelial cells. Our observations suggest that Duox1 and Duox2 are novel H2O2 sources that can support LPO-mediated antimicrobial defense mechanisms on mucosal surfaces.


Assuntos
Flavoproteínas , Peróxido de Hidrogênio/metabolismo , Mucosa/metabolismo , Oxirredutases/metabolismo , Brônquios/anatomia & histologia , Oxidases Duais , Células Epiteliais/química , Células Epiteliais/metabolismo , Humanos , Imunidade nas Mucosas , Lactoperoxidase/análise , Lactoperoxidase/metabolismo , Modelos Biológicos , Mucosa/química , NADPH Oxidases/análise , NADPH Oxidases/metabolismo , Oxirredutases/análise , RNA Mensageiro/análise , Reto/química , Mucosa Respiratória/metabolismo , Sistema Respiratório/anatomia & histologia , Ductos Salivares/química , Glândulas Salivares/anatomia & histologia , Glândulas Salivares/metabolismo , Simportadores/análise , Simportadores/metabolismo
9.
J Neurotrauma ; 20(6): 523-32, 2003 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-12906737

RESUMO

Nociceptin/orphanin FQ (N/OFQ) is a recently identified opioid-related neuropeptide. Earlier in vitro studies revealed regulation of N/OFQ expression by injury-induced factors, such as ciliary neurotrophic factor, inflammatory cytokines, and reactive oxygen species. We have extended our studies to in vivo experiments investigating the effect of traumatic brain injury on N/OFQ gene expression and peptide levels in the rat brain. Stab wound injury to the rat cerebral cortex led to a significant increase in N/OFQ mRNA levels in the vicinity of the injury, with the largest induction being seen at 24 h post-injury. Quantitative in situ hybridization revealed an almost twofold increase in the number of cells expressing N/OFQ, and the signal intensities within cells were also elevated. Stab wound injury leads to proliferation and hypertrophy of astrocytes, which respond to injury-related factors in vitro by up-regulating N/OFQ expression. However, in vivo N/OFQ co-localized exclusively with the neuronal marker, NeuN, following injury. N/OFQ expression was not detected in caspase-3-positive neurons undergoing apoptosis following injury, and increased N/OFQ expression was spatially more extended than the secondary injury-induced responses, such as astrogliosis and neuronal degeneration. Elevation of N/OFQ immunoreactivity closely followed the increase in N/OFQ gene expression as determined by immunohistochemistry. N/OFQ selectively activates the NOP receptor (ORL-1), but we did not detect parallel changes in levels of NOP receptor mRNA following injury, indicating regulation of the nociceptin system at the peptide and not the receptor level. In summary, a profound and prolonged up-regulation of N/OFQ expression in neurons surrounding a stab wound lesion to cerebral cortex was detected. The function of N/OFQ up-regulation in injury-induced responses in the brain is currently under investigation.


Assuntos
Lesões Encefálicas/metabolismo , Córtex Cerebral/metabolismo , Regulação da Expressão Gênica/fisiologia , Neurônios/metabolismo , Receptores Opioides/biossíntese , Animais , Córtex Cerebral/química , Neurônios/química , Ratos , Ratos Sprague-Dawley , Receptores Opioides/genética , Regulação para Cima/fisiologia , Receptor de Nociceptina
10.
Brain Res ; 997(1): 24-9, 2004 Jan 30.
Artigo em Inglês | MEDLINE | ID: mdl-14715146

RESUMO

Nociceptin/orphanin FQ (N/OFQ), the endogenous agonist for the opioid receptor-like receptor 1 (ORL1), shows significant similarities to dynorphin A in structure and distribution in rat central nervous system. The distribution of N/OFQ in human brain has not been studied. We measured the concentrations of N/OFQ in 47 microdissected areas of the central nervous system of adult human brain using radioimmunoassay (RIA). Significant heterogeneity was found in the levels of N/OFQ concentration in the various analyzed regions. The highest concentrations were measured in the dorsal central gray matter (periaqueductal gray), the locus coeruleus, the ventromedial nucleus of hypothalamus, the septum and the dorsal horn of the spinal cord. High concentrations were also detected in other hypothamamic nuclei, the inferior colliculus, the ventral central gray matter, the pontine tegmentum, the amygdala, the reticular formation and the spinal trigeminal nucleus. Considerable similarity with the distribution of N/OFQ in rat CNS was observed. The widespread distribution in CNS predicts multifaceted functions for N/OFQ.


Assuntos
Química Encefálica , Encéfalo/metabolismo , Peptídeos Opioides/metabolismo , Adulto , Encéfalo/anatomia & histologia , Mapeamento Encefálico , Cromatografia em Gel/métodos , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Radioimunoensaio/métodos , Nociceptina
11.
J Lipid Res ; 50(1): 90-7, 2009 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-18815435

RESUMO

Recent data suggest that dietary fat promotes intestinal absorption of lipopolysaccharides (LPS) from the gut microflora, which might contribute to various inflammatory disorders. The mechanism of fat-induced LPS absorption is unclear, however. Intestinal-epithelial cells can internalize LPS from the apical surface and transport LPS to the Golgi. The Golgi complex also contains newly formed chylomicrons, the lipoproteins that transport dietary long-chain fat through mesenteric lymph and blood. Because LPS has affinity for chylomicrons, we hypothesized that chylomicron formation promotes LPS absorption. In agreement with our hypothesis, we found that CaCo-2 cells released more cell-associated LPS after incubation with oleic-acid (OA), a long-chain fatty acid that induces chylomicron formation, than with butyric acid (BA), a short-chain fatty acid that does not induce chylomicron formation. Moreover, the effect of OA was blocked by the inhibitor of chylomicron formation, Pluronic L-81. We also observed that intragastric triolein (TO) gavage was followed by increased plasma LPS, whereas gavage with tributyrin (TB), or TO plus Pluronic L-81, was not. Most intestinally absorbed LPS was present on chylomicron remnants (CM-R) in the blood. Chylomicron formation also promoted transport of LPS through mesenteric lymph nodes (MLN) and the production of TNFalpha mRNA in the MLN. Together, our data suggest that intestinal epithelial cells may release LPS on chylomicrons from cell-associated pools. Chylomicron-associated LPS may contribute to postprandial inflammatory responses or chronic diet-induced inflammation in chylomicron target tissues.


Assuntos
Quilomícrons/fisiologia , Lipopolissacarídeos/fisiologia , Animais , Células CACO-2 , Linhagem Celular , Quilomícrons/metabolismo , Ácidos Graxos/metabolismo , Humanos , Absorção Intestinal , Lipopolissacarídeos/metabolismo , Linfonodos/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Modelos Biológicos , Poloxâmero/farmacologia , Triglicerídeos/farmacologia
12.
J Biol Chem ; 278(22): 20006-12, 2003 May 30.
Artigo em Inglês | MEDLINE | ID: mdl-12657628

RESUMO

Superoxide production by phagocytes involves activation of a multi-component NADPH oxidase. Recently, several homologues of the catalytic component of the phagocyte oxidase, gp91phox, were identified in various tissues. Here we describe two proteins, p41 and p51, with significant homology to two cytosolic components of the phagocytic oxidase, p47phox and p67phox. Like p47phox, p41 contains an amino-terminal Phox homology domain, two SH3 domains, and a conserved carboxyl-terminal, proline-rich motif. Similarly, p51 is homologous to p67phox, containing four amino-terminal tetratrico-peptide repeats, a conserved "activation domain" motif, a PB1 domain, and a carboxyl-terminal SH3 domain. The highest levels of p41 transcript are detected in the colon and in other gastrointestinal tissues that express Nox1, the predominant gp91phox homologue in these tissues. In contrast, the p51 transcript showed a more widespread expression pattern, suggesting that it may support other tissue-specific oxidases. Mouse colon in situ hybridization detected both transcripts in the epithelial cells of colon crypts. Heterologous co-expression of p41 and p51 significantly enhances the superoxide-generating activity of Nox1-expressing cells; thus, p41 and p51 appear to be novel regulators of Nox1. These proteins also support the activity of gp91phox, albeit at much lower levels than the cytosolic phox counterparts. Our results suggest colon epithelial cells contain a multi-component NAD(P)H oxidase with a molecular architecture similar to the phagocytic oxidase.


Assuntos
Colo/metabolismo , Proteínas de Ligação a DNA/fisiologia , NADPH Oxidases/metabolismo , Fosfoproteínas/química , Fosfoproteínas/fisiologia , Superóxidos/metabolismo , Transativadores , Sequência de Aminoácidos , Animais , Colo/citologia , Colo/enzimologia , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/genética , Células Epiteliais/enzimologia , Células Epiteliais/metabolismo , Genes Supressores de Tumor , Hibridização In Situ , Camundongos , Dados de Sequência Molecular , Fosfoproteínas/genética , RNA Mensageiro/genética , Homologia de Sequência de Aminoácidos , Fatores de Transcrição , Proteínas Supressoras de Tumor
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