RESUMEN
Human parechoviruses (HPeV) are picornaviruses with a highly-ordered RNA genome contained within icosahedrally-symmetric capsids. Ordered RNA structures have recently been shown to interact with capsid proteins VP1 and VP3 and facilitate virus assembly in HPeV1. Using an assay that combines reversible cross-linking, RNA affinity purification and peptide mass fingerprinting (RCAP), we mapped the RNA-interacting regions of the capsid proteins from the whole HPeV1 virion in solution. The intrinsically-disordered N-termini of capsid proteins VP1 and VP3, and unexpectedly, VP0, were identified to interact with RNA. Comparing these results to those obtained using recombinantly-expressed VP0 and VP1 confirmed the virion binding regions, and revealed unique RNA binding regions in the isolated VP0 not previously observed in the crystal structure of HPeV1. We used RNA fluorescence anisotropy to confirm the RNA-binding competency of each of the capsid proteins' N-termini. These findings suggests that dynamic interactions between the viral RNA and the capsid proteins modulate virus assembly, and suggest a novel role for VP0.
Asunto(s)
Proteínas de la Cápside/metabolismo , Parechovirus/fisiología , ARN Viral/metabolismo , Virión/metabolismo , Ensamble de Virus , Proteínas de la Cápside/química , Reactivos de Enlaces Cruzados/química , Células HT29 , Humanos , Modelos Moleculares , ARN Viral/químicaRESUMEN
2-Amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP), formed during the cooking of foods, induces colon cancer in rodents. PhIP is metabolically activated by cytochromes P450 (P450s). To evaluate the role of hepatic P450s in the bioactivation of PhIP, we used Reductase Conditional Null (RCN) mice, in which cytochrome P450 oxidoreductase (POR), the unique electron donor to P450s, can be specifically deleted in hepatocytes by pretreatment with 3-methylcholanthrene (3-MC), resulting in the loss of essentially all hepatic P450 function. RCN mice were treated orally with 50 mg/kg b.wt. PhIP daily for 5 days, with and without 3-MC pretreatment. PhIP-DNA adducts (i.e., N-(deoxyguanosin-8-yl)-2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine [dG-C8-PhIP]), measured by liquid chromatography-tandem mass spectrometry, were highest in colon (1362 adducts/10(8) deoxynucleosides), whereas adduct levels in liver were â¼3.5-fold lower. Whereas no differences in PhIP-DNA adduct levels were found in livers with active POR versus inactivated POR, adduct levels were on average â¼2-fold lower in extrahepatic tissues of mice lacking hepatic POR. Hepatic microsomes from RCN mice with or without 3-MC pretreatment were also incubated with PhIP and DNA in vitro. PhIP-DNA adduct formation was â¼8-fold lower with hepatic microsomes from POR-inactivated mice than with those with active POR. Most of the hepatic microsomal activation of PhIP in vitro was attributable to CYP1A. Our results show that PhIP-DNA adduct formation in colon involves hepatic N-oxidation, circulation of activated metabolites via the bloodstream to extrahepatic tissues, and further activation, resulting in the formation of dG-C8-PhIP. Besides hepatic P450s, PhIP may be metabolically activated mainly by a non-P450 pathway in liver.
Asunto(s)
Aductos de ADN/metabolismo , Imidazoles/metabolismo , Microsomas Hepáticos/enzimología , NADPH-Ferrihemoproteína Reductasa/metabolismo , Animales , Western Blotting , Cromatografía Liquida , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , NADPH-Ferrihemoproteína Reductasa/genética , Espectrometría de Masa por Ionización de Electrospray , Espectrometría de Masas en TándemRESUMEN
Exposure to aristolochic acid I (AAI) is associated with aristolochic acid nephropathy, Balkan endemic nephropathy, and urothelial cancer. Individual differences in xenobiotic-metabolizing enzyme activities are likely to be a reason for interindividual susceptibility to AA-induced disease. We evaluated the reductive activation and oxidative detoxication of AAI by cytochrome P450 (P450) 1A1 and 1A2 using the Cyp1a1(-/-) and Cyp1a2(-/-) single-knockout and Cyp1a1/1a2(-/-) double-knockout mouse lines. Incubations with hepatic microsomes were also carried out in vitro. P450 1A1 and 1A2 were found to (i) activate AAI to form DNA adducts and (ii) detoxicate it to 8-hydroxyaristolochic acid I (AAIa). AAI-DNA adduct formation was significantly higher in all tissues of Cyp1a1/1a2(-/-) than Cyp1a(+/+) wild-type (WT) mice. AAI-DNA adduct levels were elevated only in selected tissues from Cyp1a1(-/-) versus Cyp1a2(-/-) mice, compared with those in WT mice. In hepatic microsomes, those from WT as well as Cyp1a1(-/-) and Cyp1a2(-/-) mice were able to detoxicate AAI to AAIa, whereas Cyp1a1/1a2(-/-) microsomes were less effective in catalyzing this reaction, confirming that both mouse P450 1A1 and 1A2 are both involved in AAI detoxication. Under hypoxic conditions, mouse P450 1A1 and 1A2 were capable of reducing AAI to form DNA adducts in hepatic microsomes; the major roles of P450 1A1 and 1A2 in AAI-DNA adduct formation were further confirmed using selective inhibitors. Our results suggest that, in addition to P450 1A1 and 1A2 expression levels in liver, in vivo oxygen concentration in specific tissues might affect the balance between AAI nitroreduction and demethylation, which in turn would influence tissue-specific toxicity or carcinogenicity.
Asunto(s)
Ácidos Aristolóquicos/farmacocinética , Carcinógenos/farmacocinética , Citocromo P-450 CYP1A1/metabolismo , Citocromo P-450 CYP1A2/metabolismo , Medicamentos Herbarios Chinos/farmacocinética , Animales , Ácidos Aristolóquicos/orina , Nefropatía de los Balcanes/enzimología , Biotransformación , Citocromo P-450 CYP1A1/deficiencia , Citocromo P-450 CYP1A1/genética , Citocromo P-450 CYP1A2/deficiencia , Citocromo P-450 CYP1A2/genética , Aductos de ADN , Susceptibilidad a Enfermedades , Femenino , Riñón/enzimología , Hígado/enzimología , Pulmón/enzimología , Ratones , Ratones Noqueados , Microsomas/enzimología , Neoplasias Urológicas/enzimologíaRESUMEN
Retrotransposition amplifies LINE-1 (L1) to high copy number in mammalian genomes. The L1 protein encoded by ORF1 (ORF1p) is required for retrotransposition. This dependence on ORF1p was investigated by mutating three highly conserved residues, R238, R284 and Y318 to alanine, thereby inactivating retrotransposition. R284A and Y318A were rescued by further substituting the alanine with the appropriate conservative amino acid, e.g. lysine or phenylalanine, respectively, whereas R238K remained inactive. Quantification of the steady-state levels of L1 RNA and ORF1p failed to discriminate active from inactive variants, indicating loss of L1 retrotransposition resulted from loss of function rather than reduced expression. The two biochemical properties known for ORF1p are high-affinity RNA binding and nucleic acid chaperone activity. Only R238A/K exhibited significantly reduced RNA affinities. The nucleic acid chaperone activities of the remaining paired mutants were assessed by single-molecule DNA stretching and found to mirror retrotransposition activity. To further examine ORF1p chaperone function, their energetic barriers to DNA annealing and melting were derived from kinetic work. When plotted against each other, the ratio of these two activities distinguished functional from non-functional ORF1p variants. These findings enhance our understanding of the requirements for ORF1p in LINE-1 retrotransposition and, more generally, nucleic acid chaperone function.
Asunto(s)
Elementos de Nucleótido Esparcido Largo , Retroelementos/genética , Sustitución de Aminoácidos , Animales , ADN/química , Ratones , Mutación , Desnaturalización de Ácido Nucleico , ARN/metabolismoRESUMEN
We immunohistochemically identified protein kinase C (PKC) isoenzymes and the receptor for activated C-kinase (RACK-1) in normal, chronically inflamed, and malignant pancreas specimens. Expression patterns were specific and consistent for each microanatomic structure. In chronic pancreatitis, the expression patterns by epithelial cells were indistinguishable from those in normal pancreas. In the stroma, there was a gain of PKC-delta (P < .05) and loss of PKC-mu (P < .0001). Expression in pancreatic duct carcinomas, compared with control normal minor ductular epithelial cells, revealed relative loss of PKC-epsilon (P < .0001), PKC-iota (P = .005), and PKC-theta (P < .0001) but no gain in any isoenzyme. Compared with control normal major duct epithelial cells, the principal differences were a relative loss in PKC-gamma (P < .05) and a relative gain in PKC-beta (P < .05), PKC-iota (P < .05), and PKC-zeta (P < .005). The stroma adjacent to ductal carcinomas was characterized by prominent expression of PKC-mu and a gain in PKC-delta (P < .0001) and PKC-zeta (P > .005). Ampullary carcinomas revealed a relative gain of PKC-iota (P < .05) and RACK-1 (P < .05). In the adjacent stroma was enhanced expression of PKC-delta (P < .005) and PKC-gamma (P < .001) and loss of PKC-mu (P < .05). Specific changes in isoenzyme expression in stroma of chronic pancreatitis and in epithelial cells and stroma of ductal and ampullary pancreatic adenocarcinomas reflect specific modulation of intracellular signaling pathways that control critical homeostatic mechanisms.
Asunto(s)
Carcinoma Ductal Pancreático/enzimología , Neoplasias Pancreáticas/enzimología , Pancreatitis/enzimología , Proteína Quinasa C/metabolismo , Carcinoma Ductal Pancreático/patología , Enfermedad Crónica , Humanos , Técnicas para Inmunoenzimas , Isoenzimas/metabolismo , Páncreas/anatomía & histología , Páncreas/enzimología , Páncreas/patología , Neoplasias Pancreáticas/patología , Pancreatitis/patología , Proteína Quinasa C/clasificación , Receptores de Cinasa C Activada , Receptores de Superficie Celular/metabolismoRESUMEN
BACKGROUND: This prospective study evaluated the effectiveness and safety of laparoscopic adjustable gastric banding (LAGB) for morbid obesity. METHODS: Ninety-five consecutive patients (89 female; median age 38 years, range 19 to 69) underwent LAGB for morbid obesity. Median weight and body mass index were 123.2 (88.9 to 228.6) kg and 45 (32.7-76.4) kg/m(2) respectively. Significant coexistent disease was present in 52 (55%) patients. RESULTS: Median excess weight loss was 53% (range 96.9% to 12.1%) and 62% (range 107.5% to 32.3%) at 1 and 2 years respectively (P <0.001). Median operative time was 90 (range 35 to 285) minutes and inpatient stay 2 (range 1 to 10) days. Early complications were seen in 17 (18%) patients most commonly nausea/vomiting or dysphagia. Late complications were seen in 25 (26.3%) patients, most frequently vomiting or reflux due to band slippage or pouch dilatation. There was 1 (1%) operative death. CONCLUSIONS: LAGB is an effective operation for morbid obesity that results in equivalent weight loss to open surgical procedures.
Asunto(s)
Gastroplastia/métodos , Laparoscopía/métodos , Obesidad Mórbida/cirugía , Adulto , Anciano , Análisis de Varianza , Índice de Masa Corporal , Femenino , Estudios de Seguimiento , Gastroplastia/efectos adversos , Humanos , Laparoscopía/efectos adversos , Tiempo de Internación , Masculino , Persona de Mediana Edad , Obesidad Mórbida/diagnóstico , Probabilidad , Estudios Prospectivos , Medición de Riesgo , Resultado del Tratamiento , Pérdida de PesoRESUMEN
Pancreatic cancer has a very poor prognosis and is a common cause of cancer death in the Western world. Certain genetic alterations may be important in the prognosis of pancreatic cancer. Activation mutations in the K- ras oncogene occur in around 90% of pancreatic cancers, and the overexpression of growth factors epidermal growth factor (EGF), transforming growth factor (TGF)alpha, TGFbetas 1-3, acidic fibroblast growth factor (aFGF), basic FGF (bFGF), and growth factor receptors c-erbB-2 and -3 and TGFRbetas 1-3 is common. High mutation levels of cell cycle control genes such as p53, p16, p21, SMAD4, and cyclin D1 are found, and there is abnormal expression of apoptotic genes, such as bcl-2, bcl-XL, and bax. The expression of several of these growth factors and their receptors has been found to be associated with poorly differentiated tumors of an advanced stage and decreased survival. However, the inactivation and loss of expression of p16, p53, and p21, and the expression of several apoptotic genes, such as bax and bcl-2, have not been found to be of any prognostic significance. The expression of wild type p53, however, may predict responsiveness to chemotherapy. TGFbeta1 expression has been shown to be associated with longer survival in patients with pancreatic cancer. Two studies (including our own) have found bcl-XL expression to be significantly associated with poor survival. These and newer molecular markers may prove to be important in the choice of future therapies for pancreatic cancer.