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This article describes primary data and resources available from the Boston Adolescent Neuroimaging of Depression and Anxiety (BANDA) study, a novel arm of the Human Connectome Project (HCP). Data were collected from 215 adolescents (14-17 years old), 152 of whom had current diagnoses of anxiety and/or depressive disorders at study intake. Data include cross-sectional structural (T1- and T2-weighted), functional (resting state and three tasks), and diffusion-weighted magnetic resonance images. Both unprocessed and HCP minimally-preprocessed imaging data are available within the data release packages. Adolescent and parent clinical interview data, as well as cognitive and neuropsychological data are also included within these packages. Release packages additionally provide data collected from self-report measures assessing key features of adolescent psychopathology, including: anxious and depressive symptom dimensions, behavioral inhibition/activation, exposure to stressful life events, and risk behaviors. Finally, the release packages include 6- and 12-month longitudinal data acquired from clinical measures. Data are publicly accessible through the National Institute of Mental Health Data Archive (ID: #2505).
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Ansiedad , Conectoma , Depresión , Humanos , Adolescente , Masculino , Femenino , Trastornos de Ansiedad , Trastorno Depresivo , Imagen por Resonancia Magnética , Encéfalo/diagnóstico por imagenRESUMEN
We asked whether brain connectomics can predict response to treatment for a neuropsychiatric disorder better than conventional clinical measures. Pre-treatment resting-state brain functional connectivity and diffusion-weighted structural connectivity were measured in 38 patients with social anxiety disorder (SAD) to predict subsequent treatment response to cognitive behavioral therapy (CBT). We used a priori bilateral anatomical amygdala seed-driven resting connectivity and probabilistic tractography of the right inferior longitudinal fasciculus together with a data-driven multivoxel pattern analysis of whole-brain resting-state connectivity before treatment to predict improvement in social anxiety after CBT. Each connectomic measure improved the prediction of individuals' treatment outcomes significantly better than a clinical measure of initial severity, and combining the multimodal connectomics yielded a fivefold improvement in predicting treatment response. Generalization of the findings was supported by leave-one-out cross-validation. After dividing patients into better or worse responders, logistic regression of connectomic predictors and initial severity combined with leave-one-out cross-validation yielded a categorical prediction of clinical improvement with 81% accuracy, 84% sensitivity and 78% specificity. Connectomics of the human brain, measured by widely available imaging methods, may provide brain-based biomarkers (neuromarkers) supporting precision medicine that better guide patients with neuropsychiatric diseases to optimal available treatments, and thus translate basic neuroimaging into medical practice.
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Encéfalo/fisiopatología , Terapia Cognitivo-Conductual , Conectoma , Fobia Social/fisiopatología , Fobia Social/terapia , Adolescente , Adulto , Terapia Cognitivo-Conductual/métodos , Femenino , Humanos , Modelos Logísticos , Imagen por Resonancia Magnética , Masculino , Persona de Mediana Edad , Vías Nerviosas/fisiopatología , Fobia Social/diagnóstico , Pronóstico , Descanso , Sensibilidad y Especificidad , Resultado del Tratamiento , Adulto JovenRESUMEN
AIM: Glucose-dependent insulinotropic peptide (GIP) is an incretin hormone that is released from intestinal K cells in response to nutrient ingestion. We aimed to investigate the therapeutic potential of the novel N- and C-terminally modified GIP analogue AC163794. METHODS: AC163794 was synthesized by solid-phase peptide synthesis. Design involved the substitution of the C-terminus tail region of the dipeptidyl peptidase IV (DPP-IV)-resistant GIP analogue [d-Ala(2) ]GIP(1-42) with the unique nine amino acid tail region of exenatide. The functional activity and binding of AC163794 to the GIP receptor were evaluated in RIN-m5F ß-cells. In vitro metabolic stability was tested in human plasma and kidney membrane preparations. Acute insulinotropic effects were investigated in isolated mouse islets and during an intravenous glucose tolerance test in normal and diabetic Zucker fatty diabetic (ZDF) rats. The biological actions of AC163794 were comprehensively assessed in normal, ob/ob and high-fat-fed streptozotocin (STZ)-induced diabetic mice. Acute glucoregulatory effects of AC163794 were tested in diet-induced obese mice treated subchronically with AC3174, the exendatide analogue [Leu(14) ] exenatide. Human GIP or [d-Ala(2) ]GIP(1-42) were used for comparison. RESULTS: AC163794 exhibited nanomolar functional GIP receptor potency in vitro similar to GIP and [d-Ala(2) ]GIP(1-42). AC163794 was metabolically more stable in vitro and displayed longer duration of insulinotropic action in vivo versus GIP and [d-Ala(2) ]GIP(1-42). In diabetic mice, AC163794 improved HbA1c through enhanced insulinotropic action, partial restoration of pancreatic insulin content and improved insulin sensitivity with no adverse effects on fat storage and metabolism. AC163794 provided additional baseline glucose-lowering when injected to mice treated with AC3174. CONCLUSIONS: These studies support the potential use of a novel GIP analogue AC163794 for the treatment of type 2 diabetes.
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Glucemia/metabolismo , Diabetes Mellitus Experimental/metabolismo , Polipéptido Inhibidor Gástrico/análogos & derivados , Polipéptido Inhibidor Gástrico/metabolismo , Hipoglucemiantes/farmacología , Incretinas/farmacología , Obesidad/metabolismo , Animales , Química Farmacéutica , Diabetes Mellitus Experimental/tratamiento farmacológico , Femenino , Polipéptido Inhibidor Gástrico/síntesis química , Polipéptido Inhibidor Gástrico/efectos de los fármacos , Polipéptido Inhibidor Gástrico/farmacología , Masculino , Ratones , Ratones Obesos , Obesidad/tratamiento farmacológico , Ratas , Ratas Sprague-Dawley , Ratas ZuckerRESUMEN
In this paper, we report the synthesis of highly conducting phosphorous doped hydrogenated nanocrystalline silicon (nc-Si:H) films at substantially low substrate temperature (200 degrees C) by hot-wire chemical vapor deposition (HW-CVD) method using pure silane (SiH4) and phosphine (PH3) gas mixture without hydrogen dilution. Structural, optical and electrical properties of these films were investigated as a function of PH3 gas-phase ratio. The characterization of these films by low-angle X-ray diffraction, Raman spectroscopy and atomic force microscopy revealed that, the incorporation of phosphorous in nc-Si:H induces an amorphization in the nc-Si:H film structure. Fourier transform infrared spectroscopy analysis indicates that hydrogen predominately incorporated in phosphorous doped n-type nc-Si:H films mainly in di-hydrogen species (Si-H2) and poly-hydrogen (Si-H2)n bonded species signifying that the films become porous, and micro-void rich. We have observed high band gap (1.97-2.37 eV) in the films, though the hydrogen content is low (< 1.4 at.%) over the entire range of PH3 gas-phase ratio studied. Under the optimum deposition conditions, phosphorous doped nc-Si:H films with high dark conductivity (sigma Dark -5.3 S/cm), low charge-carrier activation energy (E(act) - 132 meV) and high band gap (- 2.01 eV), low hydrogen content (- 0.74 at.%) were obtained at high deposition rate (12.9 angstroms/s).
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Cristalización/métodos , Gases/química , Membranas Artificiales , Nanoestructuras/química , Nanoestructuras/ultraestructura , Fósforo/química , Silicio/química , Conductividad Eléctrica , Calor , Sustancias Macromoleculares/química , Ensayo de Materiales , Conformación Molecular , Tamaño de la Partícula , Propiedades de SuperficieRESUMEN
Previously, we showed that curcumin prevents chronic kidney disease (CKD) development in ⅚ nephrectomized (Nx) rats when given within 1 wk after Nx (Ghosh SS, Massey HD, Krieg R, Fazelbhoy ZA, Ghosh S, Sica DA, Fakhry I, Gehr TW. Am J Physiol Renal Physiol 296: F1146-F1157, 2009). To better mimic the scenario for renal disease in humans, we began curcumin and enalapril therapy when proteinuria was already established. We hypothesized that curcumin, by blocking the inflammatory mediators TNF-α and IL-1ß, could also reduce cyclooxygenase (COX) and phospholipase expression in the kidney. Nx animals were divided into untreated Nx, curcumin-treated, and enalapril-treated groups. Curcumin (75 mg/kg) and enalapril (10 mg/kg) were administered for 10 wk. Renal dysfunction in the Nx group, as evidenced by elevated blood urea nitrogen, plasma creatinine, proteinuria, segmental sclerosis, and tubular dilatation, was comparably reduced by curcumin and enalapril, with only enalapril significantly lowering blood pressure. Compared with controls, Nx animals had higher plasma/kidney TNF-α and IL-1ß, which were reduced by curcumin and enalapril treatment. Nx animals had significantly elevated kidney levels of cytosolic PLA(2), calcium-independent intracellular PLA(2), COX 1, and COX 2, which were comparably reduced by curcumin and enalapril. Studies in mesangial cells and macrophages were carried out to establish that the in vivo increase in PLA(2) and COX were mediated by TNF-α and IL-1ß and that curcumin, by antagonizing the cytokines, could significantly reduce both PLA(2) and COX. We conclude that curcumin ameliorates CKD by blocking inflammatory signals even if it is given at a later stage of the disease.
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Antiinflamatorios no Esteroideos/uso terapéutico , Antihipertensivos/uso terapéutico , Curcumina/uso terapéutico , Enalapril/uso terapéutico , Inflamación/tratamiento farmacológico , Fosfolipasas/metabolismo , Prostaglandina-Endoperóxido Sintasas/metabolismo , Insuficiencia Renal/tratamiento farmacológico , Animales , Antiinflamatorios no Esteroideos/farmacología , Antihipertensivos/farmacología , Curcumina/farmacología , Enalapril/farmacología , Inflamación/metabolismo , Mediadores de Inflamación/metabolismo , Interleucina-1beta/sangre , Riñón/efectos de los fármacos , Riñón/metabolismo , Nefrectomía , Ratas , Insuficiencia Renal/enzimología , Insuficiencia Renal/metabolismo , Factor de Necrosis Tumoral alfa/sangreRESUMEN
TNF-alpha and NF-kappaB play important roles in the development of inflammation in chronic renal failure (CRF). In hepatic cells, curcumin is shown to antagonize TNF-alpha-elicited NF-kappaB activation. In this study, we hypothesized that if inflammation plays a key role in renal failure then curcumin should be effective in improving CRF. The effectiveness of curcumin was compared with enalapril, a compound known to ameliorate human and experimental CRF. Investigation was conducted in Sprague-Dawley rats where CRF was induced by 5/6 nephrectomy (Nx). The Nx animals were divided into untreated (Nx), curcumin-treated (curcumin), and enalapril-treated (enalapril) groups. Sham-operated animals served as a control. Renal dysfunction in the Nx group, as evidenced by elevated blood urea nitrogen, plasma creatinine, proteinuria, segmental sclerosis, and tubular dilatation, was significantly reduced by curcumin and enalapril treatment. However, only enalapril significantly improved blood pressure. Compared with the control, the Nx animals had significantly higher plasma and kidney TNF-alpha, which was associated with NF-kappaB activation and macrophage infiltration in the kidney. These changes were effectively antagonized by curcumin and enalapril treatment. The decline in the anti-inflammatory peroxisome proliferator-activated receptor gamma (PPARgamma) seen in Nx animals was also counteracted by curcumin and enalapril. Studies in mesangial cells were carried out to further establish that the anti-inflammatory effect of curcumin in vivo was mediated essentially by antagonizing TNF-alpha. Curcumin dose dependently antagonized the TNF-alpha-mediated decrease in PPARgamma and blocked transactivation of NF-kappaB and repression of PPARgamma, indicating that the anti-inflamatory property of curcumin may be responsible for alleviating CRF in Nx animals.
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Antiinflamatorios no Esteroideos/farmacología , Curcumina/farmacología , Fallo Renal Crónico/tratamiento farmacológico , Nefritis/tratamiento farmacológico , Inhibidores de la Enzima Convertidora de Angiotensina/farmacología , Animales , Presión Sanguínea/efectos de los fármacos , Nitrógeno de la Urea Sanguínea , Células Cultivadas , Creatinina/sangre , Modelos Animales de Enfermedad , Enalapril/farmacología , Hipertensión Renal/tratamiento farmacológico , Hipertensión Renal/inmunología , Fallo Renal Crónico/inmunología , Fallo Renal Crónico/patología , Macrófagos/patología , Células Mesangiales/citología , Células Mesangiales/efectos de los fármacos , Células Mesangiales/inmunología , FN-kappa B/genética , FN-kappa B/metabolismo , Nefrectomía , Nefritis/inmunología , Nefritis/patología , PPAR gamma/genética , PPAR gamma/metabolismo , Proteinuria/tratamiento farmacológico , Proteinuria/inmunología , Proteinuria/patología , Ratas , Ratas Sprague-Dawley , Transfección , Factor de Necrosis Tumoral alfa/genética , Factor de Necrosis Tumoral alfa/metabolismoRESUMEN
In this work, we explore the use of classification algorithms in predicting mental states from functional neuroimaging data. We train a linear support vector machine classifier to characterize spatial fMRI activation patterns. We employ a general linear model based feature extraction method and use the t-test for feature selection. We evaluate our method on a memory encoding task, using participants' subjective prediction about learning as a benchmark for our classifier. We show that the classifier achieves better than random predictions and the average accuracy is close to subject's own prediction performance. In addition, we validate our tool on a simple motor task where we demonstrate an average prediction accuracy of over 90%. Our experiments demonstrate that the classifier performance depends significantly on the complexity of the experimental design and the mental process of interest.
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The adenine nucleotide translocase (ANT) is a key component in maintaining cellular energy homeostasis, and has also been implicated in formation of the mitochondrial permeability transition pore. Human ANT-3 was cloned from a human heart cDNA library and expressed as a histidine-tagged fusion protein in the mitochondria of the Trichoplusia ni. cell line. Overexpression resulted in a concomitant decrease in the endogenous ANT content, allowing for the characterization of binding of known ANT ligands to the human protein. Binding affinities for bongkrekic acid (BKA), ADP, and atractyloside (ATR) were measured in mitochondria from the human ANT-3 expressing cell line, and compared to similar preparations from bovine heart mitochondria by use of a novel radioiodinated derivative of ATR. Binding to ANT-3 by the high affinity inhibitors BKA and ATR, as well as the lower affinity natural ligand ADP, was similar to that measured in bovine heart mitochondria, and to that previously reported for mammalian heart mitochondria. Characterizations such as these of human ANT isoforms may lead to drug development for enhanced mitochondrial function and cellular viability.
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Translocador 3 del Nucleótido Adenina/genética , Translocador 3 del Nucleótido Adenina/metabolismo , Proteínas Mitocondriales/genética , Proteínas Mitocondriales/metabolismo , Adenosina Difosfato/metabolismo , Animales , Atractilósido/análogos & derivados , Atractilósido/química , Atractilósido/metabolismo , Ácido Bongcréquico/metabolismo , Bovinos , Línea Celular , Metabolismo Energético , Expresión Génica , Humanos , Técnicas In Vitro , Radioisótopos de Yodo , Cinética , Ligandos , Mitocondrias Cardíacas/metabolismo , Mariposas Nocturnas , Unión Proteica , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismoRESUMEN
Host immune responses limit the duration of expression of transgenes introduced by recombinant adenoviruses, preclude gene transfer upon vector readministration and cause liver injury. CTLA4Ig inhibits immune response by blocking the co-stimulatory interaction between CD28 on T cells and B7 on antigen-presenting cells. We have constructed a recombinant adenovirus, Ad-hUGT1A1-CTLA4Ig that coexpresses human bilirubin-uridinediphosphoglucuronate glucuronosyltransferase (hUGT1A1) and soluble murine CTLA4Ig, both driven by CMV immediate-early promoters. After intravenous injection of this vector (6 x 10(11) p.f.u.) into UGT1A1-deficient jaundiced Gunn rats, serum CTLA4Ig levels peaked at 1.8-2.0 mg/ml on day 7 and declined thereafter to 0.2 mg/ml by day 180. Serum bilirubin declined from mean preinjection levels of 8.0 mg/dl to 0.48-0.6 mg/dl in 3 days, remained normal for 28 weeks, and then gradually increased to 8 mg/dl by day 350. A second injection of Ad-hUGT1A1-CTLA4Ig normalized serum bilirubin. In two rats in this group that were followed longer, serum bilirubin increased to 3.1 and 3.5 mg/dl in 40 weeks, but was normalized again after a third injection. The antibody and cytotoxic lymphocyte (CTL) responses were negligible, and liver biopsy showed no inflammatory cell infiltration. Rats receiving a tertiary challenge with Ad-LacZ (expressing E. coli beta-galactosidase) (5 x 10(11) p.f.u.), 2 months after the second dose of Ad-hUGT1A1-CTLA4Ig, showed beta-galactosidase expression in over 80% of hepatocytes. In contrast, after Ad-hUGT1A1 (which expresses UGT1A1 alone) injection, serum bilirubin remained normal for only 4 weeks, and returned to preinjection levels by day 120. Bilirubin levels did not decline upon reinjection, and beta-galactosidase was not expressed after Ad-LacZ. High levels of adenovirus-specific antibodies and CTL, and hepatic inflammation were found. This is the first demonstration that coexpression of CTLA4Ig permits prolonged expression and repeatable gene transfer by an adenoviral vector.
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Adenoviridae/genética , Antígenos de Diferenciación/genética , Síndrome de Crigler-Najjar/terapia , Terapia Genética/métodos , Vectores Genéticos/administración & dosificación , Inmunoconjugados , Abatacept , Animales , Antígenos CD , Bilirrubina/genética , Bilirrubina/metabolismo , Antígeno CTLA-4 , Femenino , Expresión Génica , Vectores Genéticos/genética , Glucuronosiltransferasa/genética , Humanos , Hígado/metabolismo , Masculino , Ratones , Ratas , Ratas Gunn , Factores de Tiempo , TransgenesRESUMEN
Genetic lesions of bilirubin-uridine-diphosphoglucuronate glucuronosyltransferase-1 (UGT1A1) completely or partially abolish hepatic bilirubin glucuronidation, causing Crigler-Najjar syndrome type 1 or 2, respectively. Clinical observations indicate that some mutant forms of human UGT1A1 (hUGT1A1) may be dominant-negative, suggesting their interaction with the wild-type enzyme. To evaluate intermolecular interaction of hUGT1A1, Gunn rat fibroblasts were stably transduced with hUGT1A1 cDNA. Gel permeation chromatography of solubilized microsomes suggested dimerization of hUGT1A1 in solution. Nearest-neighbor cross-linking analysis indicated that, within microsomal membranes, hUGT1A1 dimerized more efficiently at pH 7.4 than at pH 9. Two-hybrid analysis in yeast and mammalian systems demonstrated positive interaction of hUGT1A1 with itself, but not with another UGT isoform, human UGT1A6, which differs only in the N-terminal domain. Dimerization was abolished by deletion of the membrane-embedded helix from the N-terminal domain of hUGT1A1, but not by substitution of several individual amino acid residues or partial deletion of the C-terminal domain. A C127Y substitution abolished UGT1A1 activity, but not its dimerization. Coexpression of mutagenized and wild-type hUGT1A1 in COS-7 cells showed that the mutant form markedly suppressed the catalytic activity of wild-type hUGT1A1. Homodimerization of hUGT1A1 may explain the dominant-negative effect of some mutant forms of the enzyme.
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Glucuronosiltransferasa/química , Animales , Células COS , Cromatografía en Gel , Dimerización , Glucuronosiltransferasa/fisiología , Humanos , Proteínas Recombinantes/química , Relación Estructura-ActividadRESUMEN
Scarcity of donor livers is a major obstacle to the general application of hepatocytes for the development of bioartificial liver assist devices as well as intracorporeal engraftment of hepatocytes for the treatment of inherited metabolic diseases. The number of hepatocytes that can be transplanted into the liver safely in a single sitting also limits the utility of this procedure. These limitations could be addressed by providing preferential proliferative advantage to the transplanted cells. Studies using transgenic mouse recipients or donors have indicated that massive repopulation of the host liver by engrafted hepatocytes requires that the transplanted cells are subjected to a proliferative stimulus to which the host hepatocytes cannot respond. Prevention of host hepatocyte proliferation has been achieved by treatment with a plant alkaloid, retrorsine. Because retrorsine is carcinogenic, we have evaluated preparative irradiation for this purpose. The proliferative stimulus may consist of the loss of hepatic mass (e.g., partial hepatectomy, reperfusion injury or induction of Fas-mediated apoptosis by gene transfer) or administration of stimulants of hepatocellular mitosis (e.g., growth factors or thyroid hormone). Potential applications of these preparative manipulations of the host liver include the treatment of inherited metabolic disorders by transplantation of allogeneic hepatocytes, hepatocyte-mediated ex vivo gene therapy, rescuing liver cancer patients from radiation-induced liver damage, and expansion of human hepatocytes in animal livers.
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Trasplante de Células/métodos , Terapia Genética/métodos , Hepatocitos/efectos de la radiación , Hepatocitos/trasplante , Fallo Hepático/terapia , Animales , Terapia Combinada , Rechazo de Injerto/prevención & control , Reacción Huésped-Injerto , Humanos , Hígado/efectos de la radiación , Ratones , Ratones Transgénicos , Radioterapia/métodos , Ratas , Sensibilidad y EspecificidadAsunto(s)
Codón/genética , Glucuronosiltransferasa/genética , Ictericia Neonatal/genética , Kernicterus/genética , Regiones Promotoras Genéticas/genética , Adulto , Animales , Secuencia de Bases , Western Blotting , Células COS , Síndrome de Crigler-Najjar/genética , ADN/química , ADN/genética , Análisis Mutacional de ADN , Femenino , Enfermedad de Gilbert/genética , Glucuronosiltransferasa/metabolismo , Humanos , Lactante , Recién Nacido , Masculino , Mutación , Mutación Missense , Plásmidos/genéticaRESUMEN
Crigler-Najjar syndrome type 1 (CN-1) is a potentially lethal condition, and is the only inherited disorder of bilirubin metabolism that needs treatment beyond the neonatal period. Currently, orthotopic liver transplantation is the only available cure for CN-1. Because the liver architecture is not disturbed in CN-1 and partial correction of bilirubin-UDP-glucuronosyltransferase (UGT1A1) activity is expected to be sufficient for protection against kernicterus, cell and gene therapies are being developed using the Gunn rat as an animal model of the disease. Ex vivo gene therapy based on the transplantation of genetically manipulated hepatocytes and in vivo gene transfer using recombinant adenovirus and Simian virus 40 (SV40)-based vectors have yielded significant success. The novel strategy of in vivo site-directed mutagenesis has also resulted in modest, but significant, correction of the genetic abnormality. Newer viral and nonviral gene delivery methods are being explored and have been discussed in brief. In summary, effective gene therapy methods have been validated in Gunn rats. Despite considerable remaining hurdles, gene therapy for CN-1 could become a clinical reality by the turn of this decade.
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Síndrome de Crigler-Najjar/terapia , Terapia Genética , Animales , Técnicas de Transferencia de Gen , Vectores Genéticos , Glucuronosiltransferasa/genética , Humanos , Trasplante de Hígado , Ratas , Ratas Gunn , VirusRESUMEN
To probe the mitochondrial involvement in neurodegenerative processes, we have generated a high-resolution map of the mitochondrial proteome from a human neuroblastoma SH-SY5Y cell line that has been used for creating cytoplasmic hybrid cell systems. Two mitochondrial preparations were evaluated using two-dimensional (2D) gel electrophoresis and mass spectrometry; one obtained from differential centrifugation and the other by a multiple-step percoll/metrizamide gradient. The 2D gel maps prepared from these mitochondrial fractions separated over 300 distinct spots as visualized by colloidal Coomassie blue (CCB), or closer to 400 proteins with silver staining. The most abundant proteins identified in the mitochondrial fraction prepared by differential centrifugation were those of mitochondrial, cytoplasmic, and endoplasmic reticulum origin. Proteins obtained using the more intensive two-step gradient method were almost exclusively known to be associated with mitochondria. From this latter preparation, 84 of the most abundant gel spots were analyzed, out of which 61 proteins were identified. The absence of many membrane-associated proteins known to be associated with the mitochondrion and the limited number of total proteins observed in the 2D gel maps suggest that the majority of mitochondrial proteins are not being detected under these separation and staining conditions. An insoluble pellet obtained after solubilization of the mitochondrial fraction prepared with the percoll/metrizamide gradient was boiled in sodium dodecylsulfate (SDS) and separated by 1D sodium dodecylsulfate polyacrylamide gel electrophoresis (SDS-PAGE). This separation yielded some additional proteins, many of which are likely membrane-associated. These studies form the basis for the analysis of differential protein expression in cybrid cellular models of neurodegenerative disorders and in affected tissue from diseased states.
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Uridine-diphosphoglucuronate glucuronosyltransferases (UGTs) are a family of enzymes that conjugate various endogenous and exogenous compounds with glucuronic acid and facilitate their excretion in the bile. Bilirubin-UGT(1) (UGT1A1) is the only isoform that significantly contributes to the conjugation of bilirubin. Lesions in the gene encoding bilirubin-UGT(1), lead to complete or partial inactivation of the enzyme causing the rare autosomal recessively inherited conditions, Crigler-Najjar syndrome type-1 (CN-1) and type 2 (CN-2), respectively. Inactivation of the enzyme leads to accumulation of unconjugated bilirubin in the serum. Severe hyperbilirubinemia seen in CN-1 can cause bilirubin encephalopathy (kernicterus). Kernicterus can be fatal or may leave behind permanent neurological sequelae. Here, we have compiled more than 50 genetic lesions of UGT1A1 that cause CN-1 (including 9 novel mutations) or CN-2 (including 3 novel mutations) and have presented a correlation of structure to function of UGT1A1. In contrast to Crigler-Najjar syndromes, Gilbert syndrome is a common inherited condition characterized by mild hyperbilirubinemia. An insertional mutation of the TATAA element upstream to UGT1A1 results in a reduced level of expression of the gene. Homozygosity for the variant promoter is required for Gilbert syndrome, but not sufficient for manifestation of hyperbilirubinemia, which is partly dependent on the rate of bilirubin production. Several structural mutations of UGT1A1, for example, a G71R substitution, have been reported to cause mild reduction of UGT activity toward bilirubin, resulting in mild hyperbilirubinemia, consistent with Gilbert syndrome. When the normal allele of a heterozygote carrier for a Crigler-Najjar type structural mutation contains a Gilbert type promoter, intermediate levels of hyperbilirubinemia, consistent with the diagnosis of CN-2, may be observed.
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Bilirrubina/metabolismo , Síndrome de Crigler-Najjar/enzimología , Síndrome de Crigler-Najjar/genética , Enfermedad de Gilbert/enzimología , Enfermedad de Gilbert/genética , Glucuronosiltransferasa/genética , Mutación/genética , Animales , Genotipo , Humanos , Isoenzimas/genética , Datos de Secuencia Molecular , FenotipoRESUMEN
The segregation and transmission of mitochondrial genomes in humans are complicated processes, but are particularly important for understanding the inheritance and clinical abnormalities of mitochondrial disorders. This review describes three aspects of mitochondrial genetics. First, that the segregation and transmission of mitochondrial (mt)DNA molecules are likely to be determined by their physical association within the organelles and by the dynamics of mitochondrial structure and subcellular organization. Second, that the transmission of heteroplasmic mtDNA sequence changes from one generation to the next often involves rapid shifts in allele frequency. For >20 years, the standard explanation has been that there is a developmental bottleneck in which, at some stage of oogenesis, there is a reduction in the effective number of mitochondrial units of inheritance. The third aspect is that ongoing analyses of the segregation and transmission of pathogenic mtDNA mutations indicate the operation of multiple genetic processes. Thus, the segregation and transmission of mtDNA mutations occurs predominantly, but not exclusively, under conditions of random genetic drift. However, there is also evidence for bias due to incomplete ascertainment of pedigrees and for negative selection of pathogenic mutations in rapidly dividing somatic tissues such as the white blood cell population.
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Segregación Cromosómica/genética , ADN Mitocondrial/genética , Miopatías Mitocondriales/genética , Mutación/genética , ADN Mitocondrial/metabolismo , Genoma , Genotipo , Humanos , Estudios Longitudinales , Síndrome MELAS/genética , Síndrome MELAS/patología , Síndrome MERRF/genética , Síndrome MERRF/patología , Atrofias Ópticas Hereditarias/genética , Atrofias Ópticas Hereditarias/patología , Polimorfismo GenéticoRESUMEN
Although liver-directed gene therapy arrived later than gene therapy directed at bone marrow cells, intrinsic advantages of the liver as a target organ make it likely that gene therapy for liver diseases will be among the first therapeutically relevant applications of this treatment modality at the onset of the 21st century. Vectorology for gene transfer to the liver is advancing rapidly, and it is safe to predict that gene therapy vehicles that will be in clinical use a decade from now, have not yet been developed. None of the currently available modes of gene transfer to the liver is optimal for all types of applications. Nonetheless, the concerted effort of many investigators has provided a wide choice of non-viral and viral vectors for gene transfer to the liver for use in specific situations. Original strategies for liver-directed gene therapy included substitution of missing gene products, overexpression of intrinsic or extrinsic genes and inhibition of expression of specific genes. To the list is now added the possibility of site-specific correction or generation of mutations within specific genes in somatic cells of living adult animals. Thus, despite some initial faux pas, liver-directed gene therapy is poised to make an important impact on health care in the year 2000 and beyond.
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Terapia Genética , Hepatopatías/terapia , Animales , Reparación del ADN , Terapia Genética/métodos , Vectores Genéticos , Humanos , Plásmidos , Virus/genéticaRESUMEN
The mutation load of the pathogenic LHON (Leber hereditary optic neuropathy) mtDNA mutation at nucleotide 3460 has been followed over time in the WBC/platelet fraction from members of a matrilineal pedigree. Longitudinal analysis over a sampling period of five to six years indicates that, in all five heteroplasmic family members, the mutation load decreases at a mean overall rate of approximately 1% per year. There was no change in mutation load in homoplasmic wildtype or in homoplasmic mutant individuals. For the purposes of comparison, a longitudinal analysis of a silent mtDNA polymorphism at nucleotide 14560 was also carried out for members of a second matrilineal pedigree. In contrast to the results for the pathogenic mtDNA mutation, there was no change in the proportion of the silent polymorphism in the WBC/platelet fraction of four family members over a period of seven years. These results indicate that the pathogenic 3460 LHON mutation segregates under negative selection in these cell populations. One possible mechanism through which selection may operate is that, in heteroplasmic individuals, the hematopoietic stem cells are generally homoplasmic, either for the wildtype or for the mutant allele. The homoplasmic mutant stem cells, because of their mitochondrial respiratory chain defect, produce fewer mature WBCs and platelets over time than do the wildtype stem cells. Alternatively, the stem cells may be heteroplasmic and selection may act to favor proliferation of mitochondria with lower levels of the pathogenic mutation in the WBC/platelet cell populations.
Asunto(s)
Análisis Mutacional de ADN/métodos , ADN Mitocondrial/genética , Heterogeneidad Genética , Atrofias Ópticas Hereditarias/genética , Polimorfismo Genético/genética , Humanos , Estudios LongitudinalesRESUMEN
Nuclear factor-kappaB (NF-kappaB) binds to nucleotide sequences between -80 and -70 bp upstream of the transcriptional start site in the interleukin-8 (IL-8) promoter and is crucial for transcription of the IL-8 gene. We showed that exogenous nitric oxide in the form of a nitric oxide donor significantly reduced IL-8 mRNA in cytokine-activated ECV304. Similarly, nitric oxide significantly reduced migration of polymorphonuclear neutrophils through cytokine-activated ECV304 monolayers, an IL-8-dependent process. Using a luciferase reporter construct containing the NF-kappaB site of the IL-8 gene, we showed that exposing cytokine-activated ECV304 to exogenous nitric oxide resulted in significant reduction of NF-kappaB binding. Follow-up studies using a luciferase reporter construct possessing a mutated NF-kappaB site confirmed that the luciferase activity observed in the NF-kappaB reporter resulted from NF-kappaB binding. These studies demonstrate that nitric oxide, supplied exogenously into reactions containing activated endothelium, down-regulates pro-inflammatory activity, such as the secretion of chemokines, and functional activity, such as transendothelial migration of neutrophils.
Asunto(s)
ADN/metabolismo , Endotelio/metabolismo , Regulación de la Expresión Génica/fisiología , Interleucina-8/genética , FN-kappa B/antagonistas & inhibidores , Óxido Nítrico/fisiología , Secuencia de Bases , Western Blotting , Línea Celular , Movimiento Celular , Cartilla de ADN , Endotelio/citología , Endotelio/fisiología , Humanos , Mutagénesis Sitio-Dirigida , FN-kappa B/metabolismo , Neutrófilos/citología , Unión Proteica , ARN Mensajero/genética , ARN Mensajero/metabolismoRESUMEN
We describe here a nuclear mitochondrial DNA-like sequence (numtDNA) that is nearly identical in sequence to a continuous 5842 bp segment of human mitochondrial DNA (mtDNA) that spans nucleotide positions 3914 to 9755. On the basis of evolutionary divergence among modern primates, this numtDNA molecule appears to represent mtDNA from a hominid ancestor that has been translocated to the nuclear genome during the recent evolution of humans. This numtDNA sequence harbors synonymous and nonsynonymous nucleotide substitutions relative to the authentic human mtDNA sequence, including an array of substitutions that was previously found in the cytochrome c oxidase subunit 1 and 2 genes. These substitutions were previously reported to occur in human mtDNA, but subsequently contended to be present in a nuclear pseudogene sequence. We now demonstrate their exclusive association with this 5842-bp numtDNA, which we have characterized in its entirety. This numtDNA does not appear to be expressed as a mtDNA-encoded mRNA. It is present in nuclear DNA from human blood donors, in human SH-SY5Y and A431 cell lines, and in rho(0) SH-SY5Y and rho(0) A431 cell lines that were depleted of mtDNA. The existence of human numtDNA sequences with great similarities to human mtDNA renders the amplification of pure mtDNA from cellular DNA very difficult, thereby creating the potential for confounding studies of mitochondrial diseases and population genetics.