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Code-switching, switching between different languages within the same conversation, is a prominent feature in bilingual communication. This study aimed to elucidate to what extent the linguistic abilities and age of dual-language-learning preschoolers influence the frequency and purposes of code-switching (compensatory, to bridge linguistic gaps; preferential, to express content as fluently as possible; pragmatic, to phrase something appropriately for the situation). Parental code-switching ratings of 101 German/French-Turkish/Italian dual-language learners aged 32-78 months were analyzed. Generalized linear mixed models revealed positive but no negative effects of societal- and heritage-language skills on children's code-switching frequencies independent of switching purposes and with no evidence of age effects. Hence, code-switching across the preschool age mainly reflects high linguistic competences. Models with linguistically and psychometrically parallelized language scores indicated a strong switching tendency toward the societal language when proficiency in both languages is high and away from the societal language when language proficiencies are low.
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Many properties of solids result from the fact that in a periodic crystal structure, electronic wave functions are delocalized over many lattice sites. Electrons should become increasingly localized when a strong electric field is applied. So far, this Wannier-Stark regime has been reached only in artificial superlattices. Here we show that extremely transient bias over the few-femtosecond period of phase-stable mid-infrared pulses may localize electrons even in a bulk semiconductor like GaAs. The complicated band structure of a three-dimensional crystal leads to a strong blurring of field-dependent steps in the Wannier-Stark ladder. Only the central step emerges strongly in interband electro-absorption because its energetic position is dictated by the electronic structure at an atomic level and therefore insensitive to the external bias. In this way, we demonstrate an extreme state of matter with potential applications due to e.g., its giant optical non-linearity or extremely high chemical reactivity.
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OBJECTIVES: Air-polishing devices are used for the instrumentation of the root surface. Their potential of harm to the hard and/or soft tissues needs to be considered during periodontal treatment. The objective of this systematic review was to analyse the effects of air polishing devices on oral tissues. METHODS: The electronic databases MEDLINE, EMBASE and the Cochrane Library were screened for studies published through 18 November 2013. The surface modifications on human cementum, dentine or gingiva after the instrumentation were considered as outcomes. RESULTS: Of the 1266 abstracts screened, 17 studies were included in the analysis. Different air polishing powders consisting of sodium bicarbonate, calcium carbonate, pumice or glycine were used in different ex vivo or in vitro settings. Thirteen publications reported data on the effects of air polishing devices on cementum and dentine. Hard tissue modifications, including defect depths and volume, caused by sodium bicarbonate or calcium carbonate powders were significantly greater compared to powders consisting of glycine. The soft tissue modifications using different modes of instrumentation were assessed in four publications. The data demonstrate less potential of harm to the gingiva after spraying with glycine powder compared to sodium bicarbonate powder or instrumentation with curettes. CONCLUSION: Glycine powder air polishing may be safely applied to human root surfaces and gingivae.
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Pulido Dental , Bicarbonato de Sodio , Raíz del Diente , Cemento Dental , Dentina , Humanos , PolvosRESUMEN
OBJECTIVES: Air polishing devices are used as an alternative to traditional instrumentation of the root surface. The objective of the systematic review was to analyse patient perception, that is pain and discomfort during treatment with air polishing devices in periodontal therapy. METHODS: The electronic databases MEDLINE, EMBASE and the Cochrane library were screened for studies published through 18th November 2013. Patient perception served as primary outcome. RESULTS: Of the 1266 abstracts screened, nine studies reporting data on patient perception using a visual analogue scale or a patient interview were included in the analysis. Different air polishing powders consisting of sodium bicarbonate, glycine or erythritol were used. Reported discomfort of non-surgical periodontal therapy was consistently equal or lower when air polishing powders consisting of glycine or erythritol were applied compared with root surface instrumentation using hand instruments or ultrasonic devices. CONCLUSION: Air polishing with powders consisting of glycine seems to be associated with less discomfort during non-surgical periodontal therapy, that is supra- and subgingival air polishing.
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Pulido Dental/métodos , Satisfacción del Paciente , Periodoncia/métodos , Glicina , Humanos , Polvos , Raíz del DienteRESUMEN
OBJECTIVES: Endometrial cancer is the most common gynecological cancer in France. The therapeutic management is based on preoperative staging. The recommended imaging examination remains the MRI. This is to evaluate ultrasound and MRI in the staging for localized cancers. METHODS: This is a retrospective observational study, conducted from July 2012 to July 2014, at the University Hospital of Nancy, on all patients care for endometrial cancer stage I, who underwent a pelvic ultrasound and MRI for the assessment of myometrial infiltration. RESULTS: Twenty-nine patients were included with a mean age of 69 years and a BMI of 30 kg/m(2). Using ultrasound, we have a sensitivity of 58%, a specificity of 100%, a positive predictive value (PPV) of 100%, a negative predictive value (NPV) of 70% and an accuracy of 75%. Using MRI, we have a sensitivity of 83%, a specificity of 100%, a PPV of 83%, a VPN of 88%, and an accuracy of 86%. CONCLUSION: Transvaginal sonography should be performed before post-menopausal bleeding. It remains possible in the staging of localized cancers.
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Neoplasias Endometriales/diagnóstico por imagen , Neoplasias Endometriales/patología , Imagen por Resonancia Magnética , Estadificación de Neoplasias/métodos , Anciano , Anciano de 80 o más Años , Femenino , Francia , Humanos , Persona de Mediana Edad , Estudios Retrospectivos , Sensibilidad y EspecificidadRESUMEN
Granulomatous mastitis is a rare disease, often associated with Corynebacterium infection. It raises the problem of diagnosis of breast tumor with a fast evolution and inflammatory character. We report two cases of granulomatous mastitis with Corynebacterium. It concerns the clinical and radiological description, followed by the therapeutic alternatives and future of the patients. The clinical presentation is variable. The treatment consists in a surgical procedure of resection. The medical treatment based of corticosteroids also proves efficient. The association between Corynebacterium presence and this pathology seems frequent and needs a specific bacteriological search.
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Infecciones por Corynebacterium , Corynebacterium/patogenicidad , Mastitis Granulomatosa , Adulto , Infecciones por Corynebacterium/complicaciones , Infecciones por Corynebacterium/microbiología , Infecciones por Corynebacterium/terapia , Femenino , Mastitis Granulomatosa/etiología , Mastitis Granulomatosa/microbiología , Mastitis Granulomatosa/terapia , HumanosRESUMEN
BACKGROUND: Vascular access patency is of vital importance for patients requiring haemodialysis. This analysis validates potential risk factors and benefits in patients undergoing vascular access procedures. PATIENTS AND METHODS: Vascular access procedures performed over a two-year period were retrospectively analysed. Clinical data and concomitant medication were retrieved from files as were surgical data following a standardized data capture sheet. Outcome parameters were primary (PP) and secondary patency (SP) as well as freedom from repeated revascularization. Minimal follow-up with functioning access was 679 days. RESULTS: During the observation period, 244 patients (mean age 62.2 +/- 0.9 years, 60.7 % male patients, 36.1 % pre-emptive, 31.1 % late referral) underwent vascular accesses procedures. PP and SP were 35.6 % and 45.6 %, respectively, at 540 days. Presence of diabetes mellitus was associated with decreased PP (OR: 0.6, 95 %-CI: 0.3 - 1.0) and SP (OR: 0.4, 95 %-CI: 0.2 - 0.7), whereas female gender was associated with lower SP (OR: 0.6, 95 %-CI: 0.3 - 0.9) and freedom from repeated revascularization rates (OR: 0.6, 95 %-CI: 0.3 - 1.0). In contrast, presence of hyperparathyreoidism was associated with higher SP (OR: 1.7, 95 %-CI: 1.0 - 3.0) and freedom from repeated revascularization (OR: 1.7, 95 %-CI: 1.0 - 3.0) rates. CONCLUSIONS: Haemodialysis access performs worst in patients with diabetes mellitus and in women. The benefit of hyperparathyroidism should be interpreted as hypothesis generating.
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Derivación Arteriovenosa Quirúrgica , Oclusión de Injerto Vascular/etiología , Fallo Renal Crónico/terapia , Diálisis Renal , Grado de Desobstrucción Vascular , Derivación Arteriovenosa Quirúrgica/efectos adversos , Implantación de Prótesis Vascular/efectos adversos , Distribución de Chi-Cuadrado , Complicaciones de la Diabetes/etiología , Femenino , Oclusión de Injerto Vascular/cirugía , Humanos , Hiperparatiroidismo/complicaciones , Estimación de Kaplan-Meier , Modelos Logísticos , Masculino , Persona de Mediana Edad , Oportunidad Relativa , Modelos de Riesgos Proporcionales , Reoperación , Estudios Retrospectivos , Medición de Riesgo , Factores de Riesgo , Factores Sexuales , Factores de Tiempo , Resultado del TratamientoRESUMEN
The zebrafish (Danio rerio) is a sensitive non-mammalian model used for studying polycyclic aromatic hydrocarbon (PAH)-induced chemical carcinogenesis. The susceptibility of zebrafish to PAH-induced carcinogenesis may be related to the ability of the zebrafish P450s to bioactivate these procarcinogens. As a part of our overall effort to identify the various P450 enzymes that are involved in the activation and detoxification of PAHs in zebrafish, therefore, we have examined the ability of recombinant zebrafish CYP1A (zCYP1A) expressed in yeast to metabolize BaP in vitro. Comparison studies also were conducted with liver microsomes from beta-naphthoflavone (BNF)-treated rainbow trout (Oncorhynchus mykiss). Results demonstrated that the trout liver microsomes were almost twice as active as zCYP1A in oxidizing BaP, with Vmax values of 1.7 and 0.94 nmol/min/nmol P450 for trout and zebrafish preparations, respectively. Like trout CYP1A1, cDNA-expressed zCYP1A was found to oxidize BaP to phenols, quinones and diols (BaP-7,8-diol and BaP-9,10-diol) in the presence of exogenous human microsomal epoxide hydrolase (hEH). BaP-7,8-diol is the precursor of the ultimate carcinogen, BaP-7,8-diol-9,10-epoxide (BaPDE). The ability of zCYP1A to bioactivate BaP was confirmed by the formation of DNA adducts when calf thymus DNA was added to the incubation mixture. BaP-DNA binding was enhanced by the addition of hEH to the incubation mixture. HPLC analysis of the [33P]-postlabeled DNA adducts showed the formation of at least four adducts mediated by both zCYP1A and trout liver microsomes, and one of these adducts co-migrated with BaPDE-dG in HPLC analysis. The addition of hEH to the incubation mixture decreased the formation of BaPDE-dG by zCYP1A and by trout liver microsomes while increasing the formation of an unidentified DNA adduct in the case of zCYP1A. zCYP1A also mediated the binding of BaP to protein, providing further evidence that this enzyme is capable of oxidizing BaP to reactive metabolites that bind to macromolecules. It thus appears that zCYP1A may play an important role in BaP-induced carcinogenesis in the zebrafish model by catalyzing the sequential formation of the ultimate diol epoxide carcinogenic metabolite of BaP.
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Benzo(a)pireno/metabolismo , Sistema Enzimático del Citocromo P-450/metabolismo , Microsomas Hepáticos/metabolismo , Oncorhynchus mykiss/metabolismo , Proteínas Recombinantes/metabolismo , Proteínas de Pez Cebra/metabolismo , Animales , Isótopos de Carbono/análisis , ADN/metabolismo , Microsomas Hepáticos/efectos de los fármacos , Isótopos de Fósforo/análisis , Unión Proteica/efectos de los fármacos , Saccharomyces cerevisiaeRESUMEN
A full-length zebrafish (Danio rerio) cytochrome P450 (CYP) 2K6 cDNA, was obtained (GenBank accession No. AF283813) through polymerase chain reaction cloning using degenerated primers based on a consensus CYP2 sequence and the heme-binding domain. This first CYP2K family member cloned from zebrafish had 1861 bp which contained 27 bp of 5'-untranslated region (5'-UTR), an open reading frame (ORF) of 1518 bp, and a 300 bp 3'-UTR with a poly A tail. The deduced 506 amino acid sequence of CYP2K6 had 63%, 62% and 59% identity with rainbow trout CYP2K1, CYP2K4 and CYP2K3, respectively; and 45%, 42%, and 42% identity with rabbit CYP2C1, human CYP2C19 and mouse CYP2C39, respectively. CYP2K6 mapped to 107.49cR on LG3 using the LN54 radiation hybrid panel. Its mRNA was detected at 5 days post-fertilization and in the adult liver and ovary among nine tissues examined. The ORF, including the 27 bp of the 5'-UTR, was cloned into pFastBac donor vector and then transferred into the baculovirus genome (bacmid DNA) in DH10Bac competent cells. The recombinant bacmid DNA was used to infect Spodoptera frugiperda insect cells to express the CYP2K6 protein (Bv-2K6). As its ortholog, rainbow trout Bv-2K1 [Yang, Y.H., Miranda, C.L., Henderson, M.C., Wang-Buhler, J.-L., Buhler, D.R., 2000. Heterologous expression of CYP2K1 and identification of the expressed protein (Bv-2K1) as lauric acid (omega-1)-hydroxylase and aflatoxin B1 exo-epoxidase. Drug Metab. Disp. 28,1279-83.], Bv-2K6 also catalyzed the conversion of aflatoxin B1 (AFB1) to its exo-8,9-epoxide as assessed by the trapping of a glutathione (GSH) adduct in the presence of a specific mouse alpha class glutathione S-transferase. The identity of the AFB1-GSH adduct was verified by liquid chromatography-mass spectrometry (LC-MS) and mass spectrometry-mass spectrometry (MS-MS) analysis. Although rainbow trout Bv-2K1 was capable of oxidizing lauric acid, zebrafish Bv-2K6 protein showed no activity against this substrate.
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Aflatoxina B1/metabolismo , Hidrocarburo de Aril Hidroxilasas/genética , Secuencia de Aminoácidos , Animales , Hidrocarburo de Aril Hidroxilasas/biosíntesis , Baculoviridae , Secuencia de Bases , Biotransformación , Cromatografía Líquida de Alta Presión , Cromatografía Liquida , Clonación Molecular , Citocromo P-450 CYP4A/metabolismo , Familia 2 del Citocromo P450 , Embrión no Mamífero , Proteínas de Peces/genética , Biblioteca de Genes , Espectrometría de Masas , Datos de Secuencia Molecular , Reacción en Cadena de la Polimerasa , Proteínas Recombinantes/metabolismo , Alineación de Secuencia , Spodoptera , Esteroide Hidroxilasas/genética , Distribución Tisular , Pez Cebra/crecimiento & desarrolloRESUMEN
MOTIVATION: Annotation of operons in a bacterial genome is an important step in determining an organism's transcriptional regulatory program. While extensive studies of operon structure have been carried out in a few species such as Escherichia coli, fewer resources exist to inform operon prediction in newly sequenced genomes. In particular, many extant operon finders require a large body of training examples to learn the properties of operons in the target organism. For newly sequenced genomes, such examples are generally not available; moreover, a model of operons trained on one species may not reflect the properties of other, distantly related organisms. We encountered these issues in the course of predicting operons in the genome of Bacteroides thetaiotaomicron (B.theta), a common anaerobe that is a prominent component of the normal adult human intestinal microbial community. RESULTS: We describe an operon predictor designed to work without extensive training data. We rely on a small set of a priori assumptions about the properties of the genome being annotated that permit estimation of the probability that two adjacent genes lie in a common operon. Predictions integrate several sources of information, including intergenic distance, common functional annotation and a novel formulation of conserved gene order. We validate our predictor both on the known operons of E.coli and on the genome of B.theta, using expression data to evaluate our predictions in the latter.
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Algoritmos , Inteligencia Artificial , Bacteroides/genética , Mapeo Cromosómico/métodos , Escherichia coli/genética , Operón/genética , Reconocimiento de Normas Patrones Automatizadas/métodos , Genoma Bacteriano , Modelos Genéticos , Modelos Estadísticos , Programas InformáticosRESUMEN
We propose an assay to detect and quantify alternative splicing simultaneously for numerous genes in a pool of cellular mRNA. The assay exploits polymerase colonies, a recently developed method for sampling and amplifying large numbers of individual transcript molecules into discrete spots on a gel. The proposed assay combines the advantages of microarrays for transcript quantitation with the sensitivity and precision of methods based on counting single transcript molecules. Given a collection of spots s(i), each containing an unknown splice variant of some known gene G(i), we design a series of hybridizations to short oligonucleotide probes to determine in parallel which exons of G(i) are present in every spot s(i). We give algorithms to minimize the cost of such designs.
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Empalme Alternativo , Biología Computacional , Técnicas Genéticas , Exones , Etiquetas de Secuencia Expresada , Perfilación de la Expresión Génica , Análisis de Secuencia por Matrices de Oligonucleótidos , ARN Mensajero/genéticaRESUMEN
MOTIVATION: Comparing two protein databases is a fundamental task in biosequence annotation. Given two databases, one must find all pairs of proteins that align with high score under a biologically meaningful substitution score matrix, such as a BLOSUM matrix (Henikoff and Henikoff, 1992). Distance-based approaches to this problem map each peptide in the database to a point in a metric space, such that peptides aligning with higher scores are mapped to closer points. Many techniques exist to discover close pairs of points in a metric space efficiently, but the challenge in applying this work to proteomic comparison is to find a distance mapping that accurately encodes all the distinctions among residue pairs made by a proteomic score matrix. Buhler (2002) proposed one such mapping but found that it led to a relatively inefficient algorithm for protein-protein comparison. RESULTS: This work proposes a new distance mapping for peptides under the BLOSUM matrices that permits more efficient similarity search. We first propose a new distance function on peptides derived from a given score matrix. We then show how to map peptides to bit vectors such that the distance between any two peptides is closely approximated by the Hamming distance (i.e. number of mismatches) between their corresponding bit vectors. We combine these two results with the LSH-ALL-PAIRS-SIM algorithm of Buhler (2002) to produce an improved distance-based algorithm for proteomic comparison. An initial implementation of the improved algorithm exhibits sensitivity within 5% of that of the original LSH-ALL-PAIRS-SIM, while running up to eight times faster.
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Algoritmos , Bases de Datos de Proteínas , Proteínas/química , Proteoma/química , Alineación de Secuencia/métodos , Análisis de Secuencia de Proteína/métodos , Secuencia de Aminoácidos , Secuencia Conservada , Datos de Secuencia Molecular , Proteínas/clasificación , Proteoma/clasificación , Homología de Secuencia de AminoácidoRESUMEN
BACKGROUND: Proliferation and migration of vascular smooth muscle cells (SMCs) mark the key processes in the development of bypass graft disease and during neointima formation in restenosis after angioplasty. Growth factors are potent SMC mitogens as they are involved in SMC proliferation and in extracellular matrix (ECM) synthesis. Based on these premises, we examined the effect of the proliferation inhibitor rapamycin in human SMC culture and in a rabbit vascular injury model. MATERIALS AND METHODS: Injection of rapamycin or its vehicle was performed with an infusion-balloon catheter directly into the vessel wall during vascular injury. The intima/media ratio was determined histologically whereas the protein expression was analysed using the powerful two-dimensional gel electrophoresis (2D page) technique. Inhibition of proliferation after rapamycin application was estimated in a human SMC culture for time and dose dependent effects. RESULTS: Rapamycin treatment resulted in a significant reduction of intima media ratio compared to vehicle treated animals after three weeks (0.65 +/- 0.1 vs. 1.2 +/- 0.2 intima-media-ratio, p < 0.05). 2D electrophoresis analysis proved increased ECM synthesis following angioplasty (i.e., lamin, vimentin) in vehicle treated animals. Local rapamycin administration resulted in profound reduction of ECM synthesis after vascular injury. In in-vitro experiments exposure of cultured human SMCs to rapamycin resulted in a significant and dose-dependent (1 nm-100 nm) reduction of human smooth muscle cell proliferation measured by cell counting. CONCLUSION: These above mentioned results suggest that protein synthesis in addition to reduction of cellular proliferation plays an important role following vascular injury, since application of rapamycin resulted in the reduction of SMC proliferation and ECM-synthesis.
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División Celular/efectos de los fármacos , Displasia Fibromuscular/patología , Músculo Liso Vascular/efectos de los fármacos , Sirolimus/farmacología , Túnica Media/efectos de los fármacos , Angioplastia de Balón , Animales , Movimiento Celular/efectos de los fármacos , Células Cultivadas/efectos de los fármacos , Células Cultivadas/patología , Relación Dosis-Respuesta a Droga , Electroforesis en Gel Bidimensional , Humanos , Masculino , Proteínas Musculares/biosíntesis , Músculo Liso Vascular/patología , Conejos , Túnica Media/patologíaRESUMEN
In mammals the cytochrome P450 3A (CYP3A) subfamily isoforms are primarily expressed in liver and intestines with lesser amounts found in other tissues. The aim of this study was to examine the cellular localization and the expression pattern of CYP3A27 in the gastrointestinal tract (GI tract) of a freshwater teleost species, the rainbow trout (Oncorhynchus mykiss), a fish model used extensively for toxicological and carcinogenesis research. Using an avidin biotinylated enzyme complex and 3,3'-diaminobenzidine staining, strong cytoplasmic immunohistochemical staining was observed for CYP3A27 protein in hepatocytes and in enterocytes of the intestinal ceca and the proximal descending intestine when probed with a polyclonal antibody raised against rainbow trout P450 LMC5, a CYP3A protein. The intensity of epithelial staining decreased distally along the GI tract with faint staining observed in the epithelial cells examined near the vent. Western blot analysis was supportive of the immunohistochemistry results. Northern blot analysis also demonstrated that CYP3A27 mRNA was expressed along the entire GI tract. The major area of CYP3A27 mRNA expression was in the intestinal ceca, followed by the proximal descending intestine, at levels that were about three- to five-fold and two- to four-fold, respectively, greater than seen in the liver of the fish studied. Monooxygenase activities of intestinal ceca microsomes against testosterone and progesterone confirmed the presence of active CYP3A enzyme in this tissue. These results suggest that the intestine of rainbow trout may possesses substantial capacity for first-pass metabolism of xenobiotics by CYP3A27, which makes it an excellent model in which to study the consequence of such metabolism.
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Hidrocarburo de Aril Hidroxilasas , Sistema Enzimático del Citocromo P-450/metabolismo , Sistema Digestivo/enzimología , Oncorhynchus mykiss/metabolismo , Oxidorreductasas N-Desmetilantes/metabolismo , Animales , Northern Blotting , Western Blotting , Ciego/enzimología , Citocromo P-450 CYP3A , Sistema Enzimático del Citocromo P-450/biosíntesis , Sistema Enzimático del Citocromo P-450/genética , Sistema Digestivo/metabolismo , Femenino , Regulación Enzimológica de la Expresión Génica , Hidroxilación , Inmunohistoquímica , Isoenzimas/biosíntesis , Isoenzimas/genética , Isoenzimas/metabolismo , Masculino , Microsomas Hepáticos/enzimología , Oxidorreductasas N-Desmetilantes/biosíntesis , Oxidorreductasas N-Desmetilantes/genética , Progesterona/metabolismo , ARN Mensajero/biosíntesis , ARN Mensajero/genética , Testosterona/metabolismoRESUMEN
We demonstrate an integrated approach to build, test, and refine a model of a cellular pathway, in which perturbations to critical pathway components are analyzed using DNA microarrays, quantitative proteomics, and databases of known physical interactions. Using this approach, we identify 997 messenger RNAs responding to 20 systematic perturbations of the yeast galactose-utilization pathway, provide evidence that approximately 15 of 289 detected proteins are regulated posttranscriptionally, and identify explicit physical interactions governing the cellular response to each perturbation. We refine the model through further iterations of perturbation and global measurements, suggesting hypotheses about the regulation of galactose utilization and physical interactions between this and a variety of other metabolic pathways.
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Galactosa/metabolismo , Perfilación de la Expresión Génica , Genoma Fúngico , Proteoma , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae/metabolismo , Biología Computacional , Medios de Cultivo , Bases de Datos Factuales , Proteínas Fúngicas/metabolismo , Galactosafosfatos/metabolismo , Regulación Fúngica de la Expresión Génica , Modelos Biológicos , Modelos Genéticos , Proteínas de Transporte de Monosacáridos/metabolismo , Mutación , Análisis de Secuencia por Matrices de Oligonucleótidos , ARN de Hongos/genética , ARN de Hongos/metabolismo , ARN Mensajero/genética , ARN Mensajero/metabolismo , Saccharomyces cerevisiae/genéticaRESUMEN
MOTIVATION: Comparison of multimegabase genomic DNA sequences is a popular technique for finding and annotating conserved genome features. Performing such comparisons entails finding many short local alignments between sequences up to tens of megabases in length. To process such long sequences efficiently, existing algorithms find alignments by expanding around short runs of matching bases with no substitutions or other differences. Unfortunately, exact matches that are short enough to occur often in significant alignments also occur frequently by chance in the background sequence. Thus, these algorithms must trade off between efficiency and sensitivity to features without long exact matches. RESULTS: We introduce a new algorithm, LSH-ALL-PAIRS, to find ungapped local alignments in genomic sequence with up to a specified fraction of substitutions. The length and substitution rate of these alignments can be chosen so that they appear frequently in significant similarities yet still remain rare in the background sequence. The algorithm finds ungapped alignments efficiently using a randomized search technique, locality-sensitive hashing. We have found LSH-ALL-PAIRS to be both efficient and sensitive for finding local similarities with as little as 63% identity in mammalian genomic sequences up to tens of megabases in length
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Algoritmos , Alineación de Secuencia/estadística & datos numéricos , Animales , Cromosomas Humanos Par 22/genética , Biología Computacional , ADN/genética , Bases de Datos Factuales , Globinas/genética , Humanos , Región de Control de Posición , Ratones , Receptores de Antígenos de Linfocitos T alfa-beta/genética , Programas InformáticosRESUMEN
A 2,037 bp CYP1A1 cDNA (GenBank AF072899) was cloned through screening of a lambdaZipLox cDNA library constructed from the liver of a leaping mullet (Liza saliens) fish captured from Izmir Bay on the Aegean coast of Turkey using rainbow trout CYP1A1 cDNA as a probe. This clone has a 130 bp 5'-flanking region, a 1,563 bp open reading frame (ORF) encoding a 521-amino acid protein (58,972 Da), and a 344 bp 3'-untranslated region without a poly (A) tail. Alignment of the deduced amino acids of CYP1A1 cDNAs showed 58% and 69-96% identities with human and 12 other fish species, respectively. Southern blot analysis suggested that this CYP1A1 cDNA was from a single-copy gene. Based on the comparison with CYP1A1 genes reported for fish and mammals, the leaping mullet CYP1A1 gene is probably split into 7 exons. The intron insertion sites were predicted. Alignment of the CYP1A1 cDNA encoded amino acids from 13 fish and 7 mammalian species disclosed differences in highly conserved amino acids between aquatic and land vertebrates. The possible associated secondary structure; conserved motifs and substrate-binding sites were discussed. The phylogenetic relationships of CYP1A1s among 13 fish species were analyzed by a distance method.
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Aminoácidos/metabolismo , Citocromo P-450 CYP1A1/genética , Hígado/metabolismo , Smegmamorpha/genética , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Northern Blotting , Southern Blotting , Clonación Molecular , Citocromo P-450 CYP1A1/química , Citocromo P-450 CYP1A1/metabolismo , ADN Complementario , Humanos , Datos de Secuencia Molecular , Filogenia , Estructura Secundaria de Proteína , Homología de Secuencia de AminoácidoRESUMEN
LMC2 is the most abundant constitutively expressed hepatic cytochrome P450 found in sexually immature rainbow trout (Onchorynchus mykiss) and is also the isozyme that activates the carcinogen aflatoxin B1 (AFB1). This P450 has been cloned, sequenced, and designated as CYP2K1. The present report describes the heterologous expression of enzymatically active CYP2K1 (BV-CYP2K1) in baculovirus Spodoptera frugiperda (Sf9) insect cells and its catalytic and immunoreactivity characterization in comparison with that of the previously purified LMC2 P450. Homogenates of Sf9 cells expressing the CYP2K1 enzyme and LMC2 both catalyzed the hydroxylation of lauric acid and the epoxidation of AFB1 in the presence of rat NADPH-cytochrome P450 reductase. Both LMC2 and BV-CYP2K1 catalyzed the oxidation of lauric acid primarily at the (omega-1) position plus small amounts at the (omega-2) position. Formation of AFB1 epoxide was shown indirectly by the appearance of an AFB1 epoxide-glutathione conjugate when P450 incubation mixtures contained AFB1, glutathione (GSH) together with mouse liver cytosol or purified rat GSH-transferase. When the AFB1 epoxide-GSH conjugate produced by BV-CYP2K1 and purified LMC2 was analyzed by HPLC using a chiral column, it had a retention time identical to that produced by CYP3A4, a human P450 known to form exclusively the AFB1 exo-epoxide. These results, therefore, confirm that the cDNA-expressed CYP2K1 protein is catalytically and immunologically identical to purified trout LMC2 and that these two enzymes produce primarily the highly carcinogenic stereoisomeric exo-epoxide form of AFB1.