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1.
PLoS Comput Biol ; 16(6): e1007918, 2020 06.
Artigo em Inglês | MEDLINE | ID: mdl-32569292

RESUMO

Vocalizations are widely used for communication between animals. Mice use a large repertoire of ultrasonic vocalizations (USVs) in different social contexts. During social interaction recognizing the partner's sex is important, however, previous research remained inconclusive whether individual USVs contain this information. Using deep neural networks (DNNs) to classify the sex of the emitting mouse from the spectrogram we obtain unprecedented performance (77%, vs. SVM: 56%, Regression: 51%). Performance was even higher (85%) if the DNN could also use each mouse's individual properties during training, which may, however, be of limited practical value. Splitting estimation into two DNNs and using 24 extracted features per USV, spectrogram-to-features and features-to-sex (60%) failed to reach single-step performance. Extending the features by each USVs spectral line, frequency and time marginal in a semi-convolutional DNN resulted in a performance mid-way (64%). Analyzing the network structure suggests an increase in sparsity of activation and correlation with sex, specifically in the fully-connected layers. A detailed analysis of the USV structure, reveals a subset of male vocalizations characterized by a few acoustic features, while the majority of sex differences appear to rely on a complex combination of many features. The same network architecture was also able to achieve above-chance classification for cortexless mice, which were considered indistinguishable before. In summary, spectrotemporal differences between male and female USVs allow at least their partial classification, which enables sexual recognition between mice and automated attribution of USVs during analysis of social interactions.


Assuntos
Comunicação Animal , Fatores Sexuais , Ultrassom , Animais , Feminino , Masculino , Camundongos , Rede Nervosa , Especificidade da Espécie
2.
Pharmacogenomics J ; 17(3): 230-236, 2017 06.
Artigo em Inglês | MEDLINE | ID: mdl-26927286

RESUMO

Acetaminophen can adversely affect the liver especially when overdosed. We used whole blood as a surrogate to identify genes as potential early indicators of an acetaminophen-induced response. In a clinical study, healthy human subjects were dosed daily with 4 g of either acetaminophen or placebo pills for 7 days and evaluated over the course of 14 days. Alanine aminotransferase (ALT) levels for responders to acetaminophen increased between days 4 and 9 after dosing, and 12 genes were detected with expression profiles significantly altered within 24 h. The early responsive genes separated the subjects by class and dose period. In addition, the genes clustered patients who overdosed on acetaminophen apart from controls and also predicted the exposure classifications with 100% accuracy. The responsive genes serve as early indicators of an acetaminophen exposure, and their gene expression profiles can potentially be evaluated as molecular indicators for further consideration.


Assuntos
Acetaminofen/efeitos adversos , Analgésicos não Narcóticos/efeitos adversos , Overdose de Drogas/genética , Perfilação da Expressão Gênica/métodos , Testes Farmacogenômicos/métodos , Variantes Farmacogenômicos , RNA/genética , Transcriptoma , Acetaminofen/administração & dosagem , Administração Oral , Adolescente , Adulto , Alanina Transaminase/sangue , Analgésicos não Narcóticos/administração & dosagem , Biomarcadores/sangue , Esquema de Medicação , Overdose de Drogas/sangue , Feminino , Redes Reguladoras de Genes , Voluntários Saudáveis , Humanos , Masculino , Pessoa de Meia-Idade , Modelos Genéticos , Análise de Sequência com Séries de Oligonucleotídeos , Farmacogenética , RNA/sangue , Método Simples-Cego , Fatores de Tempo , Adulto Jovem
3.
Clin Exp Immunol ; 180(1): 40-51, 2015 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-25418487

RESUMO

Drug-induced liver injury (DILI) is often caused by innate and adaptive host immune responses. Characterization of inflammatory infiltrates in the liver may improve understanding of the underlying pathogenesis of DILI. This study aimed to enumerate and characterize leucocytes infiltrating liver tissue from subjects with acute DILI (n = 32) versus non-DILI causes of acute liver injury (n = 25). Immunostains for CD11b/CD4 (Kupffer and T helper cells), CD3/CD20 (T and B cells) and CD8/CD56 [T cytotoxic and natural killer (NK) cells] were evaluated in biopsies from subjects with acute DILI, either immunoallergic (IAD) or autoimmune (AID) and idiopathic autoimmune (AIH) and viral hepatitis (VH) and correlated with clinical and pathological features. All biopsies showed numerous CD8(+) T cells and macrophages. DILI cases had significantly fewer B lymphocytes than AIH and VH and significantly fewer NK cells than VH. Prominent plasma cells were unusual in IAD (three of 10 cases), but were associated strongly with AIH (eight of nine) and also observed in most with AID (six of nine). They were also found in five of 10 cases with VH. Liver biopsies from subjects with DILI were characterized by low counts of mature B cells and NK cells in portal triads in contrast to VH. NK cells were found only in cases of VH, whereas AIH and VH both showed higher counts of B cells than DILI. Plasma cells were associated most strongly with AIH and less so with AID, but were uncommon in IAD.


Assuntos
Doença Hepática Induzida por Substâncias e Drogas/imunologia , Doença Hepática Induzida por Substâncias e Drogas/patologia , Hepatite Autoimune/imunologia , Hepatite Autoimune/patologia , Hepatite Viral Humana/imunologia , Hepatite Viral Humana/patologia , Doença Aguda , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Antígenos CD/imunologia , Linfócitos B/imunologia , Linfócitos B/patologia , Linfócitos T CD8-Positivos/imunologia , Linfócitos T CD8-Positivos/patologia , Criança , Feminino , Humanos , Células Matadoras Naturais/imunologia , Células Matadoras Naturais/patologia , Células de Kupffer/imunologia , Células de Kupffer/patologia , Masculino , Pessoa de Meia-Idade
4.
Pharm Res ; 32(6): 1975-92, 2015 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-25504454

RESUMO

PURPOSE: MITOsym, a new mathematical model of hepatocellular respiration and bioenergetics, has been developed in partnership with the DILIsym® model with the purpose of translating in vitro compound screening data into predictions of drug induced liver injury (DILI) risk for patients. The combined efforts of these two models should increase the efficiency of evaluating compounds in drug development in addition to enhancing patient care. METHODS: MITOsym includes the basic, essential biochemical pathways associated with hepatocellular respiration and bioenergetics, including mitochondrial oxidative phosphorylation, electron transport chain activity, mitochondrial membrane potential, and glycolysis; also included are dynamic feedback signals based on perturbation of these pathways. The quantitative relationships included in MITOsym are based primarily on published data; additional new experiments were also performed in HepG2 cells to determine the effects on oxygen consumption rate as media glucose concentrations or oligomycin concentrations were varied. The effects of varying concentrations of FCCP on the mitochondrial proton gradient were also measured in HepG2 cells. RESULTS: MITOsym simulates and recapitulates the reported dynamic changes to hepatocellular oxygen consumption rates, extracellular acidification rates, the mitochondrial proton gradient, and ATP concentrations in the presence of classic mitochondrial toxins such as rotenone, FCCP, and oligomycin. CONCLUSIONS: MITOsym can be used to simulate hepatocellular respiration and bioenergetics and provide mechanistic hypotheses to facilitate the translation of in vitro data collection to predictions of in vivo human hepatotoxicity risk for novel compounds.


Assuntos
Doença Hepática Induzida por Substâncias e Drogas/etiologia , Metabolismo Energético/efeitos dos fármacos , Hepatócitos/efeitos dos fármacos , Fígado/efeitos dos fármacos , Mitocôndrias Hepáticas/efeitos dos fármacos , Modelos Biológicos , Trifosfato de Adenosina/metabolismo , Carbonil Cianeto p-Trifluormetoxifenil Hidrazona/toxicidade , Respiração Celular/efeitos dos fármacos , Doença Hepática Induzida por Substâncias e Drogas/metabolismo , Doença Hepática Induzida por Substâncias e Drogas/patologia , Simulação por Computador , Relação Dose-Resposta a Droga , Células Hep G2 , Hepatócitos/metabolismo , Hepatócitos/patologia , Humanos , Concentração de Íons de Hidrogênio , Fígado/metabolismo , Fígado/patologia , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Mitocôndrias Hepáticas/metabolismo , Mitocôndrias Hepáticas/patologia , Consumo de Oxigênio/efeitos dos fármacos , Medição de Risco , Rotenona/toxicidade , Fatores de Tempo , Desacopladores/toxicidade
5.
Nat Genet ; 9(2): 115-25, 1995 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-7719337

RESUMO

The peroxisome biogenesis disorders (PBDs) are lethal recessive diseases caused by defects in peroxisome assembly. We have isolated PXR1, a human homologue of the yeast P. pastoris PAS8 (peroxisome assembly) gene. PXR1, like PAS8, encodes a receptor for proteins with the type-1 peroxisomal targeting signal (PTS1). Mutations in PXR1 define complementation group 2 of PBDs and expression of PXR1 rescues the PTS1 import defect of fibroblasts from these patients. Based on the observation that PXR1 exists both in the cytosol and in association with peroxisomes, we propose that PXR1 protein recognizes PTS1-containing proteins in the cytosol and directs them to the peroxisome.


Assuntos
Proteínas de Membrana/genética , Doenças Metabólicas/genética , Microcorpos/metabolismo , Receptores de Superfície Celular/genética , Receptores Citoplasmáticos e Nucleares , Sequência de Aminoácidos , Proteínas de Transporte/genética , Citosol/fisiologia , Genes Fúngicos , Teste de Complementação Genética , Humanos , Microcorpos/fisiologia , Dados de Sequência Molecular , Mutação , Receptor 1 de Sinal de Orientação para Peroxissomos , Saccharomyces cerevisiae/genética , Homologia de Sequência do Ácido Nucleico , Transdução de Sinais , Leveduras/genética
6.
Nat Genet ; 17(2): 185-9, 1997 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-9326939

RESUMO

Refsum disease is an autosomal recessive disorder characterized by retinitis pigmentosa, peripheral polyneuropathy, cerebellar ataxia and increased cerebrospinal fluid protein. Biochemically, the disorder is defined by two related properties: pronounced accumulation of phytanic acid and selective loss of the peroxisomal dioxygenase required for alpha-hydroxylation of phytanoyl-CoA2. Decreased phytanic-acid oxidation is also observed in human cells lacking PEX7, the receptor for the type-2 peroxisomal targetting signal (PTS2; refs 3,4), suggesting that the enzyme defective in Refsum disease is targetted to peroxisomes by a PTS2. We initially identified the human PAHX and mouse Pahx genes as expressed sequence tags (ESTs) capable of encoding PTS2 proteins. Human PAHX is targetted to peroxisomes, requires the PTS2 receptor for peroxisomal localization, interacts with the PTS2 receptor in the yeast two-hybrid assay and has intrinsic phytanoyl-CoA alpha-hydroxylase activity that requires the dioxygenase cofactor iron and cosubstrate 2-oxoglutarate. Radiation hybrid data place PAHX on chromosome 10 between the markers D10S249 and D10S466, a region previously implicated in Refsum disease by homozygosity mapping. We find that both Refsum disease patients examined are homozygous for inactivating mutations in PAHX, demonstrating that mutations in PAHX can cause Refsum disease.


Assuntos
Oxigenases de Função Mista/genética , Doença de Refsum/genética , Sequência de Aminoácidos , Animais , Sequência de Bases , Mapeamento Cromossômico , Cromossomos Humanos Par 10/genética , Análise Mutacional de DNA , Primers do DNA/genética , DNA Complementar/genética , Expressão Gênica , Homozigoto , Humanos , Camundongos , Microcorpos/metabolismo , Oxigenases de Função Mista/metabolismo , Dados de Sequência Molecular , Receptor 2 de Sinal de Orientação para Peroxissomos , Reação em Cadeia da Polimerase , Receptores Citoplasmáticos e Nucleares/metabolismo , Doença de Refsum/metabolismo , Homologia de Sequência de Aminoácidos , Especificidade da Espécie
7.
Nat Genet ; 27(4): 383-91, 2001 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-11279519

RESUMO

Variation in the CYP3A enzymes, which act in drug metabolism, influences circulating steroid levels and responses to half of all oxidatively metabolized drugs. CYP3A activity is the sum activity of the family of CYP3A genes, including CYP3A5, which is polymorphically expressed at high levels in a minority of Americans of European descent and Europeans (hereafter collectively referred to as 'Caucasians'). Only people with at least one CYP3A5*1 allele express large amounts of CYP3A5. Our findings show that single-nucleotide polymorphisms (SNPs) in CYP3A5*3 and CYP3A5*6 that cause alternative splicing and protein truncation result in the absence of CYP3A5 from tissues of some people. CYP3A5 was more frequently expressed in livers of African Americans (60%) than in those of Caucasians (33%). Because CYP3A5 represents at least 50% of the total hepatic CYP3A content in people polymorphically expressing CYP3A5, CYP3A5 may be the most important genetic contributor to interindividual and interracial differences in CYP3A-dependent drug clearance and in responses to many medicines.


Assuntos
Sistema Enzimático do Citocromo P-450/genética , Polimorfismo de Nucleotídeo Único , Regiões Promotoras Genéticas , Alelos , Processamento Alternativo , Citocromo P-450 CYP3A , Humanos , Dados de Sequência Molecular , Grupos Raciais
8.
Nat Med ; 4(11): 1261-8, 1998 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-9809549

RESUMO

As more functional redundancy in mammalian cells is discovered, enhanced expression of genes involved in alternative pathways may become an effective form of gene therapy. X-linked adrenoleukodystrophy (X-ALD) is a peroxisomal disorder with impaired very-long-chain fatty acid metabolism. The X-ALD gene encodes a peroxisomal membrane protein (ALDP) that is part of a small family of related peroxisomal membrane proteins. We show that 4-phenylbutyrate treatment of cells from both X-ALD patients and X-ALD knockout mice results in decreased levels of and increased beta-oxidation of very-long-chain fatty acids; increased expression of the peroxisomal protein ALDRP; and induction of peroxisome proliferation. We also demonstrate that ALDP and ALDRP are functionally related, by ALDRP cDNA complementation of X-ALD fibroblasts. Finally, we demonstrate the in vivo efficacy of dietary 4-phenylbutyrate treatment through its production of a substantial reduction of very-long-chain fatty acid levels in the brain and adrenal glands of X-ALD mice.


Assuntos
Transportadores de Cassetes de Ligação de ATP/genética , Adrenoleucodistrofia/genética , Adrenoleucodistrofia/terapia , Terapia Genética , Proteínas/genética , Cromossomo X , Subfamília D de Transportador de Cassetes de Ligação de ATP , Membro 1 da Subfamília D de Transportadores de Cassetes de Ligação de ATP , Animais , Linhagem Celular , Células Cultivadas , Primers do DNA , Fibroblastos/citologia , Fibroblastos/efeitos dos fármacos , Humanos , Linfócitos/citologia , Linfócitos/efeitos dos fármacos , Camundongos , Camundongos Knockout , Microcorpos/efeitos dos fármacos , Microcorpos/fisiologia , Microcorpos/ultraestrutura , Família Multigênica , Fenilbutiratos/farmacologia , Reação em Cadeia da Polimerase Via Transcriptase Reversa
9.
Nurs Older People ; 23(5): 31-7, 2011 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-21736103

RESUMO

Skin ageing has intrinsic signs and symptoms, often complicated by extrinsic photo-ageing symptoms and concurrent disease processes. It progresses with age but varies between individuals and its symptoms are numerous. Skin ageing has physical and psychosocial repercussions that can influence coping. Emollient therapy can help to reduce two symptoms--dryness and loss of the skin's barrier function. Better understanding of skin ageing and the usefulness of emollients can be reinforced by education and encouragement from healthcare professionals. Such interventions should encourage self-management and confidence in using emollients. The partnership between healthcare professionals and older people helps to overcome co-existent ageing difficulties, such as cognitive impairment, hearing loss and impairment of manual dexterity and mobility, which enhances self-reliance.


Assuntos
Emolientes/uso terapêutico , Envelhecimento da Pele , Dermatopatias/tratamento farmacológico , Humanos , Dermatopatias/psicologia
10.
J Med Biogr ; 29(2): 103-110, 2021 May.
Artigo em Inglês | MEDLINE | ID: mdl-31475884

RESUMO

At the surrender of Singapore on 15 February 1942, over 100,000 men became prisoners of the Japanese. This included many medical officers who, over the ensuing years, treated men (and some women) suffering the ravaging effects of disease, worsened by the inhumane conditions of captivity. Many medical officers stand out for their work. One in particular, Jacob Markowitz, developed a blood transfusion service, under the most extreme conditions, for the sick working as slave labour on the Burma Railway. Although he qualified 20 years before the outbreak of war, little has been written of Markowitz's early life, or of the impact of this on his war-time contributions.


Assuntos
Transfusão de Sangue/história , Medicina Militar/história , II Guerra Mundial , Transfusão de Sangue/métodos , História do Século XX , Mianmar , Prisioneiros
11.
Proc Natl Acad Sci U S A ; 104(46): 18211-6, 2007 Nov 13.
Artigo em Inglês | MEDLINE | ID: mdl-17984051

RESUMO

To respond to potential adverse exposures properly, health care providers need accurate indicators of exposure levels. The indicators are particularly important in the case of acetaminophen (APAP) intoxication, the leading cause of liver failure in the U.S. We hypothesized that gene expression patterns derived from blood cells would provide useful indicators of acute exposure levels. To test this hypothesis, we used a blood gene expression data set from rats exposed to APAP to train classifiers in two prediction algorithms and to extract patterns for prediction using a profiling algorithm. Prediction accuracy was tested on a blinded, independent rat blood test data set and ranged from 88.9% to 95.8%. Genomic markers outperformed predictions based on traditional clinical parameters. The expression profiles of the predictor genes from the patterns extracted from the blood exhibited remarkable (97% accuracy) transtissue APAP exposure prediction when liver gene expression data were used as a test set. Analysis of human samples revealed separation of APAP-intoxicated patients from control individuals based on blood expression levels of human orthologs of the rat discriminatory genes. The major biological signal in the discriminating genes was activation of an inflammatory response after exposure to toxic doses of APAP. These results support the hypothesis that gene expression data from peripheral blood cells can provide valuable information about exposure levels, well before liver damage is detected by classical parameters. It also supports the potential use of genomic markers in the blood as surrogates for clinical markers of potential acute liver damage.


Assuntos
Acetaminofen/toxicidade , Sangue , Expressão Gênica , Alanina Transaminase/metabolismo , Algoritmos , Animais , L-Iditol 2-Desidrogenase/metabolismo , Contagem de Leucócitos , Masculino , Ratos , Ratos Endogâmicos F344
12.
J Cell Biol ; 73(2): 332-53, 1977 May.
Artigo em Inglês | MEDLINE | ID: mdl-192733

RESUMO

The nonproliferating chicken liver cell culture system described yields cell monolayers with morphological and lipogenic properties characteristic of the physiological-nutritional state of donor animals. Synthesis and secretion of fatty acid, cholesterol, and very low density lipoprotein (VLDL) occur at in vivo rates and respond to hormones and agents which affect these processes in vivo. Cells derived from fed chickens maintain high rates of synthesis of fatty acid and cholesterol for several days if insulin is present in the medium. High rates of fatty acid synthesis are correlated with the appearance of membrane-enclosed triglyceride-rich vesicles in the cytoplasm; deletion of insulin causes a decrease (T1/2 = 22 h) in fatty acid synthetic activity. Addition of glucagon or cyclic AMP (cAMP) causes an immediate cessation of fatty acid synthesis and blocks the appearance of the triglyceride-rich vesicles. Fatty acid synthesis in liver cells prepared from fasted chickens is less than 5% that of cells from fed animals. After 2-3 days in culture with serum-free medium containing insulin +/- triiodothyronine, fatty acid synthesis is restored to normal; glucagon or dibutyryl cAMP blocks this recovery. Liver cells derived from estradiol-treated chickens synthesize and secrete VLDL for at least 48 h in culture. Electron micrographs of these cells reveal more extensive development of the rough endoplasmic reticulum and Golgi complex compared to cells from untreated chickens. Whereas [3H]leucine incorporation into total protein is unaffected by estrogen treatment, [3H]leucine incorporation into cellular and secreted immunoprecipitable VLDL is markedly increased indicating specific activation of VLDL apopeptide synthesis; 8-10% of the labeled protein synthesized and secreted is VLDL. Dodecyl sulfate-acrylamide gel electrophoresis of immunoprecipitated 3H-VLDL reveals three major apopepetides of 300,000, 11,000, and 8,000 daltons corresponding to those of purified chicken VLDL.


Assuntos
Estradiol/farmacologia , Glucagon/farmacologia , Insulina/farmacologia , Lipídeos/biossíntese , Lipoproteínas VLDL/metabolismo , Fígado/citologia , Bucladesina/farmacologia , Células Cultivadas , Colesterol/biossíntese , AMP Cíclico/farmacologia , Retículo Endoplasmático/ultraestrutura , Ácidos Graxos/biossíntese , Complexo de Golgi/ultraestrutura , Lipoproteínas VLDL/biossíntese , Fígado/metabolismo , Biossíntese de Proteínas , Tri-Iodotironina/farmacologia
13.
Science ; 235(4791): 885-90, 1987 Feb 20.
Artigo em Inglês | MEDLINE | ID: mdl-2880399

RESUMO

Alzheimer's disease is a leading cause of morbidity and mortality among the elderly. Several families have been described in which Alzheimer's disease is caused by an autosomal dominant gene defect. The chromosomal location of this defective gene has been discovered by using genetic linkage to DNA markers on chromosome 21. The localization on chromosome 21 provides an explanation for the occurrence of Alzheimer's disease-like pathology in Down syndrome. Isolation and characterization of the gene at this locus may yield new insights into the nature of the defect causing familial Alzheimer's disease and possibly, into the etiology of all forms of Alzheimer's disease.


Assuntos
Doença de Alzheimer/genética , Cromossomos Humanos Par 21 , Mapeamento Cromossômico , Ligação Genética , Humanos , Linhagem , Polimorfismo de Fragmento de Restrição
14.
Science ; 237(4815): 652-4, 1987 Aug 07.
Artigo em Inglês | MEDLINE | ID: mdl-2955519

RESUMO

Trisomy 21 usually results from nondisjunction during meiosis I. In order to determine whether nondisjunction results from failure of normal chromosome pairing or premature unpairing, recombination frequencies were estimated between DNA polymorphic markers on the long arm of chromosome 21 in families containing one individual with trisomy 21. The recombination frequencies on chromosomes 21 that had undergone nondisjunction were then compared to those on chromosomes 21 that had disjoined normally. The data indicate that recombination is reduced between DNA markers on nondisjoined chromosomes 21. These results are consistent with the hypothesis that reduced chiasma formation predisposes to nondisjunction, resulting in trisomy 21 in humans.


Assuntos
Cromossomos Humanos Par 21 , Síndrome de Down/genética , Não Disjunção Genética , Recombinação Genética , Mapeamento Cromossômico , Ligação Genética , Marcadores Genéticos , Humanos
15.
Science ; 235(4791): 880-4, 1987 Feb 20.
Artigo em Inglês | MEDLINE | ID: mdl-2949367

RESUMO

The amyloid beta protein has been identified as an important component of both cerebrovascular amyloid and amyloid plaques of Alzheimer's disease and Down syndrome. A complementary DNA for the beta protein suggests that it derives from a larger protein expressed in a variety of tissues. Overexpression of the gene in brain tissue from fetuses with Down syndrome (trisomy 21) can be explained by dosage since the locus encoding the beta protein maps to chromosome 21. Regional localization of this gene by both physical and genetic mapping places it in the vicinity of the genetic defect causing the inherited form of Alzheimer's disease.


Assuntos
Doença de Alzheimer/genética , Amiloide/genética , Cromossomos Humanos Par 21 , Sequência de Aminoácidos , Amiloidose/genética , Encéfalo/fisiopatologia , Mapeamento Cromossômico , DNA/genética , Síndrome de Down/genética , Regulação da Expressão Gênica , Ligação Genética , Humanos , RNA Mensageiro/genética , Distribuição Tecidual , Transcrição Gênica
16.
J Inherit Metab Dis ; 32(1): 109-19, 2009 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-19127411

RESUMO

Zellweger spectrum disorders (ZSD) are diagnosed by biochemical assay in blood, urine and cultured fibroblasts and PEX gene mutation identification. In most cases studies in fibroblasts corroborate results obtained in body fluids. In 1996 Clayton and colleagues described a 10-year old girl with evidence of a peroxisome disorder, based on elevated bile acid metabolites and phytanate. At the time it was not possible to distinguish whether she had a ZSD or a single peroxisomal protein defect. Studies in our laboratory showed that she also had elevated plasma pipecolate, supporting the former diagnosis. Despite the abnormal metabolites detected in blood (phytanate, bile acid intermediates and pipecolate), analysis of multiple peroxisomal pathways in fibroblasts yielded normal results. In addition, she had a milder clinical phenotype than usually associated with ZSD. Since complementation analysis to determine the gene defect was not possible, we screened this patient following the PEX Gene Screen algorithm (PGS). The PGS provides a template for sequencing PEX gene exons independent of complementation analysis. Two mutations in PEX10 were identified, a frameshift mutation inherited from her father and a de novo missense mutation in a conserved functional domain on the other allele. This case highlights that molecular analysis may be essential to the diagnosis of patients at the milder end of the ZSD spectrum. Furthermore, it supports the concept that some tissues are less affected by certain PEX gene defects than brain and liver.


Assuntos
Fibroblastos/metabolismo , Peroxissomos/metabolismo , Receptores Citoplasmáticos e Nucleares/genética , Síndrome de Zellweger/diagnóstico , Adolescente , Sequência de Aminoácidos , Sequência de Bases , Células Cultivadas , Feminino , Humanos , Dados de Sequência Molecular , Mutação/fisiologia , Linhagem , Peroxinas , Peroxissomos/química , Receptores Citoplasmáticos e Nucleares/metabolismo , Síndrome de Zellweger/genética , Síndrome de Zellweger/metabolismo
18.
J Clin Invest ; 80(4): 1017-22, 1987 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-3654967

RESUMO

Among characterized forms of liver microsomal cytochromes P-450 in rats are four related isozymes (P-450f-i) notable for their lack of inducibility. Immunoblot analyses demonstrated that human livers microsomes contained several proteins related to these rat P-450s. A human liver P-450, termed HLx, was purified and found by immunochemical assays to resemble rat P-450g. Analysis of the NH2-terminal amino acid sequence of HLx indicates that it is related to rat P-450s f-i and human liver P-450MP. A monoclonal antibody was used to measure the amounts of HLx in 21 human liver specimens. No correlation between the levels of HLx protein in these specimens and the patients' environmental histories was observed. However, statistical analysis of the data suggests that the distribution of HLx is at least bimodal. We conclude that HLx is a member of a family of human liver P-450s that resembles in its structure, and possibly in its distribution, several liver P-450s found in other animals.


Assuntos
Sistema Enzimático do Citocromo P-450/isolamento & purificação , Isoenzimas/isolamento & purificação , Microssomos Hepáticos/enzimologia , Adulto , Idoso , Animais , Anticorpos Monoclonais , Ensaio de Imunoadsorção Enzimática , Feminino , Humanos , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Pessoa de Meia-Idade , Coelhos , Ratos
19.
J Clin Invest ; 80(4): 1029-36, 1987 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-3654968

RESUMO

We used monoclonal antibodies and complementary DNAs (cDNAs) to glucocorticoid-inducible liver cytochromes P-450 in rats (P-450p) and in man (HLp) to search for related cytochromes in intestinal mucosa. In rat enterocytes, we found two dexamethasone-inducible proteins related to the steroid-inducible liver cytochromes P-450. Induction of these proteins in enterocytes was associated with increases in the amount of a P-450p-related messenger RNA and of erythromycin demethylase, an activity highly characteristic of P-450p and HLp. Similar studies on human jejunal enterocytes revealed a microsomal protein indistinguishable from HLp on immunoblots and an abundance of RNA hybridizing with HLp cDNA. In human enterocytes the specific concentration of the HLp-related cytochrome (measured immunochemically or as erythromycin demethylase activity) was similar to that found in human liver and could account for all of the CO-binding hemo-protein detected. We conclude that the intestinal mucosa contains prominent form(s) of cytochromes P-450 similar to liver cytochrome P-450p in their structure, function, and some regulatory characteristics.


Assuntos
Sistema Enzimático do Citocromo P-450/biossíntese , Dexametasona/farmacologia , Mucosa Intestinal/enzimologia , Isoenzimas/biossíntese , Adulto , Animais , Anticorpos Monoclonais , DNA/análise , Indução Enzimática , Eritromicina/metabolismo , Feminino , Humanos , Mucosa Intestinal/ultraestrutura , Jejuno/enzimologia , Jejuno/ultraestrutura , Masculino , Microssomos/enzimologia , Microssomos Hepáticos/enzimologia , Pessoa de Meia-Idade , Hibridização de Ácido Nucleico , RNA Mensageiro/metabolismo , Ratos , Ratos Endogâmicos
20.
J Clin Invest ; 90(5): 1871-8, 1992 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-1430211

RESUMO

Enzymes within the P450IIIA (CYP3A) subfamily appear to account for significant "first pass" metabolism of some drugs in the intestine. To identify which of the known P450IIIA genes are expressed in intestine, enterocyte RNA was hybridized on Northern blots with synthetic oligonucleotides complementary to hypervariable regions of hepatic P450IIIA4, P450IIIA5, and P450IIIA7 cDNAs. Hybridization was detected only with the P450IIIA4-specific oligonucleotide. The identity of the hybridizing mRNA was confirmed to be P450IIIA4 by direct sequencing of a DNA fragment amplified from enterocyte cDNA by the polymerase chain reaction. To determine if enterocyte P450IIIA4 is inducible, biopsies of small bowel mucosa were obtained from five volunteers before and after they received 7d of treatment with rifampin, a known inducer of P450IIIA4 in liver. Rifampin treatment resulted in a five- or eightfold mean increase (P < 0.05) in the biopsy concentration of P450IIIA4 mRNA when normalized for content of sucrase isomaltase or intestinal fatty acid binding protein mRNAs, respectively. Rifampin also induced P450IIIA immunoreactive protein in enterocytes in each of the subjects, as judged by immunohistochemistry, and resulted in a 10-fold increase in P450IIIA4-specific catalytic activity (erythromycin N-demethylation) in the one patient studied. Our identification of inducible P450IIIA4 in enterocytes may in part account for drug interactions characteristic of P450IIIA4 substrates and suggests a strategy for controlling entry into the body of a major class of xenobiotics.


Assuntos
Sistema Enzimático do Citocromo P-450/biossíntese , Intestino Delgado/enzimologia , Rifampina/farmacologia , Adulto , Sequência de Bases , Ciclosporina/farmacologia , Citocromo P-450 CYP3A , Sistema Enzimático do Citocromo P-450/genética , Indução Enzimática , Feminino , Humanos , Masculino , Dados de Sequência Molecular , RNA Mensageiro/análise
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