RESUMO
Mutations at the alpha-globin locus are the most common class of mutations in humans, with deletion of all four adult alpha-globin genes resulting in the perinatal lethal condition haemoglobin Barts hydrops fetalis. Using gene targeting in mice, we have deleted a 16 kilobase region encompassing both adult alpha-globin genes. Animals homozygous for this deletion become hydropic and die late in gestation mimicking humans with hydrops fetalis. Introduction of a human alpha-globin transgene rescued these animals from perinatal death thus demonstrating the utility of this murine model in the development of cellular and gene based approaches for treating this human genetic disease.
Assuntos
Modelos Animais de Doenças , Marcação de Genes , Genes Letais , Terapia Genética , Globinas/genética , Hidropisia Fetal/genética , Camundongos Knockout/genética , Animais , Sequência de Bases , Quimera , Morte Fetal/etiologia , Regulação da Expressão Gênica no Desenvolvimento , Idade Gestacional , Globinas/biossíntese , Hemoglobinas Anormais , Humanos , Hidropisia Fetal/sangue , Hidropisia Fetal/embriologia , Hidropisia Fetal/prevenção & controle , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Dados de Sequência Molecular , Deleção de Sequência , Talassemia alfa/sangue , Talassemia alfa/genética , Talassemia alfa/terapiaRESUMO
Many quantitative trait loci (QTLs) contributing to genetically complex conditions have been discovered, but few causative genes have been identified. This is mainly due to the large size of QTLs and the subtle connection between genotype and quantitative phenotype associated with these conditions. Transgenic mice have been successfully used to analyse well-characterized genes suspected of contributing to quantitative traits. Although this approach is powerful for examining one gene at a time, it can be impractical for surveying the large genomic intervals containing many genes that are typically associated with QTLs. To screen for genes contributing to an asthma QTL mapped to human chromosome 5q3 (refs 6,7), we characterized a panel of large-insert 5q31 transgenics based on studies demonstrating that altering gene dosage frequently affects quantitative phenotypes normally influenced by that gene. This panel of human YAC transgenics, propagating a 1-Mb interval of chromosome 5q31 containing 6 cytokine genes and 17 partially characterized genes, was screened for quantitative changes in several asthma-associated phenotypes. Multiple independent transgenic lines with altered IgE response to antigen treatment shared a 180-kb region containing 5 genes, including those encoding human interleukin 4 (IL4) and interleukin 13 (IL13 ), which induce IgE class switching in B cells. Further analysis of these mice and mice transgenic for mouse Il4 and Il13 demonstrated that moderate changes in Il4 and Il13 expression affect asthma-associated phenotypes in vivo. This functional screen of large-insert transgenics enabled us to identify genes that influence the QTL phenotype in vivo.
Assuntos
Asma/genética , Característica Quantitativa Herdável , Animais , Asma/fisiopatologia , Testes de Provocação Brônquica , Broncoconstrição/efeitos dos fármacos , Cromossomos Artificiais de Levedura , Citometria de Fluxo , Expressão Gênica , Testes Genéticos , Humanos , Imunoglobulina E/sangue , Interleucina-13/genética , Interleucina-4/genética , Cloreto de Metacolina/farmacologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Fenótipo , RNA/genética , RNA/metabolismo , Linfócitos T/citologia , Linfócitos T/metabolismo , Células Th2/citologia , Células Th2/efeitos dos fármacos , Células Th2/metabolismoRESUMO
Using Down syndrome as a model for complex trait analysis, we sought to identify loci from chromosome 21q22.2 which, when present in an extra dose, contribute to learning abnormalities. We generated low-copy-number transgenic mice, containing four different yeast artificial chromosomes (YACs) that together cover approximately 2 megabases (Mb) of contiguous DNA from 21q22.2. We subjected independent lines derived from each of these YAC transgenes to a series of behavioural and learning assays. Two of the four YACs caused defects in learning and memory in the transgenic animals, while the other two YACs had no effect. The most severe defects were caused by a 570-kb YAC; the interval responsible for these defects was narrowed to a 180-kb critical region as a consequence of YAC fragmentation. This region contains the human homologue of a Drosophila gene, minibrain, and strongly implicates it in learning defects associated with Down syndrome.
Assuntos
Comportamento Animal/fisiologia , Síndrome de Down/genética , Aprendizagem/fisiologia , Camundongos Transgênicos/genética , Proteínas Serina-Treonina Quinases/genética , Animais , Encéfalo/patologia , Cromossomos Artificiais de Levedura , Eletrofisiologia , Olho/patologia , Dosagem de Genes , Humanos , Aprendizagem em Labirinto/fisiologia , Camundongos , Dados de Sequência Molecular , Atividade Motora/genética , Proteínas Tirosina Quinases , Homologia de Sequência do Ácido Nucleico , Transgenes , Quinases DyrkRESUMO
We have shown previously that epimorphin (EPM), a protein expressed on the surface of myoepithelial and fibroblast cells of the mammary gland, acts as a multifunctional morphogen of mammary epithelial cells. Here, we present the molecular mechanism by which EPM mediates luminal morphogenesis. Treatment of cells with EPM to induce lumen formation greatly increases the overall expression of transcription factor CCAAT/enhancer binding protein (C/EBP)beta and alters the relative expression of its two principal isoforms, LIP and LAP. These alterations were shown to be essential for the morphogenetic activities, since constitutive expression of LIP was sufficient to produce lumen formation, whereas constitutive expression of LAP blocked EPM-mediated luminal morphogenesis. Furthermore, in a transgenic mouse model in which EPM expression was expressed in an apolar fashion on the surface of mammary epithelial cells, we found increased expression of C/EBPbeta, increased relative expression of LIP to LAP, and enlarged ductal lumina. Together, our studies demonstrate a role for EPM in luminal morphogenesis through control of C/EBPbeta expression.
Assuntos
Proteínas Estimuladoras de Ligação a CCAAT/metabolismo , Células Epiteliais/citologia , Glândulas Mamárias Animais/citologia , Glândulas Mamárias Animais/fisiologia , Glicoproteínas de Membrana/genética , Animais , Comunicação Celular/fisiologia , Células Epiteliais/fisiologia , Feminino , Lactação/fisiologia , Glândulas Mamárias Animais/crescimento & desenvolvimento , Glicoproteínas de Membrana/análise , Glicoproteínas de Membrana/metabolismo , Camundongos , Camundongos Transgênicos , Proteínas do Leite/genética , Células Estromais/citologia , Células Estromais/fisiologiaRESUMO
To create mice expressing exclusively human sickle hemoglobin (HbS), transgenic mice expressing human alpha-, gamma-, and betaS-globin were generated and bred with knockout mice that had deletions of the murine alpha- and beta-globin genes. These sickle cell mice have the major features (irreversibly sickled red cells, anemia, multiorgan pathology) found in humans with sickle cell disease and, as such, represent a useful in vivo system to accelerate the development of improved therapies for this common genetic disease.
Assuntos
Anemia Falciforme/genética , Anemia Falciforme/patologia , Animais , Modelos Animais de Doenças , Feminino , Globinas/genética , Hemoglobina Falciforme/genética , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos TransgênicosRESUMO
alpha2-Adrenergic receptors (alpha2ARs) are essential components of the neural circuitry regulating cardiovascular function. The role of specific alpha2AR subtypes (alpha2a, alpha2b, and alpha2c) was characterized with hemodynamic measurements obtained from strains of genetically engineered mice deficient in either alpha2b or alpha2c receptors. Stimulation of alpha2b receptors in vascular smooth muscle produced hypertension and counteracted the clinically beneficial hypotensive effect of stimulating alpha2a receptors in the central nervous system. There were no hemodynamic effects produced by disruption of the alpha2c subtype. These results provide evidence for the clinical efficacy of more subtype-selective alpha2AR drugs.
Assuntos
Pressão Sanguínea/fisiologia , Frequência Cardíaca/fisiologia , Receptores Adrenérgicos alfa 2/fisiologia , Agonistas alfa-Adrenérgicos/farmacologia , Antagonistas Adrenérgicos alfa/metabolismo , Antagonistas Adrenérgicos alfa/farmacologia , Animais , Pressão Sanguínea/efeitos dos fármacos , Marcação de Genes , Frequência Cardíaca/efeitos dos fármacos , Imidazóis/farmacologia , Rim/metabolismo , Medetomidina , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos , Camundongos Knockout , Músculo Liso Vascular/metabolismo , Fenilefrina/farmacologia , Prazosina/farmacologia , Receptores Adrenérgicos alfa 2/genética , Ioimbina/metabolismoRESUMO
We have produced yeast artificial chromosome (YAC) transgenic mice expressing normal (YAC18) and mutant (YAC46 and YAC72) huntingtin (htt) in a developmental and tissue-specific manner identical to that observed in Huntington's disease (HD). YAC46 and YAC72 mice show early electrophysiological abnormalities, indicating cytoplasmic dysfunction prior to observed nuclear inclusions or neurodegeneration. By 12 months of age, YAC72 mice have a selective degeneration of medium spiny neurons in the lateral striatum associated with the translocation of N-terminal htt fragments to the nucleus. Neurodegeneration can be present in the absence of macro- or microaggregates, clearly showing that aggregates are not essential to initiation of neuronal death. These mice demonstrate that initial neuronal cytoplasmic toxicity is followed by cleavage of htt, nuclear translocation of htt N-terminal fragments, and selective neurodegeneration.
Assuntos
Cromossomos Artificiais de Levedura/genética , Corpo Estriado/patologia , Doença de Huntington/genética , Mutação/fisiologia , Degeneração Neural/patologia , Proteínas do Tecido Nervoso/genética , Proteínas Nucleares/genética , Adaptação Fisiológica/fisiologia , Animais , Comportamento Animal/fisiologia , Encéfalo/patologia , Citoplasma/patologia , Modelos Animais de Doenças , Eletrofisiologia , Embrião de Mamíferos/fisiologia , Proteína Huntingtina , Doença de Huntington/metabolismo , Doença de Huntington/patologia , Doença de Huntington/fisiopatologia , Camundongos , Camundongos Endogâmicos , Camundongos Transgênicos/genética , Atividade Motora/fisiologia , Proteínas do Tecido Nervoso/metabolismo , Proteínas Nucleares/metabolismoRESUMO
Activation of the alpha(1A)-adrenergic receptor (alpha(1A)-AR)/Gq pathway has been implicated as a critical trigger for the development of cardiac hypertrophy. However, direct evidence from in vivo studies is still lacking. To address this issue, transgenic mice with cardiac-targeted overexpression of the alpha(1A)-AR (4- to 170-fold) were generated, using the rodent alpha-myosin heavy chain promoter. Heterozygous animals displayed marked enhancement of cardiac contractility, evident from increases in dP/dt(max) (80%, P<0.0001), dP/dt(max)/LVP(inst) (76%, P<0.001), dP/dt(max):dP/dt(min) (104%, P<0.0001), and fractional shortening (33%, P<0.05). Moreover, changes in the dP/dt(max)-end-diastolic volume relationship provided load-independent evidence of a primary increase in contractility. Blood pressure and heart rate were largely unchanged, and there was a small increase in (-)norepinephrine-stimulated, but not basal, phospholipase C activity. Increased contractility was directly related to the level of receptor overexpression and could be completely reversed by acute alpha(1A)- but not beta-AR blockade. Despite the robust changes in contractility, transgenic animals displayed no morphological, histological, or echocardiographic evidence of left ventricular hypertrophy. In addition, apart from an increase in atrial natriuretic factor mRNA, expression of other hypertrophy-associated genes was unchanged. To our knowledge, these data provide the first in vivo evidence for an inotropic action of the alpha(1A)-AR.
Assuntos
Cardiomegalia , Expressão Gênica/fisiologia , Marcação de Genes , Contração Miocárdica/fisiologia , Receptores Adrenérgicos alfa 1/biossíntese , Adenilil Ciclases/metabolismo , Antagonistas de Receptores Adrenérgicos alfa 1 , Agonistas alfa-Adrenérgicos/farmacologia , Antagonistas Adrenérgicos alfa/farmacologia , Animais , Fator Natriurético Atrial/genética , Fator Natriurético Atrial/metabolismo , Pressão Sanguínea/genética , Ecocardiografia , Eletrocardiografia/efeitos dos fármacos , Frequência Cardíaca/genética , Heterozigoto , Fosfatos de Inositol/metabolismo , Camundongos , Camundongos Transgênicos , Contração Miocárdica/efeitos dos fármacos , Cadeias Pesadas de Miosina/genética , Tamanho do Órgão/genética , Especificidade de Órgãos/genética , Regiões Promotoras Genéticas , RNA Mensageiro/metabolismo , Receptores Adrenérgicos alfa 1/genética , Transgenes/fisiologia , Fosfolipases Tipo C/metabolismoRESUMO
Mutations at the apterous (ap) locus in Drosophila melanogaster give rise to three distinct phenotypes: aberrant wings, female sterility and precocious adult death. The wing phenotype includes five types of abnormality: blistering, deficiencies, duplications, high-order repetitions and transformation of structures. The mildest phenotype is seen with homozygous apblt animals which have either normal or slightly blistered wings. Most alleles produce, in the homozygote, a deficient wing in which part or all of the wing margin and wing blade is missing, but wing hinge and notum regions are normal. Animals hemizygous for each of 20 ap alleles, as well as apID/apXa heterozygotes, show duplication of parts of the notum associated with complete wing deficiency. Animals heterozygous for apc and the other tested ap alleles show repetitions of parts of the anterior wing margin, an engrailed-like transformation of posterior wing margin into anterior margin or both. Both apblt and apc show similar phenotypes in homozygotes and hemizygotes, yet both produce a less extreme phenotype than that of the other hemizygotes, suggesting that neither mutation causes loss of the entire ap+ function. The 15 alleles that cause precocious death and female sterility occur in six complementation groups based on complementation for these phenotypes. This supports the previous conclusion that the effects of apterous mutations on the wing do not correlate with their effects on viability and fertility. We propose an explanation for the effects of apterous mutations on the wing in which quantitative reductions in the activity of gene product give rise to qualitatively different phenotypes because of different threshold requirements of the ap+ function for critical events in wing disc development.
Assuntos
Alelos , Drosophila melanogaster/genética , Mutação , Animais , Mapeamento Cromossômico , Feminino , Fertilidade , Genes Letais , Genótipo , Homozigoto , Masculino , Fenótipo , Tórax/anatomia & histologia , Asas de Animais/anatomia & histologiaRESUMO
Mutations at the apterous (ap) locus in Drosophila melanogaster produce a variety of developmental defects, including several classes of wing abnormalities. We describe the wing phenotype produced by homozygotes and hemizygotes of three different temperature-sensitive apterous alleles grown at 16, 18, 20, 22, 25, and 29 degrees. We also describe the phenotype produced by each of these three alleles when heteroallelic with the non-temperature-sensitive apc allele. Constant-temperature and temperature-shift experiments show that each of the heteroallelic genotypes can produce several of the previously described apterous phenotypes and that the length of the temperature-sensitive period for a given phenotype depends on the allelic combinations used to measure it. We suggest that the stage-specific requirements of the tissue for gene product, rather than the time of gene expression per se, determine the temperature-sensitive periods for apterous and other loci. The results support the hypothesis that the various wing phenotypes produced by apterous mutations are due to quantitative reductions in the activity of gene product and that failure to meet specific threshold requirements for gene product can lead to qualitatively different phenotypes.
Assuntos
Drosophila melanogaster/genética , Mutação , Temperatura , Asas de Animais/anormalidades , Alelos , Animais , Regulação da Expressão Gênica , Heterozigoto , Homozigoto , FenótipoRESUMO
OBJECTIVE: To assess the cost benefits of low dose subcutaneous recombinant human erythropoietin in correcting the anaemia of end stage renal disease. DESIGN: Three year retrospective study. SETTING: Subregional nephrology service serving a mixed urban and rural population of 800,000. SUBJECTS: 60 patients with symptoms of anaemic end stage renal disease treated with erythropoietin (43 receiving haemodialysis; 11 receiving continuous ambulatory peritoneal dialysis; two with predialysis end stage renal disease; four with failing renal transplants). MAIN OUTCOME MEASURES: Costs and savings of achieving and maintaining a haemoglobin concentration of 85-105 g/l with erythropoietin. RESULTS: All patients treated with erythropoietin achieved the target haemoglobin concentration at median induction doses of 97 (95% confidence interval 95 to 108) units/kg/week, and this was maintained with 79 (75 to 95) units/kg/week at an average annual cost per patient of 2260 pounds. Admissions related to anaemia were virtually eliminated (246 v 1 inpatient days for 12 months before and after starting erythropoietin). 54 patients required no blood transfusions after starting erythropoietin, and the total requirements fell from 230 to 21 units in the 12 months before and after starting erythropoietin. Iron stores were maintained with oral or intravenous iron. All patients reported increased wellbeing, appetite, and exercise capacity. Hypertension developed or worsened in 30 patients, resulting in hospital admissions in five patients, one of whom had seizures. CONCLUSION: Low dose subcutaneous erythropoietin restores haemoglobin concentrations sufficiently to abolish blood transfusion requirements and reduce morbidity. The net cost of erythropoietin prescribed in this way (2260 pounds/patient/year) was largely offset by savings in costs of hospital admissions. The true annual cost to the NHS was around 1200 pounds per patient.
Assuntos
Anemia Hipocrômica/tratamento farmacológico , Custos de Medicamentos , Eritropoetina/uso terapêutico , Falência Renal Crônica/complicações , Adulto , Idoso , Anemia Hipocrômica/economia , Anemia Hipocrômica/etiologia , Transfusão de Sangue/economia , Análise Custo-Benefício , Inglaterra , Eritropoetina/administração & dosagem , Feminino , Hemoglobinas/análise , Hospitalização/economia , Humanos , Injeções Subcutâneas , Falência Renal Crônica/sangue , Masculino , Pessoa de Meia-Idade , Proteínas Recombinantes , Estudos RetrospectivosAssuntos
Azatioprina/uso terapêutico , Ciclosporinas/uso terapêutico , Transplante de Rim , Prednisolona/uso terapêutico , Adulto , Ensaios Clínicos como Assunto , Ciclosporinas/administração & dosagem , Esquema de Medicação , Feminino , Humanos , Rim/efeitos dos fármacos , Masculino , Transplante HomólogoAssuntos
Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Carcinoma de Células Escamosas/tratamento farmacológico , Neoplasias Nasofaríngeas/tratamento farmacológico , Carcinoma de Células Escamosas/radioterapia , Criança , Terapia Combinada , Humanos , Masculino , Neoplasias Nasofaríngeas/radioterapia , Dosagem RadioterapêuticaAssuntos
Transtornos de Deglutição/diagnóstico , Transtornos de Deglutição/enfermagem , Avaliação em Enfermagem/métodos , Postura , Transtornos de Deglutição/complicações , Transtornos de Deglutição/reabilitação , Humanos , Pneumonia Aspirativa/etiologia , Encaminhamento e Consulta , Fatores de Risco , Fonoterapia , Patologia da Fala e LinguagemRESUMO
Certain combinations of alleles at the apterous locus generate wings with extra copies of wing margin structures, some of which are located far from the normal margin. We have examined wing imaginal discs from these mutants, using position-specific antibodies as probes for two-dimensional patterning in the discs. Our results indicate that the adult phenotypes arise from unprecedented disruptions in the two-dimensional pattern of the disc epithelia. Examination of other apterous mutants suggest that pattern alterations may be a general consequence of lesions at this locus.
Assuntos
Drosophila melanogaster/embriologia , Asas de Animais/embriologia , Animais , Anticorpos Monoclonais , Sobrevivência Celular , Imunofluorescência , Morfogênese , Mutação , FenótipoRESUMO
Of nineteen patients with RPCGN who responded promptly to initial treatment with PMP or PX, and who were subsequently maintained on oral immunosuppression with prednisolone (reducing dosage from 30mg/day) and azathioprine/cyclophosphamide (1-3 mg/kg/day), five showed progressive loss of renal function within one year of responding to treatment. Both the daily dose at four weeks and the cumulative dose of prednisolone at six months were significantly lower (p less than 0.01) in the group whose renal function deteriorated. We suggest that the follow-up dosage of prednisolone may be critical in maintaining continued stable renal function in the first few months after starting PMP or PX.
Assuntos
Glomerulonefrite/tratamento farmacológico , Prednisolona/administração & dosagem , Adolescente , Adulto , Idoso , Feminino , Glomerulonefrite/terapia , Humanos , Masculino , Metilprednisolona/uso terapêutico , Pessoa de Meia-Idade , Troca PlasmáticaRESUMO
We have microinjected DNA containing the inducible mouse metallothionein-I (MT-I) promoter, coupled to the structural gene for Escherichia coli beta-galactosidase (lacZ), into the pronuclei of one-cell mouse embryos. A qualitative histochemical assay, with 5-bromo-4-chloro-3-indolyl beta-D-galactopyranoside (X-Gal) as a substrate, was used to detect expression of lacZ at several preimplantation stages. We observed staining indicative of exogenous beta-galactosidase activity in 5-17% of DNA-injected embryos assayed at preimplantation stages after 16-24 h treatment with ZnSO4. Thus, lacZ can be used as an indicator gene for promoter function during early mouse embryogenesis, and the incorporation of the MT-I promoter into fusion genes can be a useful means of controlling the expression of exogenous genes in preimplantation mouse embryos.
Assuntos
Embrião de Mamíferos/metabolismo , Escherichia coli/genética , Galactosidases/genética , Genes Bacterianos , Genes , Metalotioneína/genética , Regiões Promotoras Genéticas , beta-Galactosidase/genética , Animais , Blastocisto/metabolismo , DNA Recombinante/metabolismo , Escherichia coli/enzimologia , Feminino , Metalotioneína/biossíntese , Camundongos , Camundongos Endogâmicos C57BL , Microinjeções , Mórula/metabolismo , Mosaicismo , Plasmídeos , Proteínas Recombinantes de Fusão/biossíntese , Mapeamento por Restrição , beta-Galactosidase/biossínteseRESUMO
A comparison has been made of the difference spectra obtained by causing various aflatoxins (B(1), G(1) and G(2)) to interact with calf-thymus DNA. The effect of these toxins on RNA and protein synthesis by rat-liver slices has been measured. The extent of their inhibitory action on the synthetic reactions was proportional to the degree of spectral shift obtained with their interaction with DNA. It is proposed that their toxicity depends on this interaction. It was demonstrated that the RNA polymerase of nucleoli isolated from the livers of aflatoxin B(1)-poisoned rats was inhibited. This finding is in agreement with the proposed mechanism for the hepatotoxic action of aflatoxin.
Assuntos
Aflatoxinas/farmacologia , Fígado/metabolismo , Ácidos Nucleicos/biossíntese , Biossíntese de Proteínas , Animais , Isótopos de Carbono , Fígado/efeitos dos fármacos , Masculino , Nucleotidiltransferases/metabolismo , Ratos , Análise EspectralRESUMO
Rapidly progressive crescentic glomerulonephritis (RPGN) carries a poor prognosis, but early immunosuppression may reverse renal impairment. We have given intensive therapy to 27 patients with biopsy proven RPGN from 1977-1981. Fourteen patients received pulse methylprednisolone (PMP) and 13 patients plasma exchange (Px). These patients fared significantly better than 17 patients seen from 1972-1979 who had neither PMP nor Px. Both groups received oral prednisolone and other immunosuppressive agents. PMP and Px were equally effective in prolonging survival without dialysis and had no serious side effects; prognostic factors affecting the outcome of treatment were identified. Early aggressive immunosuppressive therapy is indicated in RPGN.