RESUMO
The gene for the X-linked Kallmann syndrome (KAL), a developmental disorder characterized by hypogonadotropic hypogonadism and anosmia, maps to Xp22.3 and has a homologous locus, KALP, on Yq11. We show here that KAL consists of 14 exons spanning 120-200 kilobases that correlate with the distribution of domains in the predicted protein including four fibronectin type III repeats. The KALP locus reveals several large deletions and a number of small insertions, deletions and base substitutions which indicate it is a non-processed pseudogene. The sequence divergence between KAL and KALP in humans, and the chromosomal location of KAL homologous sequences in other primates, suggest that KALP and the steroid sulphatase pseudogene on Yq11 were involved in the same rearrangement event on the Y chromosome during primate evolution.
Assuntos
Síndrome de Kallmann/genética , Cromossomo X , Cromossomo Y , Sequência de Aminoácidos , Animais , Sequência de Bases , Evolução Biológica , Mapeamento Cromossômico , DNA/genética , Éxons , Feminino , Ligação Genética , Humanos , Íntrons , Masculino , Dados de Sequência Molecular , Primatas , Pseudogenes , Homologia de Sequência do Ácido NucleicoRESUMO
We describe how constraints on the binding of human interferons (IFNs), alpha1 and alpha2 and alpha8 on mouse cells are partially relieved by the expression of the bovine (Bo) or human (Hu) IFN alpha/beta receptor (IFNAR) component in these cells. We show that, while the binding of all three is substantially increased by the transfection of Bo IFNAR, it is accompanied by an increase in activity only in the case of alpha2 and alpha8 (IFNs that otherwise have little activity on mouse cells). IFN alpha1, which shows some partial activity on mouse cells, responds to the presence of Bo IFNAR by acting, at low concentrations, as a competitive antagonist to IFNs alpha2 and alpha8. A review of published results on IFN hybrid scanning and on the effects of expressing Bo IFNAR in human cells led us to propose that an N-terminal segment of the IFN molecule interacts directly with IFNAR. Applying site-directed mutagenesis to an IFN hybrid; alpha8[60]alpha1[92]alpha8, we show that the point mutations K84 to E84 and Y90 to D90 act synergistically to cause the hybrid to behave as the parental IFN alpha8, switching the preference from Mu to Hu IFNAR in transfected mouse cells. The published structural models for IFN reveal that positions 84 and 90 span the exposed residues of the alpha-helix C of the IFN molecule. We derive a model of IFN-receptor interaction in which the A helix and the C helix of IFN interact with IFNAR and in which a binding phase can be distinguished from a binding/activity phase. We propose that the so-called "hot" domains of the IFN molecule (the AB loop and the D helix) are presented by IFNAR to interact with an additional component of the functional receptor.
Assuntos
Interferon-alfa/metabolismo , Receptores de Interferon/metabolismo , Animais , Sítios de Ligação , Ligação Competitiva , Bovinos , Linhagem Celular , Humanos , Proteínas de Membrana , Camundongos , Modelos Químicos , Receptor de Interferon alfa e beta , Proteínas Recombinantes/metabolismo , Relação Estrutura-AtividadeRESUMO
The structure of the murine IFNAR gene, encoding the interferon alpha/beta receptor, is reported. The gene has eleven exons dispersed in about 23 kb of genomic DNA. The nature of the rearrangements affecting this gene in interferon-resistant (IFNR) L1210 mutant cell lines is described.
Assuntos
Rearranjo Gênico , Leucemia L1210/genética , Receptores de Interferon/genética , Animais , Proteínas de Membrana , Camundongos , Dados de Sequência Molecular , Receptor de Interferon alfa e beta , Células Tumorais CultivadasRESUMO
Using cloned Epstein-Barr nuclear antigen 1 (EBNA) and oriP elements from the Epstein-Barr virus (EBV) in conjunction with liver-specific growth media, we have constructed an EBNA-producing line of well-differentiated human hepatoma cells (Hep-EBNA-2) and appropriate EBV-oriP vectors. These vectors, pBEDC1 and pBEUG1, were maintained as free extrachromosomal elements only in cells that expressed the trans-acting EBNA protein. They were readily rescued from transfected Hep-EBNA-2 cells upon transformation of recA- Escherichia coli with cellular low-Mr DNA. They are true shuttle vectors in that they can propagate as free closed circular elements in both human Hep-EBNA-2 cells and E. coli. Finally, we have demonstrated the vector capability of our shuttle system by inserting into the SV40 expression cassette of pBEUG1 a large full-length cDNA encoding coagulation factor VIII. Our data clearly show that EBV-oriP episomes are able to stably propagate in an hepatic background and that neither high levels of EBNA protein nor multiple copy episomes significantly interfere with the expression of the set of hepatic functions that have been analyzed. These results are discussed in terms of gene amplification and cloning of genes that program liver differentiation.
Assuntos
Antígenos Virais/biossíntese , Carcinoma Hepatocelular/genética , Vetores Genéticos , Herpesvirus Humano 4/imunologia , Neoplasias Hepáticas/genética , Células Tumorais Cultivadas/metabolismo , Antígenos Virais/genética , Southern Blotting , Carcinoma Hepatocelular/patologia , Clonagem Molecular , Cosmídeos , DNA , Antígenos Nucleares do Vírus Epstein-Barr , Escherichia coli/genética , Fator VIII/biossíntese , Fator VIII/genética , Imunofluorescência , Amplificação de Genes , Herpesvirus Humano 4/genética , Fígado/citologia , Neoplasias Hepáticas/patologia , Plasmídeos , Mapeamento por Restrição , TransfecçãoRESUMO
Evidence is presented, based on sequence comparison according to Hydrophobic Cluster Analysis, of a structural and evolutionary relationship between the human alpha/beta-interferon receptor and the human and mouse gamma-interferon receptor. These results predict that the human alpha/beta-interferon receptor extracellular part is organised in two homologous subdomains connected by a proline linker. They also predict that both subdomains present some homologies to the external domain of mouse and human gamma interferon receptor.
Assuntos
Proteínas de Membrana/ultraestrutura , Receptores Imunológicos/ultraestrutura , Sequência de Aminoácidos , Espaço Extracelular , Humanos , Dados de Sequência Molecular , Receptores de InterferonRESUMO
Apart from kidney, where renin synthesis takes place in all mammals, the submaxillary gland (SMG) of most mouse strains constitutes an important source of an isoenzyme, renin-2, that is highly homologous to renal renin, but unglycosylated [(1982) Nature 298, 90-92]. This unique phenotype is due to the presence of an extra copy of th renin gene. A puzzling observation is that (pro)renin-2 cannot be detected in the kidney of these animals, although both mRNAs accumulate at similar levels [(1985) Proc. Natl. Acad. Sci. USA 82, 6196-6200]. In order to investigate whether (pro)renin-2 expression is detectable in mouse heterologous cell lines we transfected the renin-2 cDNA into AtT20 (pituitary corticotrope) and BTG9A (hepatoma) cells. Stable clones expressing renin were obtained in both cases. BTG9A cells secreted only prorenin while AtT20 cells secreted prorenin and active renin. In addition, in AtT20 cells the secretion of active renin was stimulated by 8-Br cAMP. Our results show that unglycosylated (pro)renin-2 can be expressed and secreted in two murine cell lines. Moreover, it is correctly processed to active renin and secreted upon stimulation in AtT20 cells.
Assuntos
DNA/genética , Precursores Enzimáticos/genética , Regulação da Expressão Gênica , Peptídeo Hidrolases/metabolismo , Renina/genética , Glândula Submandibular/metabolismo , 8-Bromo Monofosfato de Adenosina Cíclica/farmacologia , Animais , Linhagem Celular , Clonagem Molecular , Dexametasona/farmacologia , Precursores Enzimáticos/metabolismo , Vetores Genéticos , Glicosilação , Camundongos , Hibridização de Ácido Nucleico , RNA Mensageiro/biossíntese , Renina/metabolismo , Transfecção , Células Tumorais CultivadasRESUMO
Differences in activity among the family of human IFNs alpha are much reduced if these ligands are assayed on bovine cells. In particular, the activity of IFN alpha D is much higher on bovine than on human cells. To examine these differences, the bovine counterpart of the human IFNAR has been cloned and expressed in a human cell line. The transfected cell line now recognizes the human IFN alpha D as a high-specific-activity IFN subtype, indicating that the differences in sensitivity between the bovine and human cells to the human IFN alpha lie in the structure of the IFNAR chain rather than in the other components of the functional receptor.
Assuntos
Interferon-alfa/fisiologia , Receptores de Interferon/fisiologia , Sequência de Aminoácidos , Animais , Bovinos , Clonagem Molecular , DNA/genética , Humanos , Camundongos , Dados de Sequência Molecular , Mapeamento por Restrição , Alinhamento de Sequência , Relação Estrutura-Atividade , TransfecçãoRESUMO
Recent results indicate that coherent models of how multiple interferons (IFN) are recognized and signal selectively through a common receptor are now feasible. A proposal is made that the IFN receptor, with its subunits IFNAR-1 and IFNAR-2, presents two separate ligand binding sites, and this double structure is both necessary and sufficient to ensure that the different IFN are recognized and can act selectively. The key feature is the duplication of the extracellular domain of the IFNAR-1 subunit and the configurational geometry that this imposes on the intracellular domains of the receptor subunits and their associated tyrosine kinases.
Assuntos
Receptores de Interferon/química , Sequência de Aminoácidos , Humanos , Proteínas de Membrana , Dados de Sequência Molecular , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Proteínas Tirosina Quinases/metabolismo , Receptor de Interferon alfa e beta , Receptores de Interferon/fisiologia , Homologia de Sequência de Aminoácidos , Transdução de Sinais/fisiologia , Relação Estrutura-AtividadeRESUMO
Homology was detected between the structural genes for the nitrogenase complex of K. pneumoniae (nifHDK genes) and the total DNA of several Azospirillum strains. Bacteriophage lambda gt 7-ara6 was used to construct a gene bank of A. brasilense strain 7000 DNA and a recombinant phage carrying a 6.7 kb Eco RI fragment, termed AbRI, was selected by hybridization with the K. pneumoniae nif probe. Using heteroduplex analysis the extent of the homology of the AbRI fragment and the K. pneumoniae nif genes was found to be approximately 5 kb. Proteins encoded by the AbRI fragment were examined after infection of E. coli minicells.
Assuntos
Clonagem Molecular , Cianobactérias/genética , Klebsiella pneumoniae/genética , Fixação de Nitrogênio , Microscopia Eletrônica , Hibridização de Ácido NucleicoRESUMO
From January 1975 to June 1988, 275 patients underwent mitral valve repair for mitral regurgitation, pure (148 patients) or associated with mitral stenosis (127 patients). Patients with pure mitral stenosis were excluded from this study. The cause of mitral regurgitation was rheumatic in 180 patients (aged 28.6 +/- 1.2 years, mean +/- standard error of the mean) and degenerative in 84 patients (aged 54.7 +/- 1.5 years). Fifty-nine percent of the patients were in New York Heart Association classes III and IV before the operation. Intraoperative assessment of the mitral valve led us to identify four major mechanisms of mitral regurgitation: (1) restriction of leaflet motion by fibrosis (group I, 63 patients); (2) enhancement of leaflet motion by leaflet and chordal extension and prolapse (group II, 139 patients), (3) combination of both (group III, 64 patients); and (4) isolated dilatation of the anulus (group IV, 10 patients). One hundred sixty-one patients had isolated mitral disease and 114 had associated aortic or tricuspid valve disease, or both. The hospital mortality rate was 4.0%. Follow-up was 96% complete and totaled 1247.47 patient-years. At 13 years' follow-up, the survival rate was 93.0% +/- 6.8% in group I, 90.0% +/- 6.0% in group II, and 96.6% +/- 4.6% in group III. Freedom from reoperation was 78.1% +/- 21.0%, 83.2% +/- 18.9%, and 79.6% +/- 16.2%, respectively. Freedom from embolism was 94.7% for the whole series. In patients with isolated mitral valve repair, the cumulative morbidity was significantly higher in groups I (6.3 +/- 2.0%/pt-yr) and III 6.3% +/- 1.7%/pt-yr) than in group II (2.5% +/- 0.9%/pt-yr, p less than 0.05). Multivariate analysis identified age and associated tricuspid valve disease as significant predictors of reoperation (p less than 0.01 for both factors). These results suggest that conservative surgery should be used with caution in group I and III patients. In contrast, indications for mitral valve repair should be extended in group II patients. This observation has important clinical implications since, in Western countries, valve prolapse tends to be a major cause of mitral regurgitation.
Assuntos
Insuficiência da Valva Mitral/cirurgia , Estenose da Valva Mitral/cirurgia , Valva Mitral/cirurgia , Análise Atuarial , Adolescente , Adulto , Idoso , Criança , Pré-Escolar , Humanos , Pessoa de Meia-Idade , Insuficiência da Valva Mitral/mortalidade , Insuficiência da Valva Mitral/fisiopatologia , Estenose da Valva Mitral/mortalidade , Estenose da Valva Mitral/fisiopatologia , Complicações Pós-Operatórias/mortalidade , Reoperação , Taxa de Sobrevida , Tromboembolia/etiologiaRESUMO
We report a series of 29 patients, 5 to 75 years of age (mean age, 31.8 +/- 21.4 [SD] years), with pure mitral regurgitation caused by ruptured or elongated chordae of the anterior mitral leaflet. These patients underwent mitral valve repair by segmental transposition of the posterior leaflet with its attached chordae sutured to the free edge of the flail anterior leaflet. There were 2 hospital deaths. Follow-up ranged from 1 to 35 months (mean follow-up, 14.9 +/- 8.5 months). One patient is lost to follow-up. Two patients are in New York Heart Association Functional Class II; all others are in Class I. In 17 patients there is no detectable murmur; in 5 patients a mild to moderate systolic murmur can be detected, while 4 have a marked systolic murmur. The adequacy of the repair could be confirmed by Doppler echocardiography, which has shown no evidence of prolapse in 22 patients. A mild regurgitation jet is present in 4 patients, and a marked jet, in 3. Postoperative cardiac catheterization performed in 5 patients has confirmed the Doppler echocardiographic findings. Although longer follow-up is necessary, this technique appears adequate for repairing a major prolapse of the anterior leaflet caused by multiple ruptured or elongated chordae, therefore obviating the need for a prosthetic valve substitute.
Assuntos
Cordas Tendinosas/cirurgia , Prolapso da Valva Mitral/cirurgia , Adolescente , Adulto , Idoso , Velocidade do Fluxo Sanguíneo , Criança , Pré-Escolar , Ecocardiografia , Feminino , Humanos , Masculino , Métodos , Pessoa de Meia-Idade , Prolapso da Valva Mitral/etiologia , Prolapso da Valva Mitral/fisiopatologia , Ruptura EspontâneaRESUMO
The "time motion" echocardiographic findings in 133 patients, with stenotic mitral valve defects were compared with the anatomical and haemodynamic findings. The information was derived either by monosound, or after a multiscan survey, selecting the lines in two perpendicular planes. A morphological analysis of VMA has allowed us to define a statistical profile according to the type of valve defect: a single diastolic slope in the pure uncalcified stenoses, 2 slopes with the first being more rapid than the second, in other mitral conditions. The cinetics of VMP were related to the presence of associated regurgitation and to the type of fusion. In cases with multiple diastolic slopes, the degree of stenosis was correlated only with the first slope, whilst the second was fairly closely related to the left ventricular end-diastolic pressure. The degree of valvular involvement can be predicted on the coexistence of a thick contour with multiple images, or in their absence on the diminution of amplitude of opening (less than 12 mm) and on the maximum speed of opening (less than 250 mm/s). By contrast, the sub-valvular lesions are underestimated whichever technique is used.
Assuntos
Ecocardiografia , Estenose da Valva Mitral/diagnóstico , Valva Mitral/fisiopatologia , Calcinose/fisiopatologia , Estudos de Avaliação como Assunto , Humanos , Insuficiência da Valva Mitral/fisiopatologia , Estenose da Valva Mitral/fisiopatologiaRESUMO
This study analyses the echocardiographic findings in 41 cases of severe mitral regurgitation due to chordal rupture (33 cases), elongation of chordae (4 cases) or valve trauma (4 cases). The operative findings are given. It was possible to make the diagnosis of chordal rupture in 60% of cases by recording one or more of the following signs:--For the anterior cusp: amplitude of motion equal to or greater than 38 mm; co-existence of chaotic diastolic fluttering and multiple systolic echoes; recording of several diastolic wave forms of the anterior cusp, out of phase and crossing each other;--For the posterior cusp: paradoxical movement of the cusp in systole and diastole; presence of an echo in the left atrium in systole. The group studied was compared with a group of 40 normal subjects and 48 cases of other types of mitral regurgitation which were severe and received surgery. The various signs had good specificity. The sensitivity of the different signs varied from 33 to 50% of cases. It was greater when the number of chordal ruptures was greater. Whichever cusp was affected, it was sometimes the site of high frequency and large amplitude systolic vibrations, which were found in a quarter of the patients. The specificity of this sign is discussed. The diagnosis of rupture of chordae is possible in a large proportion of cases and the causes of error are analysed.
Assuntos
Cordas Tendinosas , Ecocardiografia , Ruptura Cardíaca/complicações , Insuficiência da Valva Mitral/diagnóstico , Valva Mitral , Adolescente , Adulto , Idoso , Cordas Tendinosas/patologia , Feminino , Aneurisma Cardíaco/complicações , Humanos , Masculino , Pessoa de Meia-Idade , Valva Mitral/patologia , Insuficiência da Valva Mitral/etiologia , Insuficiência da Valva Mitral/patologiaRESUMO
Seven patients aged 8 to 62 years with massive mitral regurgitation due to anterior leaflet prolapse related to rupture or elongation of the chordae tendinae underwent reconstructive mitral valvuloplasty between June 1984 and September 1985, consisting in transposition of a bandlet of the posterior leaflet and its chordae to the free edge of the anterior leaflet. Medium term results with 2 to 16 months follow-up (average 8 months) showed all patients to have returned to Class I of the NYHA Classification; 5 patients had no systolic murmur, a mild systolic murmur 1 and 2/6 was present in 2 cases. The quality of the repair was confirmed by pulsed Doppler examination in all patients and by catheterisation and angiography in 3 cases. This surgical technique offers a good solution to the problem of mitral regurgitation due to severe prolapse of the anterior leaflet caused by rupture or elongation of the chordae tendinae.
Assuntos
Prolapso da Valva Mitral/cirurgia , Adolescente , Adulto , Angiocardiografia , Criança , Ecocardiografia , Feminino , Humanos , Métodos , Insuficiência da Valva Mitral/etiologia , Insuficiência da Valva Mitral/cirurgia , Prolapso da Valva Mitral/complicações , Prolapso da Valva Mitral/diagnóstico por imagemAssuntos
Citocinas/química , Interferon Tipo I/química , Receptores de Interferon/química , Sequência de Aminoácidos , Animais , Mapeamento Cromossômico , Citocinas/fisiologia , Humanos , Interferon Tipo I/fisiologia , Dados de Sequência Molecular , Conformação Proteica , Receptores de Interferon/fisiologia , Relação Estrutura-Atividade , Terminologia como AssuntoRESUMO
A cDNA coding for the human interferon alpha receptor has been cloned using a gene transfer approach. This consists of transferring human DNA to mouse cells and selecting for cells sensitive to human interferon alpha. The transfected cells expressed the human interferon alpha receptor, and a 5 kb human DNA was isolated from a secondary transfectant. This DNA defects an mRNA present in human cells and was used to clone a 2.7 kb cDNA from a library constructed from human Daudi cells. The sequence of the cDNA is presented. It codes for a glycoprotein of 557 amino acids with an N-terminal hydrophobic region and a single transmembrane-spanning segment. Mouse cells expressing the cDNA become sensitive to the antiviral activity of and express binding sites for human interferon alpha, demonstrating that the cloned cDNA encodes a functional human interferon alpha receptor.
Assuntos
Clonagem Molecular , DNA/genética , Expressão Gênica , Genes , Interferon Tipo I/metabolismo , Receptores Imunológicos/genética , Transfecção , Sequência de Aminoácidos , Animais , Sequência de Bases , Northern Blotting , Southern Blotting , Linhagem Celular , Vírus da Encefalomiocardite/efeitos dos fármacos , Humanos , Interferon Tipo I/farmacologia , Interferon gama/farmacologia , Cinética , Camundongos , Dados de Sequência Molecular , Conformação Proteica , Receptores Imunológicos/metabolismo , Receptores de Interferon , Vírus da Estomatite Vesicular Indiana/efeitos dos fármacosRESUMO
A full-length cDNA corresponding to a gene mapping to the D21S58 locus was cloned. The encoded protein, called CRF2-4, was shown to be a typical class II member of the cytokine receptor family. The gene encoding CRF2-4 spans more than 30 kb. Its intron/exon structure was determined and shown to be conserved with all other members of the cytokine receptor family. The physical distance between the CRF2-4 gene and its IFNAR neighbor has been narrowed to less than 35 kb.
Assuntos
Cromossomos Humanos Par 21 , Genes , Glicoproteínas de Membrana , Receptores de Citocinas , Receptores Imunológicos/genética , Sequência de Aminoácidos , Animais , Sequência de Bases , Bovinos , DNA/genética , Éxons , Ligação Genética , Humanos , Subunidade beta de Receptor de Interleucina-10 , Camundongos , Dados de Sequência Molecular , Família Multigênica , Estrutura Secundária de Proteína , Receptores de Interferon/genética , Alinhamento de Sequência , Homologia de Sequência de AminoácidosRESUMO
With the aim of producing nonviral shuttle vectors for mammalian cells, we have constructed mouse mitochondrial DNA derivatives comprising the xanthine-guanine phosphoribosyltransferase gene as a selectable marker. Complete or subcomplete mitochondrial genomes were inserted into the plasmid pBB3 and transferred into hepatoma cells in order to generate, in vivo, new recombinant molecules. A second- and a third-generation vector, p12.2b and p delta respectively, were thus isolated for their ability to shuttle from mammalian cells to recA+ E. coli. Transfection of rodent fibroblasts and hepatoma cells showed that, contrary to our expectations, p12.2b and p delta are not self-replicating episomes; their shuttling from mammalian cells to recA+ E. coli is mediated by tandem integrated copies. The relevant property of p12.2b and p delta is a ubiquitous propensity to form head-to-tail multimeric structures when they integrate into mammalian host chromosomes. This ability is missing in pBB3 and appears only following the insertion of various mitochondrial or nuclear DNA fragments into the plasmid. These data are discussed in terms of homologous recombination and shuttling of integrated vectors.
Assuntos
Elementos de DNA Transponíveis , DNA Mitocondrial/genética , Escherichia coli/genética , Vetores Genéticos , Plasmídeos , Transfecção , Animais , Linhagem Celular , Enzimas de Restrição do DNA , DNA Bacteriano/genética , Eletroforese em Gel de Ágar , Marcadores Genéticos , Humanos , Hipoxantina Fosforribosiltransferase/genética , Neoplasias Hepáticas Experimentais/genética , Camundongos , Pentosiltransferases/genética , RatosRESUMO
Two DNA-binding factors detected in pre-B and pre-T cells and absent from mature lymphocytes are described. Factor A displayed no appreciable sequence selectivity but bound only to DNA fragments longer than 120 base pairs. The minimal size of a binding site was lower on an intrinsically curved DNA, suggesting formation of tertiary structures on DNA. Factor B interacted with sequences, other than consensus recombination signals, present in the vicinity of unrearranged immunoglobulin genes. Binding of factor B inhibited the interaction of factor A with the same DNA fragment. The presence of the factor-B-binding site in an episomal V(D)J recombinase substrate lowered the frequency of recombination in vivo. We propose that the two factors described here may function as accessory proteins in V(D)J recombination, possibly modulating accessibility of genes to the recombinase.