RESUMEN
Immunoglobulin A (IgA) is the principal antibody in secretions that bathe the gastrointestinal and respiratory mucosal surfaces and acts as an important first line of defense against invasion of pathogenic micro-organisms. The reported prevalence rate of complete IgA deficiency in healthy children ranges from 1:170 to 1:400, and as a solitary condition, it is often considered of limited clinical importance. However, patients with IgA deficiency can develop recurrent respiratory and gastrointestinal infections, as well as allergic and autoimmune diseases. In children referred for recurrent respiratory tract infections, the observed prevalence rate increases more than tenfold. This review discusses several aspects of IgA deficiency in children, including immunologic and microbiome changes in early childhood and the potential consequences of this condition in later life. It illustrates the importance of early identification of children with impaired IgA production who deserve appropriate clinical care and follow-up.
Asunto(s)
Deficiencia de IgA/inmunología , Inmunoglobulina A/inmunología , Animales , Enfermedades Autoinmunes/inmunología , Niño , Humanos , Prevalencia , Infecciones del Sistema Respiratorio/inmunologíaRESUMEN
Methanogenic enrichments from hypersaline lakes at moderate thermophilic conditions have resulted in the cultivation of an unknown deep lineage of euryarchaeota related to the class Halobacteria. Eleven soda lake isolates and three salt lake enrichment cultures were methyl-reducing methanogens that utilize C1 methylated compounds as electron acceptors and H2 or formate as electron donors, but they were unable to grow on either substrates alone or to form methane from acetate. They are extreme halophiles, growing optimally at 4 M total Na+ and the first representatives of methanogens employing the 'salt-in' osmoprotective mechanism. The salt lake subgroup is neutrophilic, whereas the soda lake isolates are obligate alkaliphiles, with an optimum around pH 9.5. Both grow optimally at 50 °C. The genetic diversity inside the two subgroups is very low, indicating that the soda and salt lake clusters consist of a single genetic species each. The phylogenetic distance between the two subgroups is in the range of distant genera, whereas the distance to other euryarchaea is below 83â% identity of the 16S rRNA gene. These isolates and enriched methanogens, together with closely related environmental clones from hypersaline habitats (the SA1 group), form a novel class-level clade in the phylum Euryarchaeota. On the basis of distinct phenotypic and genetic properties, the soda lake isolates are classified into a new genus and species, Methanonatronarchaeum thermophilum, with the type strain AMET1T (DSM 28684T=NBRC 110805T=UNIQEM U982T), and the salt lake methanogens into a candidate genus and species 'Candidatus Methanohalarchaeum thermophilum'. These organisms are proposed to form novel family, order and class Methanonatronarchaeaceae fam. nov., Methanonatronarchaeales ord. nov. and Methanonatronarchaeia classis nov., within the phylum Euryarchaeota.
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Euryarchaeota/clasificación , Lagos/microbiología , Metano/metabolismo , Filogenia , Salinidad , Composición de Base , California , Crecimiento Quimioautotrófico , Egipto , Euryarchaeota/genética , Euryarchaeota/aislamiento & purificación , ARN Ribosómico 16S/genética , Federación de Rusia , Análisis de Secuencia de ADNRESUMEN
BACKGROUND & AIMS: Macrophage migration inhibitory factor (MIF) is an inflammatory cytokine and an important regulator of innate immune responses. We hypothesised that serum concentrations of MIF are associated with disease severity and outcome in patients with decompensated cirrhosis and acute-on-chronic liver failure (ACLF). METHODS: Circulating concentrations of MIF and its soluble receptor CD74 (sCD74) were determined in sera from 292 patients with acute decompensation of cirrhosis defined as new onset or worsening of ascites requiring hospitalisation. Of those, 78 (27%) had ACLF. Short-term mortality was assessed 90 days after inclusion. RESULTS: Although serum concentrations of MIF and sCD74 did not correlate with liver function parameters or ACLF, higher MIF (optimum cut-off >2.3 ng/ml) and lower concentrations of sCD74 (optimum cut-off <66.5 ng/ml) both indicated poorer 90-day transplant-free survival in univariate analyses (unadjusted hazard ratio [HR] 2.01 [1.26-3.22]; p = 0.004 for MIF; HR 0.59 [0.38-0.92]; p = 0.02 for sCD74) and after adjustment in multivariable models. Higher MIF concentrations correlated with surrogates of systemic inflammation (white blood cells, p = 0.005; C-reactive protein, p = 0.05) and were independent of genetic MIF promoter polymorphisms. Assessment of MIF plasma concentrations in portal venous blood and matched blood samples from the right atrium in a second cohort of patients undergoing transjugular intrahepatic portosystemic shunt insertion revealed a transhepatic MIF gradient with higher concentrations in the right atrial blood. CONCLUSIONS: Serum concentrations of MIF and its soluble receptor CD74 predict 90-day transplant-free survival in patients with acute decompensation of cirrhosis. This effect was independent of liver function and genetic predispositions, but rather reflected systemic inflammation. Therefore, MIF and sCD74 represent promising prognostic markers beyond classical scoring systems in patients at risk of ACLF. LAY SUMMARY: Inflammatory processes contribute to the increased risk of death in patients with cirrhosis and ascites. We show that patients with high serum levels of the inflammatory cytokine macrophage migration inhibitory factor (MIF) alongside low levels of its binding receptor sCD74 in blood indicate an increased mortality risk in patients with ascites. The cirrhotic liver is a relevant source of elevated circulating MIF levels.
RESUMEN
AIMS/HYPOTHESIS: Cannabinoid type 1 receptor (CB1R) antagonists such as rimonabant (Rim) represent a novel approach to treat obesity and related metabolic disorders. Recent data suggest that endocannabinoids are also produced by human adipocytes. Here we studied the potential involvement of endocannabinoids in the negative crosstalk between fat and muscle. METHODS: The protein level of CB1R in human skeletal muscle cells (SkM) during differentiation was analysed using western blotting. SkM were treated with adipocyte-conditioned medium (CM) or anandamide (AEA) in combination with the CB1R antagonists Rim or AM251, and insulin-stimulated Akt phosphorylation and glucose uptake were determined. Furthermore, signalling pathways of CB1R were investigated. RESULTS: We revealed an increase of CB1R protein in SkM during differentiation. Twenty-four hour incubation of SkM with CM or AEA impaired insulin-stimulated Akt(Ser473) phosphorylation by 60% and up to 40%, respectively. Pretreatment of cells with Rim or AM251 reduced the effect of CM by about one-half, while the effect of AEA could be prevented completely. The reduction of insulin-stimulated glucose uptake by CM was completely prevented by Rim. Short-time incubation with AEA activated extracellular regulated kinase 1/2 and p38 mitogen-activated protein kinase, and impaired insulin-stimulated Akt(Ser473) phosphorylation, but had no effect on Akt(Thr308) and glycogen synthase kinase 3alpha/beta phosphorylation. In addition, enhanced IRS-1 (Ser307) phosphorylation was observed. CONCLUSIONS/INTERPRETATION: Our results show that the CB1R system may play a role in the development of insulin resistance in human SkM. The results obtained with CM support the notion that adipocytes may secrete factors which are able to activate the CB1R. Furthermore, we identified two stress kinases in the signalling pathway of AEA and enhanced IRS-1(Ser307) phosphorylation, potentially underlying the development of insulin resistance.
Asunto(s)
Tejido Adiposo/fisiología , Resistencia a la Insulina/fisiología , Músculo Esquelético/fisiología , Receptor Cross-Talk/fisiología , Receptor Cannabinoide CB1/fisiología , Adipocitos/efectos de los fármacos , Adipocitos/fisiología , Tejido Adiposo/efectos de los fármacos , Ácidos Araquidónicos/farmacología , Moduladores de Receptores de Cannabinoides/farmacología , Técnicas de Cultivo de Célula , Diferenciación Celular , Endocannabinoides , Humanos , Músculo Esquelético/citología , Músculo Esquelético/efectos de los fármacos , Piperidinas/farmacología , Alcamidas Poliinsaturadas/farmacología , Pirazoles/farmacología , Receptor Cannabinoide CB1/antagonistas & inhibidores , RimonabantRESUMEN
The complete primary structure of the protein product of the proto-oncogene c-mil was deduced from the nucleotide sequence of chicken c-mil cDNA clones. The c-mil protein contains 647 amino acid residues and has a calculated molecular weight of 73,132. Based on sequence comparisons with proteins of known or presumed biochemical function, two domains were recognized on the c-mil protein. In the carboxyl-terminal half of the protein, a 250-amino acid segment displays significant homology to the protein kinase domains of the src oncogene protein or of protein kinase C. In the amino-terminal half, a cysteine-rich segment (Cys-X2-Cys-X9-Cys-X2-Cys-X7-Cys-X7-Cys) of the c-mil protein shares significant homology with two similar repetitive domains of protein kinase C. Of the two structural and presumably functional domains of the c-mil protein, only the kinase domain is contained within the carboxyl-terminal 379-amino acid polypeptide encoded by the transduced v-mil allele of avian oncogenic retrovirus MH2. Hence, truncation of the 5' coding region in the course of the transduction and the resulting lack of the authentic amino-terminal domain in the protein product of the transduced allele may be a critical event in changing mil function from physiologic to oncogenic.
Asunto(s)
Proteínas Proto-Oncogénicas , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Pollos , Clonación Molecular , Datos de Secuencia Molecular , Conformación Proteica , Proteína Quinasa CRESUMEN
Romano-Ward syndrome (RWS), the autosomal dominant form of the congenital long QT syndrome, is characterised by prolongation of the cardiac repolarisation process associated with ventricular tachyarrhythmias of the torsades de pointes type. Genetic studies have identified mutations in six ion channel genes, KCNQ1, KCNH2, SCN5A, KCNE1 and KCNE2 and the accessory protein Ankyrin-B gene, to be responsible for this disorder. Single-strand conformation polymorphism (SSCP) analysis and subsequent DNA sequence analysis have identified a KCNQ1 mutation in a family that were clinically conspicuous due to several syncopes and prolonged QTc intervals in the ECG. The mutant subunit was expressed and functionally characterised in the Xenopus oocyte expression system. A novel heterozygous missense mutation with a C to T transition at the first position of codon 343 (CCA) of the KCNQ1 gene was identified in three concerned family members (QTc intervals: 500, 510 and 530 ms, respectively). As a result, proline 343 localised within the highly conserved transmembrane segment S6 of the KCNQ1 channel is replaced by a serine. Co-expression of mutant (KCNQ1-P343S) and wild-type (KCNQ1) cRNA in Xenopus oocytes produced potassium currents reduced by approximately 92%, while IKs reconstitution experiments with a combination of KCNQ1 mutant, wild-type and KCNE1 subunits yielded currents reduced by approximately 60%. A novel mutation (P343S) identified in the KCNQ1 subunit gene of three members of a RWS family showed a dominant-negative effect on native IKs currents leading to prolongation of the heart repolarisation and possibly increases the risk of malign arrhythmias with sudden cardiac death.
Asunto(s)
Mutación/genética , Canales de Potasio con Entrada de Voltaje/genética , Canales de Potasio con Entrada de Voltaje/metabolismo , Síndrome de Romano-Ward/genética , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Clonación Molecular , Análisis Mutacional de ADN , Electrofisiología , Femenino , Expresión Génica , Humanos , Canales de Potasio KCNQ , Canal de Potasio KCNQ1 , Masculino , Datos de Secuencia Molecular , Oocitos/metabolismo , Técnicas de Placa-Clamp , Linaje , Fenotipo , Polimorfismo Conformacional Retorcido-Simple , Potasio/metabolismo , Canales de Potasio con Entrada de Voltaje/química , Prolina/genética , Alineación de Secuencia , Xenopus laevisRESUMEN
The construction of deletions, insertions and point mutations in DNA sequences is a powerful approach to analysing the function and structure of genes and their products. Here, we present a fast and efficient method using the polymerase chain reaction to introduce mutations into cDNAs coding for the alpha-, gamma- and epsilon-subunit of the rat muscle acetylcholine receptor. Two flanking primers and one mutant oligo, in conjunction with supercoiled plasmid DNA and a fragment of the target DNA are sufficient to introduce the mutation by two PCR amplifications. Our method permits directing the location of mutations anywhere in the target gene with a very low misincorporation rate, as no substitution could be detected within 9600 bp. The utility of this approach is demonstrated by the rapid introduction and analysis of eleven mutations into three different cDNAs. Any kind of mutation can be introduced with an efficiency of at least 50%.
Asunto(s)
ADN/genética , Genes , Mutación , Técnicas de Amplificación de Ácido Nucleico , Reacción en Cadena de la Polimerasa/métodos , Receptores Colinérgicos/genética , Animales , Secuencia de Bases , Deleción Cromosómica , Clonación Molecular , ADN/aislamiento & purificación , Elementos Transponibles de ADN , Músculos/metabolismo , RatasRESUMEN
The mouse muscle cell line BC3H-1 expresses an acetylcholine receptor (AChR) composed of alpha-, beta-, gamma- and delta-subunits. The functional characteristics of this AChR are comparable to the non-synaptic AChR subtype in mouse muscle. To investigate the role of the epsilon-subunit, which is believed to replace the gamma-subunit in forming the adult AChR subtype, BC3H-1 cells were stably transfected with cDNA encoding the rat muscle AChR epsilon-subunit. Expression of this cDNA was under the control of a heat shock promoter, and the plasmid carried the neomycin resistance gene for selection. Several clones were isolated that had integrated the plasmid DNA in a stable form and produced epsilon-subunit specific RNA after heat induction. Single-channel current recording from cells which contained abundant epsilon-subunit mRNA identified a novel AChR channel having a larger conductance than the native AChR in these cells. These results suggest that the rat muscle epsilon-subunit may assemble with mouse muscle alpha-, beta- and delta-subunits to form a mouse-rat hybrid AChR with properties similar to that of end-plate channels in the mature mammalian neuromuscular synapse. The novel AChR channel appears in the surface membrane within a few hours following the rise in epsilon-subunit mRNA. Thus, the notion that replacement of the gamma-subunit by the epsilon-subunit during development is the result of the postnatal rise in the level of epsilon-subunit specific mRNA is further supported.
Asunto(s)
Receptores Colinérgicos/fisiología , Animales , Northern Blotting , Southern Blotting , Línea Celular , Calor , Canales Iónicos/fisiología , Ratones , Músculos/fisiología , Fragmentos de Péptidos/fisiología , Ratas , Receptores Colinérgicos/biosíntesis , Proteínas Recombinantes/biosíntesis , Transcripción Genética , TransfecciónRESUMEN
We have isolated the murine gene encoding the 5-HT3 receptor (5-HT3R), a member of the ligand-gated ion channels, that mediates a variety of physiological effects in central and peripheral neurons. DNA sequence analysis of the 5-HT3R gene revealed its organisation in 9 exons distributed over approximately 12 kbp of DNA. Alternative use of exon 9 splice acceptor sites generated two 5-HT3R variants. The 5-HT3R gene, whose structure is closely related to neuronal and muscle AChR alpha genes, as demonstrated by four common splice junctions, was localised on human chromosome 11.
Asunto(s)
Mapeo Cromosómico , Cromosomas Humanos Par 11 , Receptores de Serotonina/genética , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Química Encefálica , ADN Complementario/aislamiento & purificación , Femenino , Expresión Génica , Técnicas de Transferencia de Gen , Humanos , Ratones , Datos de Secuencia Molecular , Músculos/química , Neuronas/química , Oocitos/metabolismo , Empalme del ARN , Receptores de Serotonina/química , Análisis de Secuencia de ADN , Homología de Secuencia , Xenopus laevisRESUMEN
We have identified Ser-1275 and Ser-1309 as novel serine autophosphorylation sites by direct sequencing of HPLC-purified tryptic phosphopeptides of the histidine-tagged insulin receptor kinase IRKD-HIS. The corresponding peptides (Ser-1275, amino acids 1272-1292; Ser-1309, amino acids 1305-1313) have been detected in the HPLC profiles of both the soluble kinase IRKD, which contains the entire cytoplasmic domain of the insulin receptor beta-subunit, and the insulin receptor purified from human placenta. In contrast, a kinase negative mutant, IRKD-K1018A, did not undergo phosphorylation at either the tyrosine or serine residues, strongly suggesting that insulin receptor kinase has an intrinsic activity to autophosphorylate serine residues.
Asunto(s)
Receptor de Insulina/metabolismo , Serina/metabolismo , Secuencia de Aminoácidos , Animales , Línea Celular , Humanos , Datos de Secuencia Molecular , Mutación , Mapeo Peptídico , Fosfopéptidos/metabolismo , Fosforilación , Receptor de Insulina/genética , Receptor de Insulina/aislamiento & purificación , Proteínas Recombinantes/metabolismo , Solubilidad , Spodoptera , Treonina/metabolismoRESUMEN
By the combination of cDNA manipulation and functional analysis of normal and mutant acetylcholine receptor (AChR) channels of Torpedo expressed in Xenopus laevis oocytes determinants of ion flow were localized in the bends bordering the putative M2 transmembrane segment (Imoto et al. 1988). We now report that in the rat muscle AChR, substitution of a threonine residue in the alpha-subunit localized in the M2 transmembrane segment increases or decreases the channel conductance, depending on the size of the amino acid side chain located at this position. This threonine residue (alpha T264) is located adjacent to the cluster of charged amino acids that form the intermediate anionic ring (Imoto et al. 1988). This effect is pronounced for the large alkali cations Cs+, Rb+, K+ whereas for Na+ the effect is much smaller. Taken together the results suggest that the threonine residues at position 264 in the two alpha-subunits together with the amino acids of the intermediate anionic ring form part of a narrow region close to the cytoplasmic mouth of the AChR channel.
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Receptores Colinérgicos/metabolismo , Animales , Cationes , ADN/genética , Conductividad Eléctrica , Femenino , Canales Iónicos/metabolismo , Mutación , Oocitos , Conformación Proteica , Ratas , Receptores Colinérgicos/química , Receptores Colinérgicos/genética , Treonina , Xenopus laevisRESUMEN
The acetylcholine receptor (AChR) channel is a pentameric protein in which every subunit contributes to the conducting parts of the pore. Recent studies of rat nicotinic AChR channels mutated in the alpha-subunit revealed that a threonine residue (alpha T264) in the transmembrane segment M2 forms part of the narrow region of the channel. We have mutated the residues at homologous positions in the beta-, gamma-, and delta-subunits and measured the resulting change in channel conductance. For all subunits the conductance is inversely related to the volume of the amino acid residue, suggesting that they form part of the channel narrow region. Exchanges of residues between subunits do not alter the conductance, suggesting a ring-like structure formed by homologous amino acids. To investigate the relative contribution of amino acid residues at these positions in determining the channel conductance, receptors carrying the same amino acid in each subunit in the narrow region were constructed. They form functional channels in which the conductance is inversely related to the volume of the amino acids in the narrow region. Channels in which the narrow region is formed by four serines and one valine have the same conductance if the valine is located in the alpha-, beta-, or gamma-subunits, but it is smaller if the valine is located in the delta-subunit. The results suggest a structural asymmetry of the AChR channel in its narrow region formed by the hydroxylated amino acids of alpha-, gamma- and delta-subunits, where the delta-subunit serine is a main determinant of the channel conductance.
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Receptores Colinérgicos/química , Secuencia de Aminoácidos , Aminoácidos/química , Animales , Clonación Molecular , Conductividad Eléctrica , Hidroxilación , Canales Iónicos/química , Canales Iónicos/genética , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Conformación Proteica , Ratas , Receptores Colinérgicos/genéticaRESUMEN
To understand molecular mechanisms that regulate formation and maintenance of cardiac IKr (rapidly activating component of the delayed rectifier K+ current), we have investigated the spatiotemporal expression pattern of two rat potassium voltage-gated channels, namely subfamily H (eag-related), member2 (KCNH2) (alias name: rERG) and Isk-related family, member2 (KCNE2) (alias name: rMiRP1) during late embryonic development by means of the in situ hybridization technique. KCNE2 is transcribed predominantly in atrial und ventricular myocardium at stages E14.5-E18.5dpc and only a minor signal emerged in the tongue at E16.5dpc. In contrast, KCNH2 transcripts appeared in a less confined pattern with intense signals in atrial and ventricular myocardium, somites, spinal cord, bowel system, central nervous system and thymus at stages E14.5-E18.5dpc. Non-cardiac expression even exceeds the intensity of the cardiac signal, indicating that KCNH2 contributes to K+ currents in non-cardiac tissue as well. Transcription of the rat b-subunit KCNE2 is present in all regions of the fetal myocardium and co-distributes perfectly with transcription of the pore forming a-subunit KCNH2. It seems likely that KCNH2 and KCNE2 are linked to form cardiac IKr channels, associated to cardiogenesis and cardiomyocyte excitability.
Asunto(s)
Corazón/embriología , Miocardio/metabolismo , Canales de Potasio con Entrada de Voltaje/biosíntesis , Animales , Canal de Potasio ERG1 , Embrión de Mamíferos/química , Desarrollo Embrionario , Canales de Potasio Éter-A-Go-Go , Expresión Génica , Hibridación in Situ , Miocardio/química , Canales de Potasio con Entrada de Voltaje/genética , RatasRESUMEN
OBJECTIVE: The increasing quality of diagnostic ultrasound has resulted in the detection of greater numbers of potentially benign hepatic lesions. Current radiological practice requires contrast enhanced ultrasound, CT or MRI to confirm the diagnosis. Acoustic radiation force impulse (ARFI) elastography is an imaging technique measuring the elasticity of biological tissues. Recent technical advances in ultrasound have made it possible to generate shear waves, whose velocity in the liver is proportional to the degree of hepatic elasticity. METHODS: This shear wave velocity (SWV) may be used as a marker for both focal and diffuse liver pathology.We used this technique to examine patients with normal livers and those with haemangiomata and metastases. RESULTS: Patients with normal ultrasound examinations and normal liver enzymes, n = 99, had SWVs of 1.24 ± 0.23 m s(-1) (mean ± standard deviation) independent of site of measurement, age or gender. Results of SWV measurements in haemangiomata, n = 35, produced values of the same order, 1.35 ± 0.48 m s(-1). In contrast, patients with metastases, n = 10, had SWVs of 4.23 ± 0.59 m s(-1). With a cut-off value of 2.5 m s(-1), the sensitivity and specificity for haemangiomata were 97.1% and 100%, respectively, with an area under the curve of 0.999. CONCLUSION: ARFI elastography with SWV measurements is a promising new technique which could replace invasive investigations for benign hepatic lesions.
Asunto(s)
Diagnóstico por Imagen de Elasticidad , Hemangioma/diagnóstico por imagen , Hemangioma/patología , Neoplasias Hepáticas/diagnóstico por imagen , Neoplasias Hepáticas/secundario , Adulto , Anciano , Anciano de 80 o más Años , Diagnóstico Diferencial , Neoplasias del Sistema Digestivo/diagnóstico por imagen , Neoplasias del Sistema Digestivo/secundario , Femenino , Humanos , Neoplasias Renales/diagnóstico por imagen , Neoplasias Renales/secundario , Neoplasias Pulmonares/diagnóstico por imagen , Neoplasias Pulmonares/secundario , Masculino , Persona de Mediana Edad , Curva ROC , Sensibilidad y EspecificidadRESUMEN
AIMS/HYPOTHESIS: Mitogenic activity of insulin and insulin analogues and the involvement of the IGF-1 receptor (IGF-1R) is still a controversial issue. We compared levels of the proteins IGF-1R and insulin receptor (InsR) in fibroblasts and smooth muscle cells from healthy donors and assessed the downstream signalling and growth-promoting activity of insulin and insulin analogues. METHODS: DNA synthesis was monitored in human fibroblasts and coronary artery smooth muscle cells. Using small interfering RNAs, the levels of IGF-1 and InsR were reduced by 95 and 75%, respectively. RESULTS: Enhanced mitogenic potency of insulin and insulin analogues was observed which correlated with increased levels of IGF-1R and/or IRS-1. A reduction in the IGF-1R level significantly blunted stimulation of Akt phosphorylation by IGF-1, AspB10 and glargine by 72, 58 and 40%, respectively. Akt phosphorylation in response to insulin remained unaffected. Silencing of InsR did not significantly alter Akt phosphorylation in response to IGF-1, AspB10 and glargine. IGF-1R knockdown reduced the stimulation of DNA synthesis in response to IGF-1 and glargine to a level identical to that produced by insulin. CONCLUSIONS/INTERPRETATION: These data show a prominent role of IGF-1R/Akt signalling in mediating the mitogenic effects of insulin analogues. Regular insulin stimulates DNA synthesis by exclusively activating InsR, whereas insulin analogues mainly signal through IGF-1R. It is suggested that inter-individual differences in the levels of proteins of the IGF-1R system may function as a critical determinant of the mitogenic potency of insulin analogues.
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Fibroblastos/efectos de los fármacos , Insulina/análogos & derivados , Insulina/farmacología , Mitosis/efectos de los fármacos , Miocitos del Músculo Liso/efectos de los fármacos , Receptor IGF Tipo 1/fisiología , Adolescente , Adulto , Proliferación Celular/efectos de los fármacos , Células Cultivadas , Niño , Femenino , Fibroblastos/fisiología , Humanos , Insulina Aspart , Insulina Glargina , Insulina Lispro , Insulina de Acción Prolongada , Masculino , Persona de Mediana Edad , Mitógenos/farmacología , Miocitos del Músculo Liso/fisiología , Receptor IGF Tipo 1/metabolismo , Transducción de Señal/efectos de los fármacosRESUMEN
We have isolated and analysed the 5' flanking region of the rat acetylcholine receptor (AChR) beta subunit gene and determined regulatory elements that confer muscle specificity. Deletion mapping revealed a minimal TATA-box-less promoter region containing an initiator motif. An 85-bp fragment has been shown to promote high muscle-specific expression of a chloramphenicol acetyltransferase (CAT) reporter construct upon transfection in primary muscle cells. This sequence can be functionally dissected in a basal muscle-specific promoter element carrying a M-CAT box that is flanked at the 5' end by an enhancer element with two binding sites for myogenic factors. Point mutations in the M-CAT box cause the loss of transcriptional activity of the basal promoter fragment. The enhancer activity depends on the presence of both E boxes that cooperate in a synergistic fashion. We therefore conclude that the control of muscle-specific and developmental expression of the rat AChR beta subunit gene requires both regulatory elements, the M-CAT box and two adjacent E boxes, located in close proximity to each other. Cotransfection experiments in NIH3T3 cells demonstrate that the rat AChR beta subunit gene can be transactivated by myogenic factors displaying a preference for myogenin, as well as MRF4 and myf5 compared to a clearly weaker responsiveness to MyoD1.
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Regulación de la Expresión Génica , Músculos/metabolismo , Receptores Nicotínicos/genética , Secuencias Reguladoras de Ácidos Nucleicos , Células 3T3 , Animales , Secuencia de Bases , Células Cultivadas , ADN , Células HeLa , Humanos , Ratones , Datos de Secuencia Molecular , Proteínas Musculares/genética , Mutación Puntual , Regiones Promotoras Genéticas , Ratas , Eliminación de Secuencia , Activación TranscripcionalRESUMEN
We describe a method which permits the detection of exon fragments. Such DNA was cloned and expressed in the promoter proximal part of the lac Z gene of Escherichia coli. The resulting antigen-beta-galactosidase chimeras are bound to their respective antibodies fixed to polyvinyl sheets. The beta-galactosidase part of the chimera permits detection of such clones by histochemical staining. As model DNA, we used the lac I gene cleaved with HaeIII, HhaI, or HpaII. Fragments were tailed with poly(dC) and inserted into the poly(dG)-tailed promoter proximal part of the lac Z gene. Recombinant clones, isolated on lactose-agar plates, were replica-plated and lysed with chloroform. Polyvinyl sheets coated with antibody against lac repressor were placed onto the top of the lysed colonies for immunoadsorption. The immune complexes were made visible after washing by incubation with 5-bromo-4-chloro-3-indolyl-beta-D-galactoside in buffered agar. The beta-galactosidase activity of the chimera cleaves the colourless histochemical compound to a blue dye at those positions where clones produce the antigen. In the case of the lac I gene two types of clones were isolated, carrying the NH2-terminal part of the lac repressor up to codons 27 and 75.
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Clonación Molecular , Escherichia coli/genética , Galactosidasas/genética , Genes Bacterianos , Genes , beta-Galactosidasa/genética , Secuencia de Aminoácidos , Secuencia de Bases , Quimera , Enzimas de Restricción del ADN , Escherichia coli/enzimología , Mutación , PlásmidosRESUMEN
A multipurpose plasmid, pUR222, was constructed. It contains six unique cloning sites (PstI, SalI, AccI, HindII, BamHI and EcoRI) in a small region of its lac Z-gene part. Insertion of foreign DNA into the plasmid can be easily detected. Bacteria harbouring recombinant plasmids generally give rise to white colonies, while those containing only vector DNA form blue colonies on indicator plates. Plasmid DNA purified by a rapid method (Birnboim, H.C. and Doly, J. (1979) Nucl. Acids. Res. 7, 1513-1523) can be used for chemical sequencing of the cloned insert DNA. Labeled fragments need not be isolated after cutting with the proper restriction enzymes and are treated directly according to the sequencing protocol of Maxam and Gilbert.
Asunto(s)
ADN Recombinante , Vectores Genéticos , Plásmidos , Secuencia de Bases , Clonación Molecular , Enzimas de Restricción del ADN , Escherichia coli/genéticaRESUMEN
Five different subunits, alpha, beta, gamma, delta and epsilon, constitute the acetylcholine receptors from mammalian skeletal muscle. Their corresponding mRNA levels are regulated differentially. In particular, mRNAs encoding the gamma- and epsilon-subunits, which specify two AChR isoforms, show a reciprocal behaviour during synapse formation and maturation. We have isolated 5' flanking sequences of the gamma- and epsilon-subunit genes that confer muscle-specific expression upon transient transfection of primary cultures of rat muscle cells. The gamma-subunit gene fragment contains two adjacent CANNTG sequence motifs that are essential for muscle-specific transcriptional activity suggesting transactivation by helix-loop-helix proteins. The epsilon-subunit gene fragment carries only a single CANNTG consensus motif which is not required for expression in transfected muscle cells. This sequence motif is, however, necessary to repress transcriptional activity in non-muscle cells and thus may control the muscle-specific expression of the epsilon-subunit gene. The results suggest that CANNTG motifs together with their 3' and 5' flanking nucleotides provide binding sites for both activating as well as repressing trans-acting factors. These elements could thus contribute to the muscle-specific expression of AChR subunit genes.