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1.
Euro Surveill ; 21(48)2016 Dec 01.
Artículo en Inglés | MEDLINE | ID: mdl-27934581

RESUMEN

In October 2016, a severe infection with swine influenza A(H1N1) virus of the Eurasian avian lineage occurred in a child with a previous history of eczema in the Netherlands, following contact to pigs. The patient's condition deteriorated rapidly and required life support through extracorporeal membrane oxygenation. After start of oseltamivir treatment and removal of mucus plugs, the patient fully recovered. Monitoring of more than 80 close unprotected contacts revealed no secondary cases.


Asunto(s)
Oxigenación por Membrana Extracorpórea , Subtipo H1N1 del Virus de la Influenza A/aislamiento & purificación , Gripe Humana/diagnóstico , Infecciones del Sistema Respiratorio/virología , Síndrome Respiratorio Agudo Grave/terapia , Animales , Antivirales/uso terapéutico , Humanos , Gripe Humana/tratamiento farmacológico , Gripe Humana/virología , Unidades de Cuidado Intensivo Pediátrico , Países Bajos , Infecciones por Orthomyxoviridae/transmisión , Infecciones por Orthomyxoviridae/veterinaria , Infecciones por Orthomyxoviridae/virología , Oseltamivir/uso terapéutico , Reacción en Cadena en Tiempo Real de la Polimerasa , Infecciones del Sistema Respiratorio/diagnóstico , Infecciones del Sistema Respiratorio/tratamiento farmacológico , Síndrome Respiratorio Agudo Grave/complicaciones , Porcinos , Enfermedades de los Porcinos/transmisión , Enfermedades de los Porcinos/virología , Resultado del Tratamiento
2.
Pharmacogenomics J ; 15(2): 144-52, 2015 Apr.
Artículo en Inglés | MEDLINE | ID: mdl-25287072

RESUMEN

Tacrolimus, a dual substrate of CYP3A4 and CYP3A5 has a narrow therapeutic index and is characterized by high between-subject variability in oral bioavailability. This study investigated the effects of the recently described CYP3A4*22 intron 6 C>T single nucleotide polymorphism on in vivo CYP3A4 activity as measured by midazolam (MDZ) clearance and tacrolimus pharmacokinetics in two cohorts of renal allograft recipients, taking into account the CYP3A5*1/*3 genotype and other determinants of drug disposition. In CYP3A5 non-expressers, the presence of one CYP3A4*22T-allele was associated with a 31.7-33.6% reduction in MDZ apparent oral clearance, reflecting reduced in vivo CYP3A4 activity. In addition, at ⩾12 months after transplantation, steady-state clearance of tacrolimus was 36.8% decreased compared with homozygous CYP3A4*22CC-wild type patients, leading to 50% lower dose requirements. Both concurrent observations in stable renal allograft recipients are consistent with a reduced in vivo CYP3A4 activity for the CYP3A4*22T-allele.


Asunto(s)
Citocromo P-450 CYP3A/genética , Midazolam/farmacocinética , Polimorfismo de Nucleótido Simple/genética , Alelos , Estudios Transversales , Femenino , Genotipo , Humanos , Trasplante de Riñón , Masculino , Persona de Mediana Edad , Tacrolimus/farmacocinética
4.
Eur Respir J ; 41(1): 203-16, 2013 Jan.
Artículo en Inglés | MEDLINE | ID: mdl-22878883

RESUMEN

In patients with cystic fibrosis, cystic fibrosis transmembrane conductance regulator (CFTR) biomarkers, such as sweat chloride concentration and/or nasal potential difference, are used as end-points of efficacy in phase-III clinical trials with the disease modifying drugs ivacaftor (VX-770), VX809 and ataluren. The aim of this project was to review the literature on reliability, validity and responsiveness of nasal potential difference, sweat chloride and intestinal current measurement in patients with cystic fibrosis. Data on clinimetric properties were collected for each biomarker and reviewed by an international team of experts. Data on reliability, validity and responsiveness were tabulated. In addition, narrative answers to four key questions were discussed and agreed by the team of experts. The data collected demonstrated the reliability, validity and responsiveness of nasal potential difference. Fewer data were found on reliability of sweat chloride concentration; however, validity and responsiveness were demonstrated. Validity was demonstrated for intestinal current measurement, but further information is required on reliability and responsiveness. For all three end-points, normal values were collected and further research requirements were proposed. This body of work adds useful information to support the promotion of CFTR biomarkers to surrogate end-points and to guide further research in the area.


Asunto(s)
Regulador de Conductancia de Transmembrana de Fibrosis Quística/análisis , Fibrosis Quística/diagnóstico , Biomarcadores/análisis , Fibrosis Quística/tratamiento farmacológico , Humanos , Reproducibilidad de los Resultados
5.
J Cyst Fibros ; 22(3): 538-547, 2023 May.
Artículo en Inglés | MEDLINE | ID: mdl-37100706

RESUMEN

BACKGROUND: Cystic fibrosis (CF) disease severity can be highly variable, even between people with CF (pwCF) with similar genotypes. Here we use patient-derived intestinal organoids to study the influence of genetic variation within the cystic fibrosis transmembrane conductance regulator (CFTR) gene on CFTR function. METHODS: Organoids of F508del/class I, F508del/S1251N and pwCF with only one detected CF-causing mutation were cultured. Allele-specific CFTR variation was investigated using targeted locus amplification (TLA), CFTR function was measured using the forskolin-induced swelling assay and mRNA levels were quantified using RT-qPCR. RESULTS: We were able to distinguish CFTR genotypes based on TLA data. Additionally, we observed heterogeneity within genotypes, which we were able to link to CFTR function for S1251N alleles. CONCLUSIONS: Our results indicate that the paired analysis of CFTR intragenic variation and CFTR function can gain insights in the underlying CFTR defect for individuals where the disease phenotype does not match the CFTR mutations detected during diagnosis.


Asunto(s)
Regulador de Conductancia de Transmembrana de Fibrosis Quística , Fibrosis Quística , Humanos , Regulador de Conductancia de Transmembrana de Fibrosis Quística/genética , Fibrosis Quística/genética , Intestinos , Mutación , Genotipo , Organoides
6.
Physiol Genomics ; 43(11): 674-84, 2011 Jun 15.
Artículo en Inglés | MEDLINE | ID: mdl-21427361

RESUMEN

To identify additional potential functions for the multi-PDZ domain containing protein Na+/H+ exchanger regulatory factor 2 (NHERF2), which is present in the apical domain of intestinal epithelial cells, proteomic studies of mouse jejunal villus epithelial cell brush border membrane vesicles compared wild-type to homozygous NHERF2 knockout FVB mice by a two-dimensional liquid chromatography-tandem mass spectrometry (LC-MS/MS)-iTRAQ approach. Jejunal architecture appeared normal in NHERF2 null in terms of villus length and crypt depth, Paneth cell number, and microvillus structure by electron microscopy. There was also no change in proliferative activity based on BrdU labeling. Four brush border membrane vesicles (BBMV) preparations from wild-type mouse jejunum were compared with four preparations from NHERF2 knockout mice. LC-MS/MS identified 450 proteins in both matched wild-type and NHERF2 null BBMV; 13 proteins were changed in two or more separate BBMV preparations (9 increased and 4 decreased in NHERF2 null mice), while an additional 92 proteins were changed in a single BBMV preparation (68 increased and 24 decreased in NHERF2 null mice). These proteins were categorized as 1) transport proteins (one increased and two decreased in NHERF2 null); 2) signaling molecules (2 increased in NHERF2 null); 3) cytoskeleton/junctional proteins (4 upregulated and 1 downregulated in NHERF2 null); and 4) metabolic proteins/intrinsic BB proteins) (2 upregulated and 1 downregulated in NHERF2 null). Immunoblotting of BBMV was used to validate or extend the findings, demonstrating increase in BBMV of NHERF2 null of MCT1, coronin 3, and ezrin. The proteome of the NHERF2 null mouse small intestinal BB demonstrates up- and downregulation of multiple transport proteins, signaling molecules, cytoskeletal proteins, tight junctional and adherens junction proteins, and proteins involved in metabolism, suggesting involvement of NHERF2 in multiple apical regulatory processes and interactions with luminal contents.


Asunto(s)
Yeyuno/metabolismo , Fosfoproteínas/genética , Proteoma/metabolismo , Intercambiadores de Sodio-Hidrógeno/genética , Animales , Cadherinas/metabolismo , Proliferación Celular , Cromatografía Liquida , Citoesqueleto/metabolismo , Regulación hacia Abajo , Técnica del Anticuerpo Fluorescente , Masculino , Ratones , Ratones Noqueados , Microvellosidades/genética , Microvellosidades/metabolismo , Fosfoproteínas/metabolismo , Intercambiadores de Sodio-Hidrógeno/metabolismo , beta Catenina/metabolismo
7.
Br J Cancer ; 105(12): 1856-63, 2011 Dec 06.
Artículo en Inglés | MEDLINE | ID: mdl-22045186

RESUMEN

BACKGROUND: High vascular endothelial growth factor (VEGFA) levels at the time of diagnosis confer a worse prognosis to multiple malignancies. Our aim was to investigate the role of VEGFA in promoting tumour growth through interaction with its environment. METHODS: HL-60 cells were transduced with VEGFA165 or control vector using retroviral constructs. Control cells (n=7) or VEGFA165 cells (n=7) were subcutaneously injected into NOD/SCID mice. Immunohistochemistry of markers for angiogenesis (CD31) and cell proliferation (Ki67) and gene expression profiling of tumours were performed. Paracrine effects were investigated by mouse-specific cytokine arrays. RESULTS: In vivo we observed a twofold increase in tumour weight when VEGFA165 was overexpressed (P=0.001), combined with increased angiogenesis (P=0.002) and enhanced tumour cell proliferation (P=0.001). Gene expression profiling revealed human genes involved in TGF-ß signalling differentially expressed between both tumour groups, that is, TGFBR2 and SMAD5 were lower expressed whereas the inhibitory SMAD7 was higher expressed with VEGFA165. An increased expression of mouse-derived cytokines IFNG and interleukin 7 was found in VEGFA165 tumours, both described to induce SMAD7 expression. CONCLUSION: These results suggest a role for VEGFA-driven tumour growth by TGF-ß signalling inhibition via paracrine mechanisms in vivo, and underscore the importance of stromal interaction in the VEGFA-induced phenotype.


Asunto(s)
Neoplasias Experimentales/patología , Transducción de Señal , Células del Estroma/patología , Factor de Crecimiento Transformador beta/metabolismo , Factor A de Crecimiento Endotelial Vascular/fisiología , Animales , Línea Celular Tumoral , Ensayo de Inmunoadsorción Enzimática , Citometría de Flujo , Perfilación de la Expresión Génica , Inmunohistoquímica , Ratones , Ratones Endogámicos NOD , Ratones SCID , Neoplasias Experimentales/metabolismo , Reacción en Cadena en Tiempo Real de la Polimerasa
8.
Pediatr Blood Cancer ; 56(2): 294-7, 2011 Feb.
Artículo en Inglés | MEDLINE | ID: mdl-20981743

RESUMEN

In AML high VEGFA protein expression correlates with poor overall and relapse-free survival (OS/RFS). To date, the relevance of the various VEGFA isoforms is unclear. We determined VEGF121, VEGF145, VEGF148, VEGF165, VEGF183, and VEGF189 mRNA expression in pediatric AML samples and investigated the relation between VEGFA isoform expression and clinicopatholologic characteristics and outcome. A significant co-expression of VEGF121, VEGF165, VEGF183, and VEGF189 isoforms was apparent (mean rho = 0.716, P < 0.0001). This co-expression justifies measuring a single VEGFA isoform (e.g., 121, 165, 183, and 189) as representative expression of all VEGFA isoforms in future studies designed to determine the prognostic importance of VEGFA isoforms.


Asunto(s)
Leucemia Mieloide Aguda/metabolismo , ARN Mensajero/biosíntesis , Factor A de Crecimiento Endotelial Vascular/biosíntesis , Adolescente , Niño , Preescolar , Supervivencia sin Enfermedad , Femenino , Expresión Génica , Perfilación de la Expresión Génica , Humanos , Lactante , Recién Nacido , Estimación de Kaplan-Meier , Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/mortalidad , Masculino , Pronóstico , Isoformas de Proteínas/análisis , Isoformas de Proteínas/biosíntesis , ARN Mensajero/análisis , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Factor A de Crecimiento Endotelial Vascular/genética
9.
Physiol Genomics ; 42A(3): 200-10, 2010 Nov 15.
Artículo en Inglés | MEDLINE | ID: mdl-20736413

RESUMEN

Na/H exchanger regulatory factor 1 (NHERF1) is a scaffold protein made up of two PDZ domains and an ERM binding domain. It is in the brush border of multiple epithelial cells where it modulates 1) Na absorption by regulating NHE3 complexes and cytoskeletal association, 2) Cl secretion through trafficking of CFTR, and 3) Na-coupled phosphate absorption through membrane retention of NaPi2a. To further understand the role of NHERF1 in regulation of small intestinal Na absorptive cell function, with emphasis on apical membrane transport regulation, quantitative proteomic analysis was performed on brush border membrane vesicles (BBMV) prepared from wild-type (WT) and homozygous NHERF1 knockout mouse jejunal villus Na absorptive cells. Jejunal architecture appeared normal in NHERF1 null; however, there was increased proliferative activity, as indicated by increased crypt BrdU staining. LC-MS/MS analysis using iTRAQ to compare WT and NHERF1 null BBMV identified 463 proteins present in both WT and NHERF1 null BBMV of simultaneously prepared and studied samples. Seventeen proteins had an altered amount of expression between WT and NHERF1 null in two or more separate preparations, and 149 total proteins were altered in at least one BBMV preparation. The classes of the majority of proteins altered included transport proteins, signaling and trafficking proteins, and proteins involved in proliferation and cell division. Affected proteins also included tight junction and adherens junction proteins, cytoskeletal proteins, as well as metabolic and BB digestive enzymes. Changes in abundance of several proteins were confirmed by immunoblotting [increased CEACAM1, decreased ezrin (p-ezrin), NHERF3, PLCß3, E-cadherin, p120, ß-catenin]. The changes in the jejunal BBMV proteome of NHERF1 null mice are consistent with a more complex role of NHERF1 than just forming signaling complexes and anchoring proteins to the apical membrane and include at least alterations in proteins involved in transport, signaling, and proliferation.


Asunto(s)
Yeyuno/metabolismo , Fosfoproteínas/genética , Proteoma/análisis , Intercambiadores de Sodio-Hidrógeno/genética , Vesículas Transportadoras/metabolismo , Animales , Cadherinas/análisis , Cromatografía por Intercambio Iónico , Femenino , Immunoblotting , Inmunohistoquímica , Yeyuno/citología , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Microscopía Electrónica , Microvellosidades/metabolismo , Microvellosidades/ultraestructura , Fosfoproteínas/metabolismo , Proteómica/métodos , Intercambiadores de Sodio-Hidrógeno/metabolismo , Espectrometría de Masas en Tándem , beta Catenina/análisis
10.
Br J Oral Maxillofac Surg ; 58(4): 427-431, 2020 05.
Artículo en Inglés | MEDLINE | ID: mdl-32115300

RESUMEN

The aim of this retrospective cohort study was to evaluate the relative amount of cancellous bone in the mandibular ramus as a predictor of lingual fracture patterns after bilateral sagittal split osteotomy (BSSO). The study including 78 consecutive patients (156 osteotomy sites). In preoperative cone-beam computed tomographic (CT) scans, the volumes of cancellous and cortical bone in the BSSO surgical field were estimated. Patients were divided into two groups based on the cancellous:cortical bone ratio. We studied postoperative cone-beam CT scans for lingual fracture lines and subcategorised them according to the lingual split scale (LSS). Generalised linear mixed models (GLMM) were estimated to evaluate the association between the cancellous:cortical bone ratio and the lingual fracture pattern. There was a significant association between the cancellous:cortical bone ratio of the mandibular angle and the lingual fracture pattern after BSSO. Mandibular angles with a relatively small amount of cancellous bone showed significantly more LSS3 fracture lines (OR=1.990, 95%CI 1.043 to 3.796, p=0.043). These mandibular angles also showed more unfavourable fractures (LSS4), although this was not significant (OR=2.352, 95%CI 0.748 to 7.392, p=0.143). The relative amount of cancellous bone in the mandibular angle is significantly associated with the lingual fracture line after BSSO.


Asunto(s)
Mandíbula , Osteotomía Sagital de Rama Mandibular , Tomografía Computarizada de Haz Cónico , Hueso Cortical/diagnóstico por imagen , Humanos , Mandíbula/diagnóstico por imagen , Mandíbula/cirugía , Estudios Retrospectivos
11.
Pflugers Arch ; 457(5): 1079-91, 2009 Mar.
Artículo en Inglés | MEDLINE | ID: mdl-18758809

RESUMEN

We investigated the role of the Na(+)/H(+) exchanger regulatory factor 1 (NHERF1) on intestinal salt and water absorption, brush border membrane (BBM) morphology, and on the NHE3 mRNA expression, protein abundance, and transport activity in the murine intestine. NHERF1-deficient mice displayed reduced jejunal fluid absorption in vivo, as well as an attenuated in vitro Na(+) absorption in isolated jejunal and colonic, but not of ileal, mucosa. However, cAMP-mediated inhibition of both parameters remained intact. Acid-activated NHE3 transport rate was reduced in surface colonocytes, while its inhibition by cAMP and cGMP was normal. Immunodetection of NHE3 revealed normal NHE3 localization in the BBM of NHERF1 null mice, but NHE3 abundance, as measured by Western blot, was significantly reduced in isolated BBM from the small and large intestines. Furthermore, the microvilli in the proximal colon, but not in the small intestine, were significantly shorter in NHERF1 null mice. Additional knockout of PDZK1 (NHERF3), another member of the NHERF family of adaptor proteins, which binds to both NHE3 and NHERF1, further reduced basal NHE3 activity and caused complete loss of cAMP-mediated NHE3 inhibition. An activator of the exchange protein activated by cAMP (EPAC) had no effect on jejunal fluid absorption in vivo, but slightly inhibited NHE3 activity in surface colonocytes in vitro. In conclusion, NHERF1 has segment-specific effects on intestinal salt absorption, NHE3 transport rates, and NHE3 membrane abundance without affecting mRNA levels. However, unlike PDZK1, NHERF1 is not required for NHE3 regulation by cyclic nucleotides.


Asunto(s)
Colon/metabolismo , Absorción Intestinal/fisiología , Yeyuno/metabolismo , Fosfoproteínas/deficiencia , Cloruro de Sodio/metabolismo , Intercambiadores de Sodio-Hidrógeno/metabolismo , Animales , Inmunohistoquímica , Mucosa Intestinal/metabolismo , Ratones , Microvellosidades/ultraestructura , Intercambiador 3 de Sodio-Hidrógeno
12.
Trends Biochem Sci ; 22(8): 307-12, 1997 Aug.
Artículo en Inglés | MEDLINE | ID: mdl-9270304

RESUMEN

cGMP-dependent protein kinases I and II conduct signals from widespread signaling systems. Whereas the type I kinase mediates numerous effects of natriuretic peptides and nitric oxide in cardiovascular cells, the type II kinase transduces signals from the Escherichia coli heat-stable enterotoxin, STa, and from the endogenous intestinal peptide, guanylin, stimulating Cl- conductance of the cystic fibrosis transmembrane conductance regulator (CFTR). Although the two kinases may be interchangeable for several functions, CFTR regulation specifically requires the type II kinase.


Asunto(s)
Proteínas Quinasas Dependientes de GMP Cíclico/fisiología , Transducción de Señal , Secuencia de Aminoácidos , Animales , GMP Cíclico/metabolismo , Proteínas Quinasas Dependientes de GMP Cíclico/química , Proteínas Quinasas Dependientes de GMP Cíclico/genética , Regulación Enzimológica de la Expresión Génica , Humanos , Especificidad de la Especie
13.
J Clin Invest ; 93(2): 461-6, 1994 Feb.
Artículo en Inglés | MEDLINE | ID: mdl-8113384

RESUMEN

Previous Ussing chamber measurements of secretagogue-provoked changes in short circuit current in rectal suction biopsies of cystic fibrosis (CF) patients showed that in a minority of patients chloride secretion in response to cholinergic agonists is reduced but not completely absent. To assess a possible relationship between this phenomenon and both the genotype and the phenotype, we performed Ussing chamber experiments on rectal suction biopsies of 51 CF patients. The CF mutation was identified in 89 out of 102 CF alleles. No apparent chloride secretion was found in 30 CF patients (group I). Low residual chloride secretion was found in 11 CF patients (group II), while a relatively high residual secretion appeared in 10 CF patients (group III). Pancreatic function was preserved more frequently in CF patients displaying residual secretion: 0% in group I, 27% in group II, and 60% in group III (P < 0.001). The age at diagnosis (mean +/- SEM) in group III (18.4 +/- 6.6) was significantly different from group I (1.2 +/- 0.4, P < 0.01) and group II (3.5 +/- 1.4, P = 0.05). Residual chloride secretion was found in some of the 28 dF508 homozygous patients (three in group II, and one in group III), disclosing that other factors than the CF gene defect itself affect the transepithelial chloride transport. The age at diagnosis correlates significantly with the magnitude of the secretory response, even within the dF508 homozygous patients (r = 0.4, P < 0.05). We conclude that residual chloride secretion in CF is the pathophysiological basis of preserved pancreatic function and delayed presentation of the disease, which is not exclusively determined by the CF genotype.


Asunto(s)
Cloruros/metabolismo , Fibrosis Quística/genética , Fibrosis Quística/fisiopatología , Mucosa Intestinal/fisiopatología , Adolescente , Adulto , Anciano , Alelos , Biopsia , Carbacol/farmacología , Niño , Preescolar , Fibrosis Quística/metabolismo , Análisis Mutacional de ADN , Epitelio/metabolismo , Epitelio/fisiología , Epitelio/fisiopatología , Potenciales Evocados/efectos de los fármacos , Potenciales Evocados/fisiología , Femenino , Genotipo , Humanos , Lactante , Mucosa Intestinal/efectos de los fármacos , Mucosa Intestinal/patología , Masculino , Persona de Mediana Edad , Reacción en Cadena de la Polimerasa , Recto
14.
J Clin Invest ; 98(6): 1304-12, 1996 Sep 15.
Artículo en Inglés | MEDLINE | ID: mdl-8823295

RESUMEN

The most prevalent mutation (delta F508) in cystic fibrosis patients inhibits maturation and transfer to the plasma membrane of the mutant cystic fibrosis transmembrane conductance regulator (CFTR). We have analyzed the properties of a delta F508 CFTR mouse model, which we described recently. We show that the mRNA levels of mutant CFTR are normal in all tissues examined. Therefore the reduced mRNA levels reported in two similar models may be related to their intronic transcription units. Maturation of mutant CFTR was greatly reduced in freshly excised oviduct, compared with normal. Accumulation of mutant CFTR antigen in the apical region of jejunum crypt enterocytes was not observed, in contrast to normal mice. In cultured gallbladder epithelial cells from delta F508 mice, CFTR chloride channel activity could be detected at only two percent of the normal frequency. However, in mutant cells that were grown at reduced temperature the channel frequency increased to over sixteen percent of the normal level at that temperature. The biophysical characteristics of the mutant channel were not significantly different from normal. In homozygous delta F508 mice we did not observe a significant effect of genetic background on the level of residual chloride channel activity, as determined by the size of the forskolin response in Ussing chamber experiments. Our data show that like its human homologue, mouse delta F508-CFTR is a temperature sensitive processing mutant. The delta F508 mouse is therefore a valid in vivo model of human delta F508-CFTR. It may help us to elucidate the processing pathways of complex membrane proteins. Moreover, it may facilitate the discovery of new approaches towards therapy of cystic fibrosis.


Asunto(s)
Regulador de Conductancia de Transmembrana de Fibrosis Quística/genética , Regulador de Conductancia de Transmembrana de Fibrosis Quística/metabolismo , Fibrosis Quística/genética , Animales , Western Blotting , Células Cultivadas , Regulador de Conductancia de Transmembrana de Fibrosis Quística/inmunología , Modelos Animales de Enfermedad , Trompas Uterinas/metabolismo , Femenino , Vesícula Biliar/citología , Vesícula Biliar/metabolismo , Inmunohistoquímica , Yeyuno/metabolismo , Ratones , Ratones Noqueados , Técnicas de Placa-Clamp , Mutación Puntual , ARN Mensajero/genética , ARN Mensajero/metabolismo , Temperatura
15.
J Clin Invest ; 108(11): 1705-15, 2001 Dec.
Artículo en Inglés | MEDLINE | ID: mdl-11733566

RESUMEN

To investigate the impact of chloride (Cl(-)) permeability, mediated by residual activity of the cystic fibrosis transmembrane conductance regulator (CFTR) or by other Cl(-) channels, on the manifestations of cystic fibrosis (CF), we determined Cl(-) transport properties of the respiratory and intestinal tracts in Delta F508 homozygous twins and siblings. In the majority of patients, cAMP and/or Ca(2+)-regulated Cl(-) conductance was detected in the airways and intestine. Our finding of cAMP-mediated Cl(-) conductance suggests that, in vivo, at least some Delta F508 CFTR can reach the plasma membrane and affect Cl(-) permeability. In respiratory tissue, the expression of basal CFTR-mediated Cl(-) conductance, demonstrated by 30% of Delta F508 homozygotes, was identified as a positive predictor of milder CF disease. In intestinal tissue, 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid-insensitive (DIDS-insensitive) Cl(-) secretion, which is indicative of functional CFTR channels, correlated with a milder phenotype, whereas DIDS-sensitive Cl(-) secretion was observed mainly in more severely affected patients. The more concordant Cl(-) secretory patterns within monozygous twins compared with dizygous pairs imply that genes other than CFTR significantly influence the manifestation of the basic defect.


Asunto(s)
Cloruros/metabolismo , Regulador de Conductancia de Transmembrana de Fibrosis Quística/genética , Fibrosis Quística/genética , Enfermedades en Gemelos , Adolescente , Adulto , Niño , Fibrosis Quística/metabolismo , Femenino , Homocigoto , Humanos , Masculino , Fenotipo
16.
J Clin Invest ; 96(2): 822-30, 1995 Aug.
Artículo en Inglés | MEDLINE | ID: mdl-7543493

RESUMEN

Certain pathogenic bacteria produce a family of heat stable enterotoxins (STa) which activate intestinal guanylyl cyclases, increase cGMP, and elicit life-threatening secretory diarrhea. The intracellular effector of cGMP actions has not been clarified. Recently we cloned the cDNA for a rat intestinal type II cGMP dependent protein kinase (cGK II) which is highly enriched in intestinal mucosa. Here we show that cGK II mRNA and protein are restricted to the intestinal segments from the duodenum to the proximal colon, with the highest amounts of cGK II protein in duodenum and jejunum. cGK II mRNA and protein decreased along the villus to crypt axis in the small intestine, whereas substantial amounts of both were found in the crypts of cecum. In intestinal epithelia, cGK II was specifically localized in the apical membrane, a major site of ion transport regulation. In contrast to cGK II, cGK I was localized in smooth muscle cells of the villus lamina propria. Short circuit current (ISC), a measure of Cl- secretion, was increased to a similar extent by STa and by 8-Br-cGMP, a selective activator of cGK, except in distal colon and in monolayers of T84 human colon carcinoma cells in which cGK II was not detected. In human and mouse intestine, the cyclic nucleotide-regulated Cl- conductance can be exclusively accounted for by the cystic fibrosis transmembrane conductance regulator (CFTR) Cl- channel. Viewed collectively, the data suggest that cGK II is the mediator of STa and cGMP effects on Cl- transport in intestinal-epithelia.


Asunto(s)
Cloruros/farmacocinética , Proteínas Quinasas Dependientes de GMP Cíclico/biosíntesis , Mucosa Intestinal/enzimología , Isoenzimas/biosíntesis , Proteínas de la Membrana/biosíntesis , Proteínas de la Membrana/metabolismo , ARN Mensajero/biosíntesis , 8-Bromo Monofosfato de Adenosina Cíclica/farmacología , Animales , Transporte Biológico/efectos de los fármacos , Carcinoma/patología , Ciego/enzimología , Ciego/ultraestructura , Colon/enzimología , Colon/ultraestructura , Neoplasias del Colon/patología , GMP Cíclico/fisiología , Proteínas Quinasas Dependientes de GMP Cíclico/genética , Regulador de Conductancia de Transmembrana de Fibrosis Quística , Enterotoxinas/farmacología , Inducción Enzimática , Esófago/enzimología , Humanos , Hibridación in Situ , Mucosa Intestinal/ultraestructura , Intestino Delgado/enzimología , Intestino Delgado/ultraestructura , Isoenzimas/genética , Masculino , Proteínas de la Membrana/genética , Microvellosidades/enzimología , Músculo Liso/enzimología , Especificidad de Órganos , ARN Mensajero/genética , Ratas , Ratas Sprague-Dawley , Estómago/enzimología , Células Tumorales Cultivadas
17.
J Chromatogr A ; 1154(1-2): 319-30, 2007 Jun 22.
Artículo en Inglés | MEDLINE | ID: mdl-17442326

RESUMEN

High temperature asymmetrical flow field-flow fractionation (HTAF4) coupled to infrared (IR), multi-angle light scattering (MALS), and viscometry (Visc) detection is introduced as a tool for the characterization of high molecular weight polyethylenes. The high molecular weight fraction strongly affects the rheological behaviour and processability of polyethylene materials and can often not be accurately resolved by current technology such as high temperature size-exclusion chromatography (HTSEC). Molecular weight (M), radius of gyration (Rg), and intrinsic viscosity [eta] of linear high density polyethylene (HDPE) and branched low density polyethylene (LDPE) samples are studied in detail by HTAF4 and are compared to HTSEC. HTAF4 showed a better separation and mass recovery than HTSEC for very high molecular weight fractions in HDPE and LDPE samples. As no stationary phase is present in an HTAF4 channel, the technique does not show the typical drawbacks associated with HTSEC analysis of high molecular weight polyethylenes, such as, exclusion effects, shear degradation, and anomalous late elution of highly branched material. HTAF4 is applied to study the relation between the molecular weight and the zero shear viscosity eta0 for high molecular weight HDPE. It was found that the zero shear viscosity values predicted from HTAF4 results are in good qualitative agreement with measured values obtained from dynamic mechanical spectroscopy (DMS) experiments, whereas eta0 values predicted from HTSEC do not show a strong correlation. The low molecular weight cutoff of HTAF4 is approximately 5x10(4) as a result of relatively large pores in the HTAF4 channel membrane. HTAF4 is, therefore, currently not suited to analyze low molecular weight materials.


Asunto(s)
Fraccionamiento de Campo-Flujo/métodos , Polietilenos/aislamiento & purificación , Fraccionamiento de Campo-Flujo/instrumentación , Calor , Luz , Peso Molecular , Dispersión de Radiación , Espectrofotometría Infrarroja , Viscosidad
18.
Mol Biol Cell ; 6(12): 1707-19, 1995 Dec.
Artículo en Inglés | MEDLINE | ID: mdl-8590800

RESUMEN

Studies on physiological modulation of intercellular communication mediated by protein kinases are often complicated by the fact that cells express multiple gap junction proteins (connexins; Cx). Changes in cell coupling can be masked by simultaneous opposite regulation of the gap junction channel types expressed. We have examined the effects of activators and inhibitors of protein kinase A (PKA), PKC, and PKG on permeability and single channel conductance of gap junction channels composed of Cx45, Cx43, or Cx26 subunits. To allow direct comparison between these Cx, SKHep1 cells, which endogenously express Cx45, were stably transfected with cDNAs coding for Cx43 or Cx26. Under control conditions, the distinct types of gap junction channels could be distinguished on the basis of their permeability and single channel properties. Under various phosphorylating conditions, these channels behaved differently. Whereas agonists/antagonist of PKA did not affect permeability and conductance of all gap junction channels, variable changes were observed under PKC stimulation. Cx45 channels exhibited an additional conductance state, the detection of the smaller conductance states of Cx43 channels was favored, and Cx26 channels were less often observed. In contrast to the other kinases, agonists/antagonist of PKG affected permeability and conductance of Cx43 gap junction channels only. Taken together, these results show that distinct types of gap junction channels are differentially regulated by similar phosphorylating conditions. This differential regulation may be of physiological importance during modulation of cell-to-cell communication of more complex cell systems.


Asunto(s)
Comunicación Celular , Conexinas/fisiología , Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Proteínas Quinasas Dependientes de GMP Cíclico/metabolismo , Uniones Comunicantes/fisiología , Proteína Quinasa C/metabolismo , 8-Bromo Monofosfato de Adenosina Cíclica/farmacología , Secuencia de Bases , Carcinoma Hepatocelular , Conexina 26 , Conexinas/biosíntesis , Proteínas Quinasas Dependientes de AMP Cíclico/biosíntesis , Proteínas Quinasas Dependientes de AMP Cíclico/aislamiento & purificación , GMP Cíclico/análogos & derivados , GMP Cíclico/farmacología , Proteínas Quinasas Dependientes de GMP Cíclico/biosíntesis , Proteínas Quinasas Dependientes de GMP Cíclico/aislamiento & purificación , Cartilla de ADN , Inhibidores Enzimáticos/farmacología , Expresión Génica , Homeostasis , Humanos , Neoplasias Hepáticas , Potenciales de la Membrana/fisiología , Datos de Secuencia Molecular , Fosforilación , Reacción en Cadena de la Polimerasa , Proteína Quinasa C/biosíntesis , Proteína Quinasa C/aislamiento & purificación , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/aislamiento & purificación , Proteínas Recombinantes/metabolismo , Acetato de Tetradecanoilforbol/farmacología , Transfección , Células Tumorales Cultivadas
19.
Mol Biol Cell ; 7(9): 1419-27, 1996 Sep.
Artículo en Inglés | MEDLINE | ID: mdl-8885236

RESUMEN

Hypo-osmotic stimulation of human Intestine 407 cells rapidly activated compensatory CL- and K+ conductances that limited excessive cell swelling and, finally, restored the original cell volume. Osmotic cell swelling was accompanied by a rapid and transient reorganization of the F-actin cytoskeleton, affecting both stress fibers as well as apical ruffles. In addition, an increase in total cellular F-actin was observed. Pretreatment of the cells with recombinant Clostridium botulinum C3 exoenzyme, but not with mutant enzyme (C3-E173Q) devoid of ADP-ribosyltransferase activity, greatly reduced the activation of the osmo-sensitive anion efflux, suggesting a role for the ras-related GTPase p21rho. In contrast, introducing dominant negative N17-p21rac into the cells did not affect the volume-sensitive efflux. Cell swelling-induced reorganization of F-actin coincided with a transient, C3 exoenzyme-sensitive tyrosine phosphorylation of p125 focal adhesion kinase (p125FAK) as well as with an increase in phosphatidylinositol-3-kinase (PtdIns-3-kinase) activity. Pretreatment of the cells with wortmannin, a specific inhibitor of PtdIns-3-kinase, largely inhibited the volume-sensitive ion efflux. Taken together, our results indicate the involvement of a p21rho signaling cascade and actin filaments in the activation of volume-sensitive chloride channels.


Asunto(s)
Actinas/fisiología , Toxinas Botulínicas , Moléculas de Adhesión Celular/metabolismo , Canales de Cloruro/fisiología , Citoesqueleto/fisiología , Proteínas Tirosina Quinasas/metabolismo , ADP Ribosa Transferasas/farmacología , Actinas/ultraestructura , Aniones/metabolismo , Moléculas de Adhesión Celular/efectos de los fármacos , Células Cultivadas , Canales de Cloruro/efectos de los fármacos , Quinasa 1 de Adhesión Focal , Proteína-Tirosina Quinasas de Adhesión Focal , Proteínas de Unión al GTP/efectos de los fármacos , Proteínas de Unión al GTP/metabolismo , Humanos , Soluciones Hipotónicas/farmacología , Intestinos/citología , Intestinos/efectos de los fármacos , Intestinos/fisiología , Presión Osmótica , Fosfatidilinositol 3-Quinasas , Fosforilación , Fosfotransferasas (Aceptor de Grupo Alcohol)/efectos de los fármacos , Fosfotransferasas (Aceptor de Grupo Alcohol)/metabolismo , Proteínas Tirosina Quinasas/efectos de los fármacos , Tirosina/metabolismo , Proteínas de Unión al GTP rho
20.
Biochim Biophys Acta ; 381(1): 128-43, 1975 Jan 13.
Artículo en Inglés | MEDLINE | ID: mdl-1111579

RESUMEN

1. Some kinetic properties of adenylate cyclase in separately isolated upper villous and crypt cells from rat and guinea pig small intestine were compared. An apparent Km of 0.4 mM was found for both enzymes in the rat. The slight difference between the V-values measured in the fluoride-stimulated state (132 and 165 pmoles cyclic AMP formed per min per mg protein respectively) indicated an approximately equal enzyme content of both cell populations and argues strongly against a preferential localization in the brushborder region of the epithelial cell. 2. Prolonged contact of the small intestine with luminally administered choleragen led to an irreversible activation of adenylate cyclase in both villous and crypt compartments. The maximal stimulation of the upper villous enzyme (4-7 times) exceeded the maximal effect on the crypt enzyme by two-fold. 3. A lag phase of at least 30 min was found between the first luminal contact with the purified choleragen and a significant activation of the adenylate cyclase associated with isolated intestinal brushborders from the upper villous region. 4. By using a short exposure time (2 min) of the luminal surface to high amounts of choleragen, adenylate cyclase activity in the upper villus could be optimally stimulated in the absence of any alteration of crypt cell activity. 5. By comparing, in vivo, the effects of short and prolonged contact with choleratoxin on the unidirectional and net flux of ions and water in ileal and jejunal segments, it was concluded that both villous and crypt regions contribute to the secretion of water and electrolytes (sodium, chloride and bicarbonate ions) during cholera. The serosal to mucosal flux of sodium and chloride ions increased without a significant alteration of the opposite flux. These results imply that absorptive and secretory processes occur within the same epithelial compartment. 6. The view that the crypt epithelium fulfills a specific role during the choleragen-induced secretion of ions and water is incompatible with the results of the present study.


Asunto(s)
Cólera , Mucosa Intestinal/metabolismo , Intestino Delgado/metabolismo , Toxinas Biológicas/farmacología , Adenilil Ciclasas/metabolismo , Animales , Transporte Biológico , Agua Corporal/metabolismo , Cobayas , Mucosa Intestinal/efectos de los fármacos , Intestino Delgado/efectos de los fármacos , Masculino , Ratas
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