Asunto(s)

Angioplastia/efectos adversos , Hepatitis/microbiología , Infecciones por Mycobacterium no Tuberculosas/diagnóstico , Micobacterias no Tuberculosas/clasificación , Anciano , Antibacterianos/uso terapéutico , Hepatitis/tratamiento farmacológico , Humanos , Inmunocompetencia , Masculino , Infecciones por Mycobacterium no Tuberculosas/tratamiento farmacológico , Micobacterias no Tuberculosas/efectos de los fármacos , Micobacterias no Tuberculosas/genética , Micobacterias no Tuberculosas/aislamiento & purificación , Resultado del Tratamiento

RESUMEN

OBJECTIVES: A kidney transplant recipient with recurrent pleuritis underwent an open lung biopsy, the results of which revealed multiple nodular infiltrates. Grocott and periodic acid-Schiff staining were positive. Fungal and Tropheryma whipplei PCR were, however, negative. Further identification was needed. METHODS: Formalin-fixed, paraffin-embedded (FFPE) extraction was performed using an FFPE extraction kit. T. whipplei was searched for using a real-time PCR targeting the noncoding repeat specific for T. whipplei. Identification of the bacteria in the extract was done using 16S rDNA and 23S rDNA sequencing and BLAST analysis. Internal transcribed spacer PCR was used for fungal DNA identification. RESULTS: The FFPE extract was negative for fungi and T. whipplei. 16S rDNA sequence analysis of a 1375 bp fragment gave T. whipplei as the best match with 26 mismatches, resulting in only 98% agreement. Sequence analysis of the 23S rDNA gene again gave T. whipplei as the best match, but with only 91% agreement. A pan-Tropheryma 16S rDNA real-time PCR was developed, and both the biopsy sample and a respiratory sample of the patient were strongly positive. The patient received antimicrobial treatment targeting T. whipplei with good clinical outcome. CONCLUSIONS: 16S and 23S rDNA sequencing gave T. whipplei as the best hit, although with limited agreement. These findings suggest that a novel Tropheryma species that lacks the noncoding repeat, most frequently used for molecular detection of Whipple disease, might be the cause of the pulmonary disease. Adaptation of current PCR protocols is warranted in order to detect all Tropheryma species.

Asunto(s)

Infecciones por Actinomycetales/diagnóstico , Infecciones por Actinomycetales/etiología , Trasplante de Riñón/efectos adversos , Neumonía Bacteriana/diagnóstico , Neumonía Bacteriana/etiología , Receptores de Trasplantes , Tropheryma/clasificación , Biopsia , Humanos , Tipificación Molecular , ARN Ribosómico 16S/genética , ARN Ribosómico 23S/genética , Reacción en Cadena en Tiempo Real de la Polimerasa , Análisis de Secuencia de ADN , Tropheryma/genética , Tropheryma/aislamiento & purificación

RESUMEN

Molecular diagnostic testing has become an important tool in clinical laboratories. Accreditation according to the international quality standard ISO15189:2007 for medical laboratories is required for reimbursement of several molecular diagnostic tests in Belgium. Since the ISO15189:2007 standard applies to medical laboratories in general, the particular requirements for quality and competence are mentioned in general terms, not taking into account the specificities of molecular biology testing. Therefore, the working group "MolecularDiagnostics.be" described a consensus interpretation of chapter 5, Technical requirements, of the ISO standard for application in molecular diagnostic laboratories. The manuscript can be used as an instrument to prepare internal and external audits that meet the 15015189:2007 (chapter 5) criteria.

Asunto(s)

Técnicas de Diagnóstico Molecular/normas , Técnicas de Amplificación de Ácido Nucleico/normas , Bélgica , Humanos , Laboratorios de Hospital/normas , Control de Calidad

RESUMEN

Since the discovery of human bocavirus (hBoV), the virus has been detected worldwide in respiratory tract samples from young children by various polymerase chain reaction (PCR) assays and real-time PCRs (Q-PCR). Until now, no data have been reported on the presence of hBoV in Belgium and the detection of hBoV in a multiplex Q-PCR setting has not been described. The aim of this study was to develop a fast and reliable multiplex Q-PCR for the simultaneous detection of hBoV DNA and adenovirus (AdV) DNA. During the winter of 2004-2005, 445 nasopharyngeal aspirates (NPAs) were analysed from 404 Belgian children up to 5 years old with acute respiratory tract infections (ARTIs). (Co)infections with hBoV, AdV, respiratory syncytial virus (RSV), human metapneumovirus (hMPV) and influenza A virus were investigated. A viral agent was detected in 61% (n = 272/445) of the NPAs. Multiplex Q-PCR found a prevalence of 11% (n = 51/445) hBoV and 13% (n = 58/445) AdV. Coinfections were more frequently found with AdV (62%; n = 36/58) than with hBoV (49%; n = 25/51). Follow-up samples were available from 22 patients with ARTIs. In three patients, hBoV DNA persisted for one month. Multiplex Q-PCR may help in closing the diagnostic gap by addressing a broader range of potential respiratory pathogens.

Asunto(s)

Infecciones por Adenoviridae/diagnóstico , Infecciones por Adenoviridae/epidemiología , Adenovirus Humanos/aislamiento & purificación , Bocavirus Humano/aislamiento & purificación , Infecciones por Parvoviridae/diagnóstico , Infecciones por Parvoviridae/epidemiología , Reacción en Cadena de la Polimerasa/métodos , Adenovirus Humanos/genética , Virus de la Enfermedad Aleutiana del Visón/aislamiento & purificación , Bélgica/epidemiología , Preescolar , Técnicas de Laboratorio Clínico/métodos , Comorbilidad , ADN Viral/genética , ADN Viral/aislamiento & purificación , Bocavirus Humano/genética , Humanos , Lactante , Virus de la Influenza A/aislamiento & purificación , Masculino , Metapneumovirus/aislamiento & purificación , Nasofaringe/virología , Prevalencia , Virus Sincitiales Respiratorios/aislamiento & purificación , Infecciones del Sistema Respiratorio/virología , Sensibilidad y Especificidad , Factores de Tiempo

RESUMEN

Herpes simplex virus (HSV) has increasingly been associated with pulmonary disease in critically ill patients. However, the clinical relevance of HSV is still a topic of debate. Monitoring of HSV in a quantitative way could potentially give relevant information on its role in the pathogenesis of lower respiratory tract infection. A fast and reliable quantitative real-time PCR (Q-PCR) for the quantitative detection of HSV-1 and HSV-2 DNA was developed. A prospective observational study was performed in an intensive-care unit (ICU) to monitor the HSV viral load in lower respiratory tract aspirates of long-term mechanically ventilated patients. HSV was common in the lower respiratory tract (LRT) of critically ill patients with mechanical ventilation for at least 48 h (62%, n = 65/105). Detection of HSV was significantly associated with prolonged mechanical ventilation (p <0.01), prolonged ICU stay (p <0.01), and development of ventilator-associated pneumonia (p = 0.02). Corticosteroid administration (p <0.01) in the ICU and anti-HSV IgG seropositivity (p <0.01) were risk factors for the occurrence of HSV in the LRT. The fact that no HSV-seronegative patient became positive suggests that all HSV DNA-positive patients had HSV reactivations. Monitoring the HSV viral load in the LRT of critically ill patients showed a typical homogeneous pattern of HSV kinetics. HSV emerged in tracheal and bronchial aspirates after a median of 7 days of intubation (5-11 days), and this was followed by an exponential increase (c. 1 log copies/mL/day) to reach very high HSV peaks (10(6)-10(10) copies/mL) in 78% of the HSV DNA-positive patients.

Asunto(s)

Herpes Simple/virología , Herpesvirus Humano 1/aislamiento & purificación , Herpesvirus Humano 2/aislamiento & purificación , Neumonía Asociada al Ventilador/virología , Reacción en Cadena de la Polimerasa/métodos , Respiración Artificial/efectos adversos , Sistema Respiratorio/virología , Anciano , Anciano de 80 o más Años , Enfermedad Crítica , ADN Viral/aislamiento & purificación , Femenino , Herpesvirus Humano 1/genética , Herpesvirus Humano 2/genética , Humanos , Unidades de Cuidados Intensivos , Masculino , Persona de Mediana Edad , Estudios Prospectivos

RESUMEN

Viruses are an important cause of acute respiratory tract infection (ARTI) in children. This study aimed to develop and evaluate a rapid molecular diagnostic test (duplex real-time PCR) for human respiratory syncytial virus (hRSV) and human metapneumovirus (hMPV), and to determine the frequency of these two viruses as causative agents of ARTI in Belgium. Nasopharyngeal aspirates were collected over two winter and spring seasons (November 2003 to May 2004 and November 2004 to May 2005) from children aged <5 years with ARTI (n = 778). The duplex real-time PCR showed a linear range of 10(4)-10(10) copies/mL for both hMPV and hRSV. Analysis of the stability of the hRSV and hMPV genomes revealed that nasopharyngeal aspirates could be stored at room temperature for up to 1 month without significant loss of detection. hRSV was detected by antigen testing and by real-time PCR; hMPV was detected by real-time PCR only. The hRSV antigen test was less sensitive than PCR, and failed to detect one-third of the hRSV infections. Overall, 54 (6.9%) and 306 (39.3%) of the 778 samples were positive for hMPV and hRSV, respectively. Both viruses infected young infants, but the mean age of infants infected by hRSV was lower than that of infants infected by hMPV (12 months vs. 17 months, respectively).

Asunto(s)

Hospitales Pediátricos/estadística & datos numéricos , Metapneumovirus/genética , Nasofaringe/virología , Infecciones por Paramyxoviridae/epidemiología , Infecciones por Virus Sincitial Respiratorio/epidemiología , Virus Sincitial Respiratorio Humano/genética , Infecciones del Sistema Respiratorio , Antígenos Virales/aislamiento & purificación , Bélgica/epidemiología , Preescolar , Reacciones Falso Negativas , Femenino , Humanos , Lactante , Masculino , Metapneumovirus/aislamiento & purificación , Reacción en Cadena de la Polimerasa/métodos , Vigilancia de la Población , Infecciones por Virus Sincitial Respiratorio/diagnóstico , Virus Sincitial Respiratorio Humano/aislamiento & purificación , Infecciones del Sistema Respiratorio/epidemiología , Infecciones del Sistema Respiratorio/virología , Sensibilidad y Especificidad

RESUMEN

Expression of the cystic fibrosis transmembrane conductance regulator (CFTR) inhibits Ca(2+)-activated Cl- channels (CaCC) by an unknown mechanism. This inhibition does not require CFTR activation (activity-independent inhibition), but is potentiated when CFTR is activated (activity-dependent inhibition). In this study, we evaluated, in endothelial cells, possible structural determinants for this interaction. Bovine pulmonary artery endothelium (CPAE) cells, which do not express CFTR, were transfected transiently with three hybrid CFTR constructs. The functional interaction between CaCC and CFTR was assessed using the patch-clamp technique in the whole-cell configuration. CaCC was stimulated by application of adenosine 5'-triphosphate (ATP) to the bath solution. CFTR currents were evoked by application of a forskolin/3-isobutyl-l-methylxanthine (IBMX) cocktail. The inhibitory effect of CFTR was conserved when the PDZ (PSD-95/Discs large/ZO-1) binding motif was deleted (CFTR-delta PDZ). In contrast, both the CFTR activity-independent and -dependent inhibition of CaCC were abolished when the C-terminal part of the regulatory (R)-domain of CFTR was deleted (CFTR-delta R780-830). The activity-dependent inhibition of CaCC, but not the activity-independent inhibition, could be rescued by introducing the multiple drug resistance (MDR)-1 mini-linker in place of the deletion (CFTR-delta R-linker). It is concluded that the C-terminal part of the R-domain is an important determinant for CFTR-CaCC interaction.

Asunto(s)

Calcio/fisiología , Canales de Cloruro/efectos de los fármacos , Canales de Cloruro/metabolismo , Regulador de Conductancia de Transmembrana de Fibrosis Quística/química , Regulador de Conductancia de Transmembrana de Fibrosis Quística/farmacología , Secuencias de Aminoácidos/fisiología , Animales , Bovinos , Células Cultivadas , Endotelio Vascular/citología , Endotelio Vascular/metabolismo , Fragmentos de Péptidos/farmacología , Estructura Terciaria de Proteína/fisiología , Arteria Pulmonar/citología , Arteria Pulmonar/metabolismo , Proteínas Recombinantes/farmacología

RESUMEN

We expressed the human cystic fibrosis transmembrane conductance regulator (CFTR) in oocytes of the South African clawed frog Xenopus laevis. We performed simultaneous and continuous recording of membrane current (Im), conductance (Gm) and capacitance (Cm), the latter being a direct measure of membrane surface area. A cAMP-cocktail containing cAMP and isobutylmethylxanthine (IBMX) increased all parameters, demonstrating that CFTR activation was partly achieved by exocytotic delivery and insertion of preformed CFTR molecules into the plasma membrane. CFTR currents after cAMP-cocktail were correlated with the capacitance of the oocytes: oocytes with larger Cm exhibited larger currents. Expression of CFTR itself did not change the Cm of the oocytes. However, activation of CFTR with cAMP-cocktail increased Im and Gm 15- and 20-fold, respectively while membrane surface area increased by about 7%, indicating the functional insertion of preformed CFTR into the plasma membrane. While cAMP-cocktail yielded maximal CFTR stimulation, IBMX alone, but not caffeine or theophylline, was sufficient to stimulate more than half of the increases in Im and Gm as observed with cAMP-cocktail. Since Cm was not significantly stimulated by IBMX, we conclude that IBMX alone activated the CFTR channels already present in the oocyte membrane. CFTR stimulation by cAMP-cocktail was independent of external Ca2+ and ATP had no additional activating potency. The role of protein trafficking in the activation of CFTR evoked by increases of cytoplasmic cAMP was assessed by measuring the effects of brefeldin A (BFA), nocodazole and primaquine on the bioelectric parameters and membrane surface area. All these compounds that interfere with the protein trafficking machinery at different stages prevented the translocation of CFTR from intracellular pools to the plasma membrane. These data confirm and extend our previous observations that CFTR expressed in Xenopus laevis oocytes is activated via dual pathways including direct activation of CFTR already present in the membrane and exocytotic insertion of preformed CFTR channels into the membrane. Furthermore, we show that complete activation of CFTR requires an intact protein trafficking machinery.

Asunto(s)

Regulador de Conductancia de Transmembrana de Fibrosis Quística/metabolismo , Fibrosis Quística/metabolismo , Exocitosis/fisiología , Vesículas Transportadoras/metabolismo , 1-Metil-3-Isobutilxantina/farmacología , Animales , Antimaláricos/farmacología , Antineoplásicos/farmacología , Brefeldino A/farmacología , Calcio/metabolismo , Quelantes , AMP Cíclico/análogos & derivados , AMP Cíclico/farmacología , Regulador de Conductancia de Transmembrana de Fibrosis Quística/genética , Ácido Egtácico/análogos & derivados , Ácido Egtácico/farmacología , Conductividad Eléctrica , Inhibidores Enzimáticos/farmacología , Exocitosis/efectos de los fármacos , Expresión Génica , Potenciales de la Membrana/efectos de los fármacos , Potenciales de la Membrana/fisiología , Nocodazol/farmacología , Oocitos/fisiología , Técnicas de Placa-Clamp , Inhibidores de Fosfodiesterasa/farmacología , Primaquina/farmacología , Proteína Quinasa C/metabolismo , Inhibidores de la Síntesis de la Proteína/farmacología , Transporte de Proteínas/efectos de los fármacos , Transporte de Proteínas/fisiología , Tionucleótidos/farmacología , Vesículas Transportadoras/efectos de los fármacos , Xenopus laevis

RESUMEN

The cystic fibrosis transmembrane conductance regulator (CFTR) is a cAMP sensitive Cl- channel that is defective in cystic fibrosis (CF). The most frequent mutation, namely deltaF508-CFTR, accounts for 66% of CF. Here we show that cAMP-activation of CFTR occurs via at least two distinct pathways: activation of CFTR molecules already present in the plasma membrane and protein kinase A (PKA)-mediated vesicular transport of new CFTR molecules to the plasma membrane and functional insertion into the membrane. We investigated the mechanisms that are responsible for these activation pathways using the Xenopus laevis oocytes expression system. We expressed CFTR and recorded continuously membrane current (Im), conductance (Gm) and capacitance (Cm), which is a direct measure of membrane surface area. Expression of CFTR alone did not change the plasma membrane surface area. However, activation of CFTR with cAMP increased Im, Gm and Cm while deltaF508-CFTR-expressing oocytes showed no response on cAMP. Inhibition of protein kinase A or buffering intracellular Ca2+ abolished the cAMP-induced increase in Cm while increases of Im and Gm were still present. ATP or the xanthine derivative 8-cyclopentyl-1,3-dipropylxanthine (CPX) did not further activate CFTR. Insertion of pre-formed CFTR into the plasma membrane could be prevented by compounds that interfere with intracellular transport mechanisms such as primaquine, brefeldin A, nocodazole. From these data we conclude that cAMP activates CFTR by at least two distinct pathways: activation of CFTR already present in the plasma membrane and exocytotic delivery of new CFTR molecules to the oocyte membrane and functional insertion into it.

Asunto(s)

Regulador de Conductancia de Transmembrana de Fibrosis Quística/metabolismo , Oocitos/metabolismo , Teofilina/análogos & derivados , Adenosina Trifosfato/farmacología , Animales , AMP Cíclico/farmacología , Femenino , Microinyecciones , Teofilina/farmacología , Xenopus laevis

RESUMEN

The S1235R locus in CFTR was studied in combination with alleles found at the M470V and G628R loci. While R628 caused a maturational defect, R1235 did not. The impact of R1235 was found to be influenced by the alleles present at the G628R and M470V loci. At the single channel level, R1235-V (R1235 on a V470 background) was characterized by an open probability significantly higher than V470-wildtype CFTR. M470, which on its own increases CFTR chloride transport activity when compared to V470-wildtype CFTR, suppressed the activity of R1235 in such a way that a protein with an open probability not significantly different from V470-wildtype CFTR was obtained. While R628-V CFTR had similar current densities as V470-wildtype CFTR in Xenopus laevis oocytes, R1235-V resulted in current densities that were more than twofold higher than those of V470-wildtype CFTR. However, the current densities generated by R1235/R628-V (R1235 and R628 on a V470 background) CFTR were significant lower than R1235-V or R628-V CFTR.

Asunto(s)

Regulador de Conductancia de Transmembrana de Fibrosis Quística/genética , Supresión Genética , Alelos , Animales , Sitios de Unión/genética , Células COS , Fibrosis Quística/genética , Regulador de Conductancia de Transmembrana de Fibrosis Quística/fisiología , Electrofisiología , Femenino , Frecuencia de los Genes , Proteínas Fluorescentes Verdes , Haplotipos , Humanos , Proteínas Luminiscentes/genética , Masculino , Mutagénesis Sitio-Dirigida , Mutación , Oocitos/citología , Oocitos/fisiología , ARN Complementario/administración & dosificación , Proteínas Recombinantes de Fusión/genética , Xenopus laevis

RESUMEN

We investigated interactions between cystic fibrosis conductance regulator (CFTR) and endogenous Ca2+-activated Cl- channels (CaCC) in bovine pulmonary artery endothelium (CPAE). CPAE cells, which do not express CFTR, were transiently transfected with wild-type (WT) CFTR and the deletion mutant deltaF508 CFTR. Currents through CaCC were significantly reduced after expression of WT CFTR. This inhibition was increased by stimulation (isobutylmethylxanthine, forskolin) of CFTR in cells expressing WT CFTR. There were no such effects when deltaF508 mutant CFTR, which is retained in the endoplasmic reticulum, was expressed. It is concluded that CFTR and CaCC are functionally coupled probably through a direct channel-channel interaction.

Asunto(s)

Calcio/farmacología , Canales de Cloruro/fisiología , Regulador de Conductancia de Transmembrana de Fibrosis Quística/fisiología , 1-Metil-3-Isobutilxantina/farmacología , Animales , Bovinos , Línea Celular , Membrana Celular/fisiología , Colforsina/farmacología , Regulador de Conductancia de Transmembrana de Fibrosis Quística/genética , Conductividad Eléctrica , Endotelio Vascular/metabolismo , Expresión Génica , Mutación , Arteria Pulmonar/metabolismo , Transfección

RESUMEN

To improve our insight into the structure and function of the CFTR R domain, deletion and hybrid constructs in which different parts of the R domain were deleted or replaced by the MDR1 linker domain, and vice versa, were made. Replacement of the linker domain by the R domain did not result in a decrease and replacement of the CFTR R domain by the linker domain did not result in an increase of maturation efficiency, when compared to the respective wild-type proteins. This indicates that the R domain is not responsible for the high degree of degradation observed for CFTR translation products in the ER, but rather the overall structure or sequences located outside the R domain. Replacing the C-terminal part of the R domain (amino acids 780-830) by the MDR1 linker domain resulted in the appearance of PKA-dependent whole cell chloride currents which were not significantly different from wild-type CFTR currents. This might indicate that the PKA sites present in the linker domain are functional and that not the exact sequence of the C-terminal part of the R domain is important, but rather the presence of PKA sites and the length. Moreover, when this hybrid construct was PKC-stimulated, chloride currents were activated. Although these PKC-induced currents were lower than the PKA-induced ones, this again indicates that the linker domain is functional in this hybrid construct. Taken together, these results suggest that the MDR1 linker domain can substitute for part of the regulatory domain of the CFTR protein.

Asunto(s)

Miembro 1 de la Subfamilia B de Casetes de Unión a ATP/química , Regulador de Conductancia de Transmembrana de Fibrosis Quística/química , Miembro 1 de la Subfamilia B de Casetes de Unión a ATP/genética , Secuencia de Bases , Canales de Cloruro/metabolismo , Clonación Molecular , Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Regulador de Conductancia de Transmembrana de Fibrosis Quística/genética , Humanos , Activación del Canal Iónico , Datos de Secuencia Molecular , Conformación Proteica , Proteínas Recombinantes de Fusión/química , Relación Estructura-Actividad

RESUMEN

1. To investigate whether the cystic fibrosis transmembrane conductance regulator (CFTR) interacts with volume regulated anion channels (VRACs), we measured the volume-activated chloride current (ICl,swell) using the whole-cell patch-clamp technique in calf pulmonary artery endothelial (CPAE) cells and in COS cells transiently transfected with wild-type (WT) CFTR and the deletion mutant DeltaF508 CFTR. 2. ICl,swell was significantly reduced in CPAE cells expressing WT CFTR to 66.5 +/- 8.8 % (n = 13; mean +/- s. e.m.) of the control value (n = 11). This reduction was independent of activation of the CFTR channel. 3. Expression of DeltaF508 CFTR resulted in two groups of CPAE cells. In the first group IBMX and forskolin could activate a Cl- current. In these cells ICl,swell was reduced to 52.7 +/- 18.8 % (n = 5) of the control value (n = 21). In the second group IBMX and forskolin could not activate a current. The amplitude of ICl,swell in these cells was not significantly different from the control value (112.4 +/- 13.7 %, n = 11; 21 control cells). 4. Using the same method we showed that expression of WT CFTR in COS cells reduced ICl,swell to 62.1 +/- 11.9 % (n = 14) of the control value (n = 12) without any changes in the kinetics of the current. Non-stationary noise analysis suggested that there is no significant difference in the single channel conductance of VRAC between CFTR expressing and non-expressing COS cells. 5. We conclude that expression of WT CFTR down-regulates ICl, swell in CPAE and COS cells, suggesting an interaction between CFTR and VRAC independent of activation of CFTR.

Asunto(s)

Regulador de Conductancia de Transmembrana de Fibrosis Quística/fisiología , Activación del Canal Iónico/fisiología , Canales Iónicos/fisiología , Animales , Células COS/fisiología , Bovinos , Regulador de Conductancia de Transmembrana de Fibrosis Quística/genética , Estimulación Eléctrica , Electrofisiología , Endotelio Vascular/citología , Endotelio Vascular/fisiología , Endotelio Vascular/ultraestructura , Vectores Genéticos , Activación del Canal Iónico/genética , Canales Iónicos/genética , Cinética , Potenciales de la Membrana/fisiología , Técnicas de Placa-Clamp , Arteria Pulmonar/fisiología , Arteria Pulmonar/ultraestructura , Transfección

RESUMEN

We investigated CFTR channel activity of mature R-domain mutants showing single alterations at sites other than the predicted phosphorylation sites. All mutations were found in cystic fibrosis (CF) patients (H620Q, E822K and E826K). The macroscopic CFTR chloride conductance induced by phosphorylation was significantly enhanced in Xenopus oocytes injected with mRNA of H620Q but reduced in the E822K and E826K mutants compared to wild type CFTR. The anion permeability sequence for all three mutants was the same as that of wild type CFTR. Cell attached single channel studies in COS cells revealed that both open channel probability and/or the number of functional channels were either higher (H620Q) or lower (E822K and E826K) than in wild type CFTR. Single channel conductances were unchanged in all mutants. Our results suggest that additional sites in the R-domain other than phosphorylation sites influence gating of CFTR channels.

Asunto(s)

Regulador de Conductancia de Transmembrana de Fibrosis Quística/genética , Mutación , Animales , Aniones , Células COS , Regulador de Conductancia de Transmembrana de Fibrosis Quística/biosíntesis , Regulador de Conductancia de Transmembrana de Fibrosis Quística/metabolismo , Humanos , Transporte Iónico , Oocitos , Fosforilación , Conformación Proteica , Xenopus

RESUMEN

In order to get a better insight into the function of amino acid residues located in the second transmembrane domain of the cystic fibrosis transmembrane conductance regulator (CFTR) protein, all exon 18 mutations found in cystic fibrosis (CF) patients were characterized at the protein and at the electrophysiological level. Of the different mutations present in transmembrane helix 12 (M1137V, M1137R, I11139V and deltaM1140), and the intracytoplasmic loop connecting TM12 and NBD2 (D1152H and D1154G), only M1137R interfered with the proper maturation of the protein. Permeability studies performed after injection of the different wild-type and mutant cRNAs in Xenopus laevis oocytes indicated that the mutations did not alter the permeability sequence of the CFTR channels. The whole cell cAMP activated chloride currents, however, were significantly reduced for M1137V, I1139V, D1152H and D1154G and close to zero for deltaM1140, indicating that these mutations interfere with the proper gating of the chloride channels.

Asunto(s)

Regulador de Conductancia de Transmembrana de Fibrosis Quística/genética , Mutación , Animales , Células COS , Cloruros/metabolismo , Análisis Mutacional de ADN , Exones , Femenino , Humanos , Activación del Canal Iónico , Mutagénesis Sitio-Dirigida , Oocitos , Proteínas Recombinantes/metabolismo , Transfección , Xenopus laevis/genética

RESUMEN

In order to gain a better insight into the structure and function of the regulatory domain (RD) of the cystic fibrosis transmembrane conductance regulator (CFTR) protein, 19 RD missense mutations that had been identified in patients were functionally characterized. Nine of these (I601F, L610S, A613T, D614G, I618T, L619S, H620P, G628R and L633P) resulted in aberrant processing. No or a very small number of functional CFTR proteins will therefore appear at the cell membrane in cells expressing these mutants. These mutations were clustered in the N-terminal part of the RD, suggesting that this subdomain has a folding pattern that is very sensitive to amino acid changes. Mutations that caused no aberrant processing were further characterized at the electrophysiological level. First, they were studied at the whole cell level in Xenopus laevis oocytes. Mutants that induced a whole cell current that was significantly different from wild-type CFTR were subsequently analysed at the single channel level in COS1 cells transiently expressing the different mutant and wild-type proteins. Three mutant chloride channels, G622D, R792G and E822K CFTR, were characterized by significantly lower intrinsic chloride channel activities compared with wild-type CFTR. Two mutations, H620Q and A800G, resulted in increased intrinsic chloride transport activities. Finally, T665S and E826K CFTR had single channel properties not significantly different from wild-type CFTR.

Asunto(s)

Regulador de Conductancia de Transmembrana de Fibrosis Quística/genética , Regulador de Conductancia de Transmembrana de Fibrosis Quística/metabolismo , Mutación Missense , Secuencia de Aminoácidos , Animales , Células COS , Fibrosis Quística/genética , Electrofisiología/métodos , Femenino , Humanos , Datos de Secuencia Molecular , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Homología de Secuencia de Aminoácido , Especificidad de la Especie

RESUMEN

In congenital bilateral absence of the vas deferens patients, the T5 allele at the polymorphic Tn locus in the CFTR (cystic fibrosis transmembrane conductance regulator) gene is a frequent disease mutation with incomplete penetrance. This T5 allele will result in a high proportion of CFTR transcripts that lack exon 9, whose translation products will not contribute to apical chloride channel activity. Besides the polymorphic Tn locus, more than 120 polymorphisms have been described in the CFTR gene. We hypothesized that the combination of particular alleles at several polymorphic loci might result in less functional or even insufficient CFTR protein. Analysis of three polymorphic loci with frequent alleles in the general population showed that, in addition to the known effect of the Tn locus, the quantity and quality of CFTR transcripts and/or proteins was affected by two other polymorphic loci: (TG)m and M470V. On a T7 background, the (TG)11 allele gave a 2.8-fold increase in the proportion of CFTR transcripts that lacked exon 9, and (TG)12 gave a sixfold increase, compared with the (TG)10 allele. T5 CFTR genes derived from patients were found to carry a high number of TG repeats, while T5 CFTR genes derived from healthy CF fathers harbored a low number of TG repeats. Moreover, it was found that M470 CFTR proteins matured more slowly, and that they had a 1.7-fold increased intrinsic chloride channel activity compared with V470 CFTR proteins, suggesting that the M470V locus might also play a role in the partial penetrance of T5 as a disease mutation. Such polyvariant mutant genes could explain why apparently normal CFTR genes cause disease. Moreover, they might be responsible for variation in the phenotypic expression of CFTR mutations, and be of relevance in other genetic diseases.

Asunto(s)

Alelos , Regulador de Conductancia de Transmembrana de Fibrosis Quística/genética , Mutación , Animales , Células COS , Mapeo Cromosómico , Haplotipos , Humanos , Masculino , Polimorfismo Genético , ARN Mensajero/análisis , Conducto Deferente/anomalías

RESUMEN

Cystic fibrosis is caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene. The most frequent mutation is the deletion of F508 in the first nucleotide binding fold (NBF1). It induces a perturbation in the folding of NBF1, which impedes posttranslational maturation of CFTR. Determination of the three-dimensional structure of NBF1 would help to understand this defect. We present a novel model for NBF1 built from the crystal structure of bovine mitochondrial F1-ATPase protein. This model gives a reasonable interpretation of the effect of mutations on the maturation of the protein and, in agreement with the CD data, leads to reconsideration of the limits of NBF1 within CFTR.

Asunto(s)

Regulador de Conductancia de Transmembrana de Fibrosis Quística/química , Modelos Moleculares , Secuencia de Aminoácidos , Animales , Sitios de Unión , Bovinos , Dicroismo Circular , Clonación Molecular , Secuencia Conservada , Regulador de Conductancia de Transmembrana de Fibrosis Quística/genética , Regulador de Conductancia de Transmembrana de Fibrosis Quística/metabolismo , Escherichia coli/genética , Exones , Humanos , Datos de Secuencia Molecular , Mutación , Fragmentos de Péptidos/química , Fragmentos de Péptidos/genética , Fragmentos de Péptidos/aislamiento & purificación , Conformación Proteica , Pliegue de Proteína , ATPasas de Translocación de Protón/química , ATPasas de Translocación de Protón/genética , ATPasas de Translocación de Protón/metabolismo , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/aislamiento & purificación , Eliminación de Secuencia , Homología de Secuencia de Aminoácido

Asunto(s)

Espiramicina/análogos & derivados , Espiramicina/química , Antibacterianos/química , Antibacterianos/farmacología , Bacillus subtilis/efectos de los fármacos , Secuencia de Carbohidratos , Cromatografía Líquida de Alta Presión , Espectroscopía de Resonancia Magnética , Espectrometría de Masas/métodos , Datos de Secuencia Molecular , Estructura Molecular , Espiramicina/aislamiento & purificación , Espiramicina/farmacología , Streptomyces/metabolismo

RESUMEN

A novel myotropic Colorado potato beetle peptide, active in the Locusta oviduct motility assay, was isolated from a methanolic extract of 9,000 brain complexes of adult Leptinotarsa decemlineata by means of HPLC. Its sequence is Gly-Phe-Lys-Asn-Val-Ala-Leu-Ser-Thr-Ala-Arg-Gly-Phe-NH2. This peptide is identical to Lom-AG-MT-I, a myotropin previously isolated from the male accessory glands of Locusta migratoria, using the L. migratoria oviduct motility bioassay as a monitoring system. It strongly stimulated the frequency, amplitude, and tonus of the myogenic oviduct contractions, even at low concentrations.

Asunto(s)

Escarabajos , Saltamontes , Sistema Nervioso/química , Neuropéptidos/química , Neuropéptidos/aislamiento & purificación , Secuencia de Aminoácidos , Animales , Encéfalo , Cromatografía Líquida de Alta Presión , Colorado , Femenino , Genitales Masculinos/química , Masculino , Datos de Secuencia Molecular , Neuropéptidos/farmacología , Oviductos/efectos de los fármacos , Oviductos/fisiología , Homología de Secuencia de Aminoácido