RESUMEN
The Clara cell secretory protein (CC16), which is produced along the tracheal-bronchial tree, has been shown to be a sensitive marker for the detection of lung hyperpermeability. Cigarette smoke inhalation has been associated with increased lung epithelial permeability. In this study we investigated the changes in CC16 in serum and bronchoalveolar lavage fluid (BALF) from female Sprague Dawley rats after a single exposure (2 x 1 h) to diluted mainstream cigarette smoke (MS) from the Reference Cigarette 2R4F. Rats were nose-only exposed to MS at concentrations of 0 (sham exposure), 250, 500, 750, 1000 or 1250 microg total particulate matter per liter. At 2, 4, 15 and 24h after exposure, serum and BALF-samples were collected. CC16 was determined in BALF and serum. Albumin in BALF, another marker for lung permeability was also determined. A trend towards a lower CC16 recovery was observed in BALF from smoke-exposed rats. The CC16 concentration in serum showed a marked (up to five-fold) concentration- and time-dependent increase after MS exposure. The increase of CC16 in serum was most prominent at the early timepoints, i.e. 2 and 4 h after exposure, and a return to baseline concentrations was obvious at 24 h after exposure. The effect of MS exposure on the amount of albumin in BALF was limited (up to 60% increase). This study clearly showed that CC16 is a good marker for the assessment of the increased permeability of the lung/blood barrier after MS-exposure.
Asunto(s)
Pulmón/metabolismo , Contaminación por Humo de Tabaco/efectos adversos , Uteroglobina/biosíntesis , Albúminas/análisis , Albúminas/metabolismo , Animales , Biomarcadores/sangre , Líquido del Lavado Bronquioalveolar/química , Monóxido de Carbono/análisis , Células Epiteliales/metabolismo , Femenino , Permeabilidad , Ratas , Ratas Sprague-Dawley , Pruebas de Toxicidad Aguda , Uteroglobina/análisis , Uteroglobina/sangreRESUMEN
To assess the biological activity and pharmacokinetic properties of nonglycosylated ricin A-chain (RA), we have obtained the polypeptide following expression of a synthetic 842-bp RA gene in Escherichia coli. Expression of the gene was carried out using the phage T5 PN25 promoter fused to the E. coli lac operator. The RA polypeptide was synthesized in a completely soluble form and was purified in one step by immunoabsorption. It was shown to be as cytotoxic for a human cell line as both native RA and chemically deglycosylated native RA. Reconstituted whole ricin and an immunotoxin containing the recombinant RA were also biologically active. Immunotoxins made with recombinant and deglycosylated RA had similar clearance rates in vivo showing, after a short period of rapid elimination, stabilities far higher than that of an immunotoxin made with native RA. Our results show that the complete elimination of sugar side chains from the RA is not sufficient to entirely eradicate the rapid initial in vivo clearance of RA-based biologicals.
Asunto(s)
Escherichia coli/genética , Genes Sintéticos , Inmunotoxinas/genética , Ricina/genética , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Línea Celular/efectos de los fármacos , Glicosilación , Humanos , Inmunotoxinas/farmacocinética , Operón Lac , Ratones , Ratones Endogámicos BALB C , Datos de Secuencia Molecular , Regiones Promotoras Genéticas , Ricina/farmacocinéticaRESUMEN
A hybrid gene consisting of the sequences coding for the signal peptide of human growth hormone and the mature form of interleukin-1 beta (IL-1 beta) was chemically synthesized. This sequence was inserted into a eukaryotic expression vector and introduced into Chinese hamster ovary cells. The resulting stably transformed cell lines produced large amounts of recombinant IL-1 beta, which was secreted into the culture medium mainly as a 22-kDa form. Expression in the presence of tunicamycin, an inhibitor of N-glycosylation, led to the complete disappearance of the 22-kDa form and the appearance of a new form of 17.5 kDa, indicating that the hybrid protein had been both processed and N-glycosylated. However, transformed cells producing mature IL-1 beta without a signal peptide produced the predicted 17.5-kDa nonglycosylated form. These results suggest that fusion to a heterologous leader sequence allowed IL-1 beta to be translocated across the membrane of the endoplasmic reticulum and to be transported and secreted by the exocytotic pathway.
Asunto(s)
Hormona del Crecimiento/genética , Interleucina-1/genética , Señales de Clasificación de Proteína/genética , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Línea Celular , Clonación Molecular , Cricetinae , Expresión Génica , Glicosilación , Humanos , Interleucina-1/metabolismo , Datos de Secuencia Molecular , Pruebas de Precipitina , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Tunicamicina/farmacologíaRESUMEN
A cDNA library was generated from rat brain tissues and organized into 1536-well plates, using a fluorescence activated cell sorter (FACS), acting as a single cell deposition system. The organized library containing 10,000 clones, with 60% full-length cDNA inserts, allowed the generation of multiple identical membrane replicas. Each replica was hybridized with a complex probe obtained from a particular brain tissue or a given cultured cell. The signal intensity for each of the clones present on the membrane, quantified with a standard image-analysis software, is proportional both to the abundance of the corresponding mRNA in the probe and to the amount of plasmid template on the membrane. The latter value was thus used to normalize the signals produced with complex probes, to optimize the comparison of mRNA expression levels for the different systems under study. The construction of high-quality cDNA libraries, the generation of identical membrane replicas and comparable probes, and the utilization of an image-analysis software package, coupled with the normalization of the spot intensity by assaying plasmid quantity, significantly improves the differential screening approach. Altogether, these technical improvements open the possibility to compare a great number of different probes and, in consequence, to accumulate biological information for each clone present in an organized cDNA library. The functional information obtained should complement data from DNA sequencing projects.
Asunto(s)
Secuencia de Bases , Encéfalo/metabolismo , ADN Complementario , Biblioteca de Genes , Transcripción Genética , Animales , Células Cultivadas , Clonación Molecular , Citometría de Flujo , Masculino , Especificidad de Órganos , Sondas ARN , ARN Mensajero/biosíntesis , Ratas , Ratas Sprague-Dawley , Secuencias Repetitivas de Ácidos Nucleicos , Homología de Secuencia de Ácido Nucleico , Programas InformáticosRESUMEN
A human neurotensin receptor (hNTR) cDNA was cloned from the colonic adenocarcinoma cell line HT29. The cloned cDNA encodes a putative peptide of 418 amino acids with 7 transmembrane domains. The amino acid sequence of the hNTR is 84% identical to the rat NTR [Neuron, 4 (1990) 847-854]. Transfection of this cDNA into COS cells results in the expression of receptors with pharmacological properties similar to those found with HT29 cells. Northern blot analysis using the hNTR cDNA probe indicated a single transcript of 4 kb in the brain, the small intestine and blood mononuclear cells.
Asunto(s)
Neurotensina/metabolismo , Receptores de Neurotransmisores/genética , Secuencia de Aminoácidos , Secuencia de Bases , Unión Competitiva , Línea Celular , Clonación Molecular , ADN , Humanos , Leucocitos Mononucleares/metabolismo , Datos de Secuencia Molecular , ARN Mensajero/metabolismo , Receptores de Neurotensina , Receptores de Neurotransmisores/metabolismo , Homología de Secuencia de Aminoácido , Células Tumorales CultivadasRESUMEN
A gene which encodes resistance by abortive infection (Abi+) to bacteriophage was cloned from Lactococcus lactis ssp. lactis biovar. diacetylactis S94. This gene was found to confer a reduction in efficiency of plating and plaque size for prolate-headed bacteriophage phi 53 (group I of homology) and total resistance to the small isometric-headed bacteriophage phi 59 (group III of homology). The cloned gene is predicted to encode a polypeptide of 346 amino acid residues with a deduced molecular mass of 41 455 Da. No homology with any previously described genes was found. A probe was used to determine the presence of this gene in two strains on 31 tested.
Asunto(s)
Proteínas Bacterianas/genética , Bacteriófagos/patogenicidad , Lactococcus lactis/genética , Lactococcus lactis/virología , Secuencia de Bases , Southern Blotting , Clonación Molecular , Inmunidad Innata/genética , Datos de Secuencia Molecular , Plásmidos , Reacción en Cadena de la Polimerasa , Análisis de Secuencia de ADNRESUMEN
We hypothesized that charge-charge interactions may be important for the binding of the human cholecystokinin type 1 (CCK(1)) receptor-specific non-peptide full agonist SR 146131, (2-[4-(4-chloro-2, 5-dimethoxyphenyl)-5-(2-cyclohexyl-ethyl)-thiazol-2-ylcarbamoyl ]-5, 7-dimethyl-indol-1-yl-1-acetic acid), the competitive antagonist SR 27897, (1-[2-(4-(2-chlorophenyl)thiazol-2-yl) aminocarbonyl indoyl] acetic acid) and the natural octapeptide CCK-8S to the CCK(1) receptor. Alanine replacement studies of positively charged residues in the extracellular domains of the receptor showed that only the R336A mutation affected SR 146131 potency of mutated receptors transiently expressed in monkey kidney epithelial COS-7 cells. Two residues, Lys(115) and Lys(187), were implicated in SR 27897 binding. Only the replacement of Lys(115), Arg(197) and Arg(336) significantly affected CCK-8S binding or activity. These results clearly indicated the importance of certain charged residues, but not others, in SR 146131, SR 27897 and CCK-8S binding. Furthermore, although these molecules probably occupy different binding sites on the CCK(1) receptor, we show that a small non-peptide agonist, SR 146131, can stimulate the dual signaling pathways mediated by the CCK(1) receptor.
Asunto(s)
Ácidos Indolacéticos/metabolismo , Indoles/metabolismo , Receptores de Colecistoquinina/metabolismo , Sincalida/análogos & derivados , Tiazoles/metabolismo , Secuencia de Aminoácidos , Animales , Células COS , Relación Dosis-Respuesta a Droga , Humanos , Fosfatos de Inositol/metabolismo , Datos de Secuencia Molecular , Mutación , Receptor de Colecistoquinina A , Receptores de Colecistoquinina/química , Sincalida/metabolismo , Relación Estructura-ActividadRESUMEN
The free fasciocutaneous forearm flap has been described only recently. Five cases of pharyngeal or oral repair with a forearm flap (FAF) are reported and used as a basis to discuss anatomical features and the technique used for removal of the flap. The relevant literature is reviewed. The FAF appears to be the flap of choice for reparative surgery of pharynx or mouth regions in oncology, the principal advantages of this flap being: fineness, flexibility and plasticity of the skin, reliability and lack of morbidity, and function effectiveness with respect to phonation and feeding. Indications for forearm and myocutaneous flaps in oncology of upper respiratory and digestive tracts are assessed critically, other indications for the forearm flap being cutaneous repair of cervicofacial lesions due to tumor or injury. The forearm flap is of pronounced value for cervicofacial repair surgery and constitutes a new step in the plastic conception of these repair procedures.
Asunto(s)
Neoplasias de Cabeza y Cuello/cirugía , Colgajos Quirúrgicos , Adulto , Anciano , Arterias , Antebrazo/irrigación sanguínea , Humanos , Laringectomía , Masculino , Persona de Mediana Edad , Faringectomía , Cirugía Plástica/métodosRESUMEN
A study of dysosmia and dysgeusia in the elderly was undertaken in 50 patients, according to a simplified protocol based on an interview, a complete ENT examination, chemical gustrometry and recognition olfactometry. As far as olfaction was concerned, 11% of the patients interviewed said they were hyposmic, whereas following the olfactometric examination, hyposmia was found in 25% of the patients. As far as gustation was concerned, 5% of the patients complained of parageusia. The chemical gustrometric examination revealed 4% of cases of hypogeusia. As a conclusion to this study, it appears that olfactory and gustatory disorders are well tolerated in the elderly, and are not a cause for referral. The recognition olfactometric examination permits the detection of hyposmia with a relatively remarkable frequency.
Asunto(s)
Envejecimiento/fisiología , Disgeusia/diagnóstico , Trastornos del Olfato/diagnóstico , Anciano , Anciano de 80 o más Años , Femenino , Humanos , Masculino , MétodosAsunto(s)
Proteínas/análisis , Uteroglobina , Adulto , Líquidos Corporales/química , Femenino , Humanos , Masculino , Persona de Mediana Edad , Peso MolecularRESUMEN
Cadmium (Cd) exposure results in injury to the proximal tubule characterized by polyuria and proteinuria. Kidney injury molecule-1 (Kim-1) is a transmembrane glycoprotein not normally detected in the mature kidney, but is upregulated and shed into the urine following nephrotoxic injury. In this study, we determine if Kim-1 might be a useful early biomarker of Cd nephrotoxicity. Male Sprague-Dawley rats were given daily injections of Cd for up to 12 weeks. Weekly urine samples were analyzed for Kim-1, protein, creatinine, metallothionein, and Clara cell protein CC-16. Significant levels of Kim-1 were detected in the urine by 6 weeks and continued to increase throughout the treatment period. This appearance of Kim-1 occurred 4-5 weeks before the onset of proteinuria, and 1-3 weeks before the appearance of metallothionein and CC-16. Higher doses of Cd gave rise to higher Kim-1 excretion. Reverse transcriptase-polymerase chain reaction (RT-PCR) expression analysis showed that Kim-1 transcript levels were increased after 6 weeks at the low dose of Cd. Immunohistochemical analysis showed that Kim-1 was present in proximal tubule cells of the Cd-treated rats. Our results suggest that Kim-1 may be a useful biomarker of early stages of Cd-induced proximal tubule injury.
Asunto(s)
Biomarcadores/orina , Cadmio/efectos adversos , Moléculas de Adhesión Celular/orina , Proteínas de la Membrana/orina , Proteinuria/inducido químicamente , Proteinuria/orina , Animales , Peso Corporal/efectos de los fármacos , Cadmio/farmacología , Relación Dosis-Respuesta a Droga , Túbulos Renales Proximales/efectos de los fármacos , Túbulos Renales Proximales/metabolismo , Túbulos Renales Proximales/patología , Masculino , Metalotioneína/orina , Ratas , Ratas Sprague-Dawley , Uteroglobina/orinaRESUMEN
Two-hybrid screening in yeast with p73alpha isolated SUMO-1 (small ubiquitin-like modifier 1), the enzyme responsible for its conjugation, Ubc-9, and a number of novel SUMO-1-interacting proteins, including thymine DNA glycosylase, PM-Scl75, PIASx, PKY, and CHD3/ZFH. A subset of these proteins contain a common motif, hhXSXS/Taaa, where h is a hydrophobic amino acid and a is an acidic amino acid, that is shown to interact with SUMO-1 in the two-hybrid system. We show here that p73alpha, but not p73beta, can be covalently modified by SUMO-1. The major SUMO-1-modified residue in p73alpha is the C-terminal lysine (Lys(627)). The sequence surrounding this lysine conforms to a consensus SUMO-1 modification site b(X)XXhKXE, where b is a basic amino acid. SUMO-1-modified p73 is more rapidly degraded by the proteasome than unmodified p73, although SUMO-1 modification is not required for p73 degradation. SUMO-1 modification does not affect the transcriptional activity of p73alpha on an RGC-luciferase reporter gene in SK-N-AS cells. Instead, SUMO-1 modification may alter the subcellular localization of p73, because SUMO-1-modified p73 is preferentially found in detergent-insoluble fractions. Alternatively, it may modulate the interaction of p73 with other proteins that are substrates for SUMO-1 modification or which interact with SUMO-1, such as those identified here.
Asunto(s)
Proteínas de Unión al ADN/metabolismo , Proteínas Nucleares/metabolismo , Procesamiento Proteico-Postraduccional , Ubiquitinas/química , Ubiquitinas/metabolismo , Secuencias de Aminoácidos , Secuencia de Aminoácidos , Animales , Línea Celular , Secuencia Conservada , Cisteína Endopeptidasas/metabolismo , Inhibidores de Cisteína Proteinasa/farmacología , Proteínas de Unión al ADN/química , Proteínas de Unión al ADN/genética , Regulación de la Expresión Génica , Genes Reporteros , Genes Supresores de Tumor , Humanos , Leupeptinas/farmacología , Lisina/genética , Lisina/metabolismo , Datos de Secuencia Molecular , Complejos Multienzimáticos/metabolismo , Proteínas Nucleares/química , Proteínas Nucleares/genética , Complejo de la Endopetidasa Proteasomal , Unión Proteica , Transporte de Proteínas , Proteína SUMO-1 , Transfección , Células Tumorales Cultivadas , Proteína Tumoral p73 , Proteína p53 Supresora de Tumor/genética , Proteína p53 Supresora de Tumor/metabolismo , Proteínas Supresoras de Tumor , Técnicas del Sistema de Dos Híbridos , Ubiquitinas/genéticaRESUMEN
In fission yeast Schizosaccharomyces pombe, ammonium starvation induces a growth arrest, a cell cycle exit in G(1) and a further switch to meiosis. This process is regulated by the cAMP-dependent protein kinase and the Wis1-dependent MAP kinase cascade, and downstream transcription factors. In order to understand how cells adapt their genetic programme to the switch from mitotic cycling to starvation, a differential transcript analysis comparing mRNA from exponentially growing and ammonium-starved cells was performed. Genes repressed by this stimulus mainly concern cell growth, i.e. protein synthesis and global metabolism. Comparison of the expression of two of them, the ribosomal proteins Rps6 and TCTP, in many different growing conditions, evidenced a strong correlation, suggesting that their transcriptions are coordinately regulated. Nevertheless, by repeating the ammonium starvation on strains constitutively activated for the PKA pathway (Deltacgs1), or unable to activate the Wis1-dependent MAP kinase pathway (Deltawis1), or with both characteristics (Deltacgs1+Deltawis1), the transcriptional inhibition was found to be governed either by the PKA pathway, or by the Wis1 pathway, or by both. These results suggest that during the switch from exponential growth to ammonium starvation, cell homeostasis is maintained by downregulating the transcription of the most expressed genes by a PKA and a Wis1-dependent process. Accession Nos for the S30 and L14 ribosomal protein cDNA sequences are AJ2731 and AJ2732, respectively.
Asunto(s)
Biomarcadores de Tumor , Quinasas de Proteína Quinasa Activadas por Mitógenos , Compuestos de Amonio Cuaternario/metabolismo , Proteínas de Schizosaccharomyces pombe , Schizosaccharomyces/genética , Transcripción Genética , Proteínas de Unión al Calcio/genética , Proteínas Quinasas Dependientes de AMP Cíclico/fisiología , Glucosa/metabolismo , Proteínas Serina-Treonina Quinasas/fisiología , ARN Mensajero/análisis , Proteína S6 Ribosómica , Proteínas Ribosómicas/genética , Proteína Tumoral Controlada Traslacionalmente 1RESUMEN
We describe a simplified and reliable polymerase chain reaction-based method for assaying RNAs of low abundancy. The technique involves the co-amplification of cellular RNA-derived cDNA with a multispecific cDNA of synthetic origin added as an internal standard, using primer pairs common to both templates. We show that the co-amplified templates accumulate in a parallel manner throughout both the exponential and nonexponential phases of amplification, even when the starting amounts of the templates differ by up to 2 orders of magnitude. This finding means that preliminary experiments designed to determine either the late exponential region or the amplification efficiency for each pair of primers are unnecessary. This has enabled us to develop a greatly simplified quantitation protocol. We illustrate our approach by quantifying the effect of the immunosuppressor cyclosporin A on the accumulation of interleukin-4, interferon-gamma, and interleukin-2 receptor mRNAs in phytohemagglutinin-stimulated human peripheral blood mononuclear cells.
Asunto(s)
Reacción en Cadena de la Polimerasa/métodos , ARN Mensajero/análisis , Secuencia de Bases , Southern Blotting , Células Cultivadas , Ciclosporina/farmacología , ADN de Cadena Simple , Humanos , Interferón gamma/genética , Interleucina-4/genética , Cinética , Datos de Secuencia Molecular , Plásmidos , Receptores de Interleucina-2/genética , Volumetría , Factor de Necrosis Tumoral alfa/genética , Microglobulina beta-2/genéticaRESUMEN
Neurotensin is a tridecapeptide that plays several neurotransmitter or neuromodulatory roles both in the central nervous system and in the periphery. These actions are mediated by a high-affinity receptor (Ntsr). Both rat and human cDNAs encoding high-affinity receptors have been recently cloned. The availability of Ntsr probes allowed us to localize the corresponding genes on the mouse and human chromosomes. The present data demonstrate that the Ntsr gene is assigned to the H region of the mouse Chromosome (Chr) 2 and to the long arm of the human Chr 20.
Asunto(s)
Ratones/genética , Receptores de Neurotensina/genética , Animales , Mapeo Cromosómico , Cromosomas Humanos Par 20 , Genes , Humanos , Hibridación in Situ , Ratones Endogámicos , Ratones Mutantes Neurológicos/genética , Especificidad de la EspecieRESUMEN
The antagonist SR 141716A has a high specificity for the central CB1 cannabinoid receptor and negligeable affinity for the peripheral CB2 receptor, making it an excellent tool for probing receptor structure-activity relationships. From binding experiments with mutated CB1 and with chimeric CB1/CB2 receptors we have begun to identify the domains of CB1 implicated in the recognition of SR 141716A. Receptors were transiently expressed in COS-3 cells, and their binding characteristics were studied with SR 141716A and with CP 55,940, an agonist recognized equally well by the two receptors. The region delineated by the fourth and fifth transmembrane helices of CB1 proved to be crucial for high affinity binding of SR 141716A. The CB1 and CB2 second extracellular loops, e2, were exchanged, modifications that had no effect on SR 141716A binding in the CB1 variant but that eliminated CP 55,940 binding in both mutants. The replacement of the conserved cysteine residues in e2 of CB2 by serine also eliminated CP 55,940 binding, but replacement of those in CB1 resulted in the sequestration of the mutated receptors in the cell cytoplasm. The e2 domain thus plays some role in CP 55,940 binding but none in SR 141716A recognition, binding of the latter clearly implicating residues in the adjoining transmembrane helices.
Asunto(s)
Piperidinas/metabolismo , Pirazoles/metabolismo , Receptores de Droga/metabolismo , Secuencia de Aminoácidos , Animales , Línea Celular , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Unión Proteica , Conformación Proteica , Receptores de Cannabinoides , Receptores de Droga/química , Receptores de Droga/genética , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , RimonabantRESUMEN
The sequencing of endopeptidase-generated peptides from the peripheral binding site (PBS) for benzodiazepines, purified from a Chinese hamster ovary (CHO) cell line, produced internal sequence information, and confirmed and extended the NH2-terminal PBS sequence that we previously reported. Since the sequences were highly similar to the corresponding rat PBS sequences, we investigated whether they were also conserved in human PBS. Scatchard analysis of [3H]PK11195 (a derivative of isoquinoline carboxamide) binding and photoaffinity labeling with [3H]PK14105 (a nitrophenyl derivative of PK11195) revealed that CHO PBS and human PBS are closely related. Furthermore a rabbit antiserum raised against three peptides synthesized on the basis of the CHO PBS sequence immunoprecipitate the solubilized U937 PBS and also recognize the human protein in an immunoblot analysis. Based on these results, we screened a U937 cell cDNA library with four oligonucleotide probes derived from the CHO sequence. Two of the probes hybridized with several clones that we isolated and sequenced. One of these, h-pPBS11, is 831 nucleotides and contains a full-length representation of human PBS mRNA. The amino acid sequence of human PBS deduced from the cDNA is 79% identical to that reported for rat PBS, however, human PBS contains two cysteines while rat PBS is characterized by the absence of this amino acid. Using the cDNA of human PBS as a probe, the PBS gene was located in the 22q13.3 band of the human genome.
Asunto(s)
Cromosomas Humanos Par 22/ultraestructura , Linfoma de Células T Periférico/genética , Receptores de GABA-A/genética , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Mapeo Cromosómico , Clonación Molecular , Cricetinae , ADN , Endopeptidasas , Biblioteca Genómica , Humanos , Modelos Biológicos , Datos de Secuencia Molecular , Sondas de Oligonucleótidos , Pruebas de Precipitina , ARN Mensajero/química , Receptores de GABA-A/aislamiento & purificación , Tritio , Células Tumorales CultivadasRESUMEN
Amino acid sequencing of peptides obtained after proteolytic hydrolysis of Aspergillus flavus urate oxidase (uricase) permitted the design of oligodeoxynucleotide probes that were used to obtain 1.2- and 5-kilobase pair DNA fragments from A. flavus cDNA and genomic libraries, respectively. The cDNA fragment contained the entire coding region for uricase, and comparison with the genomic fragment revealed the presence of two short introns in the coding region of the gene. A. flavus uricase has around 40% overall identity with uricases from higher organisms but with many conserved amino acids. Hitherto highly conserved consensus patterns found in other uricases were found to be modified in the A. flavus enzyme, notably the sequence Val-Leu-Lys-Thr-Thr-Gln-Ser near position 150, which in the filamentous fungus is uniquely modified to Val-Leu-Lys-Ser-Thr-Asn-Ser. Silent mutations were introduced by cassette mutagenesis near the 5'-extremity of the coding sequence in order to conform with Escherichia coli codon usage, and the uricase was expressed in the E. coli cytoplasm in a completely soluble, biologically active form.
Asunto(s)
Aspergillus flavus/enzimología , Escherichia coli/genética , Expresión Génica , Urato Oxidasa/metabolismo , Secuencia de Aminoácidos , Secuencia de Bases , Cromatografía Líquida de Alta Presión , Clonación Molecular , ADN/genética , Electroforesis en Gel de Poliacrilamida , Genes Bacterianos , Vectores Genéticos , Hidrólisis , Datos de Secuencia Molecular , Alineación de SecuenciaRESUMEN
Exposure to ozone (O3) impairs lung function, induces airway inflammation and alters epithelial permeability. Whilst impaired lung function and neutrophilia have been observed at relatively low concentrations, altered lung epithelial permeability is only seen after high-dose challenges. The appearance of Clara cell protein (CC16) in serum has been proposed as a sensitive marker of lung epithelial injury. Here, the use of CC16 as an injury biomarker was evaluated under a controlled exposure to O3 and the relationship between this marker of lung injury and early lung function decrements was investigated. Subjects (n=22) were exposed on two separate occasions to 0.2 parts per million O3 and filtered air for 2 h. Blood samples were drawn and lung function assessed at 2 h pre-exposure, immediately before and immediately after exposure as well as 2 and 4 h postexposure. O3 increased CC16 serum concentrations at 2 h (12.0+/-4.5 versus 8.4+/-3.1 microg x L(-1)) and 4 h postexposure (11.7+/-5.0 versus 7.9+/-2.6 microg x L(-1)) compared with air concentrations. Archived samples from O3 studies utilising the same design indicated that this increase was sustained for up to 6 h postexposure (9.1+/-2.6 versus 7.1+/-1.7 microg x L(-1)) with concentrations returning to baseline by 18 h (7.7+/-2.9 versus 6.6+/-1.7 microg x L(-1)). In these studies, the increased plasma CC16 concentration was noted in the absence of increases in traditional markers of epithelial permeability. No association was observed between increased CC16 concentrations and lung function changes. To conclude, Clara cell protein represents a sensitive and noninvasive biomarker for ozone-induced lung epithelial damage that may have important uses in assessing the health effects of air pollutants in future epidemiological and field studies.
Asunto(s)
Exposición por Inhalación/efectos adversos , Enfermedades Pulmonares/inmunología , Oxidantes Fotoquímicos/efectos adversos , Ozono/efectos adversos , Proteínas/inmunología , Mucosa Respiratoria/inmunología , Uteroglobina , Adulto , Biomarcadores , Estudios Cruzados , Ambiente Controlado , Femenino , Humanos , Enfermedades Pulmonares/inducido químicamente , Masculino , Proteínas/análisis , Método Simple CiegoRESUMEN
AIMS: To study whether exposure to nitrogen trichloride in indoor chlorinated pools may affect the respiratory epithelium of children and increase the risk of some lung diseases such as asthma. METHODS: In 226 healthy children, serum surfactant associated proteins A and B (SP-A and SP-B), 16 kDa Clara cell protein (CC16), and IgE were measured. Lung specific proteins were measured in the serum of 16 children and 13 adults before and after exposure to NCl(3) in an indoor chlorinated pool. Relations between pool attendance and asthma prevalence were studied in 1881 children. Asthma was screened with the exercise induced bronchoconstriction test (EIB). RESULTS: Pool attendance was the most consistent predictor of lung epithelium permeability. A positive dose-effect relation was found with cumulated pool attendance and serum SP-A and SP-B. Serum IgE was unrelated to pool attendance, but correlated positively with lung hyperpermeability as assessed by serum SP-B. Changes in serum levels of lung proteins were reproduced in children and adults attending an indoor pool. Serum SP-A and SP-B were already significantly increased after one hour on the pool side without swimming. Positive EIB and total asthma prevalence were significantly correlated with cumulated pool attendance indices. CONCLUSIONS: Regular attendance at chlorinated pools by young children is associated with an exposure dependent increase in lung epithelium permeability and increase in the risk of developing asthma, especially in association with other risk factors. We therefore postulate that the increasing exposure of children to chlorination products in indoor pools might be an important cause of the rising incidence of childhood asthma and allergic diseases in industrialised countries. Further epidemiological studies should be undertaken to test this hypothesis.