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Seven National Metrology Institutes (NMIs) from France, United States, United Kingdom, Russia, Mexico, China and Germany participated in an inter-laboratory comparison on thermal conductivity measurements by the Guarded Hot Plate method. This action was part of a series of supplementary inter-laboratory comparisons (including infrared spectral emittance and thermal diffusivity) sponsored by the Consultative Committee on Thermometry (CCT) Task Group on Thermophysical Quantities (TG-ThQ). The objective of this collaborative work was to strengthen the consistency of thermal conductivity measurements carried out worldwide on low conductive materials. Measurements were conducted successively by all participants on the same sets of specimens of insulating materials (mineral wool and expanded polystyrene) at temperatures ranging from 10 °C to 40 °C, according to the International Standard ISO 8302. This protocol aimed to minimize issues of material variability by circulating the same pairs of specimens among the laboratories following the strict format of a round-robin test program. More than 120 data points (combinations of material, thickness and temperature) were compared. 92 % of the data points were in agreement, with differences to weighted mean values less than the expanded uncertainties calculated from the individual NMI uncertainties and uncertainties related to the comparison process.
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BACKGROUND: Since 2006 an assessment of the neurodevelopmental outcome of very low birth weight infants (VLBWI) at a corrected age of 2 years is mandatory for every perinatal centre in Germany. The aim of our study was to check how complete these assessments were performed in our population of infants born at our perinatal centre and receiving treatment within our local neonatal network. Furthermore, the data obtained will be used for prenatal consultations. Another objective was to identify risk factors for adverse neurodevelopmental outcomes. METHODS: All VLBWI were invited for a follow-up exam using the Bayley Scales of Infant Development II (BSID-II) or III (BSID-III), or Griffiths Mental Developmental Scales) at 2 years corrected age. The results of children assessed by other institutions were collected. RESULTS: 142 (69.3%) of the 205 VLBWI, born and finally discharged alive at the perinatal centre in Ulm were assessed at a median (minimum - maximum) corrected age of 23 (18-27) months. The BSID-II Psychomotor Development Index (PDI) 91 was (< 50-128) (n=115), the BSID-II Mental Development Index (MDI) was 87 (< 50-134) (n=96), BSID-III MDI 95 (60-112) (n=29) and the Griffiths Score was 93 (67-140) (n=17). Severe disability was diagnosed in 36 (25.4%) of the children studied. Gestational age and higher grade intraventricular haemorrhage were associated independently with severe disability. CONCLUSIONS: It is very difficult to achieve a high rate of follow-up examinations in preterm infants <1,500 g in a neonatal network. Severe impairment in VLBWI is not rare. Improving neurodevelopmental outcome remains a challenge.
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Discapacidades del Desarrollo/diagnóstico , Discapacidades del Desarrollo/epidemiología , Enfermedades del Prematuro/diagnóstico , Enfermedades del Prematuro/epidemiología , Recién Nacido de muy Bajo Peso , Enfermedades del Sistema Nervioso/diagnóstico , Enfermedades del Sistema Nervioso/epidemiología , Femenino , Alemania/epidemiología , Humanos , Incidencia , Recién Nacido , Unidades de Cuidado Intensivo Neonatal , Masculino , Medición de RiesgoRESUMEN
BACKGROUND: When facing the well-known demographic development with an increasing number of people suffering from dementia, there is a need of programmes to support nursing relatives and care at home. Many support services have been established in the past few years but they are rarely used by the relatives and the patients. The purpose of the Lighthouse Project Ulm (ULTDEM Study) was to prove the effectiveness of a single advisory approach in order to provide support services after care level classification and to relieve the burden placed on relatives caring for family members suffering from dementia ("initial case management"). METHODS: The ULTDEM Study is a prospective, open, randomized, controlled, interventional study with different parallel outcome measures (burden of caring, quality of life and mood). After the randomization, the interventional group was given comprehensive, individual advice about available treatment possibilities for dementia patients. Control group participants received standard treatment. Inclusion criteria were application of a care level (0 or 1) as well as dementia diagnosis. All participants (patients/relatives) underwent an initial and a 6 month comprehensive assessment. RESULTS: Our results show that a single advisory approach does not lead to a significant difference in outcome measures in interventional and control groups. Those tendencies described have to be interpreted as clinically not relevant. Although utilization of support services increases, it remains similar in both study groups. A confirmatory interpretation has not been possible due to a lack of adjustment to the findings regarding multiple testing and an insufficient degree of recruitment. Possible causes will be discussed such as premature intervention during the course of the disease, a lack of intervention blinding, recruitment bias and lack of an influence on adherence with regard to the use of support services. IMPLICATIONS: The study demonstrates that there is a substantial information deficit for persons affected by dementia and their relatives. Innovative ways still have to be developed to ensure that this information actually reaches the target audience.
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Cuidadores/psicología , Manejo de Caso/estadística & datos numéricos , Demencia/epidemiología , Demencia/enfermería , Servicios de Atención de Salud a Domicilio/estadística & datos numéricos , Derivación y Consulta/estadística & datos numéricos , Cuidados Intermitentes/psicología , Adulto , Anciano , Anciano de 80 o más Años , Femenino , Alemania/epidemiología , Humanos , Masculino , Persona de Mediana Edad , Prevalencia , Resultado del Tratamiento , Adulto JovenRESUMEN
Cell proliferation and cell death are essential yet opposing cellular processes. Crosstalk between these processes promotes a balance between proliferation and death, and it limits the growth and survival of cells with oncogenic mutations. New insights into the mechanisms by which strong signals to proliferate and activation of cyclin-dependent kinases promote apoptosis have recently been published, and a novel cell cycle regulated caspase inhibitor, Survivin, has been described.
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Apoptosis , Proteínas Portadoras , Proteínas de Ciclo Celular , División Celular , Proteínas de Unión al ADN , Proteínas Asociadas a Microtúbulos , Animales , Ciclo Celular/fisiología , Quinasas Ciclina-Dependientes/antagonistas & inhibidores , Quinasas Ciclina-Dependientes/fisiología , Factores de Transcripción E2F , Sustancias de Crecimiento/fisiología , Proteínas Inhibidoras de la Apoptosis , Proteínas de Neoplasias , Oncogenes/fisiología , Proteínas/metabolismo , Proteínas/fisiología , Proteína de Retinoblastoma/fisiología , Proteína 1 de Unión a Retinoblastoma , Transducción de Señal , Survivin , Factores de Transcripción/fisiologíaRESUMEN
Chronic obstructive pulmonary disease (COPD) is the single greatest risk factor for lung cancer in smokers and is found in 50-90% of lung cancer cases. The link between COPD and lung cancer may stem in part from the matrix remodelling and repair processes underlying COPD, and the development of epithelial-mesenchymal transition (EMT) that underlies lung carcinogenesis. The Hedgehog-interacting protein (HHIP), which mediates the epithelial response (EMT) to smoking, has been implicated in COPD and lung cancer. Recent genome-wide and candidate gene studies of COPD implicate genetic variants on the chromosomal 4q31 (HHIP/glycophorin A (GYPA)) locus. In a case-control study of smokers with normal lung function, COPD and lung cancer (subphenotyped for COPD), we show the GG genotype of the rs 1489759 HHIP single-nucleotide polymorphism (SNP) and the CC genotype of the rs 2202507 GYPA SNP confers a "protective" effect on COPD (OR 0.59, p = 0.006 for HHIP and OR = 0.65, p = 0.006 for GYPA) and lung cancer (OR = 0.70 (p = 0.05) for HHIP and OR 0.70 (p = 0.02) for GYPA). This study suggests that, in smokers, genetic variants of the 4q31 locus conferring a protective effect for COPD are also protective in lung cancer. We conclude that genetic susceptibility to lung cancer includes COPD-related gene variants.
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Proteínas Portadoras/genética , Cromosomas Humanos Par 4/genética , Glicoforinas/genética , Neoplasias Pulmonares/genética , Glicoproteínas de Membrana/genética , Enfermedad Pulmonar Obstructiva Crónica/genética , Anciano , Estudios de Casos y Controles , Femenino , Sitios Genéticos , Humanos , Masculino , Persona de Mediana Edad , Polimorfismo de Nucleótido Simple , FumarRESUMEN
A comparative analysis of the genomes of Drosophila melanogaster, Caenorhabditis elegans, and Saccharomyces cerevisiae-and the proteins they are predicted to encode-was undertaken in the context of cellular, developmental, and evolutionary processes. The nonredundant protein sets of flies and worms are similar in size and are only twice that of yeast, but different gene families are expanded in each genome, and the multidomain proteins and signaling pathways of the fly and worm are far more complex than those of yeast. The fly has orthologs to 177 of the 289 human disease genes examined and provides the foundation for rapid analysis of some of the basic processes involved in human disease.
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Caenorhabditis elegans/genética , Drosophila melanogaster/genética , Genoma , Proteoma , Saccharomyces cerevisiae/genética , Animales , Apoptosis/genética , Evolución Biológica , Caenorhabditis elegans/química , Caenorhabditis elegans/fisiología , Adhesión Celular/genética , Ciclo Celular/genética , Drosophila melanogaster/química , Drosophila melanogaster/fisiología , Proteínas Fúngicas/química , Proteínas Fúngicas/genética , Genes Duplicados , Enfermedades Genéticas Congénitas/genética , Genética Médica , Proteínas del Helminto/química , Proteínas del Helminto/genética , Humanos , Inmunidad/genética , Proteínas de Insectos/química , Proteínas de Insectos/genética , Familia de Multigenes , Neoplasias/genética , Estructura Terciaria de Proteína , Saccharomyces cerevisiae/química , Saccharomyces cerevisiae/fisiología , Transducción de Señal/genéticaRESUMEN
AIMS: To investigate the comparative elimination of three different human enterically transmitted viruses [i.e. hepatitis A virus (HAV), norovirus (NoV) and poliovirus (PV)] and inactivation of HAV and PV by Pacific oysters. METHODS AND RESULTS: New Zealand grown Pacific oysters (Crassostrea gigas) were allowed to bioaccumulate HAV, NoV and PV. Samples of oyster gut, faeces and pseudofaeces were then analysed by using real-time RT-PCR to determine the amount of viral RNA and cell culture methods to identify changes in the number of plaque forming units. The results suggest that the majority of the PV present in the oyster gut and oyster faeces is noninfectious, while in contrast, most of the HAV detected in the oyster gut are infectious. Depuration experiments identified a large drop in the count of PV in the gut over a 23-h cleansing period, whereas the levels of HAV and NoV did not significantly decrease. CONCLUSIONS: Human enterically transmitted viruses are eliminated and inactivated at different rates by Pacific oysters. SIGNIFICANCE AND IMPACT OF STUDY: The research presented in this article has implications for risk management techniques that are used to improve the removal of infectious human enteric viruses from bivalve molluscs.
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Crassostrea/virología , Virus de la Hepatitis A , Norovirus , Poliovirus , Infecciones por Virus ARN/transmisión , Infecciones por Virus ARN/virología , Mariscos/virología , Inactivación de Virus , Animales , Crassostrea/fisiología , Humanos , Nueva Zelanda , Poliovirus/genética , Reacción en Cadena de la Polimerasa , ARN Viral/análisis , ARN Viral/genéticaRESUMEN
AIMS: To examine the uptake and tissue distribution of norovirus (NoV) and poliovirus (PV) experimentally bioaccumulated in feeding Pacific oysters (Crassostrea gigas). METHODS AND RESULTS: Pacific oysters were allowed to bioaccumulated either PV or NoV under tidally synchronized feeding conditions in laboratory tanks. Oysters were then either fixed and paraffin wax embedded prior to localizing virus within tissues by immunohistochemistry (IHC), or they were dissected into digestive tract (stomach, intestine and digestive diverticula), gill and labial palp tissues, and the viral load determined by quantitative RT-PCR. Both PV and NoV immunoreactivities were predominantly found in the lumen and within cells of the digestive tract tissues; however, PV was also found within cells of nondigestive tract tissues, and in the gills and labial palp. Quantitative RT-PCR of tissue extracts corroborate the immunohistochemical data in that the major site for virus localization is the gut, but significant amounts of viral RNA were identified in the gills and labial palp. CONCLUSIONS: The human enteric viruses, PV and NoV, are readily bioaccumulated by feeding Pacific oysters and that some of the virus is internalized within cells of both digestive and nondigestive tissues. SIGNIFICANCE AND IMPACT OF THE STUDY: Oysters that have been virally contaminated even after depuration (cleaning) in uncontaminated seawater could pose a human health risk if consumed.
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Norovirus/aislamiento & purificación , Ostreidae/virología , Poliovirus/aislamiento & purificación , Animales , Anticuerpos Antivirales/inmunología , Microbiología de Alimentos , Tracto Gastrointestinal/virología , Branquias/virología , Inmunohistoquímica , Norovirus/genética , Norovirus/inmunología , Poliovirus/genética , Poliovirus/inmunología , ARN Viral/análisis , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
BACKGROUND: Epidemiological and family studies suggest that lung cancer results from the combined effects of age, smoking and genetic factors. Chronic obstructive pulmonary disease (COPD) is also an independent risk factor for lung cancer and coexists in 40-60% of lung cancer cases. METHODS: In a two-stage case-control association study, genetic markers associated with either susceptibility or protection against lung cancer were identified. In a test cohort of 439 Caucasian smokers or ex-smokers, consisting of healthy smokers and lung cancer cases, 157 candidate single nucleotide polymorphisms (SNPs) were screened. From this, 30 SNPs were identified, the genotypes (codominant or recessive model) of which were associated with either the healthy smokers (protective) or lung cancer (susceptibility) phenotype. After genotyping of this 30-SNP panel in a second validation cohort of 491 subjects and using the same protective and susceptibility genotypes from our test cohort, a 20-SNP panel was selected on the basis of independent univariate analyses. RESULTS: Using multivariate logistic regression, including the 20 SNPs, it was also found that age, history of COPD, family history of lung cancer and gender were significantly and independently associated with lung cancer. CONCLUSIONS: When numeric scores were assigned to both the SNP and demographic data, and sequentially combined by a simple algorithm in a risk model, the composite score was found to be linearly related to lung cancer risk with a bimodal distribution. Genetic data may therefore be combined with other risk variables from smokers or ex-smokers to identify individuals who are most susceptible to developing lung cancer.
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Neoplasias Pulmonares/genética , Enfermedad Pulmonar Obstructiva Crónica/genética , Fumar/efectos adversos , Adulto , Anciano , Métodos Epidemiológicos , Femenino , Marcadores Genéticos , Predisposición Genética a la Enfermedad , Humanos , Neoplasias Pulmonares/complicaciones , Masculino , Persona de Mediana Edad , Polimorfismo de Nucleótido Simple/genética , Enfermedad Pulmonar Obstructiva Crónica/complicaciones , Fumar/genéticaRESUMEN
We present a procedure dedicated to the calibration of a scanning thermal microscopy probe operated in an active mode and a modulated regime especially for the measurement of solid material thermal conductivities. The probe used is a microthermocouple wire mounted on a quartz tuning fork. Measurements on reference samples are performed successively in vacuum and ambient air conditions revealing a clear difference in the dependence of the thermal interaction between the probe and the sample on the sample properties. Analytical modeling based on the resolution of the heat equation in the wire probe and a description of the thermal interaction using a network of thermal conductances are used to fit experimental data and analyze this difference. We have experimentally verified that the effective thermal contact radius of the probe tip depends on the sample thermal conductivity in ambient conditions, whereas it remains constant in vacuum. We have defined the measurement range of the technique based on the decrease in the probe sensitivity at high thermal conductivities. Considering the experimental noise of our electrical device, it is shown that the maximum measurable value of thermal conductivity is near 23 W m-1 K-1 in vacuum and 37 W m-1 K-1 in ambient air conditions. Moreover, the lowest uncertainties are obtained for thermal conductivities below 5 W m-1 K-1 typically.
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Recently, several large genome-wide association studies have identified a putative "lung cancer" locus in the nicotinic acetylcholine receptor subunit genes (nAChR) on 15q25. However, these findings may be confounded by the presence of chronic obstructive pulmonary disease (COPD), which is also strongly associated with smoking exposure and lung cancer. This is likely as the prevalence of COPD in lung cancer cohorts is as much as two-fold greater than that reported in smoking control populations (50 versus 20%). The present authors compared the genotype frequencies of the most strongly associated single nucleotide polymorphism (rs16969968) in the alpha5 subunit of the nAChR gene cluster between three matched smoking cohorts. The AA genotype was found to be more frequent and was seen in 437 (16%) lung cancer cases and 445 (14%) COPD cases compared with 475 (9%) healthy smoking controls. More importantly, when 429 lung cancer cases were divided according to spirometry results (performed within 3 months of diagnosis, prior to surgery and in the absence of effusions or collapse), the AA genotype was present in 19 and 11% of cases with and without COPD, respectively. These findings suggest that the association between the alpha5 subunit nicotinic acetylcholine receptor single nucleotide polymorphism and lung cancer may, in part, be confounded by chronic obstructive pulmonary disease.
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Predisposición Genética a la Enfermedad , Neoplasias Pulmonares/complicaciones , Neoplasias Pulmonares/genética , Enfermedad Pulmonar Obstructiva Crónica/complicaciones , Enfermedad Pulmonar Obstructiva Crónica/genética , Anciano , Anciano de 80 o más Años , Cromosomas Humanos Par 15 , Estudios de Cohortes , Femenino , Genoma , Humanos , Neoplasias Pulmonares/etnología , Masculino , Persona de Mediana Edad , Polimorfismo de Nucleótido Simple , Enfermedad Pulmonar Obstructiva Crónica/etnología , Receptores Nicotínicos/genética , FumarRESUMEN
The estimation and control of the thermal power released by the radioactive waste packages are a key parameter in the management of radioactive waste geological repository sites. In the framework of the European project "Metrology for decommissioning nuclear facilities," the French National Agency of Radioactive Waste Management (ANDRA) collaborates with Laboratoire National de Métrologie et D'essais in order to measure the thermal power up to 500 W of typical real size radioactive waste packages (of at least 0.175 m3) with an uncertainty better than 5% by using a measurement method traceable to the international system of units. One of the selected metrological approaches is based on the principles of air flow calorimetry. This paper describes in detail the development of the air flow calorimeter prototype as well as the design of a radioactive waste package simulator used for its calibration. Results obtained from the calibration of the calorimeter and from the determination of thermal powers are presented here with an investigation of the measurement uncertainties.
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Considerable evidence suggests that the scrapie prion protein (PrP) is a component of the infectious particle. We studied the biogenesis and transmembrane orientation of an integral-membrane form of PrP in a cell-free transcription-linked translation-coupled translocation system programmed with a full-length PrP cDNA cloned behind the SP6 promoter. Translation of SP6 transcripts of the cDNA or of native mRNA from either normal or infected hamster brain in the absence of dog pancreas membranes resulted in the synthesis of a single PrP immunoreactive polypeptide (each polypeptide was the same size; Mr, 28,000), as predicted from the known sequence of the coding region. In the cotranslational presence of membranes, two additional forms were observed. Using peptide antisera specific to sequences from the amino- or the carboxy-terminal domain of PrP together with proteinase K or endoglycosidase H digestion or both, we showed that one of these forms included an integrated and glycosylated form of PrP (Mr = 33,000) which spans the bilayer twice, with domains of both the amino and carboxy termini in the extracytoplasmic space. By these criteria, the other form appeared to be an unglycosylated intermediate of similar transmembrane orientation. The PrP cell-free translation products did not display resistance to proteinase K digestion in the presence of nondenaturing detergents. These results suggest that the PrP cell-free translation products most closely resemble the normal cellular isoform of the protein, since its homolog from infected brain was proteinase K resistant. The implications of these findings for PrP structure and function are discussed.
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Proteínas de la Membrana , Priones , Membrana Celular/ultraestructura , Sistema Libre de Células , Clonación Molecular , ADN/genética , Endopeptidasa K , Endopeptidasas/metabolismo , Glicoproteínas/biosíntesis , Glicosilación , Procesamiento Proteico-PostraduccionalRESUMEN
Apoptosis is an active form of cell suicide that results in the orderly death and phagocytosis of cells during normal development and in the adult. Many death signals lead to the activation of members of a family of cysteine proteases known as caspases. These proteins act to transduce death signals from different cellular compartments and they cleave a number of cellular proteins, leading ultimately to many of the biochemical and morphological events associated with death. Many mechanisms act to inhibit cell death upstream of caspase activation. However, only one family of cellular proteins, the inhibitors of apoptosis (IAPs), has been identified that inhibit caspase activation and/or activity. The observations that IAP function is essential for cell survival in Drosophila, and that IAP expression is deregulated in many forms of cancer in humans, argue that IAPs are important cell death inhibitors and that deregulation of their function is likely to be important in human disease. Here we review IAP function, with particular reference to insights that study of the Drosophila IAPs has provided. We also discuss some directions for future study.
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Apoptosis/fisiología , Proteínas Bacterianas/metabolismo , Inhibidores de Caspasas , Proteínas de Drosophila , Drosophila melanogaster/fisiología , Proteínas Asociadas a Microtúbulos , Secuencia de Aminoácidos , Animales , Proteínas Bacterianas/química , Proteínas Bacterianas/genética , Caspasas/metabolismo , Inhibidores de Cisteína Proteinasa/genética , Inhibidores de Cisteína Proteinasa/metabolismo , Humanos , Proteínas Inhibidoras de la Apoptosis , Proteínas de Insectos/metabolismo , Datos de Secuencia Molecular , Proteínas de Neoplasias , Neuropéptidos/metabolismo , Péptidos/metabolismo , Estructura Terciaria de Proteína , Proteínas/genética , Proteínas/metabolismo , Survivin , Ubiquitinas/metabolismoAsunto(s)
Agotamiento Profesional/psicología , Trastorno Depresivo/psicología , Psiquiatría , Adulto , Anciano , Agotamiento Profesional/epidemiología , Agotamiento Profesional/etiología , Trastorno Depresivo/epidemiología , Trastorno Depresivo/etiología , Femenino , Alemania/epidemiología , Humanos , Masculino , Persona de Mediana Edad , Escalas de Valoración Psiquiátrica , Psiquiatría/estadística & datos numéricos , RecompensaRESUMEN
We have combined the high cloning efficiency of the lambda bacteriophage vectors with the surface expression screening method for the display of combinatorial antibody fragment (Fab) libraries on the surface of filamentous phage particles. The utility of the herein described ImmunoZAP 13 system for the isolation of Fabs that specifically bind antigen is demonstrated using two phagemid display libraries prepared from a previously characterized human combinatorial library. The percentage of clones that specifically bind antigen is maintained throughout the process of subcloning the LC and VH genes into ImmunoZAP 13, in vivo mass excision to convert the lambda library to a phagemid library, and preparation of phagemid particles displaying Fabs. Specific phagemid were isolated from libraries containing 0.6% and 0.03% tetanus toxoid (TT)-binding clones after two and three rounds of biopanning, respectively. Relative binding curves determined on a small sample of isolated clones indicate that several unique immunoglobulin Fab fragments have been isolated.
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Bacteriófago lambda/genética , Clonación Molecular/métodos , Expresión Génica , Vectores Genéticos , Fragmentos Fab de Inmunoglobulinas/biosíntesis , Proteínas Recombinantes de Fusión/biosíntesis , Secuencia de Bases , Ensayo de Inmunoadsorción Enzimática , Biblioteca de Genes , Humanos , Fragmentos Fab de Inmunoglobulinas/genética , Cinética , Datos de Secuencia Molecular , OligodesoxirribonucleótidosRESUMEN
We have combined the efficiency and ease of use of bacteriophage lambda vectors with the power of phage display screening technology to create SurfZAP. The use of bacteriophage lambda allows the construction of large lambda expression libraries, which are rapidly and efficiently converted to stable plasmid libraries by mass excision. In SurfZAP, clones are expressed as fusions with amino acids 198-406 of the M13 minor coat protein (cpIII) and are displayed on the surface of filamentous phage. When produced with helper phage proteins, the fusion proteins are incorporated into the surface of phagemid particles. We demonstrate the utility of biopanning by isolating tetanus toxoid-binding mouse Fab clones from SurfZAP libraries. Approximately 10-100-fold enrichment of specific clones was observed after each panning round. The ability to create a large library of genotypes and screen the phenotypes by activity may be a potent methodology for basic research and drug discovery.
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Bacteriófago M13 , Bacteriófago lambda , Proteínas de la Cápside , Vectores Genéticos , Animales , Secuencia de Bases , Biotecnología , Cápside , Fragmentos Fab de Inmunoglobulinas , Proteínas de la Membrana , Ratones , Datos de Secuencia Molecular , Proteínas Recombinantes de FusiónRESUMEN
The antigenic epitopes of the myohemerythrin (MHr) molecule have been studied extensively. The critical amino acid residues responsible for its immune recognition have been identified by using synthetic peptides and the technique of epitope scanning. To assess the true relevance of these techniques for determining the molecular mechanism of antigenic recognition and immunogenicity, the results obtained with isolated peptides should be tested in the context of the folded protein. To this end, we have designed and constructed a synthetic MHr gene, in modular form, which will allow subsequent alterations of nucleotide sequence encoding epitopes of interest. We have produced the recombinant protein at high level, and have shown by several criteria that it possesses the chemical, physical and immunological properties of the native worm protein. Thus, we have developed a valuable system for detailed immunological studies of the structure and chemistry required for antibody binding to protein.
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Genes Sintéticos/genética , Hemeritrina/análogos & derivados , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Clonación Molecular , Epítopos/inmunología , Escherichia coli/genética , Hemeritrina/biosíntesis , Hemeritrina/genética , Hemeritrina/inmunología , Técnicas In Vitro , Datos de Secuencia Molecular , Poliquetos/inmunología , Pruebas de Precipitina , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/genética , Proteínas Recombinantes/inmunologíaRESUMEN
A new method is presented for creating antibody expression libraries in Escherichia coli. Rather than perform two cloning steps to express the heavy and light chains of the antigen binding domain, we have used a fusion-PCR method to link the coding regions for heavy and light chain sequences in a single DNA molecule prior to vector ligation. This greatly simplifies the construction of antibody expression clones.