RESUMEN
Magnetic resonance is considered to be a necessary condition for metamaterial perfect absorbers, and dual-band absorbers can be composed of a pair of metallic layers with anti-parallel surface currents. We designed and fabricated a tunable dual-band perfect absorber based on extraordinary-optical-transmission (EOT) effect and Fabry-Perot cavity resonance. The idea and the mechanism are completely different from the absorber based on the near-field interaction. The important advantage of our structure is that we can switch a single-band absorber to a dual-band absorber by changing the distance between two metallic layers and/or incident angle. The peak originating from the EOT effect becomes significantly narrower, resulting in an increase of the Q-factor from 16.88 to 49. The dual-band absorber can be optimized to be insensitive to the polarization of the incident electromagnetic wave by slightly modifying the absorber structure.
Asunto(s)
Interferometría/instrumentación , Refractometría/instrumentación , Absorción , Diseño de Equipo , Análisis de Falla de Equipo , LuzRESUMEN
The thermal stability of the alkaline protease extracellular subtilisin-type serine protease (AprP) from Pseudomonas sp. KFCC 10818 was improved by altering an amino acid residue at an autoproteolytic cleavage site. N-terminal sequence analysis of the autoproteolytic products of the protein revealed the presence of two cleavage sites, Ser-307 and Ser-331. To increase the thermal stability of the enzyme, serine residues of these sites were replaced with aspartate. The S331D mutant enzyme was successfully purified and characterized whereas the S307D mutant was not. The half-lives of the S331D mutant at 55 degrees C and 60 degrees C were 1.5 and 2.4 times longer than that of the wild-type enzyme, respectively. In addition, the catalytic efficiency was also enhanced.
Asunto(s)
Proteínas Bacterianas , Calor , Mutagénesis Sitio-Dirigida , Pseudomonas/enzimología , Serina Endopeptidasas/genética , Serina Endopeptidasas/metabolismo , Serina/genética , Serina/metabolismo , Fosfatasa Alcalina , Secuencia de Aminoácidos , Sustitución de Aminoácidos/genética , Ácido Aspártico/genética , Ácido Aspártico/metabolismo , Sitios de Unión/genética , Estabilidad de Enzimas/genética , Hidrólisis , Datos de Secuencia Molecular , Pseudomonas/genética , Serina Endopeptidasas/aislamiento & purificaciónRESUMEN
A lipase gene, lipK, and a lipase modulator gene, limK, of Pseudomonas sp. strain KFCC 10818 have been cloned, sequenced, and expressed in Escherichia coli. The limK gene is located immediately downstream of the lipK gene. Enzymatically active lipase was produced only in the presence of the limK gene. The effect of the lipase modulator LimK on the expression of active lipase was similar to those of the Pseudomonas subfamily I.1 and I.2 lipase-specific foldases (Lifs). The deduced amino acid sequence of LimK shares low homology (17 to 19%) with the known Pseudomonas Lifs, suggesting that Pseudomonas sp. strain KFCC 10818 is only distantly related to the subfamily I.1 and I.2 Pseudomonas species. Surprisingly, a lipase variant that does not require LimK for its correct folding was isolated in the study to investigate the functional interaction between LipK and LimK. When expressed in the absence of LimK, the P112Q variant of LipK formed an active enzyme and displayed 63% of the activity of wild-type LipK expressed in the presence of LimK. These results suggest that the Pro(112) residue of LipK is involved in a key step of lipase folding. We expect that the novel finding of this study may contribute to future research on efficient expression or refolding of industrially important lipases and on the mechanism of lipase folding.
Asunto(s)
Lipasa/metabolismo , Chaperonas Moleculares/metabolismo , Pseudomonas/enzimología , Proteínas Bacterianas , Activación Enzimática , Variación Genética , Glutamina/genética , Lipasa/genética , Datos de Secuencia Molecular , Mutación , Prolina/genética , Pliegue de Proteína , Pseudomonas/genéticaRESUMEN
AIMS: To assess the importance of tumour necrosis factor alpha (TNF-alpha) promoter polymorphism in relation to infection with the cytotoxin associated gene A (cagA) subtype of Helicobacter pylori within a dyspeptic Korean population. METHODS: Eighty three patients with gastric disease and 113 healthy controls were studied. The DNA from gastric biopsy specimens was analysed by H pylori specific and cagA specific polymerase chain reaction (PCR). To characterise TNF-alpha polymorphism at positions -308 and -238, PCR based restriction fragment length polymorphism analysis was performed. RESULTS: Helicobacter pylori infection was closely correlated with G to A transition at position -308 of the TNF-alpha promoter when compared with healthy controls (odds ratio (OR), 2.912; 95% confidence interval (CI), 1.082 to 7.836; p = 0.034). Although TNF-alpha -308 polymorphism in patients with H pylori was not significantly different from that in patients without H pylori, the -308A polymorphism was strongly associated with H pylori cagA subtype infection when compared with the polymorphism in cagA negative H pylori infection (OR, 8.757; 95% CI, 1.413 to 54.262; p = 0.019) and healthy controls (OR, 3.683; 95% CI, 1.343 to 10.101; p = 0.011). G to A genetic change at position -238 of the TNF-alpha gene was not significantly associated with H pylori cagA subtype infection. In addition, genetic polymorphisms at both sites of the TNF-alpha promoter in patients with H pylori infection did not correlate with the severity of disease. CONCLUSION: TNF-alpha -308A polymorphism was significantly related to infection with the H pylori cagA subtype in Korean patients with gastric disease.
Asunto(s)
Antígenos Bacterianos , Proteínas Bacterianas/genética , Infecciones por Helicobacter/genética , Helicobacter pylori/genética , Polimorfismo Genético , Gastropatías/genética , Factor de Necrosis Tumoral alfa/genética , Técnicas de Tipificación Bacteriana , Predisposición Genética a la Enfermedad , Genotipo , Infecciones por Helicobacter/complicaciones , Infecciones por Helicobacter/microbiología , Helicobacter pylori/clasificación , Humanos , Reacción en Cadena de la Polimerasa , Polimorfismo de Longitud del Fragmento de Restricción , Regiones Promotoras Genéticas , Gastropatías/microbiología , Neoplasias Gástricas/genética , Neoplasias Gástricas/microbiologíaRESUMEN
We investigated the potential association of tumor necrosis factor-alpha (TNF-alpha) promoter polymorphisms with cancers. The study included 169 patients with gastric cancer, uterine cervical cancer, colorectal cancer, or renal cell carcinoma and 92 healthy controls. The -308 and -238 polymorphisms in the TNF-alpha promoter were analyzed by PCR-restriction fragment length polymorphism (RFLP). The proportion of individuals carrying the TNF-238A allele was significantly lower in the cancer group than in the control group. The odds ratio for cancer in subjects with the TNF-238A allele was 0.25 (95% CI, 0.10-0.64). No association was found between the -308 polymorphism and cancers. These results suggest that the -238A allele has a protective function against cancers.
Asunto(s)
Predisposición Genética a la Enfermedad , Neoplasias/genética , Polimorfismo Genético , Factor de Necrosis Tumoral alfa/genética , Frecuencia de los Genes , Genotipo , Humanos , Polimorfismo de Longitud del Fragmento de Restricción , Polimorfismo de Nucleótido Simple , Regiones Promotoras GenéticasRESUMEN
Tumor necrosis factor-alpha (TNF-alpha) is a cytokine that induces apoptosis in various cell systems by binding to the TNF receptor (TNFR). To study TNF-alpha-induced apoptosis, we isolated and characterized a novel TNF-alpha-resistant variant, U937/TNF clone UA, from human monocytic leukemia U937 cells. The UA cells resist apoptosis induced by TNF-alpha and anti-Fas antibody but not by anticancer drugs, such as VP-16 and Ara-C. Somatic cell hybridization between U937 and UA showed that apoptosis resistance to TNF-alpha in UA was genetically recessive. The hybridization analysis also showed that UA and another recessive mutant clone, UC, belong to different complementation groups in TNF-alpha-induced apoptosis signaling. In UA cells, TNF-alpha-induced disruption of mitochondrial membrane potential and CPP32 activation were abrogated. Expression of TNFR, Fas, and Bcl-2 family proteins was not changed in UA cells. These results suggest that the apoptosis resistant UA cells could have a functional defect in apoptosis signaling from the TNFR to mitochondria and interleukin-1beta converting enzyme (ICE) family protease activation. UA cells could be used to study signaling linkage between cell death-inducing receptor and mitochondria.
Asunto(s)
Apoptosis/genética , Leucemia Mieloide/genética , Mutación , Factor de Necrosis Tumoral alfa/farmacología , Apoptosis/efectos de los fármacos , Genes Recesivos , Humanos , Leucemia Mieloide/patología , Transducción de Señal/efectos de los fármacos , Células Tumorales CultivadasRESUMEN
A site-directed mutagenesis in AprP, an alkaline protease isolated from Pseudomonas sp. KFCC 10818 was carried out in order to obtain increased thermostability. Sites for cysteine substitutions to form disulfide bond within AprP were chosen by comparing the sequences with aqualysin I, an alkaline thermostable serine protease whose disulfide bonds seems to be important for its thermostability. Gly199 and Phe236 residues were each replaced with cysteine by site-directed mutagenesis. The G199C/F236C mutant enzyme appeared to form a disulfide bond spontaneously during its expression. It also showed improved kinetic parameters for the hydrolysis of a synthetic peptide substrate at pH 8.5 and 10.5 compared to those of the wild-type enzyme. The half-life of the G199C/F236C mutant was found to be 2 to 4.8 times longer than that of wild-type under various experimental conditions, except when tested under reducing condition, where no significant differences in the half-life of the two types were observed. Therefore, it is concluded that the introduction of the disulfide bond enhanced the thermostability and the catalytic efficiency of the enzyme AprP.
Asunto(s)
Proteínas Bacterianas , Disulfuros/química , Pseudomonas/enzimología , Serina Endopeptidasas/metabolismo , Fosfatasa Alcalina , Secuencia de Bases , Calcio/metabolismo , Catálisis , Estabilidad de Enzimas , Semivida , Calor , Concentración de Iones de Hidrógeno , Cinética , Datos de Secuencia Molecular , Homología de Secuencia de Aminoácido , Serina Endopeptidasas/químicaRESUMEN
Large crystals of carboxylesterase from Pseudomonas fluorescens have been grown in the presence of dioxane using ammonium sulfate and lithium sulfate as precipitant. They belong to the tetragonal space group P4(1)2(1)2 (or P4(3)2(1)2), with unit cell dimensions of a = 82.01 (+/-0.06) A and c = 145.44 (+/- 0.08) A. The presence of one carboxylesterase dimer in the asymmetric unit gives the crystal volume per protein mass (VM) of 2.56 A3/Da and solvent fraction of 52.0% by volume. The crystals diffract to at least 2.3 A Bragg spacing when exposed to CuK alpha X rays from a rotating anode generator. X-ray data (nearly complete to 2.6 A Bragg spacing) have been collected from a native crystal.
Asunto(s)
Hidrolasas de Éster Carboxílico/química , Pseudomonas fluorescens/enzimología , Hidrolasas de Éster Carboxílico/genética , Hidrolasas de Éster Carboxílico/aislamiento & purificación , Clonación Molecular , Cristalización , Escherichia coli/genética , Proteínas Recombinantes/química , Proteínas Recombinantes/aislamiento & purificación , Difracción de Rayos XRESUMEN
In order to develop a reliable and inexpensive serodiagnostic method, part of the transmembrane glycoprotein gene of HIV-1, gp41', (HIV-env 548-646) was cloned into an expression vector, pCT10 with a sequence encoding a hydroxylamine cleavage site and with a part of Lac Z gene (Lac 2": 834 base pairs) as a fusion partner. Overexpression of Lac Z"-gp41' was induced in E. coli and the gp41' fusion protein was purified to homogeneity by centrifugation, hydroxylamine cleavage and an ion-exchange chromatography. Western blot analysis and enzyme-linked immunosorbant assay (ELISA) using the purified gp41 fragment showed high sensitivity and specificity of gp41 as an antigen to detect anti HIV-1 antibodies in testing human sera. These results suggest that this simple and rapid purification method is reliable for obtaining a large quantity of purified gp41'.
Asunto(s)
Proteína gp41 de Envoltorio del VIH/aislamiento & purificación , VIH-1/inmunología , Virología/métodos , Secuencia de Bases , Clonación Molecular , ADN Viral/genética , Ensayo de Inmunoadsorción Enzimática , Epítopos/genética , Epítopos/aislamiento & purificación , Escherichia coli/genética , Genes env , Vectores Genéticos , Proteína gp41 de Envoltorio del VIH/genética , Infecciones por VIH/diagnóstico , VIH-1/genética , Humanos , Datos de Secuencia Molecular , PlásmidosRESUMEN
The Pseudomonas fluorescens gene (estB) that encodes a novel esterase (esterase II) was cloned into Escherichia coli JM83. DNA sequencing found a single open reading frame of 654 nucleotides. The open reading frame was confirmed by N-terminal amino acid sequence analysis of the esterase protein. A potential Shine-Dalgarno sequence is followed by the coding sequence of the estB gene. The amino acid sequence deduced from the nucleotide sequence contains the consensus active site sequence, G-X-S-X-G, of serine esterases. The enzyme expressed in an E. coli clone was purified by ion-exchange chromatography and gel filtration. Homogeneity of the purified enzyme was confirmed using SDS-polyacrylamide gel electrophoresis. The native enzyme exists as a dimer consisting of two identical subunits, each with a molecular weight of 23,000. The results of the experiments for identifying substrate specificity and the inhibitor studies suggest that this enzyme is a carboxylesterase (EC 3.1.1.1) and a serine residue is present at the active site of the esterase, as in the esterases of animal tissues.