RESUMEN
BACKGROUND: Thymus-and-activation-regulated chemokine (TARC; CCL17) is related to both allergy and pregnancy, but the relationships of maternal and umbilical cord blood CCL17 to atopic dermatitis (AD) development have not yet been examined. Objective Seventy paired full-term and normal vaginal delivery newborns and their mothers were enrolled in this study. METHODS: To elucidate the pathogenesis and fetomaternal inheritance of AD in infancy, CCL17, IFN-γ-inducible protein 10 kDa (IP-10; CXCL10), soluble HLA-G (sHLA-G), IgE and eosinophil counts were examined using sera from 70 paired umbilical cord and maternal blood samples. RESULTS: Serum CCL17 (r(s) =0.340, P<0.001) and sHLA-G (r(s) =0.600, P<0.001) levels showed high correlations between umbilical cord and maternal blood. Umbilical cord serum levels of CCL17 from neonates destined to develop AD in infancy were higher than in those from neonates who showed no signs of AD during infancy (median 1586.9 vs. 819.6 pg/mL, P<0.001). Serum levels of CCL17 were higher in mothers with AD than in those without AD (median 909.6 vs. 214.1 pg/mL, P<0.001). High umbilical cord serum levels of CCL17 were associated with infantile AD development even in 62 neonates born to mothers without AD (median 1514.4 vs. 740.6 pg/mL, P<0.001) and 38 neonates born to mothers with no allergies (median 1624.2 vs. 740.6 pg/mL, P<0.001). The summary estimates for umbilical cord serum CCL17 in the diagnosis of infantile AD were: sensitivity 85.7% (95% confidence interval: 72.8-98.7), specificity 73.8% (60.5-87.1), positive predictive value 68.6% (53.2-84.0) and negative predictive value 88.6% (78.0-99.1). CONCLUSION AND CLINICAL RELEVANCE: These findings suggest that the umbilical cord blood CCL17 may be involved in the pathogenesis of infantile AD and in fetomaternal inheritance. Serum levels of CCL17 from umbilical cord blood may be a predictive marker for AD in infancy.
Asunto(s)
Quimiocina CCL17/sangre , Dermatitis Atópica/sangre , Dermatitis Atópica/congénito , Sangre Fetal/química , Adulto , Edad de Inicio , Biomarcadores/sangre , Estudios de Casos y Controles , Preescolar , Femenino , Humanos , Lactante , Recién Nacido , Masculino , Valor Predictivo de las Pruebas , Pronóstico , Reproducibilidad de los ResultadosRESUMEN
BACKGROUND: Seasonal allergic rhinitis (SAR) induced by Japanese cedar pollens is a major problem in Japan. Omalizumab, a humanized monoclonal anti-IgE antibody, improves symptoms associated with SAR, but a comparative study with an anti-allergy drug has not yet been conducted. OBJECTIVE: To compare the efficacy and safety of omalizumab with suplatast tosilate, a selective T-helper type 2 (Th2) cytokine inhibitor, in patients with Japanese cedar pollen-induced SAR. METHODS: A randomized, double-blind, double-dummy study was conducted in 308 Japanese patients with a history of moderate-to-severe SAR who showed a CAP-RAST value (> or =2+) specifically to Japanese cedar pollens. Patients were treated for 12 weeks with omalizumab plus placebo of suplatast tosilate or suplatast tosilate plus placebo of omalizumab. RESULTS: The mean daily nasal symptom medication scores (sum of the daily nasal symptom severity score and daily nasal rescue medication score) were significantly lower in the omalizumab group than in the suplatast tosilate group during three evaluation periods (P<0.001). The omalizumab group also had significantly lower mean daily nasal severity scores, each of the mean daily nasal and ocular symptom severity scores (sneezing, runny nose, stuffy nose, itchy nose, itchy eyes, watery eyes, and red eyes). Omalizumab reduced rescue medication requirements, and the proportion of days with any rescue medication use in the omalizumab group was significantly lower. Serum-free IgE levels markedly decreased in the omalizumab group and it was associated with clinical efficacy. The adverse reaction profiles were similar between the two groups. The overall incidence of injection site reactions was higher in the omalizumab group than in the suplatast tosilate group, but all these events were of mild degree. No anti-omalizumab antibodies were detected. CONCLUSION: Omalizumab showed significantly greater improvements than suplatast tosilate in the treatment of SAR induced by Japanese cedar pollens.
Asunto(s)
Antialérgicos/uso terapéutico , Anticuerpos Monoclonales/uso terapéutico , Arilsulfonatos/uso terapéutico , Polen/inmunología , Rinitis Alérgica Estacional/tratamiento farmacológico , Compuestos de Sulfonio/uso terapéutico , Adulto , Anticuerpos Antiidiotipos , Anticuerpos Monoclonales Humanizados , Pueblo Asiatico , Método Doble Ciego , Femenino , Humanos , Inmunoglobulina E/sangre , Japón , Masculino , Persona de Mediana Edad , Omalizumab , Rinitis Alérgica Estacional/inmunología , Resultado del TratamientoRESUMEN
BACKGROUND: Gammadelta T cells have been described as one of immune regulators in patients with infection, malignancy, and allergy. OBJECTIVE: To elucidate the ability of gammadelta T cells as an allergen immunotherapy candidate, the effectiveness of human gammadelta T cells in allergen-specific T-helper type 2 (Th2)-type T cells was evaluated in vitro. METHODS: House dust mite-specific Th2-type T cell clones, Bacillus Calmette-Guerin (BCG)-specific Th1-type T cell clones, and gammadelta T cell lines were established from the peripheral blood mononuclear cells of two patients with allergic rhinitis. The effectiveness of gammadelta T cells and BCG-specific Th1-type T cell clones in the modulation of allergen-specific Th2 cells in terms of their cytokine productions was evaluated. RESULTS: In response to cognate antigens, the gammadelta T cell lines demonstrated a proliferation and production of IFN-gamma that exceeded that of BCG-specific Th1-type T cell clones (mean stimulation index: 14.5 vs. 2.8, mean IFN-gamma: 130.5 vs. 10.0 pg/mL). When the gammadelta T cell lines and mite-allergen-specific Th2 clones were co-cultured with each other, only the levels of IL-4 (mean, -87%) decreased, but not the levels of IL-5 and IL-13, with an increasing concentration of gammadelta T cell antigen and IFN-gamma production (mean, +730%). CONCLUSION: These results demonstrated that gammadelta T cells derived from allergic patients might thus have a partial ability to modulate allergen-specific Th2-skewed immunity.
Asunto(s)
Antígenos Dermatofagoides/inmunología , Receptores de Antígenos de Linfocitos T gamma-delta/inmunología , Subgrupos de Linfocitos T/inmunología , Células Th2/inmunología , Adulto , Animales , Células Presentadoras de Antígenos/citología , Células Presentadoras de Antígenos/inmunología , Células Presentadoras de Antígenos/metabolismo , Antígenos/inmunología , Antígenos/farmacología , Antígenos Dermatofagoides/farmacología , Proteínas de Artrópodos , Proliferación Celular/efectos de los fármacos , Células Clonales/citología , Células Clonales/inmunología , Células Clonales/metabolismo , Técnicas de Cocultivo , Humanos , Inmunofenotipificación , Interferón gamma/metabolismo , Interleucina-13/metabolismo , Interleucina-4/metabolismo , Interleucina-5/metabolismo , Leucocitos Mononucleares/citología , Leucocitos Mononucleares/inmunología , Leucocitos Mononucleares/metabolismo , Activación de Linfocitos/efectos de los fármacos , Activación de Linfocitos/inmunología , Masculino , Rinitis Alérgica Perenne/sangre , Rinitis Alérgica Perenne/inmunología , Subgrupos de Linfocitos T/citología , Subgrupos de Linfocitos T/metabolismo , Linfocitos T/citología , Linfocitos T/inmunología , Linfocitos T/metabolismo , Células TH1/inmunología , Células TH1/metabolismo , Células Th2/citología , Células Th2/metabolismo , Tuberculina/inmunología , Tuberculina/farmacologíaRESUMEN
OBJECTIVE: Aseptic loosening is one of the most important problems that can occur after total hip arthroplasty (THA). In this study, we analysed levels of large tenascin-C (TN-C) variants and compared them in pseudosynovial fluid from patients with aseptic loosening after THA with those in synovial fluid from patients undergoing primary THA (control). METHODS: Pseudosynovial fluid samples (n = 24) were obtained by aspiration at the time of revision THA performed due to aseptic loosening. Synovial fluid samples (n = 12) were obtained by aspiration at the time of primary THA. Expression of TN-C splice variants was examined using immunoblotting. TN-C levels were measured using an enzyme-linked immunosorbent assay (ELISA) system that we developed previously. RESULTS: Western blotting showed the presence of large TN-C variants in pseudosynovial fluid of artificial joints with loosening. TN-C levels were approximately three times higher in pseudosynovial fluid of loose artificial joints (median 151.9 ng/mL) than in synovial fluid controls (median 50.1 ng/mL) (p = 0.035). CONCLUSION: Levels of TN-C including large variant subunits are elevated in pseudosynovial fluid of loose artificial joints, indicating that TN-C is a useful novel biochemical marker of loose hip prostheses.
Asunto(s)
Prótesis de Cadera , Osteoartritis de la Cadera/metabolismo , Falla de Prótesis , Líquido Sinovial/metabolismo , Tenascina/metabolismo , Anciano , Anciano de 80 o más Años , Artroplastia de Reemplazo de Cadera/efectos adversos , Western Blotting , Ensayo de Inmunoadsorción Enzimática , Femenino , Humanos , Masculino , Persona de Mediana Edad , Osteoartritis de la Cadera/cirugíaRESUMEN
Glucagon-like peptide-1 (GLP-1) is an incretin, which induces glucose-dependent insulin secretion. GLP-1 is rapidly degraded by dipeptidyl peptidase IV (DPPIV) after its release. We investigated whether DPPIV-deficient F344/DuCrj rats show improved glucose tolerance when compared with DPPIV-positive F344/Jcl rats. Oral glucose tolerance test indicated improved glucose tolerance in F344/DuCrj rats, but blood glucose levels of the two strains were almost the same 120 min after the glucose bolus. Valine-pyrrolidide, a DPPIV inhibitor, had no effect on the glucose tolerance of F344/DuCrj rats, but improved that of F344/Jcl rats. Enhanced insulin secretion and high plasma active GLP-1 levels were detected in an intraduodenal glucose tolerance test. Glucose tolerance is improved in DPPIV-deficient F344/DuCrj rats via enhanced insulin release mediated by high active GLP-1 levels. Our results suggest that DPPIV inhibition is a rational strategy to treat diabetic patients by improving glucose tolerance with low risk of hypoglycemia.
Asunto(s)
Dipeptidil Peptidasa 4/metabolismo , Glucosa/metabolismo , Insulina/metabolismo , Animales , Glucemia/efectos de los fármacos , Glucemia/genética , Dipeptidil Peptidasa 4/efectos de los fármacos , Dipeptidil Peptidasa 4/genética , Duodeno/metabolismo , Inhibidores Enzimáticos/farmacología , Glucagón/sangre , Péptido 1 Similar al Glucagón , Glucosa/administración & dosificación , Prueba de Tolerancia a la Glucosa , Secreción de Insulina , Intubación Gastrointestinal , Masculino , Fragmentos de Péptidos/sangre , Precursores de Proteínas/sangre , Pirroles/farmacología , Ratas , Ratas Endogámicas F344 , Ratas Mutantes , Especificidad de la Especie , Valina/farmacologíaRESUMEN
Isoprostane (8-epi-prostaglandin F2alpha) is synthesized non-enzymatically from arachidonate and active oxygen. We examined the relationship of smoking and excretion of isoprostane in urine with gas chromatography-mass spectrometry selected ion monitoring assay and the stable isotope dilution method. Urine isoprostane concentrations were significantly higher in smokers (n=81, 605.24+/-59.01 ng/mg creatinine) than in non-smokers (n=39, 424.07+/-70.37 ng/mg creatinine), but concentrations in ex-smokers (n=21, 487.27+/-98.48 ng/mg creatinine) did not differ significantly from those in the other groups. In smokers, age, the duration of smoking, and the number of cigarettes per day were not correlated with urine isoprostane concentrations. However, urine isoprostane concentrations were negatively correlated with time since quitting in ex-smokers and with age in non-smokers. These results indicate that smoking increases isoprostane concentration in urine and suggest that smoking causes lipid peroxidation by oxidant stress.
Asunto(s)
Dinoprost/orina , Cromatografía de Gases y Espectrometría de Masas/métodos , Estrés Oxidativo , Fumar/orina , Adulto , Dinoprost/análogos & derivados , Humanos , Masculino , Persona de Mediana EdadAsunto(s)
Sangre Fetal/citología , Mastocitos/citología , Mastocitos/inmunología , Azul Alcián , Anticuerpos Antiidiotipos/farmacología , Calcimicina/farmacología , Células Cultivadas , Fijadores , Formaldehído , Liberación de Histamina , Humanos , Inmunoglobulina E/inmunología , Ionóforos/farmacología , Pulmón/citología , Coloración y EtiquetadoRESUMEN
The radial artery is currently used as a viable arterial conduit for myocardial revascularization. The aims of this study were to identify phosphodiesterase 3 isoenzyme in the human radial artery isolated for coronary artery bypass grafting, and to examine the vasorelaxant effect of a cardiotonic and vasodilating phosphodiesterase 3 inhibitor, 1, 2-dihydro-6-methyl-2-oxo-5-(imidazo[1,2-a]pyridin-6-yl)-3-pyridine carbonitrile hydrochloride monohydrate (olprinone). The phosphodiesterase 3 isoenzyme was separated from the radial artery by DEAE-Sepharose chromatography. Olprinone inhibited the phosphodiesterase 3 activity with an IC(50) value of 1.25 microM. Olprinone relaxed the phenylephrine-induced contractions of endothelium-denuded arterial strips with an EC(50) value of 0. 107+/-0.029 microM (n=5). These findings indicate that the human radial artery possesses phosphodiesterase 3 isoenzyme activity and olprinone causes potent relaxation of the arterial strip in vitro through inhibition of phosphodiesterase 3 isozyme activity.
Asunto(s)
Imidazoles/farmacología , Músculo Liso Vascular/efectos de los fármacos , Inhibidores de Fosfodiesterasa/farmacología , Piridonas/farmacología , Arteria Radial/efectos de los fármacos , Vasodilatadores/farmacología , Endotelio Vascular/efectos de los fármacos , Humanos , Técnicas In Vitro , Indicadores y Reactivos , Isoenzimas/antagonistas & inhibidores , Isoenzimas/aislamiento & purificación , Isoenzimas/metabolismo , Hidrolasas Diéster Fosfóricas/aislamiento & purificaciónRESUMEN
When oncomiracidia of Neobenedenia girellae (Monogenea, Capsalidae) were incubated in wells with lyophilized extracts of fish skin epithelia on the bottom, some attached to the well bottom with the haptor unfolded and shed the ciliated epidermal cells. Based on these morphological changes in oncomiracidia, we developed a new assay method to examine the attachment-inducing capacities of various fish extracts for oncomiracidia. Attachment-inducing capacities were found only in extracts of fish skin epithelium and not in other fish extracts. No significant difference in capacities was observed among extracts of skin epithelia of 4 fish species. Wheat germ lectin and concanavalin A suppressed capacities in extracts of skin epithelia of Japanese flounder and yellowtail, respectively. Suppressed capacities were recovered by adding sugars that bound specifically to these lectins. These results indicate that some sugar-related chemical substances that exist specifically in fish epithelium induce the attachment of N. girellae oncomiracidia.
Asunto(s)
Piel/parasitología , Extractos de Tejidos/fisiología , Trematodos/fisiología , Adhesividad , Animales , Metabolismo de los Hidratos de Carbono , Cilios/efectos de los fármacos , Cilios/fisiología , Relación Dosis-Respuesta a Droga , Epitelio/química , Epitelio/efectos de los fármacos , Epitelio/parasitología , Lenguado , Lectinas/farmacología , Oncorhynchus mykiss , Perciformes , Proteínas/fisiología , Piel/química , Piel/efectos de los fármacos , Extractos de Tejidos/química , Trematodos/ultraestructuraRESUMEN
Omega-3 polyunsaturated fatty acids have anti-inflammatory effects in vitro, and high dietary levels are associated with a lower incidence of inflammatory diseases. However, only limited effects have been demonstrated in asthma. The effects of dietary supplementation with fish oil for 10 months in 29 children with bronchial asthma was investigated in a randomized controlled fashion. In order to minimize the effects of environmental inhaled allergens and diet, this study was performed in a long-term treatment hospital. Subjects received fish oil capsules containing 84 mg eicosapentaenoic acid (EPA) and 36 mg docosahexaenoic acid (DHA) or control capsules containing 300 mg olive oil. The daily dosages of EPA and DHA were 17.0-26.8 and 7.3-11.5 mg x kg body weight(-1), respectively. Asthma symptom scores decreased and responsiveness to acetylcholine decreased in the fish oil group but not in the control group. In addition, plasma EPA levels increased significantly only in the fish oil group (p<0.0088). No significant side-effects were observed. The present results suggest that dietary supplementation with fish oil rich in the omega-3 polyunsaturated fatty acids eicosapentaenoic acid and docosahexaenoic acid is beneficial for children with bronchial asthma in a strictly controlled environment in terms of inhalant allergens and diet.
Asunto(s)
Asma/tratamiento farmacológico , Ácidos Docosahexaenoicos/administración & dosificación , Aceites de Pescado/administración & dosificación , Acetilcolina , Asma/fisiopatología , Pruebas de Provocación Bronquial , Niño , Suplementos Dietéticos , Ácidos Docosahexaenoicos/uso terapéutico , Método Doble Ciego , Ácido Eicosapentaenoico/administración & dosificación , Ácido Eicosapentaenoico/sangre , Ácido Eicosapentaenoico/uso terapéutico , Femenino , Aceites de Pescado/sangre , Aceites de Pescado/uso terapéutico , Humanos , Masculino , Aceite de Oliva , Aceites de Plantas/administración & dosificación , Aceites de Plantas/uso terapéuticoRESUMEN
A method of simultaneous analysis of prostaglandins (PGs) and thromboxane (TX) B2 in cerebrospinal fluid (CSF) with GC-MS-SIM was established. Deuterated PGs and TXB2 were used as internal standards: tetra-deuterated PGE2 (d4-PGE2) for PGE2, PGE1 and PGD2; d5-PGF2alpha for PGF2alpha and 9alpha,11beta-PGF2 and 8-epi PGF2alpha; d4-TXB2 for TXB2; and d4-6-keto PGF1alpha for 6-keto PGF1alpha. The PGs and TXB2 were derivatized to the methyl ester of the methoxim dimethyisopropylsilyl (DMiPSi) ether form or the methyl ester of the DMiPSi ether form with simultaneous preparation. Samples were extracted with octadecyl silica gel and purified in two steps with silisic acid gel chromatography between derivatization steps. The calibration curve of each PG and TXB2 was linear from 10 pg to 10 ng with the isotope dilution method. The levels of the seven types of PG and of TXB2 were assayed simultaneously in the cerebrospinal fluid (CSF) from patients with aseptic meningitis. The CSF pattern of the PG and TXB2 concentrations in mumps meningitis differed from those in other types of aseptic meningitis and in disease controls.
Asunto(s)
Cromatografía de Gases y Espectrometría de Masas/métodos , Prostaglandinas/líquido cefalorraquídeo , Tromboxano B2/líquido cefalorraquídeo , Calibración , Niño , Preescolar , Humanos , Estándares de ReferenciaRESUMEN
BACKGROUND: Eicosapentaenoic acid (EPA) is catalysed by cyclo-oxygenase (COX), as is arachidonic acid, and is a competitive inhibitor of arachidonate metabolism. OBJECTIVES: We examined the effect of EPA on prostaglandin (PG) D2 generation in the cultured human mast cells with IgE-anti-IgE challenge incubation. METHODS: Cultured human mast cells were incubated with EPA (1 micromol/L) for 20 h, then challenged with anti-IgE incubation after treatment with IgE. At the same time, COX inhibitors were tested to identify COX-1 and COX-2 activity. PGD2 synthetic activity was also assayed in a cell-free homogenate of cultured mast cells with COX inhibitors and EPA. Histamine in the culture medium and in cells was assayed with the HPLC-fluorescent method. PGD2 and PGD3 were assayed with gas chromatography-mass spectrometry and the stable isotope dilution method. RESULTS: Although EPA incubation did not affect histamine release by cultured human mast cells in response to IgE-anti-IgE challenge incubation, it did decrease PGD2 generation by inhibiting the COX-2 pathway. In contrast, in the cell-free homogenate of cultured human mast cells, EPA inhibited both COX-1 and COX-2 activities. CONCLUSION: Pre-incubation with EPA primarily affects the COX-2 pathway in cultured human mast cells and reduces PGD2 generation in response to IgE-anti-IgE challenge incubation. These findings suggest that COX-1 and COX-2 have different substrate flow systems in mast cells. They also suggest that endogenous EPA diet supplementation would reduce PGD2 production and could serve as an anti-inflammatory substrate in human mast cells.
Asunto(s)
Inhibidores de la Ciclooxigenasa/farmacología , Ácido Eicosapentaenoico/farmacología , Isoenzimas/metabolismo , Mastocitos/enzimología , Prostaglandina D2/metabolismo , Prostaglandina-Endoperóxido Sintasas/metabolismo , Anticuerpos/inmunología , Células Cultivadas , Ciclooxigenasa 2 , Inhibidores de la Ciclooxigenasa 2 , Cromatografía de Gases y Espectrometría de Masas/métodos , Liberación de Histamina , Humanos , Inmunoglobulina E/inmunología , Mastocitos/efectos de los fármacos , Proteínas de la MembranaRESUMEN
BACKGROUND: 9Alpha,11beta-prostaglandin (PG) F2 is an initial metabolite of PGD2 which has a potent bronchoconstrictive activity. OBJECTIVES: We measured the urinary levels of 9alpha,11beta-PGF2 in asthmatic children to investigate its role in not only acute asthmatic attack in a time course study but also in exercise-induced asthma (EIA). METHODS: In the acute asthma study, 30 asthmatic children were examined. Urine samples were collected on the first, third, and sixth days. Urinary levels of 9alpha,11beta-PGF2 were measured with gas chromatography mass spectrometry using the electron impact method. In the exercise challenge study, 14 children with EIA and 14 children without EIA were studied. Urine samples were collected before exercise challenge, and at 1 h, and 5 h after exercise challenge. Urinary levels of 9alpha,11beta-PGF2 were measured. RESULTS: Elevated urinary levels of 9alpha,11beta-PGF2, which were observed on the first day when treatment was started in the hospital, were gradually decreased on the third day (P < 0.05), and on the sixth day (P < 0.01). A significant correlation between urinary levels of 9alpha,11beta-PGF2 and symptom scores (P < 0.005) was observed on the first day. In EIA, there was a significant increase in urinary levels of 9alpha,11beta-PGF2 at 1 h (P < 0.01) and at 5 h (P < 0.01) after exercise challenge, but not in the children without EIA. CONCLUSION: 9Alpha,11beta-PGF2 may be involved in the pathogenesis of acute and exercise-induced asthma in children.
Asunto(s)
Asma Inducida por Ejercicio/orina , Asma/orina , Dinoprost/orina , Cromatografía de Gases y Espectrometría de Masas/métodos , Enfermedad Aguda , Adolescente , Niño , Preescolar , Femenino , Cromatografía de Gases y Espectrometría de Masas/estadística & datos numéricos , Humanos , Masculino , Factores de TiempoRESUMEN
Monoclonal antibody alpha110 recognizes Leu-456 in the alpha subunit of the Escherichia coli F1-ATPase. Binding of this antibody to the alpha subunit or mutation of this residue to Pro caused enhancement of the ATPase activity, suggesting that this residue is involved in the catalytic mechanism of this molecule (H. Kanazawa et al. (1995) Arch. Biochem. Biophys. 317, 348-356). Leu-456 together with Gly-454 and Tyr-455 are the only residues in the carboxy-terminal 75 amino acids conserved among various species, suggesting that these three residues play important roles in catalysis by the ATPase. Here, we introduced site-directed mutations into these residues. Not only L456P but also G454L, Y455K, Y455L, and L456N mutations caused enhancement of the ATPase activity. Surprisingly, Y455V, L456H, and L456S caused assembly defects of F1 subunits on the membrane. Reconstitution of the alpha betagamma complex from the wild-type beta and gamma subunits with the mutant alpha subunit (L4gamma6P) exhibited enhanced ATPase activity. Addition of delta or epsilon fused to glutathione S-transferase which are functionally similar to the delta and epsilon subunits, respectively, to the reconstituted F1-ATPase did not cause significant enhancement of its activity. Decreased interaction between alpha and beta subunits with the L456P mutation was detected by the yeast two-hybrid system. According to the deduced three-dimensional structure of the bovine a subunit, Leu-456, Gly-454, and Tyr-455 are included in a small alpha helix. These results suggest that this alpha helix affects interaction of the alpha subunit with the beta subunit but not with delta or epsilon, which may be important for the catalytic mechanism and F1 assembly.
Asunto(s)
Escherichia coli/enzimología , ATPasas de Translocación de Protón/química , ATPasas de Translocación de Protón/metabolismo , Secuencia de Aminoácidos , Animales , Anticuerpos Monoclonales , Sitios de Unión , Catálisis , Bovinos , Secuencia Conservada , Cartilla de ADN/genética , Escherichia coli/genética , Modelos Moleculares , Datos de Secuencia Molecular , Estructura Molecular , Mutagénesis Sitio-Dirigida , Mutación Puntual , Conformación Proteica , ATPasas de Translocación de Protón/genética , Saccharomyces cerevisiae/genética , Homología de Secuencia de Aminoácido , Especificidad de la EspecieRESUMEN
To elucidate the characterization of cultured human mast cells, the generation of prostaglandin D2 (PGD2) upon stimulation with IgE-anti IgE challenge was determined by using gas chromatography/mass spectrometry/selected ion monitoring (GC/MS/SIM) assay. Mononuclear cells obtained from human umbilical cord blood were cultured in the presence of Steel Factor, IL-6, and PGE2. After 8 weeks of culture, approximately 90 to 95% of cultured cells became tryptase-positive and included basophilic granules. At 12 weeks of culture, cells were harvested and incubated with 1 microgram/ml of human IgE for 1 hr and then challenged with 10 micrograms/ml of anti-IgE. The level of PGD2 release into the supernatant was measured by GC/MS/SIM with the stable isotope dilution method. Cyclooxygenase inhibitors affected the PGD2 release from the cell by IgE-anti IgE challenge incubation. Indomethacin inhibited 98% of PGD2 release. Acetylsalicylic acid (aspirin) and NS-398 (potent COX-2 inhibitor) also inhibited PGD2 release by 85 and 45% respectively.
Asunto(s)
Anticuerpos Antiidiotipos/farmacología , Inmunoglobulina E/farmacología , Mastocitos/inmunología , Mastocitos/metabolismo , Prostaglandina D2/metabolismo , Diferenciación Celular , Células Cultivadas , Quimasas , Sangre Fetal/citología , Cromatografía de Gases y Espectrometría de Masas , Humanos , Inmunohistoquímica , Mastocitos/ultraestructura , Serina Endopeptidasas/metabolismo , TriptasasRESUMEN
BACKGROUND: Allergen avoidance is recommended when treating atopic asthma. OBJECTIVES: Soft toys are often kept in close proximity to children and may serve as a source of exposure. Due to the potential danger from the mite allergen content of these toys, Der 1 from toys was measured. METHODS: We quantified the level of Der 1 (Der p 1 + Der f 1) in both 30 new and 174 old soft toys (weight 216 +/- 2.5 g, height 26.5 +/- 3.4 cm), as well as in washed and in vacuumed soft toys. Dust was collected using an electric vacuum cleaner. Der 1 was measured by monoclonal antibody-based enzyme-linked immunosorbent assay (MoAb-ELISA). RESULTS: In brand new toys Der 1 was 0.1 microgram/g of dust, and in toys used for 1 year, 9.0; 2 year, 22.2; 3 year, 18.9; and more than 4 year, 22.7 micrograms/g of dust. Der 1 in new toys was measured every 4 months for 1 year. Der 1 rapidly increased 10- to 20-fold in the first 4 months, but there was no clear seasonal change. In toys washed using a chemical detergent or cleaned using a vacuum cleaner, there was a stastistically significant (P < 0.001) decrease in Der 1 in the washed group, but not in the vacuumed group. CONCLUSION: These results confirm that mite allergens accumulate rapidly in toys to form a potentially important source of allergens and that washing toys with a chemical detergent is effective in the reduction of allergens.
Asunto(s)
Alérgenos/efectos adversos , Alérgenos/análisis , Glicoproteínas/inmunología , Juego e Implementos de Juego , Antígenos Dermatofagoides , Humanos , Factores de TiempoRESUMEN
Systems for overexpression and purification of active alpha, beta and gamma subunits of Escherichia coli H(+)-ATPase were established. The alpha and beta subunits recovered as soluble form were purified by hydroxyapatite column chromatography. Since the gamma subunit was overexpressed as the insoluble form, this subunit was purified by polyacrylamide gel-electrophoresis containing sodium dodecyl sulfate. By subsequent denaturation of this subunit with guanidine hydrochloride and renaturation, the active gamma subunit for reconstitution of the F1-ATPase activity with the purified alpha and beta subunit was obtained. The delta and epsilon subunits which were fused to the carboxy terminus of glutathione S-transferase (GST) were overproduced and purified by affinity chromatography. These fused proteins (delta-GST and epsilon-GST) were incubated with the purified alpha, beta and gamma subunits and applied to affinity chromatography. The alpha beta gamma delta-GST and alpha beta gamma epsilon-GST complex were eluted specifically by addition of glutathione and exhibited high and low ATPase activity, respectively, with a subunit stoichiometry similar to that in the native F1-ATPase, indicating that active complexes could be reconstituted with the fused proteins. These results suggested that the amino-terminal ends of the delta and epsilon subunits are not involved in formation of the active complex. The fused epsilon-GST bound the gamma subunit strongly, and the alpha subunit weakly. The delta-GST bound the gamma subunit significantly, and the alpha and beta subunits very weakly.
Asunto(s)
Escherichia coli/enzimología , Glutatión Transferasa/química , ATPasas de Translocación de Protón/química , Secuencia de Bases , Glutatión , Glutatión Transferasa/aislamiento & purificación , Ligandos , Datos de Secuencia Molecular , Plásmidos , ATPasas de Translocación de Protón/aislamiento & purificación , Proteínas Recombinantes de Fusión/químicaRESUMEN
Sensitive and specific assay methods for 9 alpha, 11 beta-prostaglandin F2 (9 alpha, 11 beta-PGF2) by gas chromatography-mass spectrometry with electron impact ionization are described. The mass spectrometric assay for 9 alpha, 11 beta-PGF2 was based on the use of the methyl ester-dimethylisopropylsilyl ether derivative, and pentadeuterated PGF2 alpha as a convenient internal standard. The calibration graph for 9 alpha, 11 beta-PGF2 was linear from 5 pg to 100 ng for both the standard and spiked biological samples. The limit of detection was 50 pg/ml for urine and 25 pg/ml for plasma (signal-to-noise ratio = 2.3). The method was applied to the determination of 9 alpha, 11 beta-PGF2 in urine and plasma samples from patients with bronchial asthma.