RESUMEN
BACKGROUND: Recent evidence indicates that TNF-α is a key mediator of the development of dsRNA-enhanced Th2 cell response to inhaled allergens. Natural killer T (NKT) cells may be a candidate source of Th2-polarizing cytokines. OBJECTIVE: The objective of this study was to evaluate the role of lung NKT cells on the development of TNF-α-mediated Th2 cell response. METHODS: A virus-associated asthma mouse model was generated by the administration of ovalbumin (OVA, 75 µg) and poly[I:C] (0.1 µg). Role of NKT and type I NKT cells was evaluated using CD1d- and Jα18-deficient mice. TNF-α receptors (TNFRs) were antagonized by using TNFR blocking peptides. RESULTS: The number of infiltrated NKT cells was increased in a virus-associated asthma mouse model. Increase in Th2 and Th17 cytokine levels in wild-type mice were abolished in both CD1d- and Jα18-deficient mice. In vitro co-culture experiments with alveolar macrophages and NKT cells showed that TNF-α produced by macrophages in the presence of poly[I:C] acts on NKT cells, inducing production of Th2-polarizing cytokines. Moreover, the induction of Th2-polarizing cytokines by poly[I:C] or recombinant TNF-α was impaired in both CD1d- and Jα18-deficient mice and that the above effect was reversed by a TNF-α receptor-2 (TNFR2) blocking peptide, but not by a TNFR1 blocker. CONCLUSIONS: These findings suggest that NKT cells play a key role in the development of Th2 cell response to inhaled allergens and that TNF-α produced by alveolar macrophages induces Th2 cell response, via TNFR2 on NKT cells.
Asunto(s)
Asma/inmunología , Células T Asesinas Naturales/inmunología , Receptores Tipo II del Factor de Necrosis Tumoral/inmunología , Células Th2/inmunología , Factor de Necrosis Tumoral alfa/inmunología , Alérgenos/inmunología , Animales , Hiperreactividad Bronquial/inmunología , Técnicas de Cocultivo , Modelos Animales de Enfermedad , Ensayo de Inmunoadsorción Enzimática , Citometría de Flujo , Hipersensibilidad/inmunología , Activación de Linfocitos/inmunología , Macrófagos Alveolares/inmunología , Ratones , Neumonía/inmunología , ARN Bicatenario/inmunologíaRESUMEN
BACKGROUND: Chronic rhinosinusitis (CRS) is an inflammatory process in the nasal cavity and paranasal sinuses, and bacteria have been considered to be a cause. Indeed, recent evidence indicates that bacteria-derived extracellular vesicles (EV) appear to be an important causative agent of inflammatory diseases. Here, we aimed to evaluate the diversity of nasal microbiota and their secreted EV in patients with CRS. METHODS: Nasal lavage (NAL) fluid samples were obtained from five patients with CRS with polyposis, three patients with CRS without polyposis, and three non-CRS controls. After preparation of bacteria and EV from samples using differential centrifugation, genomic DNA was extracted and 16S-rDNA amplicons were subjected to high-throughput pyrosequencing on a Roche 454 GS-FLX platform. RESULTS: Metagenomics showed that bacteria composition was positively correlated with EV composition. Samples from patients with CRS had greater bacterial abundance and lower diversity, both from bacteria and the EV portion of samples, compared with non-CRS samples. At each phylogenetic level, Bacteroidetes decreased while Proteobacteria increased in the CRS group at the phylum level. At the genus level, Prevotella spp. decreased in the CRS group, while Staphylococcus spp. increased from both bacteria and EV. Moreover, Staphylococcus aureus and its secreting EV compositions were higher in samples from CRS with polyps compared with CRS without polyps. CONCLUSIONS: These results suggest that patients with CRS have altered nasal microbiota and decreased diversity in bacterial compositions as well as increased S. aureus abundance in those patients with polyps.
Asunto(s)
Microbiota , Mucosa Nasal/microbiología , Rinitis/microbiología , Sinusitis/microbiología , Adulto , Anciano , Bacterias/clasificación , Bacterias/genética , Biodiversidad , Exosomas , Femenino , Humanos , Masculino , Metagenoma , Persona de Mediana Edad , Mucosa Nasal/patología , Filogenia , ARN Ribosómico 16S , Rinitis/patología , Sinusitis/patología , Adulto JovenRESUMEN
BACKGROUND: Viral pathogen-associated molecular patterns, such as dsRNA, disrupt airway tolerance to inhaled allergens. Specifically, the Th2 and Th17 cell responses are induced by low-dose dsRNA and the Th1-dominant response by high-dose dsRNA. OBJECTIVE: In this model, we evaluate the role of TNF-α in the development of adaptive immune dysfunction to inhaled allergens induced by airway sensitization with dsRNA-containing allergens. METHODS: A virus-associated asthma mouse model was generated via simultaneous airway administration of ovalbumin (OVA) and low (0.1 µg) or high (10 µg) doses of polyinosine-polycytidylic acid (poly[I:C]). The effect of TNF-α on Th2 airway inflammation was evaluated using TNF-α-deficient mice and recombinant TNF-α. RESULTS: TNF-α production was enhanced by airway exposure to low and high doses of poly[I:C]. After airway sensitization with OVA plus low-dose poly[I:C], TNF-α-deficient mice exhibited less OVA-induced airway inflammation than did wild-type (WT) mice. However, this did not occur upon sensitization with high-dose poly[I:C]. In terms of T-cell response, the production of IL-4 from lung T cells after OVA challenge was enhanced by airway sensitization with OVA plus low-dose poly[I:C] in WT mice, and this phenotype was inhibited by the absence of TNF-α. Moreover, the Th2 cell response induced by sensitization with OVA plus low-dose poly[I:C], which was abolished in TNF-α-deficient mice, was restored in these mice upon addition of recombinant TNF-α. CONCLUSION: The results of this study suggest that TNF-α produced by airway exposure to low-dose dsRNA is a key mediator in the development of Th2 cell response to inhaled allergens.
Asunto(s)
Alérgenos/inmunología , Asma/inmunología , Asma/fisiopatología , ARN Bicatenario/inmunología , ARN Viral/inmunología , Células Th2/inmunología , Factor de Necrosis Tumoral alfa/metabolismo , Administración por Inhalación , Alérgenos/administración & dosificación , Animales , Asma/virología , Hiperreactividad Bronquial/inmunología , Hiperreactividad Bronquial/fisiopatología , Hiperreactividad Bronquial/virología , Modelos Animales de Enfermedad , Ratones , Ratones Endogámicos C57BL , Factor de Necrosis Tumoral alfa/inmunologíaRESUMEN
BACKGROUND: Recent evidence indicates that Staphylococcus aureus, one of the most important human pathogens, secretes vesicles into the extracellular milieu. OBJECTIVE: To evaluate whether inhalation of S. aureus-derived extracellular vesicles (EV) is causally related to the pathogenesis of inflammatory pulmonary diseases. METHODS: Staphylococcus aureus EV were prepared by sequential ultrafiltration and ultracentrifugation. The innate immune response was evaluated in vitro after the application of EV to airway epithelial cells and alveolar macrophages. In vivo innate and adaptive immune responses were evaluated after airway exposure to EV. Adjuvant effects of EV on the development of hypersensitivity to inhaled allergens were also evaluated after airway sensitization with S. aureus EV and ovalbumin (OVA). RESULTS: Staphylococcus aureus and S. aureus EV were detected in house dust. Alveolar macrophages produced both tumor necrosis α (TNF-α) and interleukin 6 (IL-6) after in vitro stimulation with S. aureus EV, whereas airway epithelial cells produced only IL-6. Repeated airway exposure to S. aureus EV induced both Th1 and Th17 cell responses and neutrophilic pulmonary inflammation, mainly via a Toll-like receptor 2 (TLR2)-dependent mechanism. In terms of adjuvant effects, airway sensitization with S. aureus EV and OVA resulted in neutrophilic pulmonary inflammation after OVA challenge alone. This phenotype was partly reversed by the absence of interferon γ (IFN-γ) or IL-17. CONCLUSION: Staphylococcus aureus EV can induce Th1 and Th17 neutrophilic pulmonary inflammation, mainly in a TLR2-dependent manner. Additionally, S. aureus EV enhance the development of airway hypersensitivity to inhaled allergens.
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Vesículas Citoplasmáticas/inmunología , Neumonía , Staphylococcus aureus , Células TH1/inmunología , Células Th17/inmunología , Vesículas Citoplasmáticas/ultraestructura , Humanos , Infiltración Neutrófila/inmunología , Neutrófilos/inmunología , Neumonía/inmunología , Neumonía/fisiopatología , Staphylococcus aureus/inmunología , Staphylococcus aureus/ultraestructuraRESUMEN
BACKGROUND: Recently, we found that Staphylococcus aureus produces extracellular vesicles (EV) that contain pathogenic proteins. Although S. aureus infection has been linked with atopic dermatitis (AD), the identities of the causative agents from S. aureus are controversial. We evaluated whether S. aureus-derived EV are causally related to the pathogenesis of AD. METHODS: Extracellular vesicles were isolated by the ultracentrifugation of S. aureus culture media. The EV were applied three times per week to tape-stripped mouse skin. Inflammation and immune dysfunction were evaluated 48 h after the final application in hairless mice. Extracellular vesicles-specific IgE levels were measured by ELISA in AD patients and healthy subjects. RESULTS: The in vitro application of S. aureus EV increased the production of pro-inflammatory mediators (IL-6, thymic stromal lymphopoietin, macrophage inflammatory protein-1α, and eotaxin) by dermal fibroblasts. The in vivo application of S. aureus EV after tape stripping caused epidermal thickening with infiltration of the dermis by mast cells and eosinophils in mice. These changes were associated with the enhanced cutaneous production of IL-4, IL-5, IFN-γ, and IL-17. Interestingly, the serum levels of S. aureus EV-specific IgE were significantly increased in AD patients relative to healthy subjects. CONCLUSION: These results indicate that S. aureus EV induce AD-like inflammation in the skin and that S. aureus-derived EV are a novel diagnostic and therapeutic target for the control of AD.
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Dermatitis Atópica/inmunología , Exosomas/inmunología , Staphylococcus aureus/inmunología , Adolescente , Animales , Anticuerpos Antibacterianos/sangre , Niño , Citocinas/inmunología , Dermatitis Atópica/microbiología , Dermatitis Atópica/patología , Modelos Animales de Enfermedad , Epidermis/inmunología , Fibroblastos/metabolismo , Humanos , Inmunoglobulina E/sangre , Ratones , Factores de TiempoRESUMEN
BACKGROUND: Previous evidence indicates that inhalation of lipopolysaccharide (LPS)-containing with allergens induced mixed Th1 and Th17 cell responses in the airways. Extracellular vesicles (EVs) are nanometer-sized spherical, lipid-bilayered structures and are recently in the public eye as an intercellular communicator in immune responses. OBJECTIVE: To evaluate the role of EVs secreted by LPS inhalation in the development of airway immune dysfunction in response to allergens. METHODS: Extracellular vesicles in bronchoalveolar lavage fluids of BALB/c mice were isolated and characterized 24 h after applications to the airway of 10 µg of LPS for 3 days. To evaluate the role of LPS-induced EVs on the development of airway immune dysfunction, in vivo and in vitro experiments were performed using the isolated LPS-induced EVs. RESULTS: The inhalation of LPS enhanced EVs release into the BAL fluid, when compared to the application of PBS. Airway sensitization with allergens and LPS-induced EVs resulted in a mixed Th1 and Th17 cell responses, although that with allergens and PBS-induced EVs induced immune tolerance. In addition, LPS-induced EVs enhanced the production of Th1- and Th17-polarizing cytokines (IL-12p70 and IL-6, respectively) by lung dendritic cells. Moreover, the immune responses induced by the LPS-induced EVs were blocked by denaturation of the EV-bearing proteins. CONCLUSION: These data suggest that EVs (especially, the protein components) secreted by LPS inhalation are a key intercellular communicator in the development of airway immune dysfunction to inhaled LPS-containing allergens.
Asunto(s)
Alérgenos/farmacología , Micropartículas Derivadas de Células/inmunología , Sistema Respiratorio/inmunología , Animales , Líquido del Lavado Bronquioalveolar/inmunología , Interleucina-12/biosíntesis , Interleucina-6/biosíntesis , Lipopolisacáridos/farmacología , Ratones , Ratones Endogámicos BALB C , Células TH1/inmunología , Células Th17/inmunologíaRESUMEN
BACKGROUND: Innate immune response by a viral pathogen-associated molecular pattern dsRNA modulates the subsequent development of adaptive immune responses. Although virus-associated asthma is characterized by noneosinophilic inflammation, the role of Th17 cell response in the development of virus-associated asthma is still unknown. OBJECTIVE: To evaluate the role of the Th17 cell response and its underlying polarizing mechanisms in the development of an experimental virus-associated asthma. METHODS: An experimental virus-associated asthma was created via airway sensitization with ovalbumin (OVA, 75 µg) and a low (0.1 µg) or a high (10 µg) doses of synthetic dsRNA [polyinosine-polycytidylic acid; poly(I:C)]. Transgenic (IL-17-, IL-6-deficient mice) and pharmacologic [a vascular endothelial growth factor receptor (VEGFR) inhibitor] approaches were used to evaluate the roles of Th17 cell responses. RESULTS: After cosensitization with OVA and low-dose poly(I:C), but not with high-dose poly(I:C), inflammation scores after allergen challenge were lower in IL-17-deficient mice than in wild-type (WT) mice. Moreover, inflammation enhanced by low-dose poly(I:C), but not by high-dose poly(I:C), was impaired in IL-6-deficient mice; this phenotype was accompanied by the down-regulation of IL-17 production from T cells from both lymph nodes and lung tissues. Airway exposure of low-dose poly(I:C) enhanced the production of VEGF and IL-6, and the production of IL-6 was blocked by treatment with a VEGFR inhibitor (SU5416). Moreover, the allergen-specific Th17 cell response and subsequent inflammation in the low-dose poly(I:C) model were impaired by the VEGFR inhibitor treatment during sensitization. CONCLUSIONS: Airway exposure of low-level dsRNA induces an allergen-specific Th17 cell response, which is mainly dependent on VEGF and IL-6.
Asunto(s)
Asma/virología , Interleucina-6/biosíntesis , ARN Bicatenario/efectos adversos , ARN Viral/efectos adversos , Células Th17/inmunología , Factor A de Crecimiento Endotelial Vascular/biosíntesis , Inmunidad Adaptativa , Alérgenos/inmunología , Animales , Asma/inmunología , Relación Dosis-Respuesta a Droga , Inmunidad Innata , Ratones , Ratones Transgénicos , Ovalbúmina/farmacología , Poli I-C/farmacología , Sistema Respiratorio/inmunología , Sistema Respiratorio/virología , Especificidad del Receptor de Antígeno de Linfocitos T/efectos de los fármacos , Especificidad del Receptor de Antígeno de Linfocitos T/inmunologíaRESUMEN
BACKGROUND: We recently demonstrated that the T-helper type 1 (Th1) immune response plays an important role in the development of non-eosinophilic inflammation induced by airway exposure of an allergen plus double-stranded RNA (dsRNA). However, the role of lipoxygenase (LO) metabolites in the development of Th1 inflammation is poorly understood. OBJECTIVE: To evaluate the role of LO metabolites in the development of Th1 inflammation induced by sensitization with an allergen plus dsRNA. METHODS: A Th2-allergic inflammation mouse model was created by an intraperitoneal injection of lipopolysaccharide-depleted ovalbumin (OVA, 75 microg) and alum (2 mg) twice, and the Th1 model was created by intranasal application of OVA (75 microg) and synthetic dsRNA [10 microg of poly(I : C)] four times, followed by an intranasal challenge with 50 microg of OVA four times. The role of LO metabolites was evaluated using two approaches: a transgenic approach using 5-LO(-/-) and 15-LO(-/-) mice, and a pharmacological approach using inhibitors of cysteinyl leucotriene receptor-1 (cysLTR1), LTB4 receptor (BLT1), and 15-LO. RESULTS: We found that the Th1-allergic inflammation induced by OVA+dsRNA sensitization was similar between 5-LO(-/-) and wild-type (WT) control mice, although Th2 inflammation induced by sensitization with OVA+alum was reduced in the former group. In addition, dsRNA-induced Th1 allergic inflammation, which is associated with down-regulation of 15-hydroxyeicosateraenoic acids production, was not affected by treatment with cysLTR1 or BLT1 inhibitors, whereas it was significantly lower in 12/15-LO(-/-) mice compared with WT control mice. Moreover, dsRNA-induced allergic inflammation and the recruitment of T cells following an allergen challenge were significantly inhibited by treatment with a specific 15-LO inhibitor (PD146176). CONCLUSION: 15-LO metabolites appear to be important mediators in the development of Th1-allergic inflammation induced by sensitization with an allergen plus dsRNA. Our findings suggest that the 15-LO pathway is a novel therapeutic target for the treatment of virus-associated asthma characterized by Th1 inflammation.
Asunto(s)
Alérgenos/inmunología , Araquidonato 15-Lipooxigenasa/metabolismo , Hipersensibilidad/inmunología , Inflamación/inmunología , ARN Bicatenario/inmunología , Células TH1/inmunología , Acetatos/farmacología , Compuestos de Alumbre/farmacología , Animales , Araquidonato 15-Lipooxigenasa/genética , Araquidonato 15-Lipooxigenasa/inmunología , Araquidonato 5-Lipooxigenasa/genética , Araquidonato 5-Lipooxigenasa/inmunología , Araquidonato 5-Lipooxigenasa/metabolismo , Ciclopropanos , Modelos Animales de Enfermedad , Alcoholes Grasos/farmacología , Fluorenos/farmacología , Glicoles/farmacología , Hipersensibilidad/enzimología , Inflamación/metabolismo , Antagonistas de Leucotrieno/farmacología , Inhibidores de la Lipooxigenasa , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Ratones Noqueados , Ovalbúmina/farmacología , Poli I-C/inmunología , Quinolinas/farmacología , Receptores de Leucotrienos/efectos de los fármacos , Receptores de Leucotrienos/inmunología , Receptores de Leucotrienos/metabolismo , Receptores de Leucotrieno B4/antagonistas & inhibidores , Receptores de Leucotrieno B4/inmunología , Receptores de Leucotrieno B4/metabolismo , Sulfuros , Células TH1/enzimología , Células Th2/enzimología , Células Th2/inmunologíaRESUMEN
BACKGROUND AND OBJECTIVE: Treatment with angiotensin-converting enzyme (ACE) inhibitors can induce chronic cough in many patients. Genetic variations in the neurokinin 2 receptor gene (NK2R) are significantly associated with cough sensitivity to capsaicin. METHODS: This study assessed the relationship between genetic polymorphisms in the NK2R gene and chronic cough in 91 patients taking ACE inhibitors. Patients included in the study did not have chest abnormalities, postnasal drip, gastroesophageal reflux or a recent history of upper respiratory infection. RESULTS: We detected two single nucleotide polymorphisms in the NK2R gene (i.e., Gly231Glu and Arg375His). The allelic frequencies at amino acid 231 were 36.3% for Gly/Gly, 49.5% for Gly/Glu and 14.3% for Glu/Glu. The allelic frequencies at amino acid 375 were 74.7% for Arg/Arg, 24.2% for Arg/His and 1.1% for His/His. The prevalence of chronic cough in patients with the amino acid 231 genotype was 33.3% in Gly/Gly homozygotes, 24.4% in Gly/Glu heterozygotes and 0% in Glu/Glu homozygotes. There was a statistically significant association between chronic cough and the Glu/Glu allele (P = 0.028) when the data were analyzed with a recessive model. In addition, there was a significant inverse linear association between the number of Glu231 alleles and ACE inhibitor-related cough (P = 0.026). The prevalence of chronic cough in patients with the amino acid 375 genotype was 22.1% in Arg/Arg homozygotes, 31.8% in Arg/His heterozygotes and 0% in His/His homozygotes, although none of these association were statistically significant. CONCLUSION: Our findings indicate that the Gly231Glu polymorphism is associated with a lower prevalence of ACE inhibitor-related cough.
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Inhibidores de la Enzima Convertidora de Angiotensina/efectos adversos , Tos/inducido químicamente , Polimorfismo de Nucleótido Simple , Receptores de Neuroquinina-2/genética , Adulto , Anciano , Alelos , Enfermedad Crónica , Tos/genética , Femenino , Predisposición Genética a la Enfermedad , Variación Genética , Humanos , Masculino , Persona de Mediana Edad , Estudios ProspectivosRESUMEN
BACKGROUND: Recent studies showed that high levels of transforming growth factor (TGF)-beta1 in the airways reduced airway responsiveness, which was reversed in conditions of basic fibroblast growth factor (FGF2) deficiency, whereas high levels of vascular endothelial growth factor (VEGF) enhanced airway sensitization to allergens and airway hyperresponsiveness (AHR). OBJECTIVE: We investigated the effect of single-nucleotide polymorphisms (SNPs) in the VEGF, TGF-beta1, and FGF2 receptors on the expression of atopy and AHR in the general population. METHODS: Atopy and AHR were evaluated in a cohort of 2055 children and adolescents. Direct sequencing was used to identify informative SNPs (minor allele frequency >5%) in the receptors of candidate genes. Tagging SNPs were scored using the high-throughput single-base pair extension method, and the statistical significance of these scores was assessed via haplotype analysis. RESULTS: Informative SNPs were identified for VEGF receptors 1 (Flt-1); TGF-beta receptor 3 (TGFBR3); and FGR receptors 1, 2, and 4 (FGFR1, FGFR2, and FGFR4), and 13 tagging SNPs were scored in the cohort. Atopy was significantly associated with haplotypes of TGFBR3, FGFR1, and FGFR2. Meanwhile, AHR was significantly associated with haplotypes of Flt-1, FGFR1, and FGFR4. However, atopy was not associated with genetic variations of Flt-1 and FGFR4, whereas AHR not associated with TGFBR3 and FGFR2. CONCLUSION: The expression of atopy and AHR is distinctly associated with genetic variations in VEGF, TGF-beta1, and FGFR in the Korean population.
Asunto(s)
Hiperreactividad Bronquial/genética , Hipersensibilidad Inmediata/genética , Polimorfismo de Nucleótido Simple , Proteínas Serina-Treonina Quinasas/genética , Receptor Tipo 2 de Factor de Crecimiento de Fibroblastos/genética , Receptores de Factores de Crecimiento Transformadores beta/genética , Receptores de Factores de Crecimiento Endotelial Vascular/genética , Adolescente , Hiperreactividad Bronquial/diagnóstico , Hiperreactividad Bronquial/epidemiología , Pruebas de Provocación Bronquial , Niño , Predisposición Genética a la Enfermedad , Genotipo , Humanos , Hipersensibilidad Inmediata/diagnóstico , Hipersensibilidad Inmediata/epidemiología , Corea (Geográfico)/epidemiología , Cloruro de Metacolina , Fenotipo , Receptor Tipo I de Factor de Crecimiento Transformador betaRESUMEN
It is well established that GABA degradation may play a key role in epileptogenesis. However, whether or not the expression of GABA-transaminase (GABA-T), which catalyzes GABA degradation and participates in the neuronal metabolism via GABA shunt, changes chronologically after on-set of seizure remains to be clarified. To identify the change of GABA-T expression in seizure, GABA-T expression in the gerbil hippocampus, associated with different sequelae of spontaneous seizures, was investigated. The distribution pattern of GABA-T immunoreactive neurons in the hippocampus between the seizure-resistant and pre-seizure group of seizure sensitive gerbils was similar. Interestingly, at 30 min postictal, the enhancement of GABA-T immunoreactivity in the perikarya was apparently observed. This contrasted with the decline in GABA-T immunoreactivity in the granular and pyramidal layer. At 12-24 h postictal, GABA-T immunoreactivity in the hilar neurons had declined significantly. However, the GABA-T immunoreactivity in the granular layer increased. These findings suggest that in the gerbil, the alteration in GABA-T expressions may play an important role in the self-recovery mechanism from seizure attack via both GABA degradation and regulation of neuronal metabolism.
Asunto(s)
4-Aminobutirato Transaminasa/metabolismo , Hipocampo/enzimología , Convulsiones/enzimología , Animales , Western Blotting , Gerbillinae , Hipocampo/fisiopatología , InmunohistoquímicaRESUMEN
We isolated a cDNA that encodes the bovine brain gamma-aminobutyrate transaminase (GABA-T; EC 2.6.1.19) from the lambda gt 11 cDNA library, which showed a high degree of sequence similarity to the corresponding enzymes from various sources. Northern blot analysis revealed two differentially expressed GABA-T transcripts of approximately 2.0 and 6.0 kb in the bovine tissues. Southern blot analysis indicates that the two GABA-T transcripts are encoded in a greater-than 10-kb, single-copy gene. Bovine GABA-T cDNA was expressed in E. coli using the pGEX bacterial- expression vector system. The overexpressed GABA-T was enzymatically active after purification, and it had very similar kinetic parameters when compared with those of other mammalian GABA-Ts.
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4-Aminobutirato Transaminasa/metabolismo , Encéfalo/enzimología , 4-Aminobutirato Transaminasa/química , 4-Aminobutirato Transaminasa/genética , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Southern Blotting , Encéfalo/fisiología , Bovinos , Clonación Molecular , Humanos , Datos de Secuencia Molecular , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/metabolismo , Alineación de Secuencia , Distribución TisularRESUMEN
Six monoclonal antibodies that recognize porcine brain pyridoxal kinase have been selected and designated as PK67, PK86, PK91, PK144, PK252 and PK275. A total of six monoclonal antibodies recognizing different epitopes of the enzyme were obtained, of which four inhibited the enzyme activity. When total proteins of porcine brain homogenate separated by SDS-PAGE were subjected to monoclonal antibodies, a single reactive protein band of molecular weight 39 kDa which comigrated with purified porcine pyridoxal kinase was detected. Using the anti-pyridoxal kinase antibodies as probes, the cross reactivities of brain pyridoxal kinase from human and other mammalian tissues and from avian sources were also investigated. Among human and all animal tissues tested, immunoreactive bands on Western blots appeared to have the same molecular mass of 39 kDa. These results indicate that mammalian brains contain only one major type of immunologically similar pyridoxal kinase, although some properties of the enzymes reported previously differed from one another.
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Anticuerpos Monoclonales/biosíntesis , Encéfalo/enzimología , Piridoxal Quinasa/inmunología , Animales , Anticuerpos Monoclonales/inmunología , Especificidad de Anticuerpos , Electroforesis en Gel de Poliacrilamida , Epítopos/inmunología , Femenino , Glioblastoma/enzimología , Humanos , Immunoblotting , Ratones , Ratones Endogámicos BALB C , Neuroblastoma/enzimología , Piridoxal Quinasa/análisis , Porcinos , Células Tumorales CultivadasRESUMEN
80% Aqueous MeOH extracts from the wood of Caesalpinia sappan, which showed remarkable anticonvulsant activity, were fractionated using EtOAc, n-BuOH, and H2O. Among them, the EtOAc fraction significantly inhibited the activities of two GABA degradative enzymes, succinic semialdehyde dehydrogenase (SSADH) and succinic semialdehyde reductase (SSAR). Repeated column chromatographies for the fraction guided by activity test led to the isolation of the two active principal components. Their chemical structures were determined to be sappanchalcone and brazilin based on spectral data. The pure compounds, sappanchalcone (1) and brazilin (2), inactivated the SSAR activities in a dose dependent manner, whereas SSADH was inhibited partially by sappanchalcone and not by brazilin.
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Anticonvulsivantes/aislamiento & purificación , Plantas Medicinales/química , Aldehído Oxidorreductasas/antagonistas & inhibidores , Animales , Bovinos , Inhibidores Enzimáticos/aislamiento & purificación , Hidroxibutirato Deshidrogenasa/antagonistas & inhibidores , Succionato-Semialdehído Deshidrogenasa , MaderaRESUMEN
In our search for the anticonvulsant constituent of Gastrodia elata repeated column chromatographies guided by activity assay led to isolation of an active compound, which was identified as gastrodin on the basis of spectral data. Brain succinic semialdehyde dehydrogenase (SSADH) was inactivated by preincubation with gastrodin in a time-dependent manner and the reaction was monitored by absorption and fluorescence spectroscopic methods. The inactivation followed pseudo-first-order kinetics with the second-rate order constant of 1.2 x 10(3) M-1min-1. The time course of the reaction was significantly affected by the coenzyme NAD+, which affected complete protection against the loss of the catalytic activity, whereas substrate succinic semialdehyde failed to prevent the inactivation of the enzyme. It is postulated that the gastrodin is able to elevate the neurotransmitter GABA levels in central nervous system by inhibitory action on one of the GABA degradative enzymes, SSADH.
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Aldehído Oxidorreductasas/antagonistas & inhibidores , Anticonvulsivantes/aislamiento & purificación , Alcoholes Bencílicos , Inhibidores Enzimáticos/aislamiento & purificación , Glucósidos/aislamiento & purificación , Plantas Medicinales/química , Animales , Bovinos , Succionato-Semialdehído Deshidrogenasa , Ácido gamma-Aminobutírico/metabolismoRESUMEN
Human brain gamma-aminobutyrate transaminase is differentially expressed in a tissue-specific manner. mRNA master dot-blot analysis for 50 different human tissues, including different brain regions and fetal tissues, provided a complete map of the tissue distribution. Genomic Southern analysis revealed that the gamma-aminobutyrate transaminase gene is a single copy, at least 15 kb in size. In addition, human brain gamma-aminobutyrate transaminase cDNA was expressed in Escherichia coli using a pGEX expression vector system. Catalytically active gamma-aminobutyrate transaminase was expressed in large quantities and the purified recombinant enzyme had kinetic parameters that were indistinguishable from those isolated from other mammalian brains. The human enzyme was inactivated by a well-known antiepileptic drug vigabatrin. Values of Ki and kinact were 1 mM and 0.35 min-1, respectively. Results from inactivation kinetics suggested that human gamma-aminobutyrate transaminase is more sensitive to the vigabatrin drug than the enzyme isolated from bovine brain.
Asunto(s)
4-Aminobutirato Transaminasa/metabolismo , Encéfalo/enzimología , 4-Aminobutirato Transaminasa/antagonistas & inhibidores , 4-Aminobutirato Transaminasa/genética , Southern Blotting , ADN Complementario , Inhibidores Enzimáticos/farmacología , Escherichia coli/genética , Humanos , Cinética , ARN Mensajero/genética , Proteínas Recombinantes/antagonistas & inhibidores , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Vigabatrin/farmacologíaRESUMEN
The structural differences between two types of glutamate dehydrogenase (GDH) isoproteins (GDH I and GDH II), homogeneously isolated from bovine brain, were investigated using a biosensor technology and monoclonal antibodies. A total of seven monoclonal antibodies raised against GDH II were produced, and the antibodies recognized a single protein band that comigrates with purified GDH II on sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblot. Of seven anti-GDH II monoclonal antibodies tested in the immunoblot analysis, all seven antibodies interacted with GDH II, whereas only four antibodies recognized the protein band of the other GDH isoprotein, GDH I. When inhibition tests of the GDH isoproteins were performed with the seven anti-GDH II monoclonal antibodies, three antibodies inhibited GDH II activity, whereas only one antibody inhibited GDH I activity. The binding affinity of anti-GDH II monoclonal antibodies for GDH II (K(D) = 1.0 nM) determined using a biosensor technology (Pharmacia BIAcore) was fivefold higher than for GDH I (K(D) = 5.3 nM). These results, together with epitope mapping analysis, suggest that there may be structural differences between the two GDH isoproteins, in addition to their different biochemical properties. Using the anti-GDH II antibodies as probes, we also investigated the cross-reactivities of brain GDHs from some mammalian and an avian species, showing that the mammalian brain GDH enzymes are related immunologically to each other.
Asunto(s)
Antígenos/inmunología , Encéfalo/metabolismo , Glutamato Deshidrogenasa/inmunología , Isoenzimas/inmunología , Animales , Anticuerpos Monoclonales/inmunología , Bovinos , Pollos , Reacciones Cruzadas/inmunología , Perros , Humanos , Persona de Mediana Edad , Conejos , PorcinosRESUMEN
An NAD+ dependent succinic semialdehyde dehydrogenase from bovine brain was inactivated by pyridoxal-5'- phosphate. Spectral evidence is presented to indicate that the inactivation proceeds through formation of a Schiff's base with amino groups of the enzyme. After NaBH(4) reduction of the pyridoxal-5'-phosphate inactivated enzyme, it was observed that 3.8 mol phosphopyridoxyl residues were incorporated/enzyme tetramer. The coenzyme, NAD+, protected the enzyme against inactivation by pyridoxal-5'-phosphate. The absorption spectrum of the reduced and dialyzed pyridoxal-5'-phosphate-inactivated enzyme showed a characteristic peak at 325 nm, which was absent in the spectrum of the native enzyme. The fluorescence spectrum of the pyridoxyl enzyme differs completely from that of the native enzyme. After tryptic digestion of the enzyme modified with pyridoxal-5'-phosphate followed by [3H]NaBH4 reduction, a radioactive peptide absorbing at 210 nm was isolated by reverse-phase HPLC. The sequences of the peptide containing the phosphopyridoxyllysine were clearly identical to sequences of other mammalian succinic semialdehyde dehydrogenase brain species including human. It is suggested that the catalytic function of succinic semialdehyde dehydrogenase is modulated by binding of pyridoxal-5'-phosphate to specific Lys(347) residue at or near the coenzyme-binding site of the protein.