RESUMO
The intestinal permeability of low-molecular-weight hyaluronan (LMW-HA) was investigated by using cultured monolayers of Caco-2 cells. The amount of LMW-HA that permeated the Caco-2 monolayers was measured by a carbazole assay. The permeability of LMW-HA increased inversely with the molecular size and was dose-dependent. The transport was observed to be energy-independent, and was correlated with the tight junction (TJ) permeability. These results suggest that LMW-HA permeated the Caco-2 cell monolayers via the paracellular pathway.
Assuntos
Espaço Extracelular/metabolismo , Ácido Hialurônico/química , Ácido Hialurônico/metabolismo , Células CACO-2 , Cromatografia Líquida de Alta Pressão , Humanos , Peso Molecular , Oligossacarídeos/metabolismo , PermeabilidadeRESUMO
Calcineurin homologous protein (CHP) is an EF-hand Ca(2+)-binding protein capable of interacting with various cellular proteins including Na(+)/H(+) exchangers, kinesin-related proteins, and apoptosis-inducing protein kinase DRAK2. We investigated the role of CHP on the DRAK2 protein kinase in vitro. CHP significantly reduced (approximately 85% inhibition) the kinase activity of DRAK2 for both autophosphorylation and phosphorylation of exogenous substrate (myosin light chain). The inhibitory effect of CHP was dependent on the presence of Ca(2+), whereas the interaction between CHP and DRAK2 was not Ca(2+)-dependent. These observations suggest that CHP negatively regulates the apoptosis-inducing protein kinase DRAK2 in a manner that depends on intracellular Ca(2+)-concentration.
Assuntos
Proteínas de Ligação ao Cálcio/farmacologia , Cálcio/metabolismo , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Animais , Apoptose/fisiologia , Proteínas Reguladoras de Apoptose , Células COS , Calcineurina/genética , Cálcio/química , Proteínas de Ligação ao Cálcio/genética , Proteínas de Ligação ao Cálcio/metabolismo , Chlorocebus aethiops , Relação Dose-Resposta a Droga , Inibidores Enzimáticos/farmacologia , Cadeias Leves de Miosina/metabolismo , Fosforilação , Ligação Proteica , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Recombinantes/antagonistas & inibidores , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/farmacologiaRESUMO
Hyaluronidase (HAase) activity was detected in the culture supernatants of Penicillium purpurogenum and Penicillium funiculosum. The HAase from Penicillium spp. (HAase-P) was a hyaluronate 4-glycanohydrolase, which catalyzed the endolytic hydrolysis of the ß-1,4 glycosidic linkage, as do vertebrate HAases. The gene encoding HAase-P was cloned and expressed in Escherichia coli. According to homology analyses of the deduced amino acid sequences, HAase-P is not classified into any of the known HAase groups, but belongs to glycoside hydrolase family 16, which includes endo-ß-1,3(4)-glucanase. Regarding the substrate specificities, no chondroitinase and glucanase activities were detected. Judging from homology analyses and enzymatic properties, HAase-P seems to be a new type of HAase.
Assuntos
Proteínas Fúngicas/metabolismo , Hialuronoglucosaminidase/metabolismo , N-Glicosil Hidrolases/metabolismo , Penicillium/enzimologia , Sequência de Aminoácidos , Cromatografia Líquida de Alta Pressão , Clonagem Molecular , DNA Complementar/química , DNA Complementar/genética , Proteínas Fúngicas/genética , Glucanos/metabolismo , Ácido Hialurônico/metabolismo , Hialuronoglucosaminidase/genética , Hidrólise , Dados de Sequência Molecular , N-Glicosil Hidrolases/genética , Penicillium/classificação , Penicillium/genética , Análise de Sequência de DNA , Homologia de Sequência de Aminoácidos , Especificidade da Espécie , Espectrometria de Massas por Ionização por Electrospray , Especificidade por SubstratoRESUMO
Naringenin chalcone is the main active component of tomato skin extract, which has an antiallergic activity. In this study, naringenin chalcone was orally administered to rats, and the chemical structures and levels of the major metabolites in the plasma and urine of rats were determined. HPLC analysis indicated the presence of three major metabolites in the urine. LC-MS and NMR analyses tentatively identified these as naringenin chalcone-2'-O-beta-D-glucuronide, naringenin-7-O-beta-D-glucuronide, and naringenin-4'-O-beta-D-glucuronide. Naringenin chalcone-2'-O-beta-D-glucuronide was the only metabolite detected in the plasma, and its peak plasma level was observed 1 h after naringenin chalcone administration. Naringenin chalcone-2'-O-beta-D-glucuronide also inhibited histamine release from rat peritoneal mast cells stimulated with compound 48/80. This activity might contribute to the antiallergic activity of naringenin chalcone in vivo. To the best of the authors' knowledge, this study is the first to report determination of naringenin chalcone metabolites in rat plasma and urine.