RESUMO
Antimicrobial peptides are important components of the host innate immune responses by exerting broad-spectrum microbicidal activity against pathogenic microbes. Cy-AMP1 found in the cycad (Cycas revoluta) seeds has chitin-binding ability, and the chitin-binding domain was conserved in knottin-type and hevein-type antimicrobial peptides. The recombinant Cy-AMP1 was expressed in Escherichia coli and purified to study the role of chitin-binding domain. The mutants of Cy-AMP1 lost chitin-binding ability completely, and its antifungal activity was markedly decreased in comparison with native Cy-AMP1. However, the antimicrobial activities of the mutant peptides are nearly identical to that of native one. It was suggested that the chitin-binding domain plays an essential role in antifungal, but not antimicrobial, activity of Cy-AMP1.
Assuntos
Antifúngicos/farmacologia , Quitina/metabolismo , Cycas/química , Peptídeos/química , Peptídeos/metabolismo , Extratos Vegetais/química , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Sequência de Aminoácidos , Anti-Infecciosos/química , Anti-Infecciosos/metabolismo , Anti-Infecciosos/farmacologia , Antifúngicos/química , Dicroísmo Circular , Eletroforese em Gel de Poliacrilamida , Fungos/efeitos dos fármacos , Mutagênese Sítio-Dirigida , Peptídeos/genética , Peptídeos/farmacologia , Extratos Vegetais/genética , Extratos Vegetais/metabolismo , Extratos Vegetais/farmacologia , Ligação Proteica , Proteínas Recombinantes/genética , Proteínas Recombinantes/farmacologia , Sementes/químicaRESUMO
INTRODUCTION: Gap junctions (GJs) are intercellular channels that aid communication between coupling cells and may play a critical role in cell differentiation and growth. Connexins (Cxs) are structural proteins of GJs. Though several reports have demonstrated that Cx expression decreases in various malignant tumors, a pancreatic cancer cell line, PANC-1, was reported to express Cx43 mRNA. It is known that irsogladine malate (IM) can up-regulate gap junctional intercellular communication (GJIC). We examined the effects of IM on GJ between pancreatic cancer cells (PC cells) and the mechanism of GJ up-regulation. METHODOLOGY: GJIC between PC cells (PANC-1) was evaluated by dye transfer methods. The expression of Cx43 was estimated by Western blot analysis with immunoprecipitation sample and immunohistochemical analysis. Intracellular cAMP level was estimated by enzyme-linked immunoassay. RESULTS: IM increased cell coupling in a dose-dependent manner (0M-10 ). Western blot analysis of Cx43 revealed that PANC-1 cells expressed Cx43 protein. Treatment with IM was found to move localization of Cx43 immunoreactive spots from the cytoplasm to boundary lesions with neighboring cells, but no major change was seen in the phosphorylation state of Cx43. Intracellular cAMP level was increased by IM. The PKA inhibitor H-89 and adenylyl cyclase inhibitor SQ22536 inhibited the effects of IM. CONCLUSION: These results suggest that IM up-regulates GJIC between PC cells via regulation of the PKA pathway. It also suggests a useful adjuvant of IM to pancreatic cancer therapy.
