RESUMO
In this work, Opuntia ficus indica juice was explored as a potential source of natural antioxidant and anti-inflammatory ingredients towards intestinal inflammation. An adsorption separation process was used to produce a natural flavonoid-rich concentrate (FRC) from Opuntia ficus-indica juice. The FRC effect (co- or pre-incubation) on induced-oxidative stress and induced-inflammation was evaluated in human Caco-2 cells. The main constituents identified and present in the extract are flavonoids (namely isorhamnetins and their derivatives such as isorhamnetin 3-O-rhamnose-rutinoside and isorhamnetin 3-O-rutinoside) and phenolic acids (such as ferulic, piscidic and eucomic acids). Our results showed that co-incubation of FRC with the stress-inducer attenuates radicals production in a much more significant manner than pre-incubation. These results suggest that FRC compounds which cannot pass the cell membrane freely (isorhamnetin derivatives) have an ability to inhibit the formation of H2O2-induced radicals in the surrounding environment of intestinal epithelial cells. The capacity of FRC (co-incubation) for suppressing (at the extracellular level) free radicals chain initiation or propagation reaction was probably related with a more pronounced reduction in protein oxidation. A similar response was observed in the inflammatory state, where a marked decrease in IL-8 secretion and blocked degradation of IκBα was achieved for FRC co-incubation. Simultaneously, treatment with FRC significantly reduces NO and TNF-α expression and modulates apparent permeability in Caco-2 cells. In these cases, no significant differences were found between pre- and co-incubation treatments suggesting that bioavailable phenolics, such as ferulic, eucomic and piscidic acids and isorhamnetin, act at the intracellular environment.
Assuntos
Anti-Inflamatórios/farmacologia , Antioxidantes/farmacologia , Bebidas/análise , Flavonoides/farmacologia , Opuntia/química , Extratos Vegetais/farmacologia , Anti-Inflamatórios/química , Antioxidantes/química , Células CACO-2 , Flavonoides/química , Humanos , Interleucina-8/genética , Interleucina-8/imunologia , Extratos Vegetais/química , Fator de Necrose Tumoral alfa/genética , Fator de Necrose Tumoral alfa/imunologiaRESUMO
BACKGROUND: The alpha and beta subunits of FSH were fused to the Fc domain of IgG1 either in a single chain or a heterodimer format. These molecules were absorbed through the epithelium in lung and intestine by neonatal Fc receptor (FcRn)-mediated transcytosis. METHODS AND RESULTS: Single chain and heterodimer FSH-Fc were made recombinantly in Chinese hamster ovary cells. Treatment of rats with a single s.c. dose of single chain or heterodimer FSH-Fc resulted in greater stimulation of ovarian weight (20.8+/-3.9 and 26.9+/-6.1 mg respectively) compared to those receiving vehicle (12.1+/-1.0 mg) or an equimolar dose of recombinant human FSH (14.3+/-1.7 mg). Both FSH-Fc fusion proteins were absorbed after oral dosing of newborn rats with long terminal half-lives of approximately 60 h, and pulmonary delivery in four cynomolgus monkeys produced maximum serum concentrations between 69 and 131 ng/ml with long terminal half-lives between 55 and 210 h. Serum inhibin levels increased after pulmonary dosing with single chain FSH-Fc (1.3- and 1.4-fold) and heterodimer FSH-Fc (5.9- and 7.1-fold) and remained elevated for >12 days after treatment with heterodimer FSH-Fc. CONCLUSIONS: We have shown that FSH-Fc fusion proteins have increased stability in blood and improved bioactivity in vivo, and that heterodimer FSH-Fc is more active in rats and monkeys than single chain FSH-Fc. These data suggest that Fc fusion proteins offer the potential for oral and pulmonary delivery of FSH.
Assuntos
Hormônio Foliculoestimulante/administração & dosagem , Fragmentos Fc das Imunoglobulinas/administração & dosagem , Administração Oral , Aerossóis , Animais , Animais Recém-Nascidos , Sequência de Bases , Transporte Biológico Ativo , Células CHO , Cricetinae , DNA Complementar/genética , Feminino , Hormônio Foliculoestimulante/farmacocinética , Humanos , Fragmentos Fc das Imunoglobulinas/metabolismo , Macaca fascicularis , Masculino , Tamanho do Órgão/efeitos dos fármacos , Ovário/anatomia & histologia , Ovário/efeitos dos fármacos , Ratos , Receptores Fc/metabolismo , Proteínas Recombinantes de Fusão/administração & dosagem , Proteínas Recombinantes de Fusão/farmacocinética , Testículo/anatomia & histologia , Testículo/efeitos dos fármacosRESUMO
We have developed an assay to continuously monitor the branched amino acid preferring peptidase (BrAAP) activity of the proteasome. This assay is based on the hydrolysis of the fluorogenic peptide, Abz-Gly-Pro-Ala-Leu-Ala-Nba (Abz is 2-aminobenzoyl and Nba is 4-nitrobenzylamide) which is cleaved exclusively at the Leu-Ala bond by the 20S proteasome with a kc/Km value of 13 000 M-1 s-1. Hydrolysis of this peptide is accompanied by an increase in fluorescence intensity (lambda ex = 340 nm, lambda em = 415 nm) due to release of the internally quenched 2-aminobenzoyl fluorescence that accompanies diffusion apart of the hydrolysis products, Abz-Gly-Pro-Ala-Leu and Ala-Nba. Using this assay, we examined inhibition of the BrAAP activity of the proteasome by a series of tripeptide aldehydes, Z-Leu-Leu-Xaa-H. When Xaa = Phe, (p-Cl)Phe, and Trp we observe biphasic or partial inhibition of the BrAAP activity. In contrast, when Xaa = Nva and Leu, simple inhibition kinetics are observed and allow us to calculate Ki values of 120 nM and 12 nM, respectively. The inhibitors that exhibit simple inhibition kinetics for BrAAP activity are also approximately equipotent for inhibition of the chymotrypsin-like (ChT-L) and peptidyl-glutamyl peptide hydrolyzing (PGPH) activities, dissociation constants varying by less than 25-fold, whereas the inhibitors that exhibit biphasic inhibition kinetics for BrAAP activity are >300-fold more potent for inhibiting ChT-L activity than for PGPH activity. Inactivation of the BrAAP activity of the proteasome by clasto-lactacystin beta-lactone is also biphasic. beta-Lactone inactivates approximately 60% of the BrAAP activity rapidly, with kinetics indistinguishable from its inactivation of the chymotrypsin-like activity. The remaining 40% of the BrAAP activity is inactivated by beta-lactone at a 50-fold slower rate, with kinetics indistinguishable from its inactivation of the PGPH activity. These results suggest a mechanism in which hydrolysis of Abz-Gly-Pro-Ala-Leu-Ala-Nba (i.e., BrAAP activity) occurs at two different active sites in the 20S proteasome, and that these two active sites are the same ones that catalyze the previously described ChT-L and PGPH activities.
Assuntos
Aminoácidos de Cadeia Ramificada/metabolismo , Cisteína Endopeptidases/metabolismo , Endopeptidases/metabolismo , Lactonas/farmacologia , Complexos Multienzimáticos/metabolismo , Oligopeptídeos/farmacologia , Inibidores de Proteases/farmacologia , Aldeídos/farmacologia , Animais , Inibidores de Cisteína Proteinase/farmacologia , Endopeptidases/efeitos dos fármacos , Cinética , Espectrometria de Massas , Complexo de Endopeptidases do Proteassoma , Coelhos , Especificidade por SubstratoRESUMO
The natural product lactacystin exerts its cellular antiproliferative effects through a mechanism involving acylation and inhibition of the proteasome, a cytosolic proteinase complex that is an essential component of the ubiquitin-proteasome pathway for intracellular protein degradation. In vitro, lactacystin does not react with the proteasome; rather, it undergoes a spontaneous conversion (lactonization) to the active proteasome inhibitor, clasto-lactacystin beta-lactone. We show here that when the beta-lactone is added to mammalian cells in culture, it rapidly enters the cells, where it can react with the sulfhydryl of glutathione to form a thioester adduct that is both structurally and functionally analogous to lactacystin. We call this adduct lactathione, and like lactacystin, it does not react with the proteasome, but can undergo lactonization to yield back the active beta-lactone. We have studied the kinetics of this reaction under appropriate in vitro conditions as well as the kinetics of lactathione accumulation and proteasome inhibition in cells treated with lactacystin or beta-lactone. The results indicate that only the beta-lactone (not lactacystin) can enter cells and suggest that the formation of lactathione serves to concentrate the inhibitor inside cells, providing a reservoir for prolonged release of the active beta-lactone.
Assuntos
Acetilcisteína/análogos & derivados , Cisteína Endopeptidases/metabolismo , Inibidores de Cisteína Proteinase/farmacologia , Complexos Multienzimáticos/metabolismo , Acetilcisteína/química , Acetilcisteína/farmacologia , Transporte Biológico , Glutationa/química , Células HeLa , Humanos , Lactonas/farmacologia , Oligopeptídeos/química , Oligopeptídeos/metabolismo , Complexo de Endopeptidases do Proteassoma , Pirrolidinonas/química , Pirrolidinonas/metabolismo , Células Tumorais CultivadasRESUMO
Lactacystin is a Streptomyces metabolite that inhibits cell cycle progression and induces differentiation in a murine neuroblastoma cell line. The cellular target of lactacystin is the 20 S proteasome, also known as the multicatalytic proteinase complex, an essential component of the ubiquitin-proteasome pathway for intracellular protein degradation. In aqueous solution at pH 8, lactacystin undergoes spontaneous hydrolysis to yield N-acetyl-L-cysteine and the inactive lactacystin analog, clasto-lactacystin dihydroxy acid. We have studied the mechanism of lactacystin hydrolysis under these conditions and found that it proceeds exclusively through the intermediacy of the active lactacystin analog, clasto-lactacystin beta-lactone. Conditions that stabilize lactacystin (and thus prevent the transient accumulation of the intermediate beta-lactone) negate the ability of lactacystin to inactivate the proteasome. Together these findings suggest that lactacystin acts as a precursor for clasto-lactacystin beta-lactone and that the latter is the sole species that interacts with the proteasome.
Assuntos
Acetilcisteína/análogos & derivados , Cisteína Endopeptidases/metabolismo , Inibidores de Cisteína Proteinase/farmacologia , Lactonas/farmacologia , Complexos Multienzimáticos/metabolismo , Reticulócitos/enzimologia , Acetilcisteína/química , Acetilcisteína/farmacologia , Animais , Cromatografia Líquida de Alta Pressão , Cisteína Endopeptidases/isolamento & purificação , Concentração de Íons de Hidrogênio , Hidrólise , Cinética , Lactonas/química , Estrutura Molecular , Complexos Multienzimáticos/isolamento & purificação , Complexo de Endopeptidases do Proteassoma , Coelhos , StreptomycesRESUMO
Interaction of heat shock transcription factor-1 (HSF-1) with the seventy kilodalton heat shock cognate protein (HSC70) was examined in NIH 3T3 cells. HSF-1 was found in the cytoplasm of non-stressed cells associated with HSC70 in large (Mr 400-500,000) complexes. After heat shock, HSF-1 became concentrated in the nucleus in smaller, more stable complexes that did not contain HSC70, an indication of significant rearrangement within the complexes. These experiments show a profound effect of heat shock on the structure and stability of HSF-1 complexes during nuclear localization and support the hypothesis that HSC70 binding may control HSF-1 function.