RESUMO
The effects of shear stress on interleukin 8 (IL-8) production by human umbilical vein endothelial cells (HUVEC) were studied by subjecting the HUVEC to a steady flow laminar shear stress of up to 0.7 N/m(2) in a parallel plate flow chamber. Shear stress decreased IL-8 mRNA expression in a dose and time-dependent fashion. High glucose concentrations increased IL-8 mRNA levels in a MAPK-p38-dependent manner, which was suppressed by shear stress. Measurement of IL-8 protein in HUVEC culture media by ELISA demonstrated that IL-8 secretion was also increased by high glucose and suppressed by shear stress. These results suggest that the anti-atherogenic effect of shear stress arises partly from the suppression of the production of IL-8 which has been shown to trigger the adhesion of monocytes to a vascular endothelium and also acts as a mitogen and chemoattractant for vascular smooth muscle cells.
Assuntos
Endotélio Vascular/imunologia , Interleucina-8/biossíntese , Arteriosclerose/imunologia , Células Cultivadas , Meios de Cultura/química , Relação Dose-Resposta a Droga , Inibidores Enzimáticos/farmacologia , Ensaio de Imunoadsorção Enzimática , Glucose/farmacologia , Humanos , Imidazóis/farmacologia , Interleucina-8/genética , Proteínas Quinases Ativadas por Mitógeno/antagonistas & inibidores , Reação em Cadeia da Polimerase/métodos , Piridinas/farmacologia , RNA Mensageiro/análise , Fluxo Sanguíneo Regional , Reologia , Estimulação Química , Estresse Mecânico , Fatores de Tempo , Veias Umbilicais , Proteínas Quinases p38 Ativadas por MitógenoRESUMO
An enzymatic system for poly gamma-glutamate (PGA) synthesis in Bacillus subtilis, the PgsBCA system, was investigated. The gene-disruption experiment showed that the enzymatic system was the sole machinery of PGA synthesis in B. subtilis. We succeeded in achieving the enzymatic synthesis of elongated PGAs with the cell membrane of the Escherichia coli clone producing PgsBCA in the presence of ATP and D-glutamate. The enzyme preparation solubilized from the membrane with 8 mM Chaps catalyzed ADP-forming ATP hydrolysis only in the presence of glutamate; the D-enantiomer was the best cosubstrate, followed by the L-enantiomer. Each component of the system, PgsB, PgsC, and PgsA, was translated in vitro and the glutamate-dependent ATPase reaction was kinetically analyzed. The PGA synthetase complex, PgsBCA, was suggested to be an atypical amide ligase.
Assuntos
Bacillus subtilis/enzimologia , Glutamato Sintase/química , Glutamato Sintase/metabolismo , Ácido Poliglutâmico/biossíntese , Adenosina Trifosfatases/metabolismo , Trifosfato de Adenosina/metabolismo , Sequência de Aminoácidos , Bacillus subtilis/genética , Clonagem Molecular , Detergentes/metabolismo , Deleção de Genes , Expressão Gênica , Glutamato Sintase/genética , Cinética , Complexos Multienzimáticos/química , Complexos Multienzimáticos/genética , Complexos Multienzimáticos/metabolismo , Ácido Poliglutâmico/metabolismoRESUMO
In this study, we have investigated the in vitro effects of the immunomodulators lobenzarit and traxanox and a newly synthesized immunosuppressant, mizoribine, as well as cyclosporin A, on bone resorption using neonatal mouse calvariae labelled with 45Ca. As stimulators of bone resorption, bovine parathyroid hormone (PTH), lipopolysaccharide (LPS), interleukin 1 beta (IL-1 beta) and tumor necrosis factor-alpha (TNF-alpha) were used. Lobenzarit, traxanox, mizoribine and cyclosporin A inhibited or tended to inhibit bone resorption stimulated by PTH, LPS, IL-1 beta or TNF-alpha in a dose-dependent manner. Basal bone resorption was inhibited by immunosuppressant cyclosporin A or mizoribine, while immunomodulators lobenzarit and traxanox failed to inhibit basal bone resorption. Removal of lobenzarit from the culture medium resulted in the recovery of bone resorptive activity. These results suggest that the inhibitory effect of immunomodulators on bone resorption is reversible and nonselective. Also, it raises the possibility that immunomodulators and immunosuppressants may affect bone resorption by different mechanisms.
