Seven 3-methylidene-1H-indol-2(3H)-ones related to the multiple-receptor tyrosine kinase inhibitor sunitinib

The solid-state structures of a series of seven substituted 3-methylidene-1H-indol-2(3H)-one derivatives have been determined by single-crystal X-ray diffraction and are compared in detail. Six of the structures {(3Z)-3-(1H-pyrrol-2- ylmethylidene)-1H-indol-2(3H)-one, C13H10N2O, (2a); (3Z)-3-( 2-thienylmethylidene)-1H-indol-2(3H)-one, C13H9NOS, (2b); (3E)-3-(2-furylmethylidene)-1H-indol-2(3H)-one monohydrate, C13H9NO2 center dot H2O, (3a); 3-(1-methylethylidene)-1H-indol- 2(3H)-one, C11H11NO, (4a); 3-cyclohexylidene-1H-indol- 2(3H)-one, C14H15NO, (4c); and spiro[1,3-dioxane-2,3'-indolin]- 2'-one, C11H11NO3, (5)} display, as expected, intermolecular hydrogen bonding (N-H center dot center dot center dot O=C) between the 1H-indol-2(3H)-one units. However, methyl 3-(1-methylethylidene)- 2-oxo-2,3-dihydro-1H-indole-1-carboxylate, C13H13NO3, (4b), a carbamate analogue of (4a) lacking an N-H bond, displays no intermolecular hydrogen bonding. The structure of (4a) contains three molecules in the asymmetric unit, while (4b) and (4c) both contain two independent molecules.

High glucose decreases intracellular glutathione concentrations and upregulates inducible nitric oxide synthase gene expression in intestinal epithelial cells

Diabetes is associated with oxidative stress and increased levels of inflammatory cytokines. The aim of the study was to assess the effects of inflammatory cytokines and oxidative stress associated with raised glucose levels on inducible nitric oxide synthase (iNOS) promoter activity in intestinal epithelial cells. High glucose (25 mmol/l) conditions reduced glutathione (GSH) levels in the human intestinal epithelial cell line, DLD-1. Addition of the antioxidant alpha-lipoic acid resulted in the restoration of GSH levels to normal. Upregulation of basal iNOS promoter activity was observed when cells were incubated in high glucose alone. This effect was significantly reduced by the addition of the antioxidant, alpha-lipoic acid and completely blocked with inhibition of NFkappa B activity. Cytokine stimulation [interleukin-1 beta, tumor necrosis factor-alpha, interferon-gamma] induced iNOS promoter activity in all conditions and this was accompanied by an increase in nitric oxide (NO) production. Inhibition of NFkappa-B activity decreased but did not completely inhibit cytokine-induced iNOS promoter activity and subsequent NO production. In conclusion, high glucose-induced iNOS promoter activity is mediated in part through intracellular GSH and NFkappa-B.

Down-regulation of Rac Activity during beta 2 Integrin-mediated Adhesion of Human Neutrophils

In human neutrophils, beta2 integrin engagement mediated a decrease in GTP-bound Rac1 and Rac2. Pretreatment of neutrophils with LY294002 or PP1 (inhibiting phosphatidylinositol 3-kinase (PI 3-kinase) and Src kinases, respectively) partly reversed the beta2 integrin-induced down-regulation of Rac activities. In contrast, beta2 integrins induced stimulation of Cdc42 that was independent of Src family members. The PI 3-kinase dependency of beta2 integrin-mediated decrease in GTP-bound Rac could be explained by an enhanced Rac-GAP activity, since this activity was blocked by LY204002, whereas PP1 only had a minor effect. The fact that only Rac1 but not Rac2 (the dominating Rac) redistributed to the detergent-insoluble fraction and that it was independent of GTP loading excludes the possibility that down-regulation of Rac activities was due to depletion of GTP-bound Rac from the detergent-soluble fraction. The beta2 integrin-triggered relocalization of Rac1 to the cytoskeleton was enabled by a PI 3-kinase-induced dissociation of Rac1 from LyGDI. The dissociations of Rac1 and Rac2 from LyGDI also explained the PI 3-kinase-dependent translocations of Rac GTPases to the plasma membrane. However, these accumulations of Rac in the membrane, as well as that of p47phox and p67phox, were also regulated by Src tyrosine kinases. Inasmuch as Rac GTPases are part of the NADPH oxidase and the respiratory burst is elicited in neutrophils adherent by beta2 integrins, our results indicate that activation of the NADPH oxidase does not depend on the levels of Rac-GTP but instead requires a beta2 integrin-induced targeting of the Rac GTPases as well as p47phox and p67phox to the plasma membrane.

Inactivation of Human beta-Defensins 2 and 3 by Elastolytic Cathepsins

beta-Defensins are antimicrobial peptides that contribute to the innate immune responses of eukaryotes. At least three defensins, human beta-defensins 1, 2, and 3 (HBD-1, -2, and -3), are produced by epithelial cells lining the respiratory tract and are active toward Gram-positive (HBD-3) and Gram-negative (HBD-1, -2, and -3) bacteria. It has been postulated that the antimicrobial activity of defensins is compromised by changes in airway surface liquid composition in lungs of patients with cystic fibrosis (CF), therefore contributing to the bacterial colonization of the lung by Pseudomonas and other bacteria in CF. In this report we demonstrate that HBD-2 and HBD-3 are susceptible to degradation and inactivation by the cysteine proteases cathepsins B, L, and S. In addition, we show that all three cathepsins are present and active in CF bronchoalveolar lavage. Incubation of HBD-2 and -3 with CF bronchoalveolar lavage leads to their degradation, which can be completely (HBD-2) or partially (HBD-3) inhibited by a cathepsin inhibitor. These results suggest that beta-defensins are susceptible to degradation and inactivation by host proteases, which may be important in the regulation of beta-defensin activity. In chronic lung diseases associated with infection, overexpression of cathepsins may lead to increased degradation of HBD-2 and -3, thereby favoring bacterial infection and colonization.

Cell surface beta 1, 4-galactosyltransferase 1 promotes apoptosis by inhibiting epidermal growth factor receptor pathway.

Our previous studies have shown that overexpression of beta1,4-galactosyltransferase1 (beta1,4GT1) leads to increased apoptosis induced by cycloheximide (CHX) in SMMC-7721 human hepatocarcinoma cells. However, the role of beta1,4GT1 in apoptosis remains unclear. Here we demonstrated that cell surface beta1,4GT1 inhibited the autophosphorylation of epidermal growth factor receptor (EGFR) especially at Try 1068. The phosphorylation of protein kinase B (PKB/Akt) and extracellular signal-regulated protein kinase1/2 (ERK1/2), which are downstream molecules of EGFR, were also reduced in cell surface beta1,4GT1-overexpressing cells. Furthermore, the translocations of Bad and Bax that are regulated by PKB/Akt and ERK1/2 were also increased in these cells. As a result, the release of cytochrome c from mitochondria to cytosol was increased and caspase-3 was activated. In contrast, RNAi-mediated knockdown of beta1,4GT1 increased the autophosphorylation of EGFR. These results demonstrated that cell surface beta1,4GT1 may negatively regulate cell survival possibly through inhibiting and modulating EGFR signaling pathway.