PLoS One. 2009; 4(7): e6304Zhang J, Li C, Shi T, Chen K, Shen X, Jiang HGlucokinase (GK), a glucose sensor, maintains plasma glucose homeostasis via phosphorylation of glucose and is a potential therapeutic target for treating maturity-onset diabetes of the young (MODY) and persistent hyperinsulinemic hypoglycemia of infancy (PHHI). To characterize the catalytic mechanism of glucose phosphorylation by GK, we combined molecular modeling, molecular dynamics (MD) simulations, quantum mechanics/molecular mechanics (QM/MM) calculations, experimental mutagenesis and enzymatic kinetic analysis on both wild-type and mutated GK. Our three-dimensional (3D) model of the GK-Mg(2+)-ATP-glucose (GMAG) complex, is in agreement with a large number of mutagenesis data, and elucidates atomic information of the catalytic site in GK for glucose phosphorylation. A 10-ns MD simulation of the GMAG complex revealed that Lys169 plays a dominant role in glucose phosphorylation. This prediction was verified by experimental mutagenesis of GK (K169A) and enzymatic kinetic analyses of glucose phosphorylation. QM/MM calculations were further used to study the role of Lys169 in the catalytic mechanism of the glucose phosphorylation and we found that Lys169 enhances the binding of GK with both ATP and glucose by serving as a bridge between ATP and glucose. More importantly, Lys169 directly participates in the glucose phosphorylation as a general acid catalyst. Our findings provide mechanistic details of glucose phorphorylation catalyzed by GK, and are important for understanding the pathogenic mechanism of MODY.

Evid Based Complement Alternat Med. 2009 Jul 16; Khuda-Bukhsh AR, Bhattacharyya SS, Paul S, Dutta S, Boujedaini N, Belon PIn homeopathy, ability of ultra-high diluted drugs at or above potency 12C (diluted beyond Avogadro's limit) in ameliorating/curing various diseases is often questioned, particularly because the mechanism of action is not precisely known. We tested the hypothesis if suitable modulations of signal proteins could be one of the possible pathways of action of a highly diluted homeopathic drug, Secale cornutum 30C (diluted 10(60) times; Sec cor 30). It could successfully combat DMBA + croton oil-induced skin papilloma in mice as evidenced by histological, cytogenetical, immunofluorescence, ELISA and immunoblot findings. Critical analysis of several signal proteins like AhR, PCNA, Akt, Bcl-2, Bcl-xL, NF-kB and IL-6 and of pro-apoptotic proteins like cytochrome c, Bax, Bad, Apaf, caspase-3 and -9 revealed that Sec cor 30 suitably modulated their expression levels along with amelioration of skin papilloma. FACS data also suggested an increase of cell population at S and G2 phases and decrease in sub-G1 and G1 phages in carcinogen-treated drug-unfed mice, but these were found to be near normal in the Sec cor 30-fed mice. There was reduction in genotoxic and DNA damages in bone marrow cells of Sec Cor 30-fed mice, as revealed from cytogenetic and Comet assays. Changes in histological features of skin papilloma were noted. Immunofluorescence studies of AhR and PCNA also suggested reduced expression of these proteins in Sec cor 30-fed mice, thereby showing its anti-cancer potentials against skin papilloma. Furthermore, this study also supports the hypothesis that potentized homeopathic drugs act at gene regulatory level.

Inflammation. 2009 Jul 16; An Y, Liu K, Zhou Y, Liu BAntidiabetic effects of salicylates have been known for years, however the cellular and molecular mechanisms of the hypoglycemic activity are not well elucidated. We examined the effects of salicylate on inflammation-related changes in gene or/and protein expressions of several adipokines in 3T3-L1 adipocytes and of LPS-induced inflammatory factors in RAW 264.7 cell. Especially, we focused our attention on the cross-talk between the macrophages and adipocytes. Exposure to RAW-CM medium resulted in an increase in the gene expression or/and protein secretion of TNF-alpha, IL-6 and resistin, and at the same time, a decrease in the gene expression of PPARgamma and adiponectin in 3T3-L1 adipocytes. Salicylate effectively reversed these changes, and up-regulated glucose consumption in adipocytes. We also found salicylate inhibited phosphorylation of NF-kappaB in RAW-CM-stimulated adipocytes. We conclude salicylate blocks inflammatory process in the pathogenesis of inflammation-related insulin resistance.

J Pharm Biomed Anal. 2009 May 30; Chen D, Wang J, Jiang Y, Zhou T, Fan G, Wu YThe pressurized capillary electrochromatography (pCEC) was utilized for the separation and determination of coumarins in Fructus cnidii extracts from 12 different regions. After a thorough study of analytical parameters such as acetonitrile content of the mobile phase, the concentration and pH of the buffer, and the applied voltage, a methodology was proposed to separate and determine six coumarins of F. cnidii extracts in less than 15min. The experiments were performed in an in-house packed column with a monolithic outlet frit under the optimal conditions: pH 4.0 ammonium acetate buffer at 10mM containing 50% acetonitrile at -6kV applied voltage. The calibration curves were linear in the range of 10.0-100.0mug/mL for bergapten, 20.0-200.0mug/mL for imperatorin, 5.0-400.0mug/mL for osthole, 10.0-100.0mug/mL for 2'-acetylangelicin, 10.0-200.0mug/mL for oroselone, and 10.0-200.0mug/mL for O-acetylcolumbianetin. The correlation coefficients were between 0.9967 and 0.9995. With this pCEC system, fingerprints of F. cnidii extracts were preliminarily established to distinguish three types of coumarins by characteristic peaks, and the quality of various sources of raw materials was evaluated by determining the contents of six coumarins.