3-Hydrogenkwadaphnine, a novel diterpene ester from Dendrostellera lessertii, its role in differentiation and apoptosis of KG1 cells

3-Hydrogenkwadaphnin (3-HK) (Fig. 1) is a daphnane-type diterpene ester isolated from the leaves of Dendrostellera lessertii (Thymelaeaceae) with differentiation and apoptotic potency among several leukemic cells without any measurable adverse effects on normal cells [Moosavi, M.A., Yazdanparast, R., Sanati, M.H., Nejad, A.S., 2005a. 3-Hydrogenkwadaphnin targets inosine 5'-monophosphate dehydrogenase and triggers post-G1 arrest apoptosis in human leukemia cell lines. Int. J. Biochem. Cell Biol. 37, 2366-2379]. In this study, we evaluated differentiating and apoptotic efficiency of a second new anti-proliferating agent from the same plant relative to 3-HK using acute myeloid leukemia (AML) KG1 cell line. 3-HK at 5-30 nM inhibited proliferation of KG1 cells after 24-96 h of treatment. NBT reducing assay and expression of cell surface markers (CD11b and CD14) confirmed that the inhibition of proliferation is associated with differentiation toward macrophage-like morphology. Regarding the relatively weaker potency of 3-HK in the induction of differentiation compared to that of the crude extract, we looked for additional compound(s) with similar properties in the crude extract. This effort led to isolation of the second compound from the leaves' extract with higher differentiating potency. The new compound inhibited proliferation of KG1 cells by almost 48+/-3.1% after 72 h of treatment with a single dose of 1.5 microg/ml. The treated cells differentiated along the monocyte/macrophage lineage based on the morphological features apparent after Wright-Giemsa staining, phagocytic activity and expression of cell surface markers as analyzed by flow cytometry. On the other hand, the results indicated that exposure of KG1 cells to either 3-HK or the new compound for 3-4 days induced apoptosis as assayed qualitatively by acridine orange/ethidium bromide (Ao/EtBr) double staining, agarose gel electrophoresis and quantitatively by Annexin-V technique and sub-G1 DNA staining using flow cytometry. Based on the present data, these two active constituents of D. lessertii have the novelty of being further evaluated for pharmaceutical applications.     

The ultrastructure of infectious L-type bovine spongiform encephalopathy prions constrains molecular models

Bovine spongiform encephalopathy (BSE) is a prion disease of cattle that is caused by the misfolding of the cellular prion protein (PrP^C) into an infectious conformation (PrP^Sc). PrP^C is a predominantly α-helical membrane protein that misfolds into a β-sheet rich, infectious state, which has a high propensity to self-assemble into amyloid fibrils. Three strains of BSE prions can cause prion disease in cattle, including classical BSE (C-type) and two atypical strains, named L-type and H-type BSE. To date, there is no detailed information available about the structure of any of the infectious BSE prion strains. In this study, we purified L-type BSE prions from transgenic mouse brains and investigated their biochemical and ultrastructural characteristics using electron microscopy, image processing, and immunogold labeling techniques. By using phosphotungstate anions (PTA) to precipitate PrP^Sc combined with sucrose gradient centrifugation, a high yield of proteinase K-resistant BSE amyloid fibrils was obtained. A morphological examination using electron microscopy, two-dimensional class averages, and three-dimensional reconstructions revealed two structural classes of L-type BSE amyloid fibrils; fibrils that consisted of two protofilaments with a central gap and an average width of 22.5 nm and one-protofilament fibrils that were 10.6 nm wide. The one-protofilament fibrils were found to be more abundant compared to the thicker two-protofilament fibrils. Both fibrillar assemblies were successfully decorated with monoclonal antibodies against N- and C-terminal epitopes of PrP using immunogold-labeling techniques, confirming the presence of polypeptides that span residues 100–110 to 227–237. The fact that the one-protofilament fibrils contain both N- and C-terminal PrP epitopes constrains molecular models for the structure of the infectious conformer in favour of a compact four-rung β-solenoid fold.     

Selectivity of 3'-O-methylponkoranol for inhibition of N- and C-terminal maltase glucoamylase and sucrase isomaltase, potential therapeutics for digestive disorders or their sequelae

Human maltase glucoamylase (MGAM) and sucrase isomaltase (SI) are two human intestinal glucosidases responsible for the final step of starch hydrolysis. MGAM and SI are anchored to the small intestinal brush border epithelial cells and contain two catalytic N-terminal and C-terminal subunits. In this study, we report the inhibition profile of 3'-O-methylponkoranol for the individual recombinant N and C terminal enzymes and compare the inhibitory activities of this compound with de-O-sulfonated ponkoranol. We show that 3'-O-methylponkoranol inhibits the different subunits to different extents, with extraordinary selectivity for C-terminal SI (K(i)=7±2nM). The enzymes themselves could serve as therapeutic targets for the treatment of digestive disorders or their sequelae.     

Modulation of Starch Digestion for Slow Glucose Release through "Toggling" of Activities of Mucosal α-Glucosidases

Starch digestion involves the breakdown by α-amylase to small linear and branched malto-oligosaccharides, which are in turn hydrolyzed to glucose by the mucosal α-glucosidases, maltase-glucoamylase (MGAM) and sucrase-isomaltase (SI). MGAM and SI are anchored to the small intestinal brush-border epithelial cells, and each contains a catalytic N- and C-terminal subunit. All four subunits have α-1,4-exohydrolytic glucosidase activity, and the SI N-terminal subunit has an additional exo-debranching activity on the α-1,6-linkage. Inhibition of α-amylase and/or α-glucosidases is a strategy for treatment of type 2 diabetes. We illustrate here the concept of "toggling": differential inhibition of subunits to examine more refined control of glucogenesis of the α-amylolyzed starch malto-oligosaccharides with the aim of slow glucose delivery. Recombinant MGAM and SI subunits were individually assayed with α-amylolyzed waxy corn starch, consisting mainly of maltose, maltotriose, and branched α-limit dextrins, as substrate in the presence of four different inhibitors: acarbose and three sulfonium ion compounds. The IC(50) values show that the four α-glucosidase subunits could be differentially inhibited. The results support the prospect of controlling starch digestion rates to induce slow glucose release through the toggling of activities of the mucosal α-glucosidases by selective enzyme inhibition. This approach could also be used to probe associated metabolic diseases.     

Amelioration of diet-induced nonalcoholic steatohepatitis in rats by Mn-salen complexes via reduction of oxidative stress

Background

Nonalcoholic steatohepatitis (NASH), a progressive stage of nonalcoholic fatty liver disease (NAFLD), is characterized by steatosis (accumulation of triacylglycerols within hepatocytes) along with inflammation and ballooning degeneration. It has been suggested that oxidative stress may play an important role in the progress of NAFLD to NASH. The aim of present study was to determine whether antioxidant supplementations using EUK-8, EUK-134 and vitamin C could improve the biochemical and histological abnormalities associated with diet-induced NASH in rats.

Methods

NASH was induced in male N-Mary rats by feeding a methionine - choline deficient (MCD) diet. The rats were fed either normal chow or MCD diet for 10 weeks. After NASH development, the MCD-fed rats were randomly divided into four groups of six: the NASH group that received MCD diet, the EUK-8 group which was fed MCD diet plus EUK-8, the EUK-134 group which was fed MCD diet plus EUK-134 and the vitamin C group which received MCD diet plus vitamin C. EUK-8, EUK-134 and vitamin C (30 mg/kg body weight/day) were administered by gavage for eight weeks.

Results

Treatment of MCD-fed rats with salens reduced the sera aminotransferases, cholesterol, low density lipoprotein contents, the extent of lipid peroxidation and protein carbonylation whereas the HDL-C cholesterol levels were significantly increased. In addition, EUK-8 and EUK-134 improved steatosis, ballooning degeneration and inflammation in liver of MCD-fed rats.

Conclusion

Antioxidant (EUK-8, EUK-134 and vitamin C) supplementation reduces NASH-induced biochemical and histological abnormalities, pointing out that antioxidant strategy could be beneficial in treatment of NASH.     

Induction of megakaryocytic differentiation in chronic myelogenous leukemia cell K562 by 3-hydrogenkwadaphnin

3-Hydrogenkwadaphnin (3-HK) is a daphnane-type diterpene ester isolated from Dendrostellera lessertii (Thymelaeaceae) with high differentiation and apoptotic potency in leukemic cells without any measurable adverse effects on normal cells (Moosavi et al., 2005b). In this study, we report that 3-HK (12 nM) has the ability to cease proliferation, induce differentiation and apoptosis in chronic myelogenous leukemia (CML) K562 cell line. The treated cells lost erythroid properties and differentiated along the megakaryocytic lineage based on the morphological features apparent after Wright-Giemsa staining, DNA content analysis and the expression of cell surface marker glycoprotein IIb as analyzed by flow cytometry. Moreover, using Hoechst 33258 and Annexin V double staining indicated the occurrence of apoptosis among the treated cells. On the other hand, restoration of the depleted GTP pool size by exogenous addition of guanosine (50 microM) reduced the effect of the drug regarding the extent of differentiation while no further enhancement of 3-HK effect was obtained by addition of exogenous hypoxanthine (100 microM). These interesting results necessitate further investigation regarding the mechanism of action of this unique anti-leukemic agent.     

Design,synthesis, and biological evaluation of ketoprofen analogs as potent cyclooxygenase-2 inhibitors

A new series of ketoprofen analogs were synthesized to evaluate their biological activities as selective cyclooxygenase-2 (COX-2) inhibitors. In vitro COX-1 and COX-2 inhibition studies showed that all compounds were potent and selective inhibitors of the COX-2 isozyme with IC₅₀ values in the highly potent 0.057–0.085 μM range, and COX-2 selectivity indexes in the 115 to >1298.7 range. Compounds possessing azido pharmacophore group (8a and 8b) exhibited highly COX-2 inhibitory selectivity and potency even more than reference drug celecoxib. Molecular modeling studies indicated that the azido substituent can be inserted deeply into the secondary pocket of COX-2 active site for interactions with Arg⁵¹³.     

Alkaline phosphatase refolding assisted by sequential use of oppositely charged detergents: a new artificial chaperone system

A novel artificial chaperone system, based on combination of oppositely charged detergents, was elaborated to refold soluble alkaline phosphatase. Upon dilution of urea-denatured alkaline phosphatase to a nondenaturing urea concentration in the presence of the capturing agent, complexes of the detergent and non-native protein molecules are formed and thereby the formation of protein aggregates is prevented. The so-called captured protein is unable to refold from the detergent-protein complex states unless a stripping agent is used to gradually remove the detergent molecules. In that respect, we used detergents with variable charges and tail lengths to initiate and complete the refolding process. The results obtained from various analyses (fluorescence, UV, circular dichroism, surface tension, turbidity measurements and activity assays) indicated that the extent of refolding assistance was different due to detergents structure and also the length of hydrophobic portion of each detergent. These observed differences were attributed to the strong electrostatic interactions among the capturing and stripping detergents used in this investigation. Collectively it is expected that protein refolding process can be achieved easier, cheaper and more efficient, using the new technique reported here.     

Evolutionary mechanisms underlying the functional divergence of duplicate genes involved in vertebrates' circadian rhythm pathway

Circadian rhythms, that are governed physiologically and behaviorally by endogenous clock, have been described in many species. Living organisms use this endogenous circadian clock to anticipate environmental transitions, perform activities at biologically advantageous times during the day, and undergo characteristic seasonal responses. Gene duplication is one of the most important mechanisms in the evolution of gene diversity. After duplication, one or both of duplicates can accumulate amino acid changes, thereby promoting functional divergence through the action of natural selection. The circadian system, like many other multigene families, has undergone this genetic revolution, and so circadian genes that are found in single copies in insects are duplicated in vertebrates. We analyzed six groups of genes involved in vertebrates' circadian rhythm pathway to find signatures of molecular evolutionary processes such as gene duplication, natural selection, recombination, and functional divergence. The obtained results, then, were used to determine what evolutionary forces have influenced the fates of duplicated genes of each group. We showed in this research that recombination has not been widespread during the evolution of circadian genes and that purifying selection has been the prominent natural pressure operating on circadian genes. We also showed that the evolution of circadian genes has been depended on gene duplication and functional divergence. Finally, we put forward models best describing the evolutionary fates of circadian duplicates.     

Solid-phase Assisted Refolding of Carbonic Anhydrase Using β-Cyclodextrin-Polyurethane Polymer

Artificial chaperone-assisted refolding has been shown to be an effective approach for improving the refolding yield of denatured proteins. Independent refolding of several structurally diverse proteins by this approach has provided promising results regarding significant suppression of aggregation along with enhanced refolding yields. However, from the industrial point of view, some modifications seem to be essential for making the technique more efficient. In that regard and with a cost-cutting goal we designed, for the first time, a beta-cyclodextrin-polyurethane polymer to replace the soluble beta-cyclodextrin as the stripping agent for refolding of carbonic anhydrase. Our results indicated that under the optimally developed refolding environment, the denatured carbonic anhydrase was refolded with a yield of 75% using 15 mg/mL of the beta-cyclodextrin-polyurethane polymer, a yield near to stripping by soluble beta-CD. This new stripping approach seems to constitute an ideal approach for refolding of proteins at much lower industrial costs compared to stripping with soluble beta-cyclodextrin. However, further-improvements in solid-phase artificial chaperone assisted technique are demanded either through synthesizing better stripping agents or by optimizing and defining better refolding environments.