Nongporn Hutadilok-Towatana

Purification and characterization of an extracellular protease from alkaliphilic and thermophilic Bacillus sp. PS719

An alkaline protease was purified to apparent homogeneity from culture supernatants of Bacillus sp. PS719, a novel alkaliphilic, thermophilic bacterium isolated from a thermal spring soil sample, by ammonium sulfate precipitation followed by DEAE-cellulose and alpha-casein agarose column chromatographies. The purified enzyme migrated as a single protein band of 42 kDa during both denaturing and nondenaturing gel electrophoresis, suggesting that it consists of a single polypeptide chain. Its isoelectric point was approximately 4.8. The protease exhibited maximum activity towards azocasein at pH 9.0 and at 75 degrees C. The enzyme activity was stimulated by Ca2+, but was inhibited in the presence of Fe2+ or Cu2+. The enzyme was stable in the pH range 8.0 to 10.0 and up to 80 degrees C in the absence of Ca2+. Since phenylmethylsulfonyl fluoride (PMSF) and 3,4-dichloroisocoumarin (DCI) in addition to N-alpha-p-tosyl-L-lysine chloromethyl ketone (TLCK) completely inhibited the activity, this enzyme appears to be a trypsin-like serine protease. Among the various oligopeptidyl-p-nitroanilides tested, the protease showed a preference for cleavage at arginine residues on the carboxylic side of the scissile bond of the substrate, liberating p-nitroaniline from N-carbobenzoxy (CBZ)-L-arginine-p-nitroanilide with the K(m) and V(max) values of 0.6 mM and 1.0 micromol.min(-1).mg protein(-1), respectively.

Sesquiterpene and Xanthone Derivatives from the Sea Fan-Derived Fungus Aspergillus sydowii PSU-F154

Three new sesquiterpenes, named aspergillusenes A and B and (+)-(7S)-7-O-methylsydonic acid, and two new hydrogenated xanthone derivatives, named aspergillusones A and B, were isolated from the sea fan-derived fungus Aspergillus sydowii PSU-F154 together with 10 known compounds. Their structures were identified on the basis of spectroscopic data. The isolated compounds were evaluated for their antioxidant activity.

Antioxidative and Free Radical Scavenging Activities of Some Plants Used in Thai Folk Medicine

Abstract Ten methanol extracts from various parts of seven medicinal plants, Angiopteris evecta Hoffm.., (Marattiaceae) Citrus hystrix DC.., (Rutaceae) Laurentia longiflora (L.). Peterm.., (Campanulaceae) Nelumbo nucifera Gaertn.., (Nelumbonaceae), Piper sarmentosum Roxb.., (Piperaceae), Portulaca oleracea Linn.., (Portulacaceae) and Stachytarphera indica (L.). Vahl. (Verbenaceae), commonly used in Thai traditional medicine, were evaluated for their antioxidative and free radical scavenging activities. Among them, only that prepared from sacred lotus (N. nucifera) leaves exhibited a pronounced activity in the 1,1-diphenyl-2-picrylhydrazyl (DPPH) scavenging assay with an IC50 of 90 µg/ml, compared with an IC50 of 30 µg/ml for the butylated hydroxytoluene (BHT) control. The same extract was also found to be the most potent in removing the superoxide anion () radical and in inhibiting the 2,2′-azo-bis.-(2-amidinopropane) dihydrochloride (AAPH)-induced erythrocyte hemolysis and lipid peroxidation in a rat brain homogenate. The extract from leaves and peels of the Kaffir lime (C. hystrix.) exerted the strongest effect on production of the hydroxyl radical (OH·). They conferred a twice greater protection of deoxyribose from OH· than did tannin. However, none of the extracts examined in this study showed a significant pro-oxidant action in the bleomycin-dependent DNA oxidation system.

Human fortilin is a molecular target of dihydroartemisinin

Dehydroartemisinin (DHA) is an effective anti‐malaria agent. Fortilin is an anti‐apoptotic molecule overexpressed in many human cancers. Here, we show that DHA binds human fortilin, increases the ubiquitination of fortilin, shortens fortilins half‐life in a proteasome‐dependent fashion, and reduces cellular levels of fortilin in varieties of cells. DHA induced DNA fragmentation in U2OS cells in a fortilin‐dependent manner. The fortilin‐knocked‐down cells were less susceptible—and fortilin‐overexpressing cells more susceptible—to DHA than were wild‐type cells, suggesting that apoptotic effects of DHA are—at least partly—conferred through fortilin. Together, these data suggest that fortilin is a molecular target of DHA. DHA and its derivative may prove to be viable anti‐cancer agents in fortilin‐overexpressing cancers.

Embryonic lethality of fortilin-null mutant mice by BMP-pathway overactivation

BACKGROUND Fortilin negatively regulates apoptosis and is overexpressed in cancer. However, the role of fortilin in mammalian development is not clear. METHODS AND RESULTS In order to evaluate the physiological role of fortilin in vivo, we performed a targeted disruption of the fortilin gene in mice. Fortilin(+/-) mice have the ability to survive and exhibit normal growth, while fortilin(-/-) mice are embryonically lethal around the 3.5 days post-coital (dpc). Cultured blastocysts from fortilin(+/-) embryos undergo normal outgrowth to produce inner cell mass (ICM) and trophoblasts (TB), while ICM of fortilin(-/-) embryos either fails to outgrow or prematurely disintegrates. Mouse embryonic fibroblasts (MEF) derived from fortilin(+/-) embryos are more susceptible to noxious stimuli than are wild type embryos. It has been consistently shown in Xenopus embryos that the depletion of fortilins message severely compromises the formation of neural tissue, even in the brain, while overexpression of fortilin induces the partial double body axis in embryos and is capable of blocking BMP4-induced transcription of Vent1, Vent2, and Msx1 genes. This suggests that fortilin is an inhibitor of the BMP pathway. Strikingly, when fortilin levels are reduced by siRNA, BMP4 causes MEF to undergo extensive DNA-fragmentation, while DNA fragmentation is minimal in the presence of fortilin. In addition, BMP4 induces more Msx2 in the absence of fortilin than in its presence. Furthermore, Msx2 overexpression causes MEF to undergo apoptotic cell death. CONCLUSION We conclude that in early phase of development, fortilin functions as an inhibitor of the BMP pathway. The presence of fortilin in the very early stages of development is required for the survival of embryos. GENERAL SIGNIFICANCE Abnormalities in the fortilin gene may be associated with early pregnancy loss.

Cowaxanthone F, a new tetraoxygenated xanthone, and other anti-inflammatory and antioxidant compounds from Garcinia cowa

A new tetraoxygenated xanthone, cowaxanthone F (1), as well as four known compounds, morelloflavone (2), volkensiflavone (3), morelloflavone-7″-O-glucoside (fukugiside, 4), and 1,6-dihydroxyxanthon...

Morelloflavone blocks injury-induced neointimal formation by inhibiting vascular smooth muscle cell migration

BACKGROUND In-stent restenosis, or renarrowing within a coronary stent, is the most ominous complication of percutaneous coronary intervention, caused by vascular smooth muscle cell (VSMC) migration into and proliferation in the intima. Although drug-eluting stents reduce restenosis, they delay the tissue healing of the injured arteries. No promising alternative anti-restenosis treatments are currently on the horizon. METHODS In endothelium-denudated mouse carotid arteries, oral morelloflavone-an active ingredient of the Thai medicinal plant Garcinia dulcis-significantly decreased the degree of neointimal hyperplasia, without affecting neointimal cell cycle progression or apoptosis as evaluated by Ki-67 and TUNEL staining, respectively. At the cellular level, morelloflavone robustly inhibited VSMC migration as shown by both scratch wound and invasion assays. In addition, morelloflavone prevented VSMCs from forming lamellipodia, a VSMC migration apparatus. Mechanistically, the inhibition by morelloflavone of VSMC migration was through its negative regulatory effects on several migration-related kinases, including FAK, Src, ERK, and RhoA. Consistently with the animal data, morelloflavone did not affect VSMC cell cycle progression or induce apoptosis. RESULTS These data suggest that morelloflavone blocks injury-induced neointimal hyperplasia via the inhibition of VSMC migration, without inducing apoptosis or cell cycle arrest. GENERAL SIGNIFICANCE We propose morelloflavone to be a viable oral agent for the prevention of restenosis, without compromising effects on the integrity and healing of the injured arteries.

Benzene, coumarin and quinolinone derivatives from roots of Citrus hystrix

Two coumarins, hystrixarin (1) and (+)-hopeyhopin (2); a benzenoid derivative, hystroxene-I (3) and a quinolinone alkaloid, hystrolinone (4), along with 33 known compounds were isolated from the crude acetone extract of the roots of Citrus hystrix. Their structures were determined by analysis of 1D and 2D NMR spectroscopic data. The antioxidant, anti-HIV and antibacterial activities of the isolated compounds were also evaluated.

Morelloflavone from Garcinia dulcis as a novel biflavonoid inhibitor of HMG-CoA reductase.

Morelloflavone, a biflavonoid from Garcinia dulcis previously shown to have hypocholesterolemic activity, was examined for its effect on HMG‐CoA reductase, the rate‐limiting enzyme of the cholesterol biosynthetic pathway. By using the catalytic domain of house mouse HMG‐CoA reductase, morelloflavone was found to inhibit the enzyme activity by competing with HMG‐CoA whereas it was non‐competitive towards NADPH. The inhibition constants (Ki) with respect to HMG‐CoA and NADPH were 80.87 ± 0.06 µm and 103 ± 0.07 µm, respectively. Both flavonoid subunits of this compound, naringenin and luteolin, equally competed with HMG‐CoA with Ki of 83.58 ± 4.37 µm and 83.59 ± 0.94 µm, respectively, and were also non‐competitive with NADPH (Ki of 182 ± 0.67 µm and 188 ± 0.14 µm, respectively). Due to these findings, we suggest that each subunit of morelloflavone would occupy the active site of the enzyme, thereby blocking access of its substrate. The present study thus demonstrates the ability of morelloflavone from G. dulcis to inhibit HMG‐CoA reductase in vitro. As a result, this biflavonoid might serve as a new candidate for the future development of hypocholesterolemic agents. Copyright

Ivy gourd (Coccinia grandis L. Voigt) root suppresses adipocyte differentiation in 3T3-L1 cells

BackgroundIvy gourd (Coccinia grandis L. Voigt) is a tropical plant widely distributed throughout Asia, Africa, and the Pacific Islands. The anti-obesity property of this plant has been claimed but still remains to be scientifically proven. We therefore investigated the effects of ivy gourd leaf, stem, and root on adipocyte differentiation by employing cell culture model.MethodsDried roots, stems, and leaves of ivy gourd were separately extracted with ethanol. Each extract was then applied to 3T3-L1 pre-adipocytes upon induction with a mixture of insulin, 3-isobutyl-1-methylxanthine, and dexamethasone, for anti-adipogenesis assay. The active extract was further fractionated by a sequential solvent partitioning method, and the resulting fractions were examined for their abilities to inhibit adipogenesis in 3T3-L1 cells. Differences in the expression of adipogenesis-related genes between the treated and untreated cells were determined from their mRNA and protein levels.ResultsOf the three ivy gourd extracts, the root extract exhibited an anti-adipogenic effect. It significantly reduced intracellular fat accumulation during the early stages of adipocyte differentiation. Together with the suppression of differentiation, expression of the genes encoding PPARγ, C/EBPα, adiponectin, and GLUT4 were down-regulated. Hexane-soluble fraction of the root extract also inhibited adipocyte differentiation and decreased the mRNA levels of various adipogenic genes in the differentiating cells.ConclusionsThis is the first study to demonstrate that ivy gourd root may prevent obesity based mainly on the ability of its active constituent(s) to suppress adipocyte differentiation in vitro. Such an inhibitory effect is mediated by at least down-regulating the expression of PPARγ-the key transcription factor of adipogenesis in pre-adipocytes during their early differentiation processes.