Antimicrobial and cytotoxic activity of 18 prenylated flavonoids isolated from medicinal plants: Morus alba L., Morus mongolica Schneider, Broussnetia papyrifera (L.) Vent, Sophora flavescens Ait and Echinosophora koreensis Nakai

Antimicrobial activity of the 18 prenylated flavonoids, which were purified from five different medicinal plants, was evaluated by determination of MIC using the broth microdilution methods against four bacterial and two fungal microorganisms (Candida albicans, Saccaromyces cerevisiae, Escherichia coli, Salmonella typhimurium, Staphylococcus epidermis and S. aureus). Papyriflavonol A, kuraridin, sophoraflavanone D and sophoraisoflavanone A exhibited a good antifungal activity with strong antibacterial activity. Kuwanon C, mulberrofuran G, albanol B, kenusanone A and sophoraflavanone G showed strong antibacterial activity with 5–30 μg/ml of MICs. Morusin, sanggenon B and D, kazinol B, kurarinone, kenusanone C and isosophoranone were effective to only gram positive bacteria, and broussochalcone A was effective to C. albicans. IC₅₀ values of papyriflavonol A, kuraridin, sophoraflavanone D, sophoraisoflavanone A and broussochalcone A in HepG2 cells were 20.9, 37.8, 39.1, 22.1, and 22.0 μg/ml, respectively. These results support the use of prenylated flavonoids in Asian traditional medicine to treat microbial infection and indicate a high potential for prenylated flavonoids as antimicrobial agents as well as anti-inflammatory agents.     

Regioselective hydroxylation of daidzein using P450 (CYP105D7) from Streptomyces avermitilis MA4680

Regiospecific 3′‐hydroxylation reaction of daidzein was performed with CYP105D7 from Streptomyces avermitilis MA4680 expressed in Escherichia coli. The apparent K_m and k_cat values of CYP105D7 for daidzein were 21.83 ± 6.3 µM and 15.01 ± 0.6 min^?1 in the presence of 1 µM of CYP105D7, putidaredoxin (CamB) and putidaredoxin reductase (CamA), respectively. When CYP105D7 was expressed in S. avermitilis MA4680, its cytochrome P450 activity was confirmed by the CO‐difference spectra at 450 nm using the whole cell extract. When the whole‐cell reaction for the 3′‐hydroxylation reaction of daidzein was carried out with 100 µM of daidzein in 100 mM of phosphate buffer (pH 7.5), the recombinant S. avermitilis grown in R2YE media overexpressing CYP105D7 and ferredoxin FdxH (SAV7470) showed a 3.6‐fold higher conversion yield (24%) than the corresponding wild type cell (6.7%). In a 7 L (working volume 3 L) jar fermentor, the recombinants S. avermitilis grown in R2YE media produced 112.5 mg of 7,3′,4′‐trihydroxyisoflavone (i.e., 29.5% conversion yield) from 381 mg of daidzein in 15 h. Biotechnol. Bioeng. 2010. 105: 697–704. © 2009 Wiley Periodicals.     

AMP-activated protein kinase (AMPK) activators from Myristica fragrans (nutmeg) and their anti-obesity effect

AMP-activated protein kinase (AMPK) is a potential therapeutic target for the treatment of metabolic syndrome including obesity and type-2 diabetes. As part of an ongoing search for new AMPK activators from plants, this study found that the total extract of Myristica fragrans (nutmeg) activated the AMPK enzyme in differentiated C2C12 cells. As active constituents, seven 2,5-bis-aryl-3,4-dimethyltetrahydrofuran lignans, tetrahydrofuroguaiacin B (1), saucernetindiol (2), verrucosin (3), nectandrin B (4), nectandrin A (5), fragransin C₁ (6), and galbacin (7) were isolated from this extract. Among the isolates, compounds 1, 4, and 5 at 5 μM produced strong AMPK stimulation in differentiated C2C12 cells. In addition, the preventive effect of a tetrahydrofuran mixture (THF) on weight gain in a diet-induced animal model was further examined. These results suggest that nutmeg and its active constituents can be used not only for the development of agents to treat obesity and possibly type-2 diabetes but may also be beneficial for other metabolic disorders.     

Endostatin blocks vascular endothelial growth factor-mediated signaling via direct interaction with KDR/Flk-1

Endostatin, a fragment of collagen XVIII, is a potent anti-angiogenic protein, but the molecular mechanism of its action is not yet clear. We examined the effects of endostatin on the biological and biochemical activities of vascular endothelial growth factor (VEGF). Endostatin blocked VEGF-induced tyrosine phosphorylation of KDR/Flk-1 and activation of ERK, p38 MAPK, and p125(FAK) in human umbilical vein endothelial cells. Endostatin also inhibited the binding of VEGF(165) to both endothelial cells and purified extracellular domain of KDR/Flk-1. Moreover, the binding of VEGF(121) to KDR/Flk-1 and VEGF(121)-stimulated ERK activation were blocked by endostatin. The direct interaction between endostatin and KDR/Flk-1 was confirmed by affinity chromatography. However, endostatin did not bind to VEGF. Our findings suggest that a direct interaction of endostatin with KDR/Flk-1 may be involved in the inhibitory function of endostatin toward VEGF actions and responsible for its potent anti-angiogenic and anti-tumor activities in vivo.     

Isolation and characterization of a mycovirus in Lentinula edodes

A mycovirus was isolated from an edible mushroom, Lentinula edodes, that was suffering from a severe epidemic. Fractionation of the diseased cell extract by isopycnic centrifugation with 50% CsCl revealed that the diseased mushroom was infected by Lentinula edodes spherical virus (LeSV), a new spherical virus with a diameter of 55 nm. The particle of LeSV encapsidated the 12 kb RNA genome by a 120 kDa coat protein. BLAST analysis of the partially sequenced LeSV genome showed 95% sequence identity with a putative RNA-dependent RNA polymerase (RdRp) gene of the mycovirus HKB, which was previously reported as being a double-stranded RNA (dsRNA) element. In contrast to HKB, the RNA genome in LeSV is encapsidated by the 120 kDa coat protein. To confirm that the LeSV coat protein is encoded by the viral genome, the N-terminal amino acid sequence of the coat protein was determined. The resulting N-terminal amino acid sequence, N-SALDVAPVVPELYFXXLEV-C, was found to be located in the middle of the HKB ORF1, suggesting that the LeSV coat protein was indeed encoded by the virus. To detect LeSV in L. edodes, a primer set targeting the RdRp gene was designed based on the partial sequence of the LeSV genome. RT-PCR analysis showed that 56 of the 84 commercially available dikaryotic cultivars carry LeSV. The transmission pattern of the virus was determined by analysing basidiospores from LeSV-infected and LeSV-free fruiting bodies. Nine out of 10 basidiospores from the LeSV-infected cultivars contained the virus while the spores from the LeSV-free parent were free of LeSV, suggesting that vertical transmission is the primary mode of LeSV propagation.     

Incidence of Alternaria Species Associated with Watermelon Leaf Blight in Korea

Alternaria leaf blight is one of the most common diseases in watermelon worldwide. In Korea, however, the Alternaria species causing the watermelon leaf blight have not been investigated thoroughly. A total of 16 Alternaria isolates was recovered from diseased watermelon leaves with leaf blight symptoms, which were collected from 14 fields in Korea. Analysis of internal transcribed spacer (ITS) region, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), and RNA polymerase II second largest subunit (RPB2) were not competent to differentiate the Alternaria isolates. On the contrary, analysis of amplicon size of the histone H3 (HIS3) gene successfully differentiated the isolates into three Alternaria subgroups, and further sequence analysis of them identified three Alternaria spp. Alternaria tenuissima, A. gaisen, and A. alternata. Representative Alternaria isolates from three species induced dark brown leaf spot lesions on detached watermelon leaves, indicating that A. tenuissima, A. gaisen, and A. alternata are all causal agents of Alternaria leaf blight. Our results indicate that the Alternaria species associated watermelon leaf blight in Korea is more complex than reported previously. This is the first report regarding the population structure of Alternaria species causing watermelon leaf blight in Korea.     

The arginine regulatory protein mediates repression by arginine of the operons encoding glutamate synthase and anabolic glutamate dehydrogenase in Pseudomonas aeruginosa

The arginine regulatory protein of Pseudomonas aeruginosa, ArgR, is essential for induction of operons that encode enzymes of the arginine succinyltransferase (AST) pathway, which is the primary route for arginine utilization by this organism under aerobic conditions. ArgR also induces the operon that encodes a catabolic NAD(+)-dependent glutamate dehydrogenase (GDH), which converts l-glutamate, the product of the AST pathway, in alpha-ketoglutarate. The studies reported here show that ArgR also participates in the regulation of other enzymes of glutamate metabolism. Exogenous arginine repressed the specific activities of glutamate synthase (GltBD) and anabolic NADP-dependent GDH (GdhA) in cell extracts of strain PAO1, and this repression was abolished in an argR mutant. The promoter regions of the gltBD operon, which encodes GltBD, and the gdhA gene, which encodes GdhA, were identified by primer extension experiments. Measurements of beta-galactosidase expression from gltB::lacZ and gdhA::lacZ translational fusions confirmed the role of ArgR in mediating arginine repression. Gel retardation assays demonstrated the binding of homogeneous ArgR to DNA fragments carrying the regulatory regions for the gltBD and gdhA genes. DNase I footprinting experiments showed that ArgR protects DNA sequences in the control regions for these genes that are homologous to the consensus sequence of the ArgR binding site. In silica analysis of genomic information for P. fluorescens, P. putida, and P. stutzeri suggests that the findings reported here regarding ArgR regulation of operons that encode enzymes of glutamate biosynthesis in P. aeruginosa likely apply to other pseudomonads.     

Development of mouse enucleated oocytes receiving a nucleus from different stages of the second cell cycle.

The influence of the stage of the cell cycle of donor nuclei on the development of mouse oocytes enucleated at telophase I was examined. After nuclear transplantation and activation, a high proportion of the oocytes remodelled a nucleus, emitted a polar body and formed a pronuclear-like nucleus. Most of the reconstituted embryos that received an interphase nucleus 30-32 h or 34-36 h after treatment with human chorionic gonadotrophin (hCG) arrested at the 2-cell stage. The reconstituted embryos were able to develop to blastocysts when nuclei from late 2-cell embryos (44-46 and 48-50 h after hCG) were transferred to the oocytes. The resulting blastocysts were transferred to recipients and ten live young were obtained from the embryos that formed a pronuclear-like nucleus after extrusion of a polar body. Thus, the developmental ability of the reconstituted embryos was critically influenced by the stage of the cell cycle of the donor nuclei.     

The role of transient receptor potential channel blockers in human gastric cancer cell viability

Transient receptor potential cation channel, subfamily M, receptor 7 (TRPM7) is a ubiquitous divalent-selective ion channel with its own kinase domain. Human gastric cancer cells express the TRPM7 channel, and the presence of this channel is essential for cell survival. Recent studies have suggested that 5-lipoxygenase (5-LOX) inhibitors are potent blockers of the TRPM7 channels. The aim of this study was to show the effects of 5-LOX inhibitors on the growth and survival of gastric cancer cells. Among 5-LOX inhibitors, nordihydroguaiaretic acid (NDGA), 2,3,5-trimethyl-6-(12-hydroxy-5,10-dodecadiynyl)-1,4-benzoquinone (AA861), and 3-[1-(p-chlorobenzyl)-5-(isopropyl)-3-tert-butylthioindol-2-yl]-2,2-dimethylpropanoic acid (MK886) were potent blockers of TRPM7-like currents in gastric cancer cells and also induced cell death. However, zileuton was ineffective in suppressing TRPM7-like current activity and inducing cell death. Moreover, a specific transient receptor potential cation channel, subfamily C, member 3 (TRPC3) inhibitor, a pyrazole compound (Pyr3), and a specific melastatin TRP (TRPM4) inhibitor, 9-phenanthrol, did not affect TRPM7-like currents or induce cell death. We conclude that TRPM7 has an important role in the growth and survival of gastric cancer cells and a likely potential target for the pharmacological treatment of gastric cancer.