Zanthoxylum ailanthoides

Zanthoxylum ailanthoides (ZA) has been used as folk medicines in East Asian and recently reported to have several bioactivity; however, the studies of ZA on the regulation of triacylglycerol (TG) biosynthesis have not been elucidated yet. In this study, we examined whether the methanol extract of ZA (ZA-M) could reduce oleic acid- (OA-) induced intracellular lipid accumulation and confirmed its mode of action in HepG2 cells. ZA-M was shown to promote the phosphorylation of AMPK and its upstream LKB1, followed by reduction of lipogenic gene expressions. As a result, treatment of ZA-M blocked de novo TG biosynthesis and subsequently mitigated intracellular neutral lipid accumulation in HepG2 cells. ZA-M also inhibited OA-induced production of reactive oxygen species (ROS) and TNF-α, suggesting that ZA-M possess the anti-inflammatory feature in fatty acid over accumulated condition. Taken together, these results suggest that ZA-M attenuates OA-induced lipid accumulation and inflammation through the activation of LKB1/AMPK signaling pathway in HepG2 cells

Differential effects of luteolin and its glycosides on invasion and apoptosis in MDA-MB-231 triple-negative breast cancer cells

Luteolin is known to have anticancer activity in various ca ncers. Recent studies have shown that luteolin glyco- sides, such as luteolin-8- C - β -fucopyranoside, 7-methoxy-luteolin-8-C- β -(6- deoxyxylopyranos-3-uloside) and lu- teolin-8-C- β - D -glucopyranoside, flavonoid s that are present in Arthraxon hispidus , exert antimigratory and anti- invasive effects, but no cytotoxic effect in estrogen receptor-positive MCF7 breast cancer cells. In the present study, we further investigated and compared differential effects of luteolin and its glycosides in MDA-MB-231 triple-negative breast cancer cells. Lute olin suppressed the expression of matrix metalloproteinase-9 and inhibited migration and invasion in MDA-MB-231 cells treated with the tumor promotor 12-O-tetradecanoylphorbol-13- acetate at non-cytotoxic concentrations (0, 5, and 10 μ M). Furthermore, at cytotoxic concentrations (20 and 40 μ M), luteolin induced apoptosis via extrinsic and intrinsic pathways in MDA-MB-231 cells. However, luteolin glycosides did not exert any cytotoxic, antimigratory, or anti-invasive effect in MDA-MB-231 cells . In brief, l u- teolin had both antimetastatic and cytotoxic effects on MDA-MB-231 cells, whereas luteolin glycosides had no effect on this cell line. Taking together the present results and our previous findings on the differential effects of luteolin and its glycosides on MDA-MB-231 and MCF-7 br east cancer cells, luteolin and its glycosides can be suggested as a potential candida te for breast cancer therapy

Effects of Hanwoo (Korean cattle) manure as organic fertilizer on plant growth, feed quality, and soil bacterial community

IntroductionThe development of organic manure from livestock excreta is a useful source for sustainable crop production in environment-friendly agriculture. Organic manure increases soil microbial activity and organic matter (OM) supply. The excessive use of chemical fertilizers (CFs) leads to air and water pollution caused by toxic chemicals and gases, and soil quality degradation via nutrient imbalance due to supplying specific chemical components. Thus, the use of organic manure will serve as a long-term supply of various nutrients in soil via OM decomposition reaction as well as the maintenance of environment.MethodsIn this study, we aimed to analyze the diverse effects of Hanwoo manure (HM) on plant growth, feed quality, and soil bacterial communities in comparison with CFs, commercial poultry manure (CM), and the combined use of chemical fertilizer and Hanwoo manure (HM+CF). We analyzed the contents of crude matter (protein, fat, fiber, and ash), P, acid detergent fiber (ADF), and neutral detergent fiber (NDF) through feed quality analysis, and the contents or activities of total phenol, total flavonoid, ABTS, nitrite scavenging, and reducing power via the antioxidant assay. Furthermore, the soil microbial communities were determined using 16S rRNA sequencing. We compared the soil bacteria among different soil samples by using amplicon sequence variant (ASV) analysis.Results and discussionWe observed increased OM in the soil of the HM group compared to that of the CF and non-treated groups over a period of two years. Moreover, HM+CF treatment enormously improved plant growth. Organic manure, especially HM, caused an increase in the content of crude ash and phosphorus in plants. There were no significant differences in total polyphenol, total flavonoid, ABTS, nitrite scavenging, and reducing power in plants between HM and CF groups. Finally, we detected 13 soil bacteria (Acidibacter, Algisphaera, Cystobacter, Microvirga, Ohtaekwangia, Panacagrimonas, Pseudarthrobacter, Reryanella, Rhodoligotrophos, Solirubrobacter, Stenotrophobacter, Tellurimicrobium, and Thermomarinilinea) that were considerably correlated with OM and available phosphorus, and three considerably correlated bacteria were specifically distributed in CF or organic manure. The results suggest that HM is a valuable source of organic manure that can replace CF for sustainable crop production

Haplotypes and a Novel Defective Allele of CES2 Found in a Japanese Population

ABSTRACT: Human carboxylesterase 2 (hCE-2) is a member of the serine esterase superfamily and is responsible for hydrolysis of a wide variety of xenobiotic and endogenous esters. hCE-2 also activates an anticancer drug, irinotecan (7-ethyl-10-[4-(1-piperidino)-1-piperidino]-carbonyloxycamptothecin, CPT-11), into its active metabolite, 7-ethyl-10-hydroxycamptothecin (SN-38). In this study, a comprehensive haplotype analysis of the CES2 gene, which encodes hCE-2, in a Japanese population was conducted. Human carboxylesterases are members of the serine esterase superfamily and are responsible for hydrolysis of a wide variety of xenobiotic and endogenous esters. They metabolize esters, thioesters, carbamates, and amides to yield soluble acids and alcohols or amines Although both hCE-1 and hCE-2 show broad substrate specificities, hCE-2 is relatively specific for heroin, cocaine (benzoyl ester), 6-acetylmorphine, procaine, and oxybutynin 1865 camptothecin (SN-38), a topoisomerase inhibitor, by carboxylesterases Previously, 12 exons and their flanking regions of CES2 were sequenced from 153 Japanese subjects, who received irinotecan or steroidal drugs, and 12 novel SNPs, including the nonsynonymous SNP, 100CϾT (Arg 34 Trp), and the SNP at the splice acceptor site of intron 8 (IVS8-2AϾG) were found Materials and Methods Chemicals. Irinotecan, SN-38, and SN-38G were kindly supplied by Yakult Honsha Co. Ltd. (Tokyo, Japan). Patients. A total of 262 Japanese subjects analyzed in this study consisted of 85 patients with allergies who received steroidal drugs and 177 patients with cancer who received irinotecan. The ethical review boards of the National Cancer Center, National Center for Child Health and Development, and National Institute of Health Sciences approved this study. Written informed consent was obtained from all participants. DNA Sequencing. Total genomic DNA was extracted from blood leukocytes or Epstein-Barr virus-transformed lymphocytes and used as a template in the polymerase chain reaction (PCR). Sequence data of the CES2 gene from 72 patients and 81 cancer patients were described previously Linkage Disequilibrium and Haplotype Analyses. LD analysis was performed by the SNPAlyze software (version 5.1; Dynacom Co., Yokohama, Japan), and a pairwise two-dimensional map between SNPs was obtained for the DЈ and rho square (r 2 ) values. All allele frequencies were in HardyWeinberg equilibrium. Some haplotypes were unambiguously assigned in the subjects with homozygous variations at all sites or a heterozygous variation at only one site. Separately, the diplotype configurations (combinations of haplotypes) were inferred by LDSUPPORT software, which determines the posterior probability distribution of the diplotype configuration for each subject on the basis of estimated haplotype frequencies Administration of Irinotecan and Pharmacokinetic Analysis. The demographic data and eligibility criteria for 177 cancer patients who received irinotecan in the National Cancer Center Hospitals (Tokyo and Chiba, Japan) were described elsewhere Each patient received a 90-min i.v. infusion at doses of 60 to 150 mg/m 2 , which varied depending on regimens/coadministered drugs: i.e., irinotecan dosages were 100 or 150 mg/m 2 for monotherapy and combination with 5-FU, 150 mg/m 2 for combination with mitomycin C (MMC), and 60 (or 70) mg/m 2 for combination with platinum anticancer drugs. Heparinized blood was collected before administration of irinotecan and at 0 min (end of infusion), 20 min, 1 h, 2 h, 4 h, 8 h, and 24 h after infusion. Plasma concentrations of irinotecan, SN-38, and SN-38G were determined as described previously Expression of Wild-Type and Variant CES2 Proteins in COS-1 Cells. Expression of wild-type and variant CES2 proteins in COS-1 cells was examined as described previously and ZERO-Dscan software (Raytest, Straubenhardt, Germany). The relative expression levels are shown as the means Ϯ S.D. of three separate transfection experiments. Determination of CES2 mRNA by Real-Time RT-PCR. Total RNA was isolated from transfected COS-1 cells using the RNeasy Mini Kit (QIAGEN, Tokyo, Japan). After RNase-free DNase treatment of samples to minimize plasmid DNA contamination, first-strand cDNA was prepared from 1 g of total RNA using the High-Capacity cDNA Archive Kit (Applied Biosystems, Foster City, CA) with random primers. Real-time PCR assays were performed with the ABI7500 Real Time PCR System (Applied Biosystems) using the TaqMan Gene Expression Assay for CES2 (Hs01077945_m1; Applied Biosystems) according to the manufacturer's instructions. The relative mRNA levels were determined using calibration curves obtained from serial dilutions of the pooled wild-type CES2 cDNA. Samples without reverse transcriptase were routinely included in the RT-PCR reactions to measure possible contributions of contaminating DNA, which was usually less than 1% of the mRNA-derived amplification. Transcripts of ␤-actin were quantified as internal controls using TaqMan ␤-Actin Control Reagent (Applied Biosystems), and normalization of CES2 mRNA levels were based on ␤-actin concentrations. Enzyme Assay. CPT-11 hydrolyzing activity of the postmitochondrial supernatants (microsomal fraction plus cytosol) was assayed over the substrate concentration range of 0.25 to 50 M as described previously Statistical Analysis. Statistical analysis of the differences in the AUC ratios among CES2 diplotypes, coadministered drugs. or irinotecan dosages was performed using the Kruskal-Wallis test, Mann-Whitney test, or Spearman rank correlation test (Prism 4.0, GraphPad Software, Inc., San Diego, CA). The t test (Prism 4.0) was applied to the comparison of the average values of protein expression and mRNA levels between wild-type and variant CES2. Results CES2 Variations Detected in a Japanese Population. Previously, the promoter region, all 12 exons, and their flanking introns of the CES2 gene were sequenced from 72 allergic patients and 81 cancer patients and resulted in the identification of 12 novel SNPs The nonsynonymous SNP 424GϾA (V142M) reported by our group LD and Haplotype Analysis. Using the detected SNPs, LD analysis was performed, and the pairwise values of r 2 and DЈ were obtained. A perfect linkage (r 2 ϭ 1.00) was observed between SNPs Ϫ363CϾG and IVS10-87GϾA. A close association (r 2 ϭ 0.85) was found between SNPs IVS10-108GϾA and 1749AϾG. Other associations were much lower (r 2 Ͻ 0.1). Therefore, the entire CES2 gene was analyzed as one LD block. The determined/inferred haplotypes are summarized i

DJ-1 Null Dopaminergic Neuronal Cells Exhibit Defects in Mitochondrial Function and Structure: Involvement of Mitochondrial Complex I Assembly

DJ-1 is a Parkinson's disease-associated gene whose protein product has a protective role in cellular homeostasis by removing cytosolic reactive oxygen species and maintaining mitochondrial function. However, it is not clear how DJ-1 regulates mitochondrial function and why mitochondrial dysfunction is induced by DJ-1 deficiency. In a previous study we showed that DJ-1 null dopaminergic neuronal cells exhibit defective mitochondrial respiratory chain complex I activity. In the present article we investigated the role of DJ-1 in complex I formation by using blue native-polyacrylamide gel electrophoresis and 2-dimensional gel analysis to assess native complex status. On the basis of these experiments, we concluded that DJ-1 null cells have a defect in the assembly of complex I. Concomitant with abnormal complex I formation, DJ-1 null cells show defective supercomplex formation. It is known that aberrant formation of the supercomplex impairs the flow of electrons through the channels between respiratory chain complexes, resulting in mitochondrial dysfunction. We took two approaches to study these mitochondrial defects. The first approach assessed the structural defect by using both confocal microscopy with MitoTracker staining and electron microscopy. The second approach assessed the functional defect by measuring ATP production, O2 consumption, and mitochondrial membrane potential. Finally, we showed that the assembly defect as well as the structural and functional abnormalities in DJ-1 null cells could be reversed by adenovirus-mediated overexpression of DJ-1, demonstrating the specificity of DJ-1 on these mitochondrial properties. These mitochondrial defects induced by DJ-1mutation may be a pathological mechanism for the degeneration of dopaminergic neurons in Parkinson's disease

Twelve pil Genes Are Required for Biogenesis of the R64 Thin Pilus

The IncI1 plasmid R64 produces two kinds of sex pili: a thin pilus and a thick pilus. The thin pilus, which belongs to the type IV family, is required only for liquid matings. Fourteen genes, pilI to -V, were found in the DNA region responsible for the biogenesis of the R64 thin pilus (S.-R. Kim and T. Komano, J. Bacteriol. 179:3594–3603, 1997). In this study, we introduced frameshift mutations into each of the 14 pil genes to test their requirement for R64 thin pilus biogenesis. From the analyses of extracellular secretion of thin pili and transfer frequency in liquid matings, we found that 12 genes, pilK to -V, are required for the formation of the thin pilus. Complementation experiments excluded the possible polar effects of each mutation on the expression of downstream genes. Two genes, traBC, were previously shown to be required for the expression of the pil genes. In addition, the rci gene is responsible for modulating the structure and function of the R64 thin pilus via the DNA rearrangement of the shufflon. Altogether, 15 genes, traBC, pilK through pilV, and rci, are essential for R64 thin pilus formation and function