The contribution of soluble epoxide hydrolase (sEH) to atherosclerosis has been well defined. However, less is understood about the role of sEH and its underlying mechanism in the cholesterol metabolism of macrophages. The expression of sEH protein was increased in atherosclerotic aortas of apolipoprotein E-deficient mice, primarily in macrophage foam cells. Oxidized low-density lipoprotein (oxLDL) increased sEH expression in macrophages. Genetic deletion of sEH (sEH(-/-) ) in macrophages markedly exacerbated oxLDL-induced lipid accumulation and decreased the expression of ATP-binding cassette transporters-A1 (ABCA1) and apolipoprotein AI-dependent cholesterol efflux following oxLDL treatment. The down-regulation of ABCA1 in sEH(-/-) macrophages was due to an increase in the turnover rate of ABCA1 protein but not in mRNA transcription. Inhibition of phosphatase activity, but not hydrolase activity, of sEH decreased ABCA1 expression and cholesterol efflux following oxLDL challenge, which resulted in increased cholesterol accumulation. Additionally, oxLDL increased the phosphatase activity, promoted the sEH-ABCA1 complex formation and decreased the phosphorylated level of ABCA1 at threonine residues. Overexpression of phosphatase domain of sEH abrogated the oxLDL-induced ABCA1 phosphorylation and further increased ABCA1 expression and cholesterol efflux, leading to the attenuation of oxLDL-induced cholesterol accumulation. Our findings suggest that the phosphatase domain of sEH plays a crucial role in the cholesterol metabolism of macrophages.
Title: Cognitive Improving Effects by Highbush Blueberry (Vaccinium crymbosum L.) Vinegar on Scopolamine-Induced Amnesia Mice Model Hong SM, Soe KH, Lee TH, Kim IS, Lee YM, Lim BO Ref: Journal of Agricultural and Food Chemistry, 66:99, 2018 : PubMed
The present study aimed to evaluate the preventive effects of highbush blueberry (Vaccinium corymbosum L.) vinegar (BV) on cognitive functions in a scopolamine (Sco)-induced amnesia model in mice. In this study, Sco (1 mg/kg, intraperitoneal injection) was used to induce amnesia. ICR mice were orally administered donepezil (5 mg/kg), blueberry extract (120 mg/kg), and BV (120 mg/kg) for 7 days. After inducing cognitive impairment by Sco, a behavioral assessment using behavior tests (i.e., Y-maze and passive avoidance tests) was performed. The BV group showed significantly restored cognitive function in the behavioral tests. BV facilitated cholinergic activity by inhibiting acetylcholinesterase activity, and enhanced antioxidant enzyme activity. Furthermore, BV was found to be rehabilitated in the cornu ammonis 1 neurons of hippocampus. In our study, we demonstrated that the memory protection conferred by BV was linked to activation of brain-derived neurotrophic factor (BDNF)/cAMP response element binding protein (CREB)/serine-threonine kinase (AKT) signaling.
Fatty acid synthase (FASN) predominantly generates straight-chain fatty acids using acetyl-CoA as the initiating substrate. However, monomethyl branched-chain fatty acids (mmBCFAs) are also present in mammals but are thought to be primarily diet derived. Here we demonstrate that mmBCFAs are de novo synthesized via mitochondrial BCAA catabolism, exported to the cytosol by adipose-specific expression of carnitine acetyltransferase (CrAT), and elongated by FASN. Brown fat exhibits the highest BCAA catabolic and mmBCFA synthesis fluxes, whereas these lipids are largely absent from liver and brain. mmBCFA synthesis is also sustained in the absence of microbiota. We identify hypoxia as a potent suppressor of BCAA catabolism that decreases mmBCFA synthesis in obese adipose tissue, such that mmBCFAs are significantly decreased in obese animals. These results identify adipose tissue mmBCFA synthesis as a novel link between BCAA metabolism and lipogenesis, highlighting roles for CrAT and FASN promiscuity influencing acyl-chain diversity in the lipidome.
We aimed to investigate the antioxidant and acetylcholinesterase inhibitory activities of the anthocyanin rich extract of grape skin. Grape skin anthocyanin (GSA) neutralized free radicals in different test systems, such as 2,-2'-azinobis-(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) and 2,2-diphenyl-1-picrylhydrazyl (DPPH) assays, to form complexes with Fe2+ preventing 2,2'-azobis(2-amidinopropane) dihydrochloride (AAPH)-induced erythrocyte hemolysis and oxidative DNA damage. Moreover, GSA decreased reactive oxygen species (ROS) generation in isolated mitochondria thus inhibiting 2',-7'-dichlorofluorescin (DCFH) oxidation. In an in vivo study, female BALB/c mice were administered GSA, at 12.5, 25, and 50 mg per kg per day orally for 30 consecutive days. Herein, we demonstrate that GSA administration significantly elevated the level of antioxidant enzymes in mice sera, livers, and brains. Furthermore, GSA inhibited acetylcholinesterase (AChE) in the in vitro assay with an IC50 value of 363.61 microg/mL. Therefore, GSA could be an excellent source of antioxidants and its inhibition of cholinesterase is of interest with regard to neurodegenerative disorders such as Alzheimer's disease.
Title: Effect of pharmaceuticals exposure on acetylcholinesterase (AchE) activity and on the expression of AchE gene in the monogonont rotifer, Brachionus koreanus Rhee JS, Kim BM, Jeong CB, Park HG, Leung KM, Lee YM, Lee JS Ref: Comparative Biochemistry & Physiology C Pharmacol Toxicol, 158:216, 2013 : PubMed
Pharmaceuticals are widely used in human and veterinary medicine. However, they are emerging as a significant contaminant in aquatic environments through wastewater. Due to the persistent and accumulated properties of pharmaceuticals via the food web, their potential harmful effects on aquatic animals are a great concern. In this study, we investigated the effects of six pharmaceuticals: acetaminophen, ATP; atenolol, ATN; carbamazepine, CBZ; oxytetracycline, OTC; sulfamethoxazole, SMX; and trimethoprim, TMP on acetylcholinesterase (AChE; EC 3.1.1.7) activity and its transcript expression with chlorpyrifos (as a positive control) in the monogonont rotifer, Brachionus koreanus. ATP, CBZ, and TMP exposure also remarkably inhibited Bk-AChE activity at 100mug/L (24h) and 1000mug/L (12h and 24h). ATP, CBZ, and TMP exposure showed a significant decrease in the Bk-AChE mRNA level in a concentration-dependent manner. However, in the case of OTC and SMX, a slight decrease in Bk-AChE mRNA expression was found but only at the highest concentration. The time-course experiments showed that ATP positively induced Bk-AChE mRNA 12h after exposure at both 100 and 1000mug/L, while the Bk-AChE mRNA expression was significantly downregulated over 6 to 24h after exposure to 1000mug/L of CBZ, OTC, SMX, and TMP. Our findings suggest that Bk-AChE would be a useful biomarker for risk assessment of pharmaceutical compounds as an early signal of their toxicity in aquatic environments. Particularly, ATP, CBZ, and TMP may have a toxic cholinergic effect on rotifer B. koreanus by inhibiting AChE activity.
To identify effective herb to treat obesity, we screened 115 herbal extracts for inhibition of porcine pancreatic lipase (triacylg-ycerol acylhydrolase, EC 3.1.1.3) activity in vitro. Of the extracts tested, Cudrania tricuspidata leaves exhibited the most pronounced inhibitory effect on lipase activity with an IC(50) value of 9.91 mug/mL. Antilipid absorption effects of C. tricuspidata leaves were examined in rats after oral administration of lipid emulsions containing 50 or 250 mg C. tricuspidata/kg body weight. Plasma triacylglycerol levels 2 h after the oral administration of emulsions containing C. tricuspidata were significantly reduced compared to the untreated group (P < 0.05). These results suggest that C. tricuspidata leaves may be useful for the treatment of obesity.
A new coumaroyl triterpene, 3-O-trans-p-coumaroyl actinidic acid (1), as well as five known triterpenes, ursolic acid (2), 23-hydroxyursolic acid (3), corosolic acid (4), asiatic acid (5) and betulinic acid (6) were isolated from an EtOAc-soluble extract of the roots of Actinidia arguta. The structure of compound 1 was elucidated from interpretation of the spectroscopic data, particularly by extensive 1D and 2D NMR studies. All the isolates (1-6) were evaluated in vitro for their inhibitory activities on pancreatic lipase (PL). Of the isolates, the new compound 1 possessed the highest inhibitory activity on PL, with an IC(50) of 14.95 microM, followed by ursolic acid (2, IC(50) = 15.83 microM). The other four triterpenes (3-6) also showed significant PL inhibitory activity, with IC(50) values ranging from 20.42 to 76.45 microM.
Title: The complete mitochondrial genome of the Korean soft-shelled turtle Pelodiscus sinensis (Testudines, Trionychidae) Jungt SO, Lee YM, Kartavtsev Y, Park IS, Kim DS, Lee JS Ref: DNA Sequence, 17:471, 2006 : PubMed
We isolated Korean soft-shelled turtle, Pelodiscus sinensis, mitochondrial DNA by long-polymerase chain reaction (long-PCR) with conserved primers and sequenced this mitochondrial genome (mitogenome) with primer walking using flanking sequences. The P. sinensis mitochondrial DNA has 17,042 bp and its structural organization is conserved compared to those of other reptiles and mammals. To unveil the phylogenetic relationship of the turtles, we used the NJ, MP, and ML analysis methods after inferring those sequences from the mitochondrial 16S rRNA gene. We also compared two P. sinensis variants from Korea and China using the mitochondrial genome. In this study, we report the basic characteristics of the P. sinensis mitochondrial genome, including structural organization and base composition of the rRNAs, tRNAs and protein-coding genes, as well as characteristics of tRNAs. These features are applicable for the study of phylogenetic relationships in turtles.
Title: Molecular cloning, characterization, and expression of a redox-responsive cutinase from Monilinia fructicola (Wint.) Honey Wang GY, Michailides TJ, Hammock BD, Lee YM, Bostock RM Ref: Fungal Genet Biol, 35:261, 2002 : PubMed
cDNA clones encoding a cutinase expressed in cutin-induced cultures of the plant pathogen Monilinia fructicola were isolated using a protein-based strategy. The largest cDNA (Mfcut1) was found to contain an open reading frame of 603 bp that predicted a 20.2-kDa protein of 201 amino acids with a 20-amino-acid secretory signal peptide and a pI of 8.4. The predicted protein contained cutinase/lipase consensus sequences with active site serines and potential protein kinase phosphorylation sites. Comparison of the deduced amino sequence from Mfcut1 with other fungal cutinase sequences revealed new features, which include conserved cysteines, C-terminal aromatic residues, and a novel histidine substitution in the D-H active site motif. The presence in the growth medium of antioxidants, such as caffeic acid, suppressed mRNA accumulation and enzyme activity of a cutinase from M. fructicola. MFCUT1 was expressed at high levels as a His-tagged fusion protein in Pichia pastoris and purified to apparent homogeneity in a single step by Ni(2+)-nitrilotriacetic acid affinity chromatography. Analysis of variant MFCUT1 mutants in which the novel serine and histidine residues were replaced by site-directed mutagenesis indicated that these residues had an important effect on enzyme activity.
Title: Affinity purification and characterization of a cutinase from the fungal plant pathogen Monilinia fructicola (Wint.) honey Wang GY, Michailides TJ, Hammock BD, Lee YM, Bostock RM Ref: Archives of Biochemistry & Biophysics, 382:31, 2000 : PubMed
Trifluoromethyl ketones (TFK) are potent inhibitors of a variety of serine hydrolases. The TFK inhibitor, 3-(4-mercaptobutylthio)-1,1,1-trifluoro-2-propanone (MBTFP), was found to competitively inhibit cutinase activity (I50 = 9.4 x 10(-3)) from the fungal plant pathogen Monilinia fructicola and to serve as an effective affinity ligand for the purification of cutinases from culture filtrates. The TFK inhibitors, 3-n-octylthio-1,1,1-trifluoro-2-propanone (OTFP) and 3-n-pentylthio-1,1,1-trifluoro-2-propanone (PTFP), also inhibited cutinase activity with I50 values of 1.6 x 10(-6) and 2.3 x 10(-4) M, respectively. Buffer containing OTFP was the strongest eluant for cutinases of M. fructicola and provided the best purification factor and yield, although buffers containing OTFP, detergent, and salt were found to be effective for eluting cutinases bound to MBTFP-Sepharose. Buffer containing 0.5% Triton X-100 also selectively eluted cutinases from the affinity column. Two-dimensional electrophoretic analysis by SDS-PAGE and isoelectric focusing of the affinity-purified cutinase fraction indicated activity associated with proteins of pI 8.2 and molecular masses of approximately 18.6 and 20.8 kDa. These proteins hydrolyzed [3H]cutin and artificial substrates such as p-nitrophenylbutyrate and related esters, typical of other cutinases, but differ from previously characterized cutinases in molecular mass. The two low-molecular-weight proteins resolved by 2-D gel electrophoresis were subjected to in-gel digestion with Lys-C and the resulting peptide fragments were separated by Microbore-HPLC. The amino acid sequences of several internal peptide fragments had high homology with cutinase sequences from other fungi, particularly the plant pathogen Botrytis cinerea. Our study illustrates the potential of TFK ligands for the affinity purification of cutinases and indicates that the cutinases from M. fructicola have novel features warranting further study.
Title: Simultaneous determination of flupyrazofos and its metabolite 1-phenyl-3-trifluoromethyl-5-hydroxypyrazole and flupyrazofos oxon in rat plasma by high-performance liquid chromatography with ultraviolet absorbance detection Shin HC, Shim HO, Lee YM, Song SW, Kim JH, Chung MK, Han SS, Roh JK Ref: Journal of Chromatography B Biomed Sci Appl, 718:61, 1998 : PubMed
An isocratic high-performance liquid chromatography (HPLC) system with UV detection was developed for simultaneous determination of flupyrazofos and its metabolites, 1-phenyl-3-trifluoromethyl-5-hydroxypyrazole and flupyrazofos oxon, in rat plasma. Optimal analytical conditions involved an analytical cartridge column consisting of a phenyl bonded phase, a mobile phase of 50 mM phosphate buffer (pH3.0)-acetonitrile (40:60, v/v) and a UV detection wavelength of 232 nm. Under these conditions the peaks of flupyrazofos and its metabolites were all well separated and the total time for complete separation was less than 12 min. The limit of quantitation was 40 ng/ml for flupyrazofos and 20 ng/ml for PTMHP. Recoveries from rat plasma were higher than 90%. Following intravenous administration of flupyrazofos, the method has been successfully applied in a toxicokinetic study in rats involving plasma samples. Therefore, the current method is a valuable analytical tool for investigating the metabolism and toxicokinetics of flupyrazofos.
