Common Repository of FBS Proteins (cRFP) To Be Added to a Search Database for Mass Spectrometric Analysis of Cell Secretome.

We propose to use cRFP (common Repository of FBS Proteins) in the MS (mass spectrometry) raw data search of cell secretomes. cRFP is a small supplementary sequence list of highly abundant fetal bovine serum proteins added to the reference database in use. The aim behind using cRFP is to prevent the contaminant FBS proteins from being misidentified as other proteins in the reference database, just as we would use cRAP (common Repository of Adventitious Proteins) to prevent contaminant proteins present either by accident or through unavoidable contacts from being misidentified as other proteins. We expect it to be widely used in experiments where the proteins are obtained from serum-free media after thorough washing of the cells, or from a complex media such as SILAC, or from extracellular vesicles directly.

Circular permutation of a bacterial tyrosinase enables efficient polyphenol-specific oxidation and quantitative preparation of orobol.

Tyrosinase is a type 3 copper oxygenase that catalyzes a phenol moiety into ortho-diphenol, and subsequently to ortho-quinone. Diverse tyrosinases have been observed across the kingdom including Animalia, Bacteria, Plantae, and Fungi. Among the tyrosinases, bacterial, and mushroom tyrosinases have been extensively exploited to prepare melanin, ortho-hydroxy-polyphenols, or novel plant secondary metabolites during the past decade. And their use as a biocatalyst to prepare various functional biocompounds have drawn great attention worldwide. Herein, we tailored a bacterial tyrosinase from Bacillus megaterium (BmTy) using circular permutation (CP) engineering technique which is a novel enzyme engineering technique to covalently link original N and C termini and create new termini on the middle of its polypeptide. To construct a smart rationally-designed CP library, we introduced 18 new termini at the edge of each nine loops that link α-helical secondary structure in BmTy. Among the small library, seven functional CP variants were successfully identified and they represented dramatic change in their enzyme characteristics including kinetic properties and substrate specificity. Especially, cp48, 102, and 245 showed dramatically decreased tyrosine hydroxylase activity, behaving like a catechol oxidase. Exploiting the dramatic increased polyphenol oxidation activity of cp48, orobol (3'-hydroxy-genistein) was quantitatively synthesized with 1.48 g/L, which was a 6-fold higher yield of truncated wild-type. We examined their kinetic characters through structural speculation, and suggest a strategy to solubilize the insoluble artificial variants effectively.

RiSLnet: Rapid identification of smart mutant libraries using protein structure network. Application to thermal stability enhancement.

A key point of protein stability engineering is to identify specific target residues whose mutations can stabilize the protein structure without negatively affecting the function or activity of the protein. Here, we propose a method called RiSLnet (Rapid identification of Smart mutant Library using residue network) to identify such residues by combining network analysis for protein residue interactions, identification of conserved residues, and evaluation of relative solvent accessibility. To validate its performance, the method was applied to four proteins, that is, T4 lysozyme, ribonuclease H, barnase, and cold shock protein B. Our method predicted beneficial mutations in thermal stability with ~62% average accuracy when the thermal stability of the mutants was compared with the ones in the Protherm database. It was further applied to lysine decarboxylase (CadA) to experimentally confirm its accuracy and effectiveness. RiSLnet identified mutations increasing the thermal stability of CadA with the accuracy of ~60% and significantly reduced the number of candidate residues (~99%) for mutation. Finally, combinatorial mutations designed by RiSLnet and in silico saturation mutagenesis yielded a thermally stable triple mutant with the half-life (T 1/2 ) of 114.9 min at 58°C, which is approximately twofold higher than that of the wild-type.

Inhibitory Effects of Roseoside and Icariside E4 Isolated from a Natural Product Mixture (No-ap) on the Expression of Angiotensin II Receptor 1 and Oxidative Stress in Angiotensin II-Stimulated H9C2 Cells.

Hypertension is a major risk factor for the development of cardiovascular diseases. This study aimed to elucidate whether the natural product mixture No-ap (NA) containing Pine densiflora, Annona muricate, and Monordica charantia, or its single components have inhibitory effects on hypertension-related molecules in Angiotensin II (Ang II)-stimulated H9C2 cells. Individual functional components were isolated and purified from NA using various columns and solvents, and then their structures were analyzed using ESI⁻MS, ¹H-NMR, and 13H-NMR spectra. H9C2 cells were stimulated with 300 nM Ang II for 7 h. NA, telmisartan, ginsenoside, roseoside (Roseo), icariside E4 (IE4), or a combination of two components (Roseo and IE4) were administered to the cells 1 h before Ang II stimulation. The expression and activity of hypertension-related molecules or oxidative molecules were determined using RT-PCR, western blot, and ELISA. Ang II stimulation increased the expression of Ang II receptor 1 (AT1), tumor necrosis factor-α (TNF-α), monocyte chemoattractant protein-1 (MCP-1), tumor growth factor-ß (TGF-ß) mRNA, and nicotinamide adenine dinucleotide phosphate (NADPH) oxidase activity and the levels of hydrogen peroxide (H2O2) and superoxide anion (•O2-) and reduced anti-oxidant enzyme activity. NA significantly improved the expression or activities of all hypertension-related molecules altered in Ang II-stimulated cells. Roseo or IE4 pretreatment either decreased or increased the expression or activities of all hypertension-related molecules similar to NA, but to a lesser extent. The pretreatment with a combination of Roseo and IE4 (1:1) either decreased or increased the expression of all hypertension-related molecules, compared to each single component, revealing a synergistic action of the two compounds. Thus, the combination of single components could exert promising anti-hypertensive effects similar to NA, which should be examined in future animal and clinical studies.

Inhibition of Osteoarthritis-Related Molecules by Isomucronulatol 7-O-ß-d-glucoside and Ecliptasaponin A in IL-1ß-Stimulated Chondrosarcoma Cell Model.

Osteoarthritis (OA) is the common form of arthritis and is characterized by disability and cartilage degradation. Although natural product extracts have been reported to have anti-osteoarthritic effects, the potential bioactivity of Ryupunghwan (RPH), a traditional Korean medicinal botanical formula that contains Astragalus membranaceus, Turnera diffusa, Achyranthes bidentata, Angelica gigas, Eclipta prostrata, Eucommia ulmoides, and Ilex paraguariensis, is not known well. Therefore, the inhibitory effects of single compounds isolated from RPH on the OA-related molecules were investigated using IL-1ß-stimulated chondrosarcoma SW1353 (SW1353) cell model. Two bioactive compounds, isomucronulatol 7-O-ß-d-glucoside (IMG) and ecliptasaponin A (ES) were isolated and purified from RPH using column chromatography, and then the structures were analyzed using ESI-MS, ¹H-NMR, and 13C-NMR spectrum. The expression or amount of matrix metalloproteinase 13 (MMP13), COX1/2, TNF-α, IL-1ß or p65 was determined by RT-PCR, Western blot, and enzyme-linked immunosorbent assay (ELISA). RPH pretreatment reduced the expression and amounts of MMP13, and the expression of collagen II, COX1/2, TNF-α, IL-1ß or p65, which were increased in IL-1ß-stimulated SW1353 cells. IMG reduced the expression of all OA-related molecules, but the observed inhibitory effect was less than that of RPH extract. The other single compound ES showed the reduced expression of all OA-related molecules, and the effect was stronger than that in IMG (approximately 100 fold). Combination pretreatment of both single components remarkably reduced the expression of MMP13, compared to each single component. These synergic effects may provide potential molecular modes of action for the anti-osteoarthritic effects of RPH observed in clinical and animal studies.

Artemisia Iwayomogi Extract Attenuates High-Fat Diet-Induced Hypertriglyceridemia in Mice: Potential Involvement of the Adiponectin-AMPK Pathway and Very Low Density Lipoprotein Assembly in the Liver.

This study aimed to examine the protective effect of Artemisia iwayomogi extract (AI) against hypertriglyceridemia induced by a high-fat diet (HFD) in mice and to uncover the underlying molecular mechanisms. C57BL/6N mice were fed chow, HFD, HFD + 0.1% AI, HFD + 0.25% AI, or HFD + 0.5% AI for 10 weeks. The addition of 0.25% and 0.5% AI resulted in dose-dependent improvements in the major parameters of hypertriglyceridemia, including plasma triglyceride, free fatty acids, apolipoprotein B, and lipoprotein lipase, with parallel reductions in body weight gain, hepatic lipid accumulation, and insulin resistance. These beneficial effects were accompanied by the activation of adiponectin-adenosine monophosphate-activated protein kinase (AMPK) mediated signaling cascades in the liver, which downregulated molecules involved in lipogenesis and concurrently upregulated molecules related to fatty acid oxidation. The downregulation of molecules involved in very low density lipoprotein assembly, which was associated with improved hepatic insulin signaling, also appeared to contribute to the AI-induced attenuation of hypertriglyceridemia.

Mume Fructus reduces interleukin-1 beta-induced cartilage degradation via MAPK downregulation in rat articular chondrocytes.

Osteoarthritis is the most prevalent type of degenerative arthritis. It is characterized by persistent pain, joint dysfunction, and physical disability. Pain relief and inflammation control are prioritised during osteoarthritis treatment Mume Fructus (Omae), a fumigated product of the Prunus mume fruit, is used as a traditional medicine in several Asian countries. However, its therapeutic mechanism of action and effects on osteoarthritis and articular chondrocytes remain unknown. In this study, we analyzed the anti-osteoarthritis and articular regenerative effects of Mume Fructus extract on rat chondrocytes. Mume Fructus treatment reduced the interleukin-1ß-induced expression of matrix metalloproteinase 3, matrix metalloproteinase 13, and a disintegrin and metalloproteinase with thrombospondin type 1 motifs 5. Additionally, it enhanced collagen type II alpha 1 chain and aggrecan accumulation in rat chondrocytes. Furthermore, Mume Fructus treatment regulated the inflammatory cytokine levels, mitogen-activated protein kinase phosphorylation, and nuclear factor-kappa B activation. Overall, our results demonstrated that Mume Fructus inhibits osteoarthritis progression by inhibiting the nuclear factor-kappa B and mitogen-activated protein kinase pathways to reduce the levels of inflammatory cytokines and prevent cartilage degeneration. Therefore, Mume Fructus may be a potential therapeutic option for osteoarthritis.

EGT022, an RGD-containing recombinant disintegrin, inhibits the VEGF-induced angiogenic process by targeting integrin ß3 in endothelial cells.

OBJECTIVES: EGT022, an RGD-containing recombinant disintegrin from human ADAM metallopeptidase domain 15 (ADAM15), has been reported to stimulate vascular maturation of retinal blood vessels with promotion of pericyte coverage through binding to integrin αIIbß3. Previous studies have reported that angiogenesis can be inhibited by several RGD motif-containing disintegrins; however, the effect of EGT022 on Vascular endothelial growth factor (VEGF)-induced angiogenesis has not yet been determined. This study was conducted in order to evaluate the anti-angiogenic function of EGT022 in VEGF-induced endothelial cells. METHODS: A proliferation and migration assay was performed using human umbilical vein endothelial cells (HUVEC) cells stimulated with VEGF to determine whether the angiogenic process was suppressed by EGT022. An in vitro trans-well assay and Mile's permeability assay were performed to determine the effect of EGT022 on permeability. Western blot was performed in order to further determine whether EGT022 can inhibit phosphorylation of VEGF receptor-2 (VEGFR2) and Phospholipase C gamma1 (PLC-γ1). An integrin binding assay and luciferase assay were performed for identification of the integrin target of EGT022. RESULTS: Angiogenesis including proliferation, migration, tube formation, and permeability was significantly inhibited by EGT022 in HUVEC cells. Our findings also demonstrated that EGT022 binds directly to integrin αvß3, induces dephosphorylation of integrin ß3, and inhibits phosphorylation of VEGFR2. In addition, phosphorylation of PLC-γ1 and activation of Nuclear Factor of Activated T-cell (NFAT), a downstream pathway of VEGF, are inhibited by EGT022 in HUVEC cells. CONCLUSION: These results clearly demonstrate the anti-angiogenic role played by EGT022 as a potent antagonist of integrin ß3 in endothelial cells.

Polygonatum sibiricum improves menopause symptoms by regulating hormone receptor balance in an ovariectomized mouse model.

Female menopause is a hormone deficiency phenomenon that causes hot flashes, vaginal dryness, depression, nervous tension, insomnia, obesity, and bone loss. There are various hormone replacement therapy (HRT)-based menopausal treatments, but they are accompanied by side effects such as endometrial cancer and hyperplasia. To confirm the menopausal improvement effect of Polygonatum sibiricum (PS), we prepared an ovariectomized animal model, administered 17ß-estradiol (E2) and PS, and analyzed various menopausal symptoms. PS restored vaginal epithelium thickness, by increasing the expression of estrogen receptors ERα (ESR1) and ERß (ESR2), and increased serotonin concentration by reducing serotonin receptor 1 A (5-HT1A) and glucocorticoid receptor (Gr) expression. In addition, PS suppressed obesity by increasing HDL-C and decreasing LDL-C levels and improved the osteoporosis induced by ovariectomy. In particular, by controlling Hand2, Fgf2, and Faf9 expression through PR, the antiproliferative signal was suppressed in uterine epithelium, thereby reducing the risk of side effects of the administration of E2 alone. These results demonstrate that PS alleviates menopausal symptoms without causing endometrial hyperplasia.

Natural Thiols, but Not Thioethers, Attenuate Patulin-Induced Endoplasmic Reticulum Stress in HepG2 Cells.

Patulin, a mycotoxin, is known to have cytotoxic effects, but few studies have focused on the involvement of the endoplasmic reticulum (ER) stress response in patulin toxicity and the natural compounds that attenuate it in HepG2 cells. This study tested the ability of patulin to induce ER stress, and that of four thiols and three thioethers to attenuate patulin-induced ER stress in HepG2 cells. Patulin dose-dependently inhibited cell proliferation (IC50, 8.43 µM). Additionally, patulin was found to increase the expression levels of ER stress-related genes and/or protein markers, including BiP, CHOP, and spliced XBP1, in HepG2 cells compared to the vehicle control, indicating its potential in ER stress induction. Patulin-induced cytotoxicity in HepG2 cells was reduced by naturally occurring thiol compounds (glutathione, L-acetyl-L-cysteine, cysteine, and captopril), but not by thioether compounds (sulforaphane, sulforaphene, and S-allyl-L-cysteine). Patulin-thiol co-treatment decreased CHOP expression and BiP and CHOP levels in HepG2 cells but did not alter BiP expression. Spliced XBP1 expression was decreased by patulin-thiol co-treatment. Thus, patulin induced ER stress in HepG2 cells and thiols, but not in thioethers, attenuated patulin-induced ER stress.

Rational engineering of ornithine decarboxylase with greater selectivity for ornithine over lysine through protein network analysis.

Ornithine decarboxylase (ODC) converts C5 ornithine into C4 putrescine, a monomer for polyamide synthesis. However, ODC also has minor activity towards cell metabolite C6 lysine and yields C5 cadaverine. The accumulation of cadaverine in the reaction solution causes increase in the operational cost of subsequent distillation process for putrescine purification. Here, to increase ODC substrate specificity toward ornithine over lysine, molecular modelling and protein network analysis, specifically k-clique community analysis, around the substrate tunnel were performed. This resulted in a mutant with two-fold increase in substrate specificity (ornithine versus lysine) without losing its original activity towards ornithine (kcat/KM  = 61.5 s-1  mM-1), compared to the native enzyme. When this mutant was used for putrescine synthesis, 31.6 g/L putrescine (based on 51.5 g/L ornithine) titer was achieved, while 0.007 g/L (based on 2.57 g/L lysine) cadaverine was produced. This corresponds to four-fold decrease in cadaverine yield compared to the native ODC.

Development of a Highly Active Fluorescence-Based Detector for Yeast G Protein-Coupled Receptor Ste2p.

Twenty analogs of [Orn6,D-Ala9]α-factor were synthesized and assayed for their biological activities: seven analogs of [Orn6,X9]α-factor, seven analogs of [X6,D-Ala9]α-factor, five analogs of [X5,X6,D-Ala9]α-factor, and native α-factor (X = amino acids). Their biological activities (halo, gene induction, and affinity) were measured using S. cerevisiae Y7925 and LM102 and compared with those of native α-factor (100%). G protein-coupled receptor was expressed in strain LM102 containing pESC-LEU-STE2 vector. [Dap6,D-Ala9]α-factor with weak halo activity (10%) showed the highest receptor affinity (＞ 230%) and the highest gene induction activity (167%). [Arg6,D-Ala9]α-factor showed the highest halo activity (2,000%). The number of active binding sites per cell (about 20,000 for strain LM102) was determined using a newly-designed fluorescence-based detector, [Arg6,D-Ala9]α-factor-Edan, with high sensitivity (12,500-fold higher than the absorption-based detector [Orn6]α-factor-[Cys]3).

Inhibitory Effect of Chemical Constituents Isolated from Artemisia iwayomogi on Polyol Pathway and Simultaneous Quantification of Major Bioactive Compounds.

Blocking the polyol pathway plays an important role preventing diabetic complications. Therefore, aldose reductase (AR) and advanced glycation endproducts (AGEs) formation has significant effect on diabetic complications. Artemisia iwayomogi has long been used as treatment of various diseases in Korea. However, no literatures have reported on AR and AGEs formation inhibitory activities of A. iwayomogi. For these reasons, we aimed to assess that A. iwayomogi had potential as anti-diabetic complications agents. We led to isolation of two coumarins (1 and 2), nine flavonoids (3-11), five caffeoylquinic acids (12-16), three diterpene glycosides (17-19), and one phenolic compound (20) from A. iwayomogi. Among them, hispidulin (4), 6-methoxytricin (6), arteanoflavone (7), quercetin-3-gentiobioside (10), 1,3-di-O-caffeoylquinic acid (13), and suavioside A (18) were first reported on the isolation from A. iwayomogi. Not only two coumarins (1 and 2), nine flavonoids (3-11), and five caffeoylquinic acids (12-16) but also extracts showed significant inhibitor on AR and AGEs formation activities. We analyzed contents of major bioactive compounds in Korea's various regions of A. iwayomogi. Overall, we selected Yangyang, Gangwon-do, from June, which contained the highest amounts of bioactive compounds, as suitable areas for cultivating A. iwayomogi as preventive or therapeutic agent in the treatment of diabetic complications.

Simultaneously Enhancing the Stability and Catalytic Activity of Multimeric Lysine Decarboxylase CadA by Engineering Interface Regions for Enzymatic Production of Cadaverine at High Concentration of Lysine.

Cadaverine (1,5-diaminopentane) is a major source of many industrial polyamides such as nylon and chelating agents. Currently, cadaverine is produced by the microbial fermentation of glucose to lysine, which is then decarboxylated by lysine decarboxylase (CadA). However, utilizing CadA for cadaverine production causes enzyme instability. In order to stabilize the CadA homo-decamer structure for in vitro decarboxylation reaction, mutants are designed. Of the four disulfide bond mutants in the multimeric interfacial region, B1 (F14C/K44C) showed a 216-folds increase in the half-life of CadA at 60 °C. On top of B1, another round of mutant screening is performed around F14C and K44C to generate B1/L7M/N8G, which is then examined for cadaverine production (2M lysine and 10% v/v of cell-extract at 50 °C). The reaction pH increased from 4.9 to 8.3, and the final titer of the mutant is 157 g L-1 , that is, 76.7% conversion yield in 9.5 h, whereas the wild-type gave 119 g L-1 , that is, 58.2% conversion yield in 9.5 h.

Scopolin ameliorates high-fat diet induced hepatic steatosis in mice: potential involvement of SIRT1-mediated signaling cascades in the liver.

The present study aimed to investigate whether scopolin exhibits beneficial effects on high-fat diet (HFD)-induced hepatic steatosis in mice. The involvement of sirtuin 1 (SIRT1) as a molecular target for scopolin was also explored. Scopolin decreased the Km of SIRT1 for p53 and nicotinamide adenine dinucleotide without altering Vmax in a cell-free system. Scopolin alleviated oleic acid-induced lipid accumulation and downregulation of SIRT1 activity in HepG2 cells, and these beneficial effects of scopolin were abolished in the presence of SIRT1 inhibitor. Mice administered 0.02% scopolin for 8 weeks exhibited improved phenotypes of HFD-induced hepatic steatosis along with increased hepatic SIRT1 activity and protein expression. Scopolin resulted in increased deacetylation of sterol regulatory element-binding protein 1c with subsequent downregulation of lipogenic genes, and enhanced deacetylation of protein peroxisome proliferator-activated receptor-γ coactivator 1α with upregulation of fatty acid oxidation genes in livers. Scopolin also enhanced deacetylation of nuclear factor-kappa enhancer binding protein and liver kinase B1 (LKB1), facilitating LKB1/AMP-activated protein kinase signaling cascades. Scopolin attenuated hepatic steatosis through activation of SIRT1-mediated signaling cascades, a potent regulator of lipid homeostasis. Increased hepatic SIRT1 activity and protein expression appeared to be associated with these beneficial effects of scopolin.

Harmonized Collaborative Validation of Aflatoxins and Sterigmatocystin in White Rice and Sorghum by Liquid Chromatography Coupled to Tandem Mass Spectrometry.

An interlaboratory study was performed in eight laboratories to validate a liquid chromatography-tandem mass spectrometry (LC/MS/MS) method for the simultaneous determination of aflatoxins and sterigmatocystin (STC) in white rice and sorghum (Sorghum bicolor). Fortified samples (at three different levels) of white rice and sorghum were extracted, purified through a solid-phase extraction (SPE) column, and then analyzed by LC/MS/MS. The apparent recoveries (ARs) ranged from 78.8% to 95.0% for aflatoxins and from 85.3% to 96.7% for STC. The relative standard deviations for repeatability (RSDr) and reproducibility (RSDR) of aflatoxins were in the ranges 7.9%-33.8% and 24.4%-81.0%, respectively. For STC, the RSDr ranged from 7.1% to 40.2% and the RSDR ranged from 28.1% to 99.2%. The Horwitz ratio values for the aflatoxins and STC ranged from 0.4 to 1.2 in white rice and from 0.3 to 1.0 in sorghum, respectively. These results validated this method for the simultaneous determination of aflatoxins and STC by LC/MS/MS after SPE column cleanup. The percentages of satisfactory Z-score values (|Z| ≤ 2) were the following: for white rice, 100% for aflatoxins and STC; for sorghum, 100%, except in data from two laboratories for STC (0.3 µg/kg). This validated that the LC/MS/MS method was successfully applied for the determination of aflatoxins and STC in 20 white rice and 20 sorghum samples sourced from Korean markets.

Effects of ginseol k-g3, an Rg3-enriched fraction, on scopolamine-induced memory impairment and learning deficit in mice.

BACKGROUND: Although ginsenosides such as Rg1, Rb1 and Rg3 have shown promise as potential nutraceuticals for cognitive impairment, their use has been limited due to high production cost and low potency. In particular, the process of extracting pure Rg3 from ginseng is laborious and expensive. METHODS: We described the methods in preparing ginseol k-g3, an Rg3-enriched fraction, and evaluated its effects on scopolamine-induced memory impairment in mice. RESULTS: Ginseol k-g3 (25-200 mg/kg) significantly reversed scopolamine-induced cognitive impairment in the passive avoidance, but not in Y-maze testing. Ginseol k-g3 (50 and 200 mg/kg) improved escape latency in training trials and increased swimming times within the target zone of the Morris water maze. The effect of ginseol k-g3 on the water maze task was more potent than that of Rg3 or Red ginseng. Acute or subchronic (6 d) treatment of ginseol k-g3 did not alter normal locomotor activity of mice in an open field. Ginseol k-g3 did not inhibit acetylcholinesterase activity, unlike donezepil, an acetylcholinesterase inhibitor. Rg3 enrichment through the ginseol k-g3 fraction enhanced the efficacy of Rg3 in scopolamine-induced memory impairment in mice as demonstrated in the Morris water maze task. CONCLUSION: The effects of ginseol k-g3 in ameliorating scopolamine-induced memory impairment in the passive avoidance and Morris water maze tests indicate its specific influence on reference or long-term memory. The mechanism underlying the reversal of scopolamine-induced amnesia by ginseol k-g3 is not yet known, but is not related to anticholinesterase-like activity.

Luteolin mediates the antidepressant-like effects of Cirsium japonicum in mice, possibly through modulation of the GABAA receptor.

Cirsium japonicum (CJ) has been shown to possess antidepressant-like properties. In the present study, we sought to identify which constituent of CJ might be responsible for its antidepressant effects and determine probable mechanism of action. The ethanol extract of CJ was administered to mice then behavioral changes were evaluated in the forced-swimming test (FST) and open-field test (OFT). In addition, its effects on norepinephrine (NE) reuptake and intracellular chloride (Cl(-)) flux were determined, in vitro. The effects of CJ's major constituents (linarin, pectolinarin, chlorogenic acid, luteolin) were also evaluated. CJ showed antidepressant-like effect by significantly reducing immobile behavior of mice in the FST, without increasing locomotor activity in the OFT. CJ had no effect on monoamine (NE) uptake, but it significantly promoted Cl(-) ion influx in human neuroblastoma cells. This CJ-induced Cl(-) influx was significantly blocked by co-administration of the competitive GABAA receptor antagonist, bicuculline. Among the major constituents of the CJ extract, only luteolin produced similar antidepressant-like effect, in vivo, and Cl(-) ion influx, in vitro. Altogether, the present results suggest that the antidepressant-like effect of CJ was most probably induced by its constituent luteolin, mediated through potentiation of the GABAA receptor-Cl(-) ion channel complex.