Soluble epoxide hydrolase (sEH) is a target enzyme for the treatment of inflammation and cardiovascular disease. A Glycyrrhiza uralensis extract exhibited ~50% inhibition of sEH at 100 microg/mL, and column chromatography yielded compounds 1-11. Inhibitors 1, 4-6, 9, and 11 were non-competitive; inhibitors 3, 7, 8, and 10 were competitive. The IC(50) value of inhibitor 10 was below 2 microM. Molecular simulation was used to identify the sEH binding site. Glycycoumarin (10) requires further evaluation in cells and animals.
Title: Anti-Cancer Effect of Neural Stem Cells Transfected with Carboxylesterase and sTRAIL Genes in Animals with Brain Lesions of Lung Cancer Kim JH, Ahn JS, Lee DS, Hong SH, Lee HJ Ref: Pharmaceuticals (Basel), 16:, 2023 : PubMed
A metastatic brain tumor is the most common type of malignancy in the central nervous system, which is one of the leading causes of death in patients with lung cancer. The purpose of this study is to evaluate the efficacy of a novel treatment for metastatic brain tumors with lung cancer using neural stem cells (NSCs), which encode rabbit carboxylesterase (rCE) and the secretion form of tumor necrosis factor-related apoptosis-inducing ligand (sTRAIL). rCE and/or sTRAIL were transduced in immortalized human fetal NSCs, HB1.F3. The cytotoxic effects of the therapeutic cells on human lung cancer cells were evaluated in vitro with the ligands and decoy receptor expression for sTRAIL in the presence of CPT-11. Human NSCs encoding rCE (F3.CE and F3.CE.sTRAIL) significantly inhibited the growth of lung cancer cells in the presence of CPT-11 in vitro. Lung cancer cells were inoculated in immune-deficient mice, and therapeutic cells were transplanted systematically through intracardiac arterial injection and then treated with CPT-11. In resting state, DR4 expression in lung cancer cells and DcR1 in NSCs increased to 70% and 90% after CPT-11 addition, respectively. The volumes of the tumors in immune-deficient mice were reduced significantly in mice with F3.CE.sTRAIL transplantation and CPT-11 treatment. The survival was also significantly prolonged with treatment with F3.sTRAIL and F3.CE plus CPT-11 as well as F3.CE.sTRAIL plus CPT-11. NSCs transduced with rCE and sTRAIL genes showed a significant anti-cancer effect on brain metastatic lung cancer in vivo and in vitro, and the effect may be synergistic when rCE/CPT-11 and sTRAIL are combined. This stem-cell-based study using two therapeutic genes of different biological effects can be translatable to clinical application.
This study was conducted to evaluate the cognitive dysfunction improvement effect of aqueous extract of Codium fragile (AECF) by regulating the imbalance of the gut-brain axis in chronic particulate matter (PM)(2.5)-exposed mice. The physiological compounds of AECF were identified as hexadecanamide, oleamide, octadecanamide, stearidonic acid, and linolenic acid by the ultra-performance liquid chromatography-quadrupole time of flight mass spectrometry (UPLC Q-TOF MS(E)) analysis. To evaluate the effect of PM(2.5) on the antioxidant system, superoxide dismutase (SOD) contents, reduced glutathione (GSH) contents, and malondialdehyde (MDA) contents were measured in colon and brain tissues. AECF significantly ameliorated the imbalance of the antioxidant systems. Also, AECF improved intestinal myeloperoxidase (MPO) activity, the abundance of the gut microbiome, short-chain fatty acids (SCFAs) contents, and tight junction protein expression against PM(2.5)-induced damage. In addition, AECF prevented PM(2.5)-induced inflammatory and apoptotic expression via the toll-like receptor-4 (TLR-4)/myeloid differentiation primary response 88 (MyD88) pathway in colon and brain tissues. Additionally, AECF enhanced the mitochondrial function, including the mitochondrial membrane potential (MMP) and reactive oxygen species (ROS) contents in brain tissues. Furthermore, AECF regulated the cholinergic system, such as acetylcholine (ACh) contents, acetylcholinesterase (AChE) activity, and protein expression levels of AChE and choline acetyltransferase (ChAT) in brain tissues. To evaluate the effect of cognitive dysfunction caused by PM(2.5)-induced intestinal dysfunction, behavior tests such as Y-maze, passive avoidance, and Morris water maze tests were performed. From the results of the behavior tests, AECF ameliorated spatial learning and memory, short-term memory, and long-term learning and memory function. This study confirmed that AECF reduced PM(2.5)-induced cognitive dysfunction by regulating gut microbiome and inflammation, apoptosis, and mitochondrial function by enhancing the gut-brain axis. Based on these results, this study suggests that AECF, which contains fatty acid amides, might be a potential material for ameliorating PM(2.5)-induced cognitive dysfunction via gut-brain axis improvement.
OBJECTIVE: Diagnosing parathyroid carcinoma (PC) is complicated and controversial that early diagnosis and intervention are often difficult. Therefore, we aimed to elucidate the protein signatures of PC through quantitative proteomic analyses to aid in the early and accurate diagnosis of PC. DESIGN: Retrospective cohort study. METHODS: We performed liquid chromatography with tandem mass spectrometry using formalin-fixed paraffin-embedded samples. For the analyses, 23 PC and 15 parathyroid adenoma (PA) tissues were collected from 6 tertiary hospitals in South Korea. RESULTS: The mean age of the patients was 52 years, and 63% were women. Proteomic expression profiling revealed 304 differentially expressed proteins (DEPs) with a cutoff of p < 0.05, and fold change >1.5. Among DEPs, we identified a set of 5 proteins that can discriminate PC from PA: carbonic anhydrase 4 [CA4], alpha/beta hydrolase domain-containing protein 14B [ABHD14B], laminin subunit beta-2 [LAMB2], CD44 antigen [CD44], and alpha-1-acid glycoprotein 1 [ORM1], that exhibited the highest area under the curve of 0.991 in neural network model. The nuclear percentage of CA4 and LAMB2 in immunohistochemistry were significantly lower in PC tissue than in the PA (CA4: 2.77 +/- 1.96%, 26.2 +/- 3.45%, p < 0.001; LAMB2: 6.86 +/- 3.46%, 38.54 +/- 4.13%, p < 0.001). The most enriched canonical pathways in PC included glycoprotein-6 signaling and mammalian target of rapamycin (mTOR). CONCLUSION: We identified key proteins differentially expressed between PC and PA using proteomic analyses of parathyroid neoplasms. These findings may help to diagnose PC accurately and elucidate potential therapeutic targets.
Title: Isocucurbic Acid Derivaties and Soluble Epoxide Hydroxylase Inhibitors from the Flowers of Chrysanthemum indicum L Thi Thu Trang B, Kim JH, Luyen BTT Ref: Chem Biodivers, :e202301242, 2023 : PubMed
Soluble epoxide hydrolase (sEH) inhibitory activity guilded fractionation and isolation of two new isocucurbic acid derivatives (1 and 2) and nine known compounds (3-11) from the flowers of Chrysanthemum indicum L. Their structures were elucidated on the basis of spectroscopic data interpretation and comparison with those reported in previous studies. Luteolin (3), acacetin-7-O-beta-D-glucopyranoside (6), and methyl 3,4-di-O-caffeoylquinate (10) displayed sEH inhibitory activities with IC50 values ranging from 13.7 +/- 3.6 to 20.8 +/- 0.4 microM. Enzyme kinetic analysis revealed that 3, 6, and 10 were non-competitive inhibitors with Ki values of 14.8 +/- 0.5, 31.2 +/- 0.8, and 3.9 +/- 0.2 microM, respectively. Additionally, molecular docking studies indicated compound 10 had the ability to form six hydrogen bonds at sEH active site, resulting binding engery as low as -9.58 Kcal/mol.
Anti-dementia medications are widely prescribed to patients with Alzheimer's dementia (AD) in South Korea. This study investigated the pattern of medical management in newly diagnosed patients with AD using a standardized data format-the Observational Medical Outcome Partnership Common Data Model from five hospitals. We examined the anti-dementia treatment patterns from datasets that comprise > 5 million patients during 2009-2019. The medication utility information was analyzed with respect to treatment trends and persistence across 11 years. Among the 8653 patients with newly diagnosed AD, donepezil was the most commonly prescribed anti-dementia medication (4218; 48.75%), followed by memantine (1565; 18.09%), rivastigmine (1777; 8.98%), and galantamine (494; 5.71%). The rising prescription trend during observation period was found only with donepezil. The treatment pathways for the three cholinesterase inhibitors combined with N-methyl-D-aspartate receptor antagonist were different according to the drugs (19.6%; donepezil; 28.1%; rivastigmine, and 17.2%; galantamine). A 12-month persistence analysis showed values of approximately 50% for donepezil and memantine and approximately 40% for rivastigmine and galantamine. There were differences in the prescribing pattern and persistence among anti-dementia medications from database using the Observational Medical Outcome Partnership Common Data Model on the Federated E-health Big Data for Evidence Renovation Network platform in Korea.
Title: Carboxyl Esterase-TRAIL Expressing Human Adipose Stem Cells Inhibit Tumor Growth in Castration-Resistant Prostate Cancer-Bearing Mice with Less Toxicity Kim JH, Oh E, Yun CW, Lee SH, Song YS Ref: Technol Cancer Research Treat, 21:15330338221093146, 2022 : PubMed
It has been proposed that CRPC treatment with reduced systemic toxicity can be achieved by employing genes that express enzymes that activate pharmacological agents. In this paper, we report our study that used human adipose-derived stem cells (ADSC), rabbit CE, and human TRAIL with reduced toxicity to explore how tumor development can be suppressed in CRPC-bearing mouse models. In vitro and in vivo directional migration of ADSC.CE.sTRAIL cells toward PC3 cells was significantly stimulated.ADSC.CE.sTRAIL showed higher suicide effects than did ADSC, ADSC.CE, or ADSC.sTRAIL under CPT-11 treatment. PC3 cells co-cultured with ADSC.CE.TRAIL showed higher cytotoxicity than did CPT-11 monotherapy, ADSC.CE, or ADSC.sTRAIL under CPT-11 treatment. ADSC.CE.sTRAIL showed higher apoptosis than did CPT-11 monotherapy, ADSC.CE, or ADSC.sTRAIL under CPT-11 treatment. In the in vivo study, ADSC.CE.sTRAIL inhibited tumor growth more than did CPT-11 monotherapy, ADSC.CE, or ADSC.sTRAIL under CPT-11 treatment. The evidence suggests that patients' own ADSC could be used in clinical trials for CRPC treatment based on therapeutic stem cells that express CE and TRAIL complex genes.
The quaternary isoquinoline alkaloids of palmatine (1), berberine (2), and jatrorrhizine (3) were evaluated in terms of their ability to inhibit soluble epoxide hydrolase (sEH). They had similar inhibitory activities, with IC(50) values of 29.6 +/- 0.5, 33.4 +/- 0.8, and 27.3 +/- 0.4 microM, respectively. Their respective Ki values of 26.9, 46.8, and 44.5 microM-determined by enzyme kinetics-indicated that they inhibited the catalytic reaction by binding noncompetitively with sEH. The application of computational chemistry to the in vitro results revealed the site of the receptor to which the ligand would likely bind. Accordingly, three alkaloids were identified as having a suitable basic skeleton for lead compound development of sEH inhibitors.
Title: Polyurethane biodegradation by Serratia sp. HY-72 isolated from the intestine of the Asian mantis Hierodula patellifera Kim JH, Choi SH, Park MG, Park DH, Son KH, Park HY Ref: Front Microbiol, 13:1005415, 2022 : PubMed
Polyurethane (PU), currently replacing existing synthetic materials worldwide, is a synthetic polymer derived from polyols, isocyanates, and a chain extender added by condensation reactions. PU wastes which are difficult to recycle, are commonly discarded in landfills and flow into ecosystems, thereby causing serious environmental problems. In recent years, insect-associated microbes have become a promising, eco-friendly strategy as an alternative to plastic recycling. This study aimed to evaluate the potential of Serratia sp. HY-72 strain isolated from the intestine of the Asian mantis (Hierodula patellifera) for PU degradation. The 65 kDa family I.3 lipase which degrades PU was identified and characterized, with a specific activity of 2,883 U mg(-1). The bacterial filtrates and the recombinant lipase degraded Impranil (a colloidal polyester-PU dispersion, 100 g l(-1)) by 85.24 and 78.35% after 72 h incubation, respectively. Fourier transform infrared spectroscopy analysis revealed changes in Impranil functional groups, with decreased C=O functional group and aliphatic chain signals, and increased N-H bending with C-N stretching and C-O stretching. The current study also revealed that the HY-72 strain biodegraded the commercial PU foams (polyester- and polyether- PU) with 23.95 and 10.95% weight loss after 2 weeks, respectively with changes in surface morphology and structure such as cracks, roughness, and surface roughening. Altogether, this is one of the few studies reporting biodegradation of PU by the insect-associated microbe. These findings suggest that the insect-associated microbe could be a promising resource for biodegradation and recycling of plastic waste.
Title: Comparison of the Effect of Sugammadex and Pyridostigmine on Postoperative Catheter-Related Bladder Discomfort: A Retrospective Matched Cohort Analysis Kwon YS, Kim JH, Hwang SM, Choi JW, Kang SS Ref: Medicina (Kaunas), 58:, 2022 : PubMed
Background and Objectives: As the use of sugammadex for reversing neuromuscular blockade during general anesthesia increases, additional effects of sugammadex have been reported compared to cholinesterase inhibitors. Here, we compare the incidence of postoperative catheter-related bladder discomfort (CRBD) between sugammadex and pyridostigmine/glycopyrrolate treatments for reversing neuromuscular blockade. Materials and Methods: We retrospectively analyzed patients aged <= 18 years who underwent surgery under general anesthesia, received sugammadex or pyridostigmine with glycopyrrolate to reverse neuromuscular blockade, and had a urinary catheter in the post-anesthesia care unit between March 2019 and February 2021. After applying the exclusion criteria, 1179 patients were included in the final analysis. The incidence and severity of CRBD were collected from post-anesthesia recovery records. Results: The incidence was 13.7% in the sugammadex group (n = 211) and 24.7% in the pyridostigmine group (n = 968). Following propensity score matching, 211 patients each were included in the pyridostigmine and sugammadex matched group (absolute standardized difference (ASD), 0.01-0.05). Compared to the pyridostigmine group, the odds ratio for CRBD occurring in the sugammadex group was 0.568 (95% confidential interval, 0.316-1.021, p = 0.059). Conclusions: Sugammadex has a similar effect on the occurrence of postoperative CRBD compared with pyridostigmine.
Title: Toxic effects of waterborne cadmium exposure on hematological parameters, oxidative stress, neurotoxicity, and heat shock protein 70 in juvenile olive flounder, Paralichthys olicaceus Lee DC, Choi YJ, Kim JH Ref: Fish Shellfish Immunol, :, 2022 : PubMed
Cadmium-induced toxicity can affect fish embryo development, ion homeostasis regulation, energy metabolism, maturation and growth, stress response, and immunity. However, studies on the toxic effects of cadmium exposure to aquatic animals, particularly olive flounder (Paralichthys olivaceus), are limited. In this study, juvenile P. olivaceus (mean length, 12.9 +/- 1.3 cm; mean weight, 23.1 +/- 3.2 g) was exposed to waterborne cadmium (0, 50, 100, 200, and 400 microg/L) for 10 d. Hematological parameters, including hematocrit value and hemoglobin level, in P. olivaceus were significantly decreased after waterborne cadmium exposure. Plasma components such as calcium, glucose, cholesterol, glutamic-oxaloacetic transaminase, and glutamic-pyruvic transaminase were significantly altered via cadmium exposure. The activities of antioxidant enzymes, such as superoxide dismutase, catalase, and glutathione S-transferase, were significantly altered in P. olivaceus after cadmium exposure. Acetylcholinesterase activity was significantly inhibited upon waterborne cadmium exposure. Hepatic heat shock protein 70 was significantly upregulated in P. olivaceus after waterborne cadmium exposure. Therefore, waterborne cadmium at concentrations of >100 or 200 microg/L can induce physiological toxicity in P. olivaceus via changes in hematological parameters, antioxidant enzymes, neurotransmitters, and stress indicators.
Title: Toxic Effects on Oxidative Stress, Neurotoxicity, Stress, and Immune Responses in Juvenile Olive Flounder, Paralichthys olivaceus, Exposed to Waterborne Hexavalent Chromium Lee JW, Kim JH, Lee DC, Lim HJ, Kang JC Ref: Biology (Basel), 11:, 2022 : PubMed
Juvenile Paralichthys olivaceus were exposed to waterborne hexavalent chromium at various concentrations (0, 0.5, 1.0, and 2.0 mg/L) for 10 days. After chromium exposure, the activities of superoxide dismutase and glutathione S-transferase, which are oxidative stress indicators, were significantly increased; however, the glutathione level was significantly reduced. Acetylcholinesterase activity as a neurotoxicity marker was significantly inhibited upon chromium exposure. Other stress indicators, including plasma cortisol and heat shock protein 70, were significantly increased. The immune response markers (lysozyme and immunoglobulin M) were significantly decreased after chromium exposure. These results suggest that exposure to environmental toxicity in the form of waterborne chromium at concentrations higher than 1.0 mg/L causes significant alterations in antioxidant responses, neurotransmitters, stress, and immune responses in juvenile olive flounders. This study will provide a basis for an accurate assessment of the toxic effects of hexavalent chromium on aquatic organisms.
Title: Characterization of molecular and kinetic properties of two acetylcholinesterases from the Colorado potato beetle, Leptinotarsa decemlineata Yoon KA, Kim JH, Nauen R, Alyokhin A, Clark JM, Lee SH Ref: Pestic Biochem Physiol, 185:105137, 2022 : PubMed
The molecular and biochemical properties of two acetylcholinesterases (LdAChE1 and LdAChE2) from the Colorado potato beetle, Leptinotarsa decemlineata, were investigated in this study. Polyacrylamide gel electrophoresis in conjunction with western blotting with LdAChE1- or LdAChE2-specific antibodies suggested that LdAChE1 exists in a soluble form, whereas LdAChE2 exists in both soluble and amphiphilic forms with a glycophosphatidylinositol anchor. Both LdAChEs exist as homodimers with each monomer connected with a disulfide bond. LdAChE1 was the most highly expressed in the thorax followed by the head, leg, and abdomen, whereas LdAChE2 was the most highly expressed in the head, followed by the thorax, leg, and abdomen. The overall expression levels of LdAChE1, however, were higher than those of LdAChE2 in all examined tissues. Kinetic analysis using recombinant LdAChE1 and LdAChE2 showed that LdAChE2 has a 4.8-fold higher catalytic efficiency toward acetylthiocholine iodide compared to LdAChE1. LdAChE2 was more sensitive to organophosphorus and carbamate insecticides than LdAChE1. The addition of irreversibly phosphorylated LdAChE1 via paraoxon titration significantly reduced LdAChE2 inhibition by insecticides and glycoalkaloids, suggesting a sequestration role of soluble LdAChE1 in the chemical defense against xenobiotics. Taken together, LdAChE2 may be the main enzyme for synaptic transmission, thus serving as a toxicologically more relevant target, whereas the soluble LdAChE1 may function as a bioscavenger.
Title: Comparison of neuromuscular blockade recovery co-administered with neostigmine and different doses of calcium gluconate: a randomized control trial Choi SR, Kim JH, Lee KH, Park SY Ref: BMC Anesthesiol, 21:93, 2021 : PubMed
BACKGROUND: Calcium increases the probability of transmitter release at the neuromuscular junction. It is not known whether there is a dose-dependent relationship between the dosage of calcium gluconate and the probability of transmitter release for non-depolarizing neuromuscular blockade (NMB) recovery by acetylcholinesterase inhibitors (AchEIs). This study compared the neuromuscular recovery time and the incidence of postoperative residual curarization (PORC) according to the dosage of calcium gluconate co-administered with neostigmine in three patient groups. METHODS: Patients were randomly allocated to a control group, a 5 mg/kg calcium gluconate group (calcium 5 group), or a 10 mg/kg calcium gluconate group (calcium 10 group). In patients with a TOF ratio (TOFr) between 0.2-0.7, 0.04 mg/kg of neostigmine was administered and both 0.2 mg of glycopyrrolate and 0.4 mg of atropine per 1 mg of neostigmine were administered. And additional 5 or 10 mg/kg of calcium gluconate were administrated to the calcium 5 and 10 groups. The primary endpoint was neuromuscular recovery time (the time between reversal and TOFr<=0.9). The secondary endpoints were the incidence of PORC at 5, 10, and 20 min after reversal administration and the train-of-four ratio (TOFr) at each time point. RESULTS: The neuromuscular recovery time was 5.3 min in the control group, 3.9 min in the calcium 5 group, and 4.1 min in the calcium 10 group, respectively (P = 0.004). The incidence of PORC at 5 min after neostigmine administration was 12 in the control group, 4 in the calcium 5 group, and 4 in the calcium 10 group, respectively, with statistical significance (P = 0.014). CONCLUSIONS: The co-administration of calcium gluconate with neostigmine safely promoted early NMB recovery, and the neuromuscular recovery time of the calcium 10 group tended to be more evenly distributed than that of the calcium 5 group. TRIAL REGISTRATION: https://cris.nih.go.kr/cris/index.jsp(KCT0004182 ). Date of registration: August 122,019.
Title: Platinum Nanoparticles Enhance Exosome Release in Human Lung Epithelial Adenocarcinoma Cancer Cells (A549): Oxidative Stress and the Ceramide Pathway are Key Players Gurunathan S, Kang MH, Jeyaraj M, Kim JH Ref: Int J Nanomedicine, 16:515, 2021 : PubMed
BACKGROUND: Several studies have demonstrated various molecular mechanisms involved in the biogenesis and release of exosomes. However, how external stimuli, such as platinum nanoparticles (PtNPs), induces the biogenesis and release of exosomes remains unclear. To address this, PtNPs were synthesized using lutein to examine their effect on the biogenesis and release of exosomes in human lung epithelial adenocarcinoma cancer cells (A549). METHODS: The size and concentration of isolated exosomes were characterized by dynamic light scattering (DLS) and nanoparticle tracking analysis system (NTA). Morphology and structure of exosomes were examined using scanning electron microscopy and transmission electron microscopy (TEM), respectively. Quantification of exosomes were analyzed by EXOCET(TM) assay and fluorescence polarization (FP). The expression of typical markers of exosomes were analyzed by quantitative reverse transcription-polymerase chain reaction (qRT-PCR) and enzyme-linked immunosorbent assay (ELISA). RESULTS: A549 cells cultured with PtNPs enhance exosome secretion by altering various physiological processes. Interestingly, A549 cells treated with PtNPs increases total protein concentration, biogenesis and release of exosomes associated with PtNPs-induced oxidative stress. GW4869 inhibits PtNPs induced biogenesis and release of exosomes and also acetylcholinesterase (AChE), neutral sphingomyelinase activity (n-SMase), and exosome counts. A549 cells pre-treated with N-acetylcysteine (NAC) significantly inhibited PtNPs induced exosome biogenesis and release. These findings confirmed that PtNPs-induced exosome release was due to the induction of oxidative stress and the ceramide pathway. These factors enhanced exosome biogenesis and release and may be useful in understanding the mechanism of exosome formation, release, and function. CONCLUSION: PtNPs provide a promising agent to increase exosome production in A549 cells. These findings offer novel strategies for enhancing exosome release, which can be applied in the treatment and prevention of cancer. Importantly, this is the first study, to our knowledge, showing that PtNPs stimulate exosome biogenesis by inducing oxidative stress and the ceramide pathway.
Title: Palladium Nanoparticle-Induced Oxidative Stress, Endoplasmic Reticulum Stress, Apoptosis, and Immunomodulation Enhance the Biogenesis and Release of Exosome in Human Leukemia Monocytic Cells (THP-1) Gurunathan S, Kang MH, Jeyaraj M, Kim JH Ref: Int J Nanomedicine, 16:2849, 2021 : PubMed
BACKGROUND: Exosomes are endosome-derived nano-sized vesicles that have emerged as important mediators of intercellular communication and play significant roles in various diseases. However, their applications are rigorously restricted by the limited secretion competence of cells. Therefore, strategies to enhance the production and functions of exosomes are warranted. Studies have shown that nanomaterials can significantly enhance the effects of cells and exosomes in intercellular communication; however, how palladium nanoparticles (PdNPs) enhance exosome release in human leukemia monocytic cells (THP-1) remains unclear. Therefore, this study aimed to address the effect of PdNPs on exosome biogenesis and release in THP-1 cells. METHODS: Exosomes were isolated by ultracentrifugation and ExoQuick(TM) and characterized by dynamic light scattering, nanoparticle tracking analysis system, scanning electron microscopy, transmission electron microscopy, EXOCET(TM) assay, and fluorescence polarization. The expression levels of exosome markers were analyzed via quantitative reverse transcription-polymerase chain reaction and enzyme-linked immunosorbent assay. RESULTS: PdNP treatment enhanced the biogenesis and release of exosomes by inducing oxidative stress, endoplasmic reticulum stress, apoptosis, and immunomodulation. The exosomes were spherical in shape and had an average diameter of 50-80 nm. Exosome production was confirmed via total protein concentration, exosome counts, acetylcholinesterase activity, and neutral sphingomyelinase activity. The expression levels of TSG101, CD9, CD63, and CD81 were significantly higher in PdNP-treated cells than in control cells. Further, cytokine and chemokine levels were significantly higher in exosomes isolated from PdNP-treated THP-1 cells than in those isolated from control cells. THP-1 cells pre-treated with N-acetylcysteine or GW4869 showed significant decreases in PdNP-induced exosome biogenesis and release. CONCLUSION: To our knowledge, this is the first study showing that PdNPs stimulate exosome biogenesis and release and simultaneously increase the levels of cytokines and chemokines by modulating various physiological processes. Our findings suggest a reasonable approach to improve the production of exosomes for various therapeutic applications.
Here, we report a bienzymatic cascade to produce beta-amino acids as an intermediate for the synthesis of the leading oral antidiabetic drug, sitagliptin. A whole-cell biotransformation using recombinant E. coli co-expressing a esterase and transaminase were developed, wherein the desired expression level of each enzyme was achieved by promotor engineering. The small-scale reactions (30 mL) performed under optimized conditions at varying amounts of substrate (100-300 mM) resulted in excellent conversions of 82-95% for the desired product. Finally, a kilogram-scale enzymatic reaction (250 mM substrate, 220 L) was carried out to produce beta-amino acid (229 mM). Sitagliptin phosphate was chemically synthesized from beta-amino acids with 82% yield and > 99% purity. This article is protected by copyright. All rights reserved.
Title: Salinity-mediated changes in hematological parameters, stress, antioxidant responses, and acetylcholinesterase of juvenile olive flounders (Paralichthys olivaceus) Kim JH, Jeong EH, Jeon YH, Kim SK, Hur YB Ref: Environ Toxicol Pharmacol, :103597, 2021 : PubMed
The purpose of this study was to confirm the limit of salinity tolerance in juvenile olive flounders (Paralichthys olivaceus) by changes in blood parameters, AChE, antioxidant and stress responses. The P. olivaceus (mean weight 38.8 +/- 4.2 g and mean length 16.4 +/- 1.2 cm) were exposed to different concentrations of salinity (seawater, 16, 8, 4, 2, and 0 psu) for 2 weeks. Plasma osmotic pressure was significantly decreased in the P. olivaceus at 0 psu. Hematological parameters such as hematocrit and hemoglobin were significantly decreased in the P. olivaceus at low salinity. Plasma components also changed significantly in the low salinity environment. As a stress indicator, cortisol was significantly increased at low salinity. SOD and GST antioxidant responses, were significantly increased. GSH level in the liver was significantly increased, whereas a significant decrease was observed in the gill GSH level. AChE was significantly increased in P. olivaceus at low salinity. The results of this study indicate that exposure to salinities lower than 8 psu leads to changes in hematological parameters, neurotransmitter, antioxidant and stress responses of P. olivaceus.
Hevin, also known as SPARC-like protein 1 (SPARCL1 or SC1), is a synaptogenic protein secreted by astrocytes and modulates the formation of glutamatergic synapses in the developing brain by interacting with synaptic adhesion proteins, such as neurexin and neuroligin. Here, we identified the neuron-specific vesicular protein calcyon as a novel interaction partner of hevin and demonstrated that this interaction played a pivotal role in synaptic reorganization after an injury in the mature brain. Astrocytic hevin was upregulated post-injury in a photothrombotic stroke model. Hevin was fragmented by MMP3 induced during the acute stage of brain injury, and this process was associated with severe gliosis. At the late stage, the functional hevin level was restored as MMP3 expression decreased. The C-terminus of hevin interacted with the N-terminus of calcyon. By using RNAi and binding competitor peptides in an ischemic brain injury model, we showed that this interaction was crucial in synaptic and functional recoveries in the sensory-motor cortex, based on histological and electrophysiological analyses. Regulated expression of hevin and calcyon and interaction between them were confirmed in a mouse model of traumatic brain injury and patients with chronic traumatic encephalopathy. Our study provides direct evidence for the causal relationship between the hevin-calcyon interaction and synaptic reorganization after brain injury. This neuron-glia interaction can be exploited to modulate synaptic reorganization under various neurological conditions.
Title: Effects on the survival rates, hematological parameters, and neurotransmitters in olive flounders, Paralichthys olivaceus, reared in bio-floc and seawater by Streptococcus iniae challenge Kim JH, Sohn S, Kim SK, Kim SR, Kim SM, Kim NY, Hur YB Ref: Fish Shellfish Immunol, :, 2021 : PubMed
Bacterial infections cause huge losses to aquaculture globally, and increased antibiotic resistance means that alternative methods of reducing mortality from bacterial diseases are required. We compared the resistance of Juvenile olive flounders, Paralichthys olivaceus, to Streptococcus iniae between those reared in biofloc and seawater conditions for ten months. Experimental fish were challenged with S. iniae at concentrations of 0, 3.36 x 10(6), 3.36 x 10(7), 3.36 x 10(8), and 3.36 x 10(9) colony forming units (CFU)/g fish for 96 h to evaluate the difference in S. iniae susceptibility of flounders reared in biofloc and seawater. The 96 h lethal concentration 50% (LC(50)) of fish injected with S. iniae was 2.41 x 10(9) CFU/g fish in biofloc and 1.51 x 10(8) CFU/g fish in seawater. Hematological parameters such as hemoglobin and hematocrit significantly decreased when fish were challenged by S. iniae. Plasma components such as calcium, glucose, cholesterol, total protein, GOT, GPT, and ALP were significantly altered by S. iniae infection and acetylcholinesterase activity was significantly inhibited. These results indicate that S. iniae infection affects the survival rates, hematological parameters, and neurotransmitter levels of flounders reared in biofloc and seawater, and that S. iniae susceptibility was higher in flounders reared in seawater than those reared in biofloc.
Title: Enhanced inhibition of tumor growth using TRAIL-overexpressing adipose-derived stem cells in combination with the chemotherapeutic agent CPT-11 in castration-resistant prostate cancer Kim JH, Oh E, Han YS, Lee SH, Song YS Ref: Prostate Int, 9:31, 2021 : PubMed
BACKGROUND: This study investigated the inhibition of tumor growth in castrate-resistant prostate cancer (CRPC)-bearing mice by tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)-overexpressing adipose-derived stem cells (ADSCs) (hTERT-ADSC.sTRAIL), which was enhanced by combined treatment with CPT-11. MATERIALS AND METHODS: An hTERT-ADSC.sTRAIL cell line was established by transfection with a lentiviral vector (CLV-Ubic) encoding the human sTRAIL gene. Quantitative polymerase chain reaction and Western blots were performed to confirm gene overexpression. An invasion study for the selective migration ability toward PC3 cells was performed. In the in vivo study, the tumor volume in mice treated with ADSC. sTRAIL and CPT-11 was measured. RESULTS: Carboxylesterase was generated from hTERT-ADSCs. The gene expression of sTRAIL from hTERT-ADSC.sTRAIL was shown. The directional migration of ADSC.sTRAIL cells toward PC3 cells was significantly stimulated by PC3 cells in vitro (P < 0.05). In the in vitro study, the viability of PC3 cells significantly decreased in the presence of ADSC.sTRAIL (62.7 +/- 2.0%) and CPT-11 compared with that of CPT-11 alone (83.0 +/- 1.0%) at a cell ratio as low as 0.05 (PC3: ADSC.sTRAIL) (P < 0.05). The proportion of apoptotic PC3 cells significantly increased in the presence of ADSC.sTRAIL (37.2 +/- 2.1%) and CPT-11 compared with that of CPT-11 alone (16.5 +/- 1.0%) (P < 0.05). In the in vivo study, the inhibition of tumor growth in CRPC-bearing mice by TRAIL-overexpressing adipose stem cells was enhanced by combined treatment with the chemotherapeutic agent CPT-11 compared with that in the treatment with cpt-11 alone. Immunohistochemical staining of the removed tumors showed anti-TRAIL-positive cells and apoptotic bodies after hTERT-ADSC.sTRAIL treatment or combined treatment with hTERT-ADSC.sTRAIL and CPT-11. CONCLUSIONS: Therapeutic stem cells expressing sTRAIL genes combined with CPT-11 can provide a new strategy for treating CRPC in clinical trials using the patients' own ADSCs.
Title: Simultaneous Quantification of Four Marker Compounds in Bauhinia coccinea Extract and Their Potential Inhibitory Effects on Alzheimer's Disease Biomarkers Kim YJ, Sohn E, Lim HS, Kim Y, Kim JH, Jeong SJ Ref: Plants (Basel), 10:, 2021 : PubMed
Bauhinia coccinea is a tropical woody plant widely distributed in Vietnam and Unnan in southern China. Although many studies have shown the biological activities of extracts from various other species in the genus, no studies have investigated the effects of B. coccinea extracts on biological systems. In the present study, a quantitative analysis of four marker compounds of ethanol extracts of B. coccinea branches (EEBC) was performed using the high performance liquid chromatography (HPLC)-photodiode array (PDA) method. Among gallic acid, (+)-catechin, ellagic acid, and quercitrin contained in EEBC, the most abundant compound was (+)-catechin (18.736 mg/g). In addition, we investigated the EEBC on neuroprotection, antioxidation, and Alzheimer's disease (AD) marker molecules, acetylcholinesterase (AChE), and amyloid-beta (Abeta). EEBC significantly inhibited hydrogen peroxide (H(2)O(2))-induced cell death in a HT22 neuronal cell line and increased 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) and 2,2-diphenyl-1-picrylhydrazyl scavenging activity markedly. EEBC also inhibited AChE and Abeta aggregation. Among the four compounds, gallic acid exhibited strong inhibitory effects against AChE activation. In the Abeta aggregation assay, the four marker compounds exhibited inhibitory effects lower than 30%. According to the results, EEBC could exert anti-AChE activation and Abeta aggregation activities based on the interactive effects of the marker compounds. Our findings suggest that EEBC are sources of therapeutic candidates for application in the development of AD medication based on AChE and Abeta dual targeting.
Title: Inhibition of soluble epoxide hydrolase by phytochemical constituents of the root bark of Ulmus davidiana var. japonica Kim JH, Park JS, Lee YJ, Choi S, Kim YH, Yang SY Ref: J Enzyme Inhib Med Chem, 36:1049, 2021 : PubMed
A novel compound 1 and nine known compounds (2-10) were isolated by open column chromatography analysis of the root bark of Ulmus davidiana. Pure compounds (1-10) were tested in vitro to determine the inhibitory activity of the catalytic reaction of soluble epoxide hydrolase (sEH). Compounds 1, 2, 4, 6-8, and 10 had IC(50) values ranging from 11.4 +/- 2.3 to 36.9 +/- 2.6 microM. We used molecular docking to simulate inhibitor binding of each compound and estimated the binding pose of the catalytic site of sEH. From this analysis, the compound 2 was revealed to be a potential inhibitor of sEH in vitro and in silico. Additionally, molecular dynamics (MD) study was performed to find detailed interaction signals of inhibitor 2 with enzyme. Finally, compound 2 is promising candidates for the development of a new sEH inhibitor from natural plants.
Among 276 herbal extracts, a methanol extract of Castanopsis cuspidata var. sieboldii stems was selected as an experimental source for novel acetylcholinesterase (AChE) inhibitors. Five compounds were isolated from the extract by activity-guided screening, and their inhibitory activities against butyrylcholinesterase (BChE), monoamine oxidases (MAOs), and beta-site amyloid precursor protein cleaving enzyme 1 (BACE-1) were also evaluated. Of these compounds, 4'-O-(alpha-L-rhamnopyranosyl)-3,3',4-tri-O-methylellagic acid (3) and 3,3',4-tri-O-methylellagic acid (4) effectively inhibited AChE with IC(50) values of 10.1 and 10.7 microM, respectively. Ellagic acid (5) inhibited AChE (IC(50) = 41.7 microM) less than 3 and 4. In addition, 3 effectively inhibited MAO-B (IC(50) = 7.27 microM) followed by 5 (IC(50) = 9.21 microM). All five compounds weakly inhibited BChE and BACE-1. Compounds 3, 4, and 5 reversibly and competitively inhibited AChE, and were slightly or non-toxic to MDCK cells. The binding energies of 3 and 4 (- 8.5 and - 9.2 kcal/mol, respectively) for AChE were greater than that of 5 (- 8.3 kcal/mol), and 3 and 4 formed a hydrogen bond with Tyr124 in AChE. These results suggest 3 is a dual-targeting inhibitor of AChE and MAO-B, and that these compounds should be viewed as potential therapeutics for the treatment of Alzheimer's disease.
OBJECTIVES: Systemic rheumatic disease is characterized by autoimmunity and systemic inflammation and affects multiple organs. Few studies have investigated whether autoimmune diseases increase the risk of dementia. Herein, we evaluate the relationship between systemic rheumatic disease and dementia through a population-based study using the Korean National Health Insurance Service (NHIS) claims database. METHODS: We conducted a nationwide population-based study using the Korean NHIS database, consisting of individuals who submitted medical claims from 2002-2013. Dementia was defined as having an acetylcholinesterase inhibitors (AChEIs) prescription along with symptoms satisfying the Alzhemier's disease (AD) International Classification of Diseases (ICD)-10 codes (F00 or G30), or vascular dementia (VaD; ICD-10 or F01) criteria. Control subjects were matched to the dementia patients by age and sex. The study group was limited to those diagnosed with rheumatic disease at least 6 months prior to diagnosis of dementia. Rheumatic disease was defined by the following ICD-10 codes: Rheumatoid arthritis (RA: M05), Sjogren's syndrome (SS: M35), systemic lupus erythematosus (SLE: M32), and Behcet's disease (BD: M35.2). RESULTS: Of the 6,028 dementia patients, 261 (4.3%) had RA, 108 (1.6%) had SS, 12 (0.2%) had SLE, and 6 (0.1%) had BD. SLE history was significantly higher in dementia patients (0.2%) than in controls (0.1%) and was associated with dementia (odds ratio [OR], 2.48; 95% confidence interval [CI], 1.19-5.15). In subgroup analysis, SLE significantly increased dementia risk, regardless of dementia type (AD: OR, 2.29; 95% CI, 1.06-4.91; VaD: OR, 4.54; 95% CI, 1.36-15.14). However, these associations were not sustained in the mild CCI or elderly group. CONCLUSION: SLE was independently associated with a higher risk of dementia, including AD and VaD when compared to the control group, even after adjustment. SLE patients (<65 years old) are a high-risk group for early vascular dementia and require screening for early detection and active prevention.
Title: Ficus erecta Thunb Leaves Alleviate Memory Loss Induced by Scopolamine in Mice via Regulation of Oxidative Stress and Cholinergic System Sohn E, Kim YJ, Kim JH, Jeong SJ Ref: Molecular Neurobiology, :, 2021 : PubMed
We examined the neuropharmacological effects of ethanol extract of Ficus erecta Thunb leaves (EEFE) on cognitive dysfunction in a scopolamine (SCO)-induced memory impairment animal model. Memory impairment was measured using the Y-maze test and passive avoidance task (PAT). For 19 days, EEFE (100 or 200 mg/kg) was treated through oral administration. Treatment with EEFE ameliorated memory impairment in behavioral tests, along with significant protection from neuronal oxidative stress and neuronal cell loss in the brain tissues of SCO-injected mice. Antioxidant and neuroprotective effects of EEFE were further confirmed using in vitro assays. Our findings indicate that the mechanisms of neuroprotection and antioxidation of EEFE are regulated by the cholinergic system, promotion of cAMP response element-binding protein (CREB) phosphorylation, and the nuclear factor erythroid-2-related factor 2 (Nrf2)/heme oxygenase (HO)-1 signaling activation. The current study proposes that EEFE could be an encouraging plant resource and serve as a potent neuropharmacological drug candidate against neurodegenerative diseases.
Title: Protein Hydrolysate of Silkworm Pupa Prevents Memory Impairment Induced by Oxidative Stress in Scopolamine-Induced Mice via Modulating the Cholinergic Nervous System and Antioxidant Defense System Baek SY, Li FY, Kim JH, Ahn C, Kim HJ, Kim MR Ref: Prev Nutr Food Sci, 25:389, 2020 : PubMed
Silkworm pupae (Bombyx mori) is an edible insect that has been reported to contain high-quality proteins, lipids, minerals, and vitamins, and to possess high antioxidant activity. However, there have been no studies on the neuroprotective effects of silkworm pupae. Therefore, we investigated a water extract of silkworm pupae with protease (WSP) as a functional and therapeutic candidate for neurodegenerative disorders. First, we evaluated the effect of WSP on oxidative stress-induced mouse hippocampal neuronal cells (HT-22 cells). Cell viability diminished by addition of glutamate but was significantly recovered by WSP treatment. Furthermore, WSP significantly decreased the release of lactate dehydrogenase and generation of intracellular reactive oxygen species in oxidative stress-induced cells. In addition, in scopolamine-treated mice, WSP attenuated memory impairment, as demonstrated in the Morris water maze and passive avoidance tests, indicating protection of neuronal cells against oxidative damage. Moreover, WSP prevented scopolamine-induced increases in acetylcholinesterase activity and decreases in choline-acetyltransferase activity. Finally, treatment with WSP enhanced the antioxidant defense system by regulating the activities of antioxidant enzymes. Overall, this study showed that WSP exerted antioxidant and memory enhancing action against oxidative stress.
Title: Inhibitory Activity of Quercetin 3-O-Arabinofuranoside and 2-Oxopomolic Acid Derived from Malus domestica on Soluble Epoxide Hydrolase Cho IS, Kim JH, Lin Y, Su XD, Kang JS, Yang SY, Kim YH Ref: Molecules, 25:, 2020 : PubMed
Flavonoids and triterpenoids were revealed to be the potential inhibitors on soluble epoxide hydrolase (sEH). The aim of this study is to reveal sEH inhibitors from Fuji apples. A flavonoid and three triterpenoids derived from the fruit of Malus domestica were identified as quercetin-3-O-arabinoside (1), ursolic acid (2), corosolic acid (3), and 2-oxopomolic acid (4). They had half-maximal inhibitory concentration of the inhibitors (IC(50)) values of 39.3 +/- 3.4, 84.5 +/- 9.5, 51.3 +/- 4.9, and 11.4 +/- 2.7 muM, respectively, on sEH. The inhibitors bound to allosteric sites of enzymes in mixed (1) and noncompetitive modes (2-4). Molecular simulations were carried out for inhibitors 1 and 4 to calculate the binding force of ligands to receptors. The inhibitors bound to the left (1) and right (4) pockets next to the enzyme's active site. Based on analyses of their molecular docking and dynamics, it was shown that inhibitors 1 and 4 can stably bind sEH at 1 bar and 300 K. Finally, inhibitors 1 and 4 are promising candidates for further studies using cell-based assays and in vivo cardiovascular tests.
Title: Acetylcholinesterase and butyrylcholinesterase inhibitory activities of khellactone coumarin derivatives isolated from Peucedanum japonicum Thurnberg Heo JH, Eom BH, Ryu HW, Kang MG, Park JE, Kim DY, Kim JH, Park D, Oh SR, Kim H Ref: Sci Rep, 10:21695, 2020 : PubMed
Cholinesterase (ChE) and monoamine oxidase (MAO) inhibitors have been attracted as candidate treatments for Alzheimer's disease (AD). Fifteen khellactone-type coumarins from the roots of Peucedanum japonicum Thunberg were tested for acetylcholinesterase (AChE), butyrylcholinesterase (BChE), and MAO inhibitory activities. Compound 3'-angeloyl-4'-(2-methylbutyryl)khellactone (PJ13) most potently inhibited AChE (IC(50) = 9.28 microM), followed by 3'-isovaleryl-4'-(2-methylbutyroyl)khellactone (PJ15) (IC(50) = 10.0 M). Compound senecioyl-4'-angeloyl-khellactone (PJ5) most potently inhibited BChE (IC(50) = 7.22 M) and had the highest selectivity index (> 5.54), followed by 3'-senecioyl-4'-(2-methylbutyryl)khellactone (PJ10) and 3',4'-disenecioylkhellactone (PJ4) (IC(50) = 10.2 and 10.7 M, respectively). Compounds PJ13, PJ15, and PJ5 showed reversible and mixed-types of inhibition with K(i) values of 5.98, 10.4 (for AChE), and 4.16 microM (for BChE), respectively. However, all 15 compounds weakly inhibited MAO-A and MAO-B. Molecular docking simulation revealed that PJ13 had a higher binding affinity (- 9.3 kcal/mol) with AChE than PJ15 (- 7.8 kcal/mol) or PJ5 (- 5.4 kcal/mol), due to the formation of a hydrogen bond with Tyr121 (distance: 2.52 ). On the other hand, the binding affinity of PJ5 (- 10.0 kcal/mol) with BChE was higher than for PJ13 (- 7.7 kcal/mol) or PJ15 (- 8.1 kcal/mol), due to the formation of a hydrogen bond with Ser198 (distance: 2.05 ). These results suggest that PJ13 and PJ5 are potential reversible selective inhibitors of AChE and BChE, respectively, for the treatment of AD.
Title: Inhibitory Activity of Flavonoids, Chrysoeriol and Luteolin-7-O-Glucopyranoside, on Soluble Epoxide Hydrolase from Capsicum chinense Kim JH, Jin CH Ref: Biomolecules, 10:E180, 2020 : PubMed
Title: Authors' Response to Association between Acetylcholinesterase Inhibitors and Osteoporotic Fractures in Older Persons With Alzheimer's Disease Kim JH, Byun SJ, Shin JY Ref: J Am Med Dir Assoc, 21:707, 2020 : PubMed
Title: Toxic effects of waterborne nitrite exposure on antioxidant responses, acetylcholinesterase inhibition, and immune responses in olive flounders, Paralichthys olivaceus, reared in bio-floc and seawater Kim JH, Kim SK, Hur YB Ref: Fish Shellfish Immunol, 97:581, 2020 : PubMed
Paralichthys olivaceus (mean weight, 280.1 +/- 10.5 g; mean length, 28.37 +/- 2.3 cm) was reared in bio-floc and seawater for 6 months to determine the toxic effects of waterborne nitrite exposure (0, 25, 50, 100, and 200 mg/L) for 1 week, compared to those observed with bio-floc and seawater only. The effects on antioxidant activity, immune responses, and acetylcholinesterase activity were measured. Following nitrite exposure, superoxide dismutase activity in the liver and gills was significantly elevated and catalase activity was significantly increased, except for in the gills of P. olivaceus reared in bio-floc. Further, glutathione S-transferase activity was significantly elevated in the liver and gills, and glutathione was significantly lower. Meanwhile, acetylcholinesterase activity in the liver and gills was significantly inhibited and plasma lysozyme activity and immunoglobulin M were considerably elevated.
Title: Effects on hematological parameters, antioxidant and immune responses, AChE, and stress indicators of olive flounders, Paralichthys olivaceus, raised in bio-floc and seawater challenged by Edwardsiella tarda Kim JH, Sohn S, Kim SK, Hur YB Ref: Fish Shellfish Immunol, 97:194, 2020 : PubMed
Studies on the resistance of fish raised in bio-floc systems against bacterial infection are limited. We aimed to evaluate the changes in hematological parameters, antioxidant and immune responses, stress indicators, and acetylcholinesterase (AChE) in olive flounder, Paralichthys olivaceus, raised in bio-floc and seawater for 10 months and, then, infected with Edwardsiella tarda at concentrations of 0 (control), 6.61 x 10(4), 6.61 x 10(5), 6.61 x 10(6), and 6.61 x 10(7) CFU/g fish for 7 days. The lethal concentration 50% was 4.32 x 10(7) in bio-floc and 3.11 x 10(6) in seawater. Hematological parameters were significantly decreased by E. tarda challenge, and plasma components were significantly changed. The superoxide dismutase, catalase, and glutathione-S-transferase activities, as antioxidant responses, were significantly increased after infection, whereas the reduced glutathione level was significantly decreased. The lysozyme activity was significantly increased and the AChE level was significantly decreased after infection. Cortisol and HSP 70, as stress indicators, were also significantly increased. The results indicate that E. tarda infection affected various physiological factors in P. olivaceus. Additionally, P. olivaceus raised in seawater were more susceptible to E. tarda infection than those raised in bio-floc.
Title: Comparison of the Effects of Sugammadex, Neostigmine, and Pyridostigmine on Postoperative Nausea and Vomiting: A Propensity Matched Study of Five Hospitals Kim JH, Lim MS, Choi JW, Kim H, Kwon YS, Lee JJ Ref: J Clin Med, 9:, 2020 : PubMed
Thus far, few studies have compared the effects of sugammadex and cholinesterase inhibitors on postoperative nausea and vomiting (PONV), and the results have been controversial. Here, we compared the effects of sugammadex, neostigmine, and pyridostigmine on PONV by means of a five hospital analysis with propensity score matching. We analyzed adults aged >= 18 years who underwent general anesthesia between January 2014 and December 2019. Following propensity score matching, 7793 patients were included in each of the neostigmine and sugammadex matched patient groups (absolute standardized difference (ASD), 0.01-0.07), and 10,197 patients were included in each of the pyridostigmine and sugammadex matched patient groups (ASD, 0.01-0.02), while 19,377 patients were included in each of the pyridostigmine and neostigmine matched patient groups. (ASD, 0.01-0.19). The odds of PONV were low in the sugammadex group (odds ratio, 0.65; 95% confidence interval, 0.59-0.72; p < 0.0001) and pyridostigmine group (odds ratio, 0.22; 95% confidence interval, 0.20-0.24; p < 0.0001) compared to the neostigmine group, while there was no difference between sugammadex and pyridostigmine (odds ratio, 0.95; 95% confidence interval, 0.86-1.04; p = 0.281). Therefore, sugammadex and pyridostigmine may lower the incidence of PONV compared to neostigmine in patients undergoing general anesthesia.
Title: Regional and seasonal detection of resistance mutation frequencies in field populations of Anopheles Hyrcanus Group and Culex pipiens complex in Korea Lee DE, Kim HC, Chong ST, Klein TA, Choi KS, Kim YH, Kim JH, Lee SH Ref: Pestic Biochem Physiol, 164:33, 2020 : PubMed
Pyrethroid (PYR) and organophosphate (OP) insecticides have been extensively used for mosquito control for several decades in South Korea, and has resulted in the rapid development of resistance in the field. In this study, quantitative sequencing (QS) protocols were developed for the frequency prediction of insecticide resistance alleles [e.g., the L1014F/C mutation on the voltage sensitive sodium channel as a PYR resistance allele and the G119S mutation on the acetylcholinesterase 1 as OP resistance alleles] in four regional populations of Anopheles Hyrcanus Group and Culex pipiens complex. Both of the L1014F/C and G119S mutations were observed in all examined regional populations of An. Hyrcanus Group, suggesting a wide distribution of both PYR and OP resistance. In contrast, populations of the Cx. pipiens complex were determined to possess almost no G119S mutation, but relatively higher frequencies of the L1014F mutation, showing differential resistance patterns between different mosquito groups. The mutation frequencies were also monitored throughout a mosquito season (May-October) at one collection site to determine the seasonal changes of resistance mutation frequency in mosquito populations. Dramatic decreases of both L1014F/C and G119S mutation frequencies were observed in the An. Hyrcanus Group toward the fall, with no mutations observed in the early spring, suggesting a connection between the fitness costs of overwintering and insecticide resistance. However, no apparent trends were detectable in the Cx. pipiens complex populations due to low or zero mutation frequencies.
Title: Simultaneous Analysis of Fenthion and Its Five Metabolites in Produce Using Ultra-High Performance Liquid Chromatography-Tandem Mass Spectrometry Lee J, Kim JH Ref: Molecules, 25:1938, 2020 : PubMed
A simultaneous analytical method for the organophosphorus insecticide fenthion and its five metabolites (fenthion oxon, fenthion oxon sulfoxide, fenthion oxon sulfone, fenthion sulfoxide, and fenthion sulfone) was developed based on ultra-high performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS). Five matrices (brown rice, chili pepper, orange, potato, and soybean) were selected to validate the method. The target compounds were analyzed using positive electrospray ionization in the multiple reaction monitoring mode. For the best sensitivity in regard to the detector response, water and methanol containing formic acid (0.1%) were selected as the mobile phase. The optimum extraction efficiency was obtained through a citrate-buffered QuEChERS (quick, easy, cheap, effective, rugged, and safe) method. Recovery tests were carried out at three spiking levels (n = 3). At all fortification levels, the accuracy and precision results were between 70% and 120% with a relative standard deviation of >=15%. The limit of quantitation was 0.01 mg/kg, and the correlation coefficients (r(2)) of the matrix-matched calibration curves were >0.99. Significant signal suppression in the detector responses were observed for all matrices, suggesting that a compensation method, such as matrix-matched calibration, is required to provide accurate quantitative results. The applicability of the presented method was confirmed for the simultaneous analysis of fenthion and its metabolites in various crops.
The interplay between glioblastoma stem cells (GSCs) and tumor-associated macrophages (TAMs) promotes progression of glioblastoma multiforme (GBM). However, the detailed molecular mechanisms underlying the relationship between these two cell types remain unclear. Here, we demonstrate that ARS2 (arsenite-resistance protein 2), a zinc finger protein that is essential for early mammalian development, plays critical roles in GSC maintenance and M2-like TAM polarization. ARS2 directly activates its novel transcriptional target MGLL, encoding monoacylglycerol lipase (MAGL), to regulate the self-renewal and tumorigenicity of GSCs through production of prostaglandin E2 (PGE2), which stimulates beta-catenin activation of GSC and M2-like TAM polarization. We identify M2-like signature downregulated by which MAGL-specific inhibitor, JZL184, increased survival rate significantly in the mouse xenograft model by blocking PGE2 production. Taken together, our results suggest that blocking the interplay between GSCs and TAMs by targeting ARS2/MAGL signaling offers a potentially novel therapeutic option for GBM patients.
Title: Identification of Catalposide Metabolites in Human Liver and Intestinal Preparations and Characterization of the Relevant Sulfotransferase, UDP-glucuronosyltransferase, and Carboxylesterase Enzymes Hwang DK, Kim JH, Shin Y, Choi WG, Kim S, Cho YY, Lee JY, Kang HC, Lee HS Ref: Pharmaceutics, 11:, 2019 : PubMed
Catalposide, an active component of Veronica species such as Catalpa ovata and Pseudolysimachion lingifolium, exhibits anti-inflammatory, antinociceptic, anti-oxidant, hepatoprotective, and cytostatic activities. We characterized the in vitro metabolic pathways of catalposide to predict its pharmacokinetics. Catalposide was metabolized to catalposide sulfate (M1), 4-hydroxybenzoic acid (M2), 4-hydroxybenzoic acid glucuronide (M3), and catalposide glucuronide (M4) by human hepatocytes, liver S9 fractions, and intestinal microsomes. M1 formation from catalposide was catalyzed by sulfotransferases (SULTs) 1C4, SULT1A1*1, SULT1A1*2, and SULT1E1. Catalposide glucuronidation to M4 was catalyzed by gastrointestine-specific UDP-glucuronosyltransferases (UGTs) 1A8 and UGT1A10; M4 was not detected after incubation of catalposide with human liver preparations. Hydrolysis of catalposide to M2 was catalyzed by carboxylesterases (CESs) 1 and 2, and M2 was further metabolized to M3 by UGT1A6 and UGT1A9 enzymes. Catalposide was also metabolized in extrahepatic tissues; genetic polymorphisms of the carboxylesterase (CES), UDP-glucuronosyltransferase (UGT), and sulfotransferase (SULT) enzymes responsible for catalposide metabolism may cause inter-individual variability in terms of catalposide pharmacokinetics.
Alzheimer's disease (AD) is linked to an extensive neuron loss via accumulation of amyloid-beta (Abeta) as senile plaques associated with reactive astrocytes and microglial activation in the brain. The objective of this study was to assess the therapeutic effect of WS-5 ethanol extract in vitro and in vivo against Abeta-induced AD in mice and to identify the extract's active constituents. In the present study, WS-5 exerted a significant inhibitory effect on acetylcholinesterase (AChE). Analysis by transmission electron microscopy (TEM) revealed that WS-5 prevented Abeta oligomerization via inhibition of Abeta 1-42 aggregation. Evaluation of antioxidant activities using 1, 1-diphenyl-2-picrylhydrazyl (DPPH) demonstrated that WS-5 possessed a high antioxidant activity, which was confirmed by measuring the total antioxidant status (TAS). Furthermore, the anti-inflammatory properties of WS-5 were examined using lipopolysaccharide-stimulated BV-2 microglial cells. WS-5 significantly inhibited the lipopolysaccharide-induced production of nitric oxide and two proinflammatory cytokines, TNF-alpha and IL-6. The memory impairment in mice with Abeta-induced AD was studied using the Morris water maze and passive avoidance test. Immunohistochemistry was performed to monitor pathological changes in the hippocampus and cortex region of the mouse brain. The animal study showed that WS-5 (250 mg/kg) treatment improved learning and suppressed memory impairment as well as reduced Abeta plaque accumulation in Abeta-induced AD. HPLC analysis identified the extract's active compounds that exert anti-AChE activity. In summary, our findings suggest that WS-5 could be applied as a natural product therapy with a focus on neuroinflammation-related neurodegenerative disorders.
Title: Isolation of soluble epoxide hydrolase inhibitor of capsaicin analogs from Capsicum chinense Jacq. cv. Habanero Kim JH, Jo YD, Jin CH Ref: Int J Biol Macromol, 135:1202, 2019 : PubMed
Capsaicin (1) and dihydrocapsaicin (2) were isolated from the aerial parts of Capsicum chinense Jacq. cv. Habanero. In in vitro studies, the exhibited potent inhibitory activity against soluble epoxide hydrolase (sEH), with IC50 values of 5.6+/-1.2 and 7.3+/-0.7muM. Enzyme kinetics suggested that the two compounds (1 and 2) were competitive inhibitors with Ki values of 2.5+/-1.0 and 4.7+/-2.3muM, respectively. Molecular docking and molecular dynamic studies showed that capsaicin analogs (1 and 2) bound strongly with Asp335, Tyr383, and Tyr466 residues in the active site. These results suggest that the two inhibitors (1 and 2) represent potentially therapeutic inhibitors of sEH.
Title: Safflower (Carthamus tinctorius L.) seed attenuates memory impairment induced by scopolamine in mice via regulation of cholinergic dysfunction and oxidative stress Kim JH, He MT, Kim MJ, Yang CY, Shin YS, Yokozawa T, Park CH, Cho EJ Ref: Food Funct, 10:3650, 2019 : PubMed
Cholinergic dysfunction and oxidative stress are the most common causes of Alzheimer's disease (AD). Safflower seed contains various anti-oxidant and cholinergic improvement compounds, such as serotonin and its derivatives. In the present study, we investigated the protective effects and mechanisms of a safflower seed extract on scopolamine-induced memory impairment in a mouse model. The safflower seed extract was orally administered at a dose of 100 mg kg-1 day-1, and then behavior tests (such as T-maze and novel object recognition tests) were conducted. Acetyl cholinesterase (AChE) activity, reactive oxygen species (ROS) production, and antioxidant enzymes in the brain were measured. In behavior tests, the novel route exploration and object recognition were improved by the administration of the safflower seed extract, which suggests that the safflower seed extract improves memory function in the scopolamine-treated mouse model. In addition, the safflower seed extract-administered group showed inhibition of the AChE activity and improved cholinergic dysfunction. Furthermore, the administration of the safflower seed extract resulted in lower ROS production and higher antioxidant enzyme levels as compared to the scopolamine-treated group, suggesting the protective role of the safflower seed extract against oxidative stress. The results of the present study suggest that the safflower seed extract improves scopolamine-induced memory deficits via the inhibition of cholinergic dysfunction and oxidative stress. Therefore, safflower seeds might become a promising agent for memory improvement in AD patients.
In this study, maltoheptaose (G7)-based sugar esters were synthesized from maltoheptaose and fatty acids (C10-C16) using a commercial lipase. With the exception of dimethyl sulfoxide (DMSO; 76.4%, w/v), G7 showed only limited solubility in organic solvents. Among the fatty acids, palmitic acid (PA) was the best substrate for G7-based ester formation. G7-PA ester was successfully synthesized as the monoester structure exclusively in 10% DMSO of t-butanol with a 22% conversion yield. NMR and enzymatic analyses of the purified monoester product revealed that the ester bond in the G7 was located at C-6 of the glucose at the reducing end. The G7-PA monoester showed the melting temperature at 56.3 degrees C that was 6.5 degrees C lower than that of the free PA and exhibited a different endothermic pattern from the free G7. The G7-PA monoester exhibited excellent emulsifier potential with more even droplet size distribution compared with the commercial sucrose esters for an oil-in-water emulsion system.
Title: Elaeagnus glabra f. oxyphylla Attenuates Scopolamine-Induced Learning and Memory Impairments in Mice by Improving Cholinergic Transmission via Activation of CREB/NGF Signaling Sohn E, Lim HS, Kim YJ, Kim BY, Kim JH, Jeong SJ Ref: Nutrients, 11:, 2019 : PubMed
We aimed to investigate the therapeutic effects of an Elaeagnus glabra f. oxyphylla (EGFO) ethanol extract in mice with scopolamine-induced memory dysfunction. Fifty male mice were randomly divided into a normal control group, a scopolamine-treated group, a scopolamine and EGFO extract-treated group, and a scopolamine and tacrine-treated group. EGFO (50 or 100 mg/kg/day) was received for 21 days. Step-through passive avoidance and Y-maze tests were performed to examine the effects of treatment on learning and memory impairments. Acetylcholine (Ach) levels and acetylcholinesterase (AchE) activity were measured via an enzyme-linked immunosorbent assay (ELISA). Levels of choline acetyltransferase (ChAT), nerve growth factor (NGF), cAMP response element-binding protein (CREB), and apoptosis-related protein expression were determined via Western blot analysis. EGFO pretreatment significantly attenuated scopolamine-induced memory impairments, relative to findings observed in the scopolamine-treated group. Levels of cholinergic factors in the brain tissues were markedly attenuated in the scopolamine-treated group. EGFO treatment also attenuated neural apoptosis in scopolamine-treated mice by decreasing the expression of apoptosis-related proteins such as Bax, Bcl2, cleaved caspase-3, and TUNEL staining. These results suggest that EGFO improves memory and cognition in a mouse model of memory impairment by restoring cholinergic and anti-apoptotic activity, possibly via activation of CREB/NGF signaling.
Title: In vitro and in silico investigation of anthocyanin derivatives as soluble epoxide hydrolase inhibitors Kim JH, Cho IS, Ryu J, Lee JS, Kang JS, Kang SY, Kim YH Ref: Int J Biol Macromol, 112:961, 2018 : PubMed
Anthocyanin derivatives are well-known secondary constituents contained in fruits. The inhibitory activity of anthocyanin derivatives toward soluble epoxide hydrolase (sEH) was tested for potential applications in the treatment of cardiovascular diseases. Anthocyanin derivatives 1-5 showed dose-dependent inhibitory activity toward sEH, with IC50 values ranging from 4.3+/-0.2 to 25.3+/-2.6muM. Lineweaver-Burk plots showed that all anthocyanin derivatives preferentially interacted with allosteric sites instead of active sites as noncompetitive (1-3) and mixed (4 and 5) inhibitors. Furthermore, the cavity located next to the active site may interact with anthocyanin derivatives (1-5) by molecular docking. Among the tested derivatives, (4) bonded with key amino acids at two loops around the binding site for 10ns. Finally, anthocyanin derivatives (1-5) are potential inhibitors of sEH, and anthocyanin-rich fruits may be useful for the targeted treatment of cardiovascular diseases via sEH inhibition.
Title: Sugammadex affects emergence agitation in children undergoing strabismus surgery Kim YS, Cha JR, Lee YS, Kim WY, Kim JH, Kim YH Ref: J Internal Medicine Res, :300060518781480, 2018 : PubMed
Objective Emergence agitation (EA) has a multifactorial origin, and the effect of sugammadex on EA has not been established. We investigated the effect of sugammadex on EA incidence and severity. Methods We performed a retrospective study of children aged 1 to 13 years who underwent strabismus surgery. Patients received sugammadex or conventional neuromuscular reversal agents. The primary outcome variables were EA incidence and severity. Secondary outcome variables were postoperative fentanyl use, postoperative nausea and vomiting, time from reversal agent administration to extubation, time from the end of surgery to arrival in the post-anesthesia care unit (PACU) and time spent in the PACU. We used propensity score matching to eliminate baseline imbalances. Results Age, sex, use of desflurane, and intraoperative fentanyl were significant predictors of agitation severity using a multivariable analysis. Sugammadex did not significantly affect EA in logistic regression and multiple regression analyses. In the propensity-matched analysis, patients in the sugammadex group showed rapid recovery, but there was no difference in the EA incidence or severity. Conclusion Sugammadex did not affect EA incidence or severity compared with conventional cholinesterase inhibitors, although it showed a favorable recovery profile in children undergoing strabismus surgery.
Title: Molecular and biochemical characterization of the bed bug salivary gland cholinesterase as an acetylcholine-sequestering enzyme Kim JH, Hwang CE, Yoon KA, Seong KM, Lee J, Lee SH Ref: Insect Biochemistry & Molecular Biology, 102:52, 2018 : PubMed
The common bed bug, Cimex lectularius, possesses a cholinesterase expressed exclusively in the salivary gland (ClSChE). In this study, we investigated the molecular forms, tissue distribution patterns and biochemical properties of ClSChE and showed that ClSChE exists as a soluble monomeric form or a soluble dimeric form connected by a disulfide bridge. Immunohistochemical analysis confirmed that ClSChE was expressed in the epithelial cells of both the salivary gland and the duct. In addition, the secretion of monomeric ClSChE through the proboscis during feeding was confirmed by western blotting using a ClSChE-specific antibody. To predict the role of ClSChE injected into the tissue of an animal host, we analyzed the extent of hydrolysis of acetylcholine (ACh) by ClSChE by ultra-performance liquid chromatography-tandem mass spectrometry. ClSChE binding to ACh was not clearly resolved in the binding assay format used in this study, probably due to the weak but detectable ACh-hydrolytic activity of ClSChE. Nevertheless, kinetic analysis revealed that ClSChE possesses extremely low Km (high affinity to ACh) and Vmax values. These findings suggest that ClSChE functions virtually as an ACh-sequestering protein by having a very strong affinity to ACh but an extremely long turnover time. Given that ACh regulates a wide variety of host physiologies, we discuss the tentative roles of ClSChE in blood vessel constriction and itch/pain regulation in the host.
Title: The insight of in vitro and in silico studies on cholinesterase inhibitors from the roots of Cimicifuga dahurica (Turcz.) Maxim Kim JH, Thao NP, Han YK, Lee YS, Luyen BTT, Oanh HV, Kim YH, Yang SY Ref: J Enzyme Inhib Med Chem, 33:1174, 2018 : PubMed
Cholinesterases (ChEs) are enzymes that break down neurotransmitters associated with cognitive function and memory. We isolated cinnamic acids (1 and 2), indolinones (3 and 4), and cycloartane triterpenoid derivatives (5-19) from the roots of Cimicifuga dahurica (Turcz.) Maxim. by chromatography. These compounds were evaluated for their inhibitory activity toward ChEs. Compound 1 was determined to have an IC50 value of 16.7 +/- 1.9 muM, and to act as a competitive inhibitor of acetylcholinesterase (AChE). Compounds 3, 4 and 14 were found to be noncompetitive with IC50 values of 13.8 +/- 1.5 and 6.5 +/- 2.5 muM, and competitive with an IC50 value of 22.6 +/- 0.4 muM, respectively, against butyrylcholinesterase (BuChE). Our molecular simulation suggested each key amino acid, Tyr337 of AChE and Asn228 of BuChE, which were corresponded with potential inhibitors 1, and 3 and 4, respectively. Compounds 1 and 4 were revealed to be promising compounds for inhibition of AChEs and BuChEs, respectively.
Title: In Vitro and In Silico Insights into sEH Inhibitors with Amide-Scaffold from the Leaves of Capsicum chinense Jacq Kim JH, Jo YD, Kim HY, Kim BR, Nam B Ref: Comput Struct Biotechnol J, 16:404, 2018 : PubMed
Two compounds termed 1 and 2 were isolated from the leaves of Capsicum chinense using column chromatography. Their structures were identified as amide scaffolds by analyzing spectroscopic signals. Compounds 1 and 2 have been confirmed to be competitive soluble epoxide hydrolase (sEH) inhibitors that suppress the catalytic reaction of sEH in a dose-dependent manner in vitro. Molecular docking was used for analyzing two binding clusters of ligand and receptor. The results confirmed that the key amino acids interacting with the ligand were Asp335, Tyr383, and Gln384. On the basis of molecular dynamics, inhibitors 1 and 2 were noted to interact at a distance of 3.5A from Asp335, Tyr383, Leu408 and Tyr466, and Asp335, Tyr383, and Tyr466, respectively. These results highlight the potential of N-trans-coumaroyltyramine (1) and N-trans-feruloyltyramine (2) as sEH inhibitors.
Title: Cholinesterases inhibition studies of biological active compounds from the rhizomes of Alpinia officinarum Hance and in silico molecular dynamics Lee JS, Kim JH, Han YK, Ma JY, Kim YH, Li W, Yang SY Ref: Int J Biol Macromol, 120:2442, 2018 : PubMed
Six diarylheptanoids (1-6) and two flavonoids (7 and 8) derived from Alpinia officinarum were evaluated for their ability to inhibit acetylcholinesterase. Compound 1 showed the highest degree of inhibition, with an IC50 of approximately 2muM, followed by moderate degrees of inhibition by 2, 4 and 7, with IC50 values ranging from 20 to 40muM. The remaining isolated compounds 3, 5, 6 and 8 had IC50 values greater than 50muM. Enzyme kinetic studies showed that the compounds with high or moderate activity were competitive inhibitors, anchored to the active site of acetylcholinesterase. In particular, compounds 1 and 2 were docked at slightly different positions from those occupied by 4 and 7. Furthermore, molecular dynamics studies showed that compound 1 maintained its interactions with residues Thr74 and Phe295 throughout the simulation trajectory. Our findings suggest that compound 1 is a potential therapeutically relevant inhibitor of acetylcholinesterase.
Title: Toxic Effects and Depuration on the Antioxidant and Neurotransmitter Responses after Dietary Lead Exposure in Starry Flounder Park HJ, Hwang IK, Kim KW, Kim JH, Kang JC Ref: J Aquat Anim Health, 30:245, 2018 : PubMed
Starry Flounder Platichthys stellatus were exposed to dietary lead (Pb) at concentrations of 0, 30, 60, 120, and 240 mg/kg for 4 weeks. Recover period was conducted for 2 weeks after the exposure. Exposure to Pb concentrations over 60 mg/kg induced significant changes in the antioxidant responses in the liver, kidney, and gill and continued even after the depuration period in the liver (over 120 mg/kg for superoxide dismutase [SOD] activity) and kidney (at 240 mg/kg for glutathione [GSH] levels). Glutathione S-transferase (GST) activity in liver, kidney, and gill were increased by dietary Pb exposure, and recovery was observed in all groups during the recovery period. Acetylcholinesterase (AChE) activity was significantly inhibited in the brain and muscle of flounder at Pb exposure over 120 mg/kg, and no restoration was observed after the depuration period. Lysozyme activity in the plasma was significantly increased at Pb exposures greater than 60 mg kg but was restored after the depuration period. The results of this study indicate that dietary Pb exposure induces toxic effects on antioxidant responses, neurotransmitter, and immune responses of Starry Flounder.
Title: Expression of acetylcholinesterase 1 is associated with brood rearing status in the honey bee, Apis mellifera Kim YH, Kim JH, Kim K, Lee SH Ref: Sci Rep, 7:39864, 2017 : PubMed
Acetylcholinesterase 1 (AmAChE1) of the honey bee, Apis mellifera, has been suggested to have non-neuronal functions. A systematic expression profiling of AmAChE1 over a year-long cycle on a monthly basis revealed that AmAChE1 was predominantly expressed in both head and abdomen during the winter months and was moderately expressed during the rainy summer months. Interestingly, AmAChE1 expression was inhibited when bees were stimulated for brood rearing by placing overwintering beehives in strawberry greenhouses with a pollen diet, whereas it resumed when the beehives were moved back to the cold field, thereby suppressing brood rearing. In early spring, pollen diet supplementation accelerated the induction of brood-rearing activity and the inhibition of AmAChE1 expression. When active beehives were placed in a screen tent in late spring, thereby artificially suppressing brood-rearing activity, AmAChE1 was highly expressed. In contrast, AmAChE1 expression was inhibited when beehives were allowed to restore brood rearing by removing the screen, supporting the hypothesis that brood rearing status is a main factor in the regulation of AmAChE1 expression. Since brood rearing status is influenced by various stress factors, including temperature and diet shortage, our finding discreetly suggests that AmAChE1 is likely involved in the stress response or stress management.
Title: Soluble epoxide hydrolase inhibitory activity of components from Leonurus japonicus Leem HH, Lee GY, Lee JS, Lee H, Kim JH, Kim YH Ref: Int J Biol Macromol, 103:451, 2017 : PubMed
One new compound, 10-methoxy-leonurine (1), and four known compounds (2-5) were purified by silica gel, C-18, and Sephadex LH-20 column chromatography from Leonurus japonicus. Their structures were elucidated using one-dimensional (1D)/two-dimensional (2D)-nuclear magnetic resonance (NMR), high-resolution (HR)-electrospray ionization (ESI) mass spectrometry (MS). The compounds were evaluated to determine their inhibition of the catalysis of soluble epoxide hydrolase (sEH). According to the results from in vitro analyses, compounds 1 and 2, which contain guanidine and flavonoid (3), were determined to be potential inhibitors of this enzyme. All compounds were revealed to be non-competitive inhibitors according to Lineweaver-Burk plots. Furthermore, in silico molecular docking indicated that compounds 1-3 are bound to sEH in a similar fashion and have stable binding energies, as calculated by AutoDock 4.2. Molecular dynamics determined the root-mean-square deviation (RMSD), total energy, RMS fluctuation (RMSF), hydrogen bonds, and distance of the complex according to time.
A series of 3,4-dihydroquinazoline derivatives consisting of the selected compounds from our chemical library on the diversity basis and the new synthetic compounds were in vitro tested for their inhibitory activities for both acetylcholinesterase (AChE, from electric eel) and butyrylcholinesterase (BChE, from equine serum) enzymes. It was discovered that most of the compounds displayed weak AChE and strong BuChE inhibitory activities. In particular, compound 8b and 8d were the most active compounds in the series against BChE with IC50 values of 45nM and 62nM, as well as 146- and 161-fold higher affinity to BChE, respectively. To understand the excellent activity of these compounds, molecular docking simulations were performed to get better insights into the mechanism of binding of 3,4-dihydroquinazoline derivatives. As expected, compound 8b and 8d bind to both catalytic anionic site (CAS) and peripheral site (PS) of BChE with better interaction energy values than AChE, in agreement with our experimental data. Furthermore, the non-competitive/mixed-type inhibitions of both compounds further confirmed their dual binding nature in kinetic studies.
Title: In silico investigation of cycloartane triterpene derivatives from Cimicifuga dahurica (Turcz.) Maxim. roots for the development of potent soluble epoxide hydrolase inhibitors Thao NP, Kim JH, Thuy Luyen BT, Dat NT, Kim YH Ref: Int J Biol Macromol, 98:526, 2017 : PubMed
In our search for natural soluble epoxide hydrolase (sEH) inhibitors from plants, we found that an ethanolic extract of the roots of Cimicifuga dahurica (Turcz.) Maxim. significantly inhibits sEH in vitro. A phytochemical study on the dichloromethane fraction of C. dahurica resulted in the isolation of two new cycloartane triterpenoids (1 and 6), together with 13 known cycloartane analogues (2-5 and 7-15). The structures of compounds were determined by spectroscopic methods. All of the triterpenoid derivatives inhibited sEH enzymatic activity in a concentration-dependent manner, and 13 of the tested compounds showed significant activity. Among them, compounds 1, 3, 5, 7, 9, and 12 showed the highest levels of inhibitory activity, with IC50 values of about 5muM or less. Kinetic analysis of compounds 1, 3, 5-9, 11, 12, and 14 revealed that compounds 3, 6, 7, 11, and 14 were non-competitive; 1, 5, 9, and 12 were mixed-type; and 8 was a competitive inhibitor. Furthermore, in silico molecular docking indicated that compounds 3, 6-9, 11, 12, and 14 bound to sEH in a similar manner and had stable binding energies, as calculated by AutoDock 4.2 and processed in a 10,000-ps molecular dynamics simulation to assess the binding stability of compounds 5, 7, and 9.
It has been of great interest to measure the activity of acetylcholinesterase (AChE) and its inhibitor, as AChE is known to accelerate the aggregation of the amyloid beta peptides that underlie Alzheimer's disease. Herein, we report the development of graphene oxide (GO) fluorescence-based biosensors for the detection of AChE activity and AChE inhibitors. To this end, GO was non-covalently functionalized with phenoxy-modified dextran (PhO-dex-GO) through hydrophobic interaction; the resulting GO showed excellent colloidal stability and intense fluorescence in various aqueous solutions as compared to pristine GO and the GO covalently functionalized with dextran. The fluorescence of PhO-dex-GO remarkably increased as AChE catalyzed the hydrolysis of acetylthiocholine (ATCh) to give thiocholine and acetic acid. It was found that the turn-on fluorescence response of PhO-dex-GO to AChE activity was induced by protonation of carboxyl groups on it from the product of the enzymatic hydrolysis reaction, acetic acid. On the basis of its turn-on fluorescence response, PhO-dex-GO was able to report kinetic and thermodynamic parameters involving a maximum velocity, a Michaelis constant, and an inhibition dissociation constant for AChE activity and inhibition. These parameters enable us to determine the activity of AChE and the efficiency of the inhibitor.
Title: Use of the Integrated Biomarker Response to Measure the Effect of Short-term Exposure to Dibenz[a,h]anthracene in Common carp (Cyprinus carpio) Kim JH, Kim WK Ref: Bulletin of Environmental Contamination & Toxicology, 96:496, 2016 : PubMed
Dibenz[a,h]anthracene (DbA) is a polycyclic aromatic hydrocarbon that is released into the environment through incomplete combustion of gasoline, cigarettes, and coal tar. The effects of short-term (10 days) exposure of common carp (Cyprinus carpio) to DbA (0-50 microg L(-1)) were evaluated using the following four biomarkers: DNA damage, 7-ethoxyresorufin-O-deethylase (EROD) activity, acetylcholinesterase (AChE) activity, and vitellogenin (VTG) levels. An integrated biomarker response (IBR) was calculated for exposure to DbA, and the results were compared with those in our previous study of two other PAHs, benzo[k]fluoranthene (BkF) and benzo[a]pyrene (BaP). DbA exposure resulted in a significant (p < 0.05) increase in DNA damage, EROD activity, and VTG levels relative to the control. By contrast, DbA did not affect AChE activity. The IBR increased as the concentration of DbA increased. Based on the IBR values, the order of toxicity for the PAHs was BkF > BaP > DbA. Our results suggest that the IBR can be used as a quantitative tool for evaluating the responses of multiple biomarkers to PAH exposure.
Title: (-)-Epicatechin derivate from Orostachys japonicus as potential inhibitor of the human butyrylcholinesterase Kim JH, Lee SH, Lee HW, Sun YN, Jang WH, Yang SY, Jang HD, Kim YH Ref: Int J Biol Macromol, 91:1033, 2016 : PubMed
Cholinesterase inhibitors block the bioconversion of neurotransmitters by cholinesterase in the nervous system. Epicatechin derivatives (1, 3 and 5), polyphenols (6 and 7) from Orostachys japonicus, and catechin derivatives (2 and 4) from our in-house library were evaluated for their inhibitory activity on cholinesterase. Compound 5 exhibited IC50 values of 58.3+/-2.4 and 17.8+/-3.8mug/mL on AChE and BuChE, respectively. Compound 5 inhibited BuChE more strongly than AChE through a competitive behavior. In silico binding positions of 5 in the active site were predicted using Autodock 4.2 and processed in a 10000-ps molecular dynamics simulation to assess the stability of compound 5 binding.
Title: The immune responses and expression of metallothionein (MT) gene and heat shock protein 70 (HSP 70) in juvenile rockfish, Sebastes schlegelii, exposed to waterborne arsenic (As3+) Kim JH, Kang JC Ref: Environ Toxicol Pharmacol, 47:136, 2016 : PubMed
Juvenile rockfish, Sebastes schlegelii (mean length 16.4+/-1.9cm, and mean weight 71.6+/-6.4g) were exposed for 20days with the different levels of waterborne arsenic concentration (0, 50, 100, 200 and 400mug/L). The plasma cortisol of S. schlegelii was significantly increased by the waterborne arsenit exposure. In the immune responses, the immunoglobulin M (Ig M) and lysozyme activity of S. schlegelii were significantly increased by the waterborne arsenic exposure. The acetylcholinesterase (AChE) activity of S. schlegelii was inhibited by the waterborne arsenic exposure. The substantial increases in the gene expression such as metallothionein (MT) and heat shock protein 70 (HSP 70) were observed by the waterborne arsenic exposure. The results demonstrated that waterborne arsenic exposure can induce the significant alterations in the immune responses and specific gene expression of S. schlegelii.
Title: Effects of sub-chronic exposure to lead (Pb) and ascorbic acid in juvenile rockfish: Antioxidant responses, MT gene expression, and neurotransmitters Kim JH, Kang JC Ref: Chemosphere, 171:520, 2016 : PubMed
Juvenile rockfish Sebastes schlegelii were exposed to varying levels of dietary lead (Pb2+) at 0, 120 and 240 mg/L, and ascorbic acid (AsA) at 100, 200 and 400 mg/L for four weeks. Antioxidant responses such as superoxide dismutase (SOD), glutathione S-transferase (GST), and glutathione (GSH) were analyzed to assess oxidative stress. SOD and GST activity in the liver and gills were considerably elevated by dietary Pb. In contrast, GSH levels in the liver and gills were significantly reduced following Pb exposure. High levels of AsA supplementation attenuated the increase in SOD and GST activity and reduction in GSH levels. The metallothionein gene (MT) in the liver was notably stimulated by Pb exposure, and AsA supplementation attenuated this increase. With respect to neurotoxicity, acetylcholinesterase (AChE) activity was substantially inhibited in the brain and muscle following Pb exposure. AsA supplementation also attenuated AChE inhibition following Pb exposure. The results of this study presented Pb exposure affected rockfish as toxicity, and AsA was effective to alleviate toxic effects of Pb.
Leaf senescence is not only primarily governed by developmental age but also influenced by various internal and external factors. Although some genes that control leaf senescence have been identified, the detailed regulatory mechanisms underlying integration of diverse senescence-associated signals into the senescence programs remain to be elucidated. To dissect the regulatory pathways involved in leaf senescence, we isolated the not oresara1-1 (nore1-1) mutant showing accelerated leaf senescence phenotypes from an EMS-mutagenized Arabidopsis thaliana population. We found that altered transcriptional programs in defense response-related processes were associated with the accelerated leaf senescence phenotypes observed in nore1-1 through microarray analysis. The nore1-1 mutation activated defense program, leading to enhanced disease resistance. Intriguingly, high ambient temperature effectively suppresses the early senescence and death phenotypes of nore1-1. The gene responsible for the phenotypes of nore1-1 contains a missense mutation in SENESCENCE-ASSOCIATED E3 UBIQUITIN LIGASE 1 (SAUL1), which was reported as a negative regulator of premature senescence in the light intensity- and PHYTOALEXIN DEFICIENT 4 (PAD4)-dependent manner. Through extensive double mutant analyses, we recently identified suppressor of the G2 Allele of SKP1b (SGT1b), one of the positive regulators for disease resistance conferred by many resistance (R) proteins, as a downstream signaling component in NORE1-mediated senescence and cell death pathways. In conclusion, NORE1/SAUL1 is a key factor integrating signals from temperature-dependent defense programs and leaf senescence in Arabidopsis. These findings provide a new insight that plants might utilize defense response program in regulating leaf senescence process, possibly through recruiting the related genes during the evolution of the leaf senescence program.
Title: Soluble Epoxide Hydrolase Inhibitory and Anti-inflammatory Components from the Leaves of Eucommia ulmoides Oliver (Duzhong) Bai MM, Shi W, Tian JM, Lei M, Kim JH, Sun YN, Kim YH, Gao JM Ref: Journal of Agricultural and Food Chemistry, 63:2198, 2015 : PubMed
Eucommia ulmoides leaves have been used as a functional food and drink in China. The purpose of this study was to identify the bioactive constituents with soluble epoxide hydrolase (sEH) inhibitory activity and anti-inflammatory properties. Twenty-seven known compounds (1-27) were isolated from the leaves of E. ulmoides Oliver, and their structures were identified by NMR and ESIMS analysis; three of these, 2,5-dimethoxy-3-glucopyranosyl cinnamic alcohol (11), foliasalacioside E2 (26), and icariside F2 (27), were obtained from this plant for the first time. Compounds 1-7 exhibited soluble epoxide hydrolase (sEH) inhibitory activity at 100 muM; among them, quercetin (1) and kaempferol (5) displayed potential activities with IC50 values of 22.5 +/- 0.9 and 31.3 +/- 2.6 muM, respectively, with noncompetitive inhibition mode. Nuclear factor kappa B (NF-kappaB) inhibitory activity of the isolated compounds was evaluated by the NF-kappaB liciferase assay in HepG2 cells. Compounds 1, 9, 20, and 27 displayed potent NF-kappaB inhibitory effects, with IC50 values of 15.14 +/- 2.29, 15.23 +/- 2.34, 16.88 +/- 2.17, and 16.25 +/- 2.19 muM, respectively, whereas other compounds showed weak inhibition of NF-kappaB transcriptional activity ranging from 17.54 to 92.6 muM. A structure-activity relationship of flavonoids 1-9 was also discussed. The results obtained in this work might contribute to the understanding of pharmacological activities of E. ulmoides leaves and further investigation on its potential application values for food and drug.
Title: In Vitro Metabolic Pathways of the New Anti-Diabetic Drug Evogliptin in Human Liver Preparations Jeong HU, Kim JH, Lee DY, Shim HJ, Lee HS Ref: Molecules, 20:21802, 2015 : PubMed
Evogliptin ((R)-4-((R)-3-amino-4-(2,4,5-trifluorophenyl)butanoyl)-3-(tert-butoxymethyl)-pipe razin-2-one), is a new dipeptidyl peptidase IV inhibitor used for the treatment of type II diabetes mellitus. The in vitro metabolic pathways of evogliptin were identified in human hepatocytes, liver microsomes, and liver S9 fractions using liquid chromatography-Orbitrap mass spectrometry (LC-HRMS). Five metabolites of evogliptin-4-oxoevogliptin (M1), 4(S)-hydroxyevogliptin (M2), 4(R)-hydroxyevogliptin (M3), 4(S)-hydroxyevogliptin glucuronide (M4), and evogliptin N-sulfate (M5)-were identified in human liver preparations by comparison with authentic standards. We characterized the cytochrome P450 (CYP) enzymes responsible for evogliptin hydroxylation to 4(S)-hydroxyevogliptin (M2) and 4(R)-hydroxyevogliptin (M3) and the UGT enzymes responsible for glucuronidation of 4(S)-hydroxyevogliptin (M2) to 4(S)-hydroxy-evogliptin glucuronide (M4). CYP3A4/5 played the major role in the hydroxylation of evogliptin to 4(S)-hydroxyevogliptin (M2) and 4(R)-hydroxyevogliptin (M3). Glucuronidation of 4(S)-hydroxy-evogliptin (M2) to 4(S)-hydroxyevogliptin glucuronide (M4) was catalyzed by the enzymes UGT2B4 and UGT2B7. These results suggest that the interindividual variability in the metabolism of evogliptin in humans is a result of the genetic polymorphism of the CYP and UGT enzymes responsible for evogliptin metabolism.
Title: BACE1 and cholinesterase inhibitory activities of Nelumbo nucifera embryos Jung HA, Karki S, Kim JH, Choi JS Ref: Arch Pharm Res, 38:1178, 2015 : PubMed
The aim of the present study was to evaluate the comparative anti-Alzheimer's disease (AD) activities of different parts of Nelumbo nucifera (leaves, de-embryo seeds, embryos, rhizomes, and stamens) in order to determine the selectivity and efficient use of its individual components. Anti-AD activities of different parts of N. nucifera were evaluated via inhibitory activities on acetylcholinesterase (AChE), butyrylcholinesterase (BChE), and beta-site amyloid precursor protein-cleaving enzyme 1 (BACE1) along with scavenging activity on peroxynitrite (ONOO(-)). Among the evaluated parts of N. nucifera, the embryo extract exhibited significant inhibitory potential against BACE1 and BChE as well as scavenging activity against ONOO(-). Thus, the embryo extract was selected for detailed investigation on anti-AD activity using BACE1- and ChEs-inhibitory assays. Among the different solvent-soluble fractions, the dichloromethane (CH2Cl2), ethyl acetate (EtOAc), and n-butanol (n-BuOH) fractions showed promising ChEs and BACE1 inhibitory activities. Repeated column chromatography of the CH2Cl2, EtOAc and n-BuOH fractions yielded compounds 1-5, which were neferine (1), liensinine (2), vitexin (3), quercetin 3-O-glucoside (4) and northalifoline (5). Compound 2 exhibited potent inhibitory activities on BACE1, AChE, and BChE with respective IC50 values of 6.37 +/- 0.13, 0.34 +/- 0.02, and 9.96 +/- 0.47 microM. Likewise, compound 1 showed potent inhibitory activities on BACE1, AChE, and BChE with IC50 values of 28.51 +/- 4.04, 14.19 +/- 1.46, and 37.18 +/- 0.59 microM, respectively; the IC50 values of 3 were 19.25 +/- 3.03, 16.62 +/- 1.43, and 11.53 +/- 2.21 microM, respectively. In conclusion, we identified potent ChEs- and BACE1-inhibitory activities of N. nucifera as well as its isolated constituents, which may be further explored to develop therapeutic and preventive agents for AD and oxidative stress related diseases.
Title: Inhibition of soluble epoxide hydrolase activity by compounds isolated from the aerial parts of Glycosmis stenocarpa Kim JH, Morgan AM, Tai BH, Van DT, Cuong NM, Kim YH Ref: J Enzyme Inhib Med Chem, :1, 2015 : PubMed
The aim of this study is to search for soluble epoxide hydrolase (sEH) inhibitors from natural plants, bioassay-guided fractionation of lipophilic n-hexane and chloroform layers of an extract of the aerial parts of Glycosmis stenocarpa led to the isolation of 12 compounds (1-12) including murrayafoline-A (1), isomahanine (2), bisisomahanine (3), saropeptate (4), (24 S)-ergost-4-en-3,6-dione (5), stigmasta-4-en-3,6-dion (6), stigmast-4-en-3-one (7), beta-sitosterol (8), 24-methylpollinastanol (9), trans-phytol (10), neosarmentol III (11) and (+)-epiloliolide (12). Their structures were elucidated on the basis of spectroscopic data. Among them, neosarmentol III (11) was isolated from nature for the first time. All the isolated compounds were evaluated for their inhibitory activity against sEH. Among isolated carbazole-type compounds, isomahanine (2) and bisisomahanine (3) were identified as a potent inhibitor of sEH, with IC50 values of 22.5 +/- 1.7 and 7.7 +/- 1.2 microM, respectively. Moreover, the inhibitory action of 2 and 3 represented mixed-type enzyme inhibition.
To overcome the poor properties of solubility and stability of cinnamic acid, cinnamate derivatives with sugar alcohols were produced using the immobilized Candida antarctica lipase with vinyl cinnamate and D-sorbitol as substrate at 45 degrees C. Immobilized C. antarctica lipase was found to synthesize 6-O-cinnamoyl-sorbitol and confirmed by HPLC and (1)H-NMR and had a preference for vinyl cinnamate over other esters such as allyl-, ethyl-, and isobutyl cinnamate as co-substrate with D-sorbitol. Contrary to D-sorbitol, vinyl cinnamate, and cinnamic acid, the final product 6-O-cinnamoyl-sorbitol was found to have radical scavenging activity. This would be the first report on the biosynthesis of 6-O-cinnamoyl-sorbitol with immobilized enzyme from C. antarctica.
Title: Mutation and duplication of arthropod acetylcholinesterase: Implications for pesticide resistance and tolerance Lee SH, Kim YH, Kwon DH, Cha DJ, Kim JH Ref: Pestic Biochem Physiol, 120:118, 2015 : PubMed
A series of common/shared point mutations in acetylcholinesterase (AChE) confers resistance to organophosphorus and carbamate insecticides in most arthropod pests. However, the mutations associated with reduced sensitivity to insecticides usually results in the reduction of catalytic efficiency and leads to a fitness disadvantage. To compensate for the reduced catalytic activity, overexpression of neuronal AChE appears to be necessary, which is achieved by a relatively recent duplication of the AChE gene (ace) as observed in the two-spotted spider mite and other insects. Unlike the cases with overexpression of neuronal AChE, the extensive generation of soluble AChE is observed in some insects either from a distinct non-neuronal ace locus or from a single ace locus via alternative splicing. The production of soluble AChE in the fruit fly is induced by chemical stress. Soluble AChE acts as a potential bioscavenger and provides tolerance to xenobiotics, suggesting its role in chemical adaptation during evolution.
Title: SUMO modification regulates the protein stability of NDRG1 Lee JE, Kim JH Ref: Biochemical & Biophysical Research Communications, 459:161, 2015 : PubMed
N-myc Downstream Regulated Gene 1 (NDRG1) is a metastasis suppressor protein which suppresses metastasis without affecting primary tumorigenesis. There have been many reports about the anti-metastatic function of NDRG1 in various cancers. However, the regulatory mechanism of NDRG1 at the protein level has not been studied widely. Here, we found that NDRG1 is posttranslationally modified by Small Ubiquitin-like Modifier (SUMO), preferentially by SUMO-2, and the major SUMO acceptor site of NDRG1 is Lys 14. Using various SUMO-2 modification status mimicking NDRG1 mutants, we characterized the role of SUMO-2 modification on NDRG1. SUMO-2 modification does not affect the subcellular distribution of NDRG1. However, the protein stability of NDRG1 is influenced by SUMO-2 modification. We found that both the wildtype and the SUMO modification site mutant form of the NDRG1 protein were very stable but the protein stability of SUMO-2 fused NDRG1 K14R had dramatically decreased. In addition, the expression of p21 is downregulated by overexpression of SUMO-2 fused NDRG1 K14R mutants. These results indicate that SUMO-2 modification is implicated in the modulation of NDRG1 protein level and function. This novel link between SUMO modification and regulation of NDRG1 could be a therapeutic target for treatment of various metastatic cancers.
Title: Soluble epoxide hydrolase inhibitory activity of anthraquinone components from Aloe Sun YN, Kim JH, Li W, Jo AR, Yan XT, Yang SY, Kim YH Ref: Bioorganic & Medicinal Chemistry, 23:6659, 2015 : PubMed
Aloe is a short-stemmed succulent herb widely used in traditional medicine to treat various diseases and as raw material in cosmetics and heath foods. In this study, we isolated and identified two new anthraquinone derivatives, aloinoside C (6) and aloinoside D (7), together with six known compounds from an aqueous dissolved Aloe exudate. Their structures were identified by spectroscopic analysis. The inhibitory effects of the isolated compounds on soluble epoxide hydrolase (sEH) were evaluated. Compounds 1-8 inhibited sEH activity potently, with IC50 values ranging from 4.1+/-0.6 to 41.1+/-4.2muM. A kinetic analysis of compounds 1-8 revealed that the inhibitory actions of compounds 1, 6 and 8 were non-competitive, whereas those of compounds 2-5 and 7 were the mixed-type. Molecular docking increases our understanding of receptor-ligand binding of all compounds. These results demonstrate that compounds 1-8 from Aloe are potential sEH inhibitors.
Title: A new approach of Integrated Health Responses (IHR(s)) modeling for ecological risk/health assessments of an urban stream Kim JH, Yeom DH, An KG Ref: Chemosphere, 108:376, 2014 : PubMed
The objective of this study was to evaluate the ecological health of an urban stream using Integrated Health Responses (IHRs). Water chemistry analysis, habitat health, and ecotoxicity tests were conducted in the stream along with analyses of molecular/biochemical, physiological biomarkers, and population-level responses in indicator species. Chemical stresses, measured as nutrient levels, ionic content and organic matter concentrations were significantly greater (p<0.01) at the downstream than the reference site (RF). The habitat health was largely impacted in the downstream reaches and had a negative relation with the land-use pattern of % urban area. Comet assay, 7-ethoxyresorufin-O-deethylase (EROD), acetylcholinesterase (AChE), and vitellogenin (VTG) were evaluated for low-level biomarker responses on DNA/physiological conditions of target species. The multi-metric fish model (Mm-F) was used to test the community-level response in relation to chemical and physical habitat stresses. The impaired responses of separate biomarker and bioindicator at the downstream sites occurred at all organizations from molecular/biochemical level to community level. Using all biomarkers/bioindicators, the star-plot model of IHRs was developed and then the integrative health/risk assessments were conducted in the urban stream. The reduced values of IHRs occurred in the downstream sites and the impacts were attributed to effluents from wastewater treatment plants (WTPs) and industrial complex. Ecological health impairments, thus, were evident in the urban reach, and reflected the long-term community responses as well as short-term responses of molecular biomarkers. The degradation of the urban stream was mainly due to a combined effect of chemical pollution and physical habitat modifications.
Title: Identification and characterization of an esterase involved in malathion resistance in the head louse Pediculus humanus capitis Kwon DH, Kim JH, Kim YH, Yoon KS, Clark JM, Lee SH Ref: Pestic Biochem Physiol, 112:13, 2014 : PubMed
Enhanced malathion carboxylesterase (MCE) activity was previously reported to be involved in malathion resistance in the head louse Pediculus humanus capitis (Gao et al., 2006 [8]). To identify MCE, the transcriptional profiles of all five esterases that had been annotated to be catalytically active were determined and compared between the malathion-resistant (BR-HL) and malathion-susceptible (KR-HL) strains of head lice. An esterase gene, designated HLCbE3, exhibited approximately 5.4-fold higher transcription levels, whereas remaining four esterases did not exhibit a significant increase in their transcription in BR-HL, indicating that HLCbE3 may be the putative MCE. Comparison of the entire cDNA sequences of HLCbE3 revealed no sequence differences between the BR-HL and KR-HL strains and suggested that no single nucleotide polymorphism is associated with enhanced MCE activity. Two copies of the HLCbE3 gene were observed in BR-HL, implying that the over-transcription of HLCbE3 is due to the combination of a gene duplication and up-regulated transcription. Knockdown of HLCbE3 expression by RNA interference in the BR-HL strain led to increases in malathion susceptibility, confirming the identity of HLCbE3 as a MCE responsible for malathion resistance in the head louse. Phylogenetic analysis suggested that HLCbE3 is a typical dietary esterase and belongs to a clade containing various MCEs involved in malathion resistance.
To investigate herbicide metabolism, human liver microsomes were incubated with threo- and erythro-isomers of flucetosulfuron. Each isomer produced one metabolite; the metabolites were unambiguously identified as enzymatic hydrolysis products by using liquid chromatography-mass spectrometry (LC-MS). These metabolites were synthesized, producing white solids characterized using LC-MS and nuclear magnetic resonance spectroscopy (1H and 13C). Using specific esterase inhibitors and activators, carboxylesterases and cholinesterases were demonstrated to be involved in flucetosulfuron metabolism. Under optimized metabolic conditions, the kinetic parameters for metabolite formation from erythro-flucetosulfuron and threo-flucetosulfuron were: Vmax, 134.38 and 151.41 nmol/min/mg protein, respectively; Km, 2798.53 and 2957.37 muM, respectively; and CLint, 48.02 and 51.20 muL/min/mg microsomes, respectively. No significant kinetic differences were observed between the two isomers. These results indicated that the primary metabolic pathway for both flucetosulfuron isomers in human liver microsomes involves hydrolysis, catalyzed by carboxylesterase and cholinesterase.
Title: Bisphenol A-associated alterations in the expression and epigenetic regulation of genes encoding xenobiotic metabolizing enzymes in human fetal liver Nahar MS, Kim JH, Sartor MA, Dolinoy DC Ref: Environmental & Molecular Mutagenesis, 55:184, 2014 : PubMed
Alterations in xenobiotic metabolizing enzyme (XME) expression across the life course, along with genetic, nutritional, and environmental regulation, can influence how organisms respond to toxic insults. In this study, we investigated the hypothesis that in utero exposure to the endocrine active compound, bisphenol A (BPA), influences expression and epigenetic regulation of phase I and II XME genes during development. Using healthy 1st to 2nd trimester human fetal liver specimens quantified for internal BPA levels, we examined XME gene expression using PCR Array (n = 8) and RNA-sequencing (n = 12) platforms. Of the greater than 160 XME genes assayed, 2 phase I and 12 phase II genes exhibited significantly reduced expression with higher BPA levels, including isoforms from the carboxylesterase, catechol O-methyltransferase, glutathione S-transferase, sulfotransferase, and UDP-glucuronosyltransferase families. When the promoters of these candidate genes were evaluated in silico, putative binding sites for the E-twenty-six (ETS) and activator protein1 (AP1) related transcription factor families were identified and unique to 97% of all candidate transcripts. Interestingly, many ETS binding sites contain cytosine-guanine dinucleotides (CpGs) within their consensus sequences. Thus, quantitative analysis of CpG methylation of three candidate genes was conducted across n = 50 samples. Higher BPA levels were associated with increased site-specific methylation at COMT (P < 0.005) and increased average methylation at SULT2A1 (P < 0.020) promoters. While toxicological studies have traditionally focused on high-dose effects and hormonal receptor mediated regulation, our findings suggest the importance of low-dose effects and nonclassical mechanisms of endocrine disruption during development. Environ. Mol. Mutagen. 55:184-195, 2014. (c) 2013 Wiley Periodicals, Inc.
Individuals aboard jet aircraft may be exposed to potentially toxic triaryl organophosphate anti-wear lubricant additives (TAPs) that are converted by cytochromes P450 into toxic metabolites. Consequences of exposure could be reduced by using less toxic TAPs. Our goal was to determine whether an in vitro assay for inhibition of butyrylcholinesterase (BChE) by bioactivated TAPs would be predictive of inhibition of serine active-site enzymes in vivo. The in vitro assay involved TAP bioactivation with liver microsomes and NADPH, followed by incubation with human BChE and measurement of BChE activity. Of 19 TAPs tested, tert-butylated isomers produced the least BChE inhibition. To determine the relevance of these results in vivo, mice were exposed to Durad 125 (D125; a commercial mixture of TAP esters) or to TAPs demonstrating low or no BChE inhibition when assayed in vitro. Inhibition of BChE by bioactivated TAPs in vitro correlated well with inhibition of other serine active-site enzymes in vivo, with the exception of brain acetylcholinesterase and neuropathy target esterase (NTE), which were not inhibited by any TAP tested following single exposures. A recombinant catalytic domain of NTE (rNEST) exhibited classical kinetic properties of NTE. The metabolite of tri-(o-cresyl) phosphate (ToCP), 2-(o-cresyl)-4H-1,3,2-benzodioxaphosphoran-2-one (CBDP), inhibited rNEST in vitro, but with an IC50 value almost 6-times higher than for inhibition of BChE. Physiologically-relevant concentrations of the flavonoid naringenin dramatically reduced D125 bioconversion in vitro. The in vitro assay should provide a valuable tool for prescreening candidate TAP anti-wear additives, identifying safer additives and reducing the number of animals required for in vivo toxicity testing.
Despite the pivotal functions of the NMDA receptor (NMDAR) for neural circuit development and synaptic plasticity, the molecular mechanisms underlying the dynamics of NMDAR trafficking are poorly understood. The cell adhesion molecule neuroligin-1 (NL1) modifies NMDAR-dependent synaptic transmission and synaptic plasticity, but it is unclear whether NL1 controls synaptic accumulation or function of the receptors. Here, we provide evidence that NL1 regulates the abundance of NMDARs at postsynaptic sites. This function relies on extracellular, NL1 isoform-specific sequences that facilitate biochemical interactions between NL1 and the NMDAR GluN1 subunit. Our work uncovers NL1 isoform-specific cis-interactions with ionotropic glutamate receptors as a key mechanism for controlling synaptic properties.
Micrometre- and submicrometre-size functionalized beads are frequently used to capture targets of interest from a biological sample for biological characterizations and disease diagnosis. The main challenge of the microbead-based assay is in the immobilization of probe molecules onto the microbead surfaces. In this paper, we report a versatile droplet microfluidics method to fabricate alginate microspheres while simultaneously immobilizing anti-Mycobacterium tuberculosis complex IgY and anti-Escherichia coli IgG antibodies primarily on the porous alginate carriers for specific binding and binding affinity tests. The binding affinity of antibodies is directly measured by fluorescence intensity of stained target bacteria on the microspheres. We demonstrate that the functionalized alginate microspheres yield specificity comparable with an enzyme-linked immunosorbent assay. The high surface area-to-volume ratio of the functionalized porous alginate microspheres improves the detection limit. By using the droplet microfluidics, we can easily modify the size and shape of alginate microspheres, and increase the concentration of functionalized alginate microspheres to further enhance binding kinetics and enable multiplexing.
Title: Mapping gene clusters within arrayed metagenomic libraries to expand the structural diversity of biomedically relevant natural products Owen JG, Reddy BV, Ternei MA, Charlop-Powers Z, Calle PY, Kim JH, Brady SF Ref: Proc Natl Acad Sci U S A, 110:11797, 2013 : PubMed
Complex microbial ecosystems contain large reservoirs of unexplored biosynthetic diversity. Here we provide an experimental framework and data analysis tool to facilitate the targeted discovery of natural-product biosynthetic gene clusters from the environment. Multiplex sequencing of barcoded PCR amplicons is followed by sequence similarity directed data parsing to identify sequences bearing close resemblance to biosynthetically or biomedically interesting gene clusters. Amplicons are then mapped onto arrayed metagenomic libraries to guide the recovery of targeted gene clusters. When applied to adenylation- and ketosynthase-domain amplicons derived from saturating soil DNA libraries, our analysis pipeline led to the recovery of biosynthetic clusters predicted to encode for previously uncharacterized glycopeptide- and lipopeptide-like antibiotics; thiocoraline-, azinomycin-, and bleomycin-like antitumor agents; and a rapamycin-like immunosuppressant. The utility of the approach is demonstrated by using recovered eDNA sequences to generate glycopeptide derivatives. The experiments described here constitute a systematic interrogation of a soil metagenome for gene clusters capable of encoding naturally occurring derivatives of biomedically relevant natural products. Our results show that previously undetected biosynthetic gene clusters with potential biomedical relevance are very common in the environment. This general process should permit the routine screening of environmental samples for gene clusters capable of encoding the systematic expansion of the structural diversity seen in biomedically relevant families of natural products.
Title: Effects of luteolin on spatial memory, cell proliferation, and neuroblast differentiation in the hippocampal dentate gyrus in a scopolamine-induced amnesia model Yoo DY, Choi JH, Kim W, Nam SM, Jung HY, Kim JH, Won MH, Yoon YS, Hwang IK Ref: Neurol Res, 35:813, 2013 : PubMed
OBJECTIVES: Luteolin, a common flavonoid from many plants, has various pharmacological activities, including a memory-improving effect. In this study, we investigated the effects of luteolin on spatial memory, cell proliferation, and neuroblast differentiation in the hippocampal dentate gyrus in a rat model of scopolamine (SCO)-induced amnesia. METHODS: Scopolamine was subcutaneously administered for 28 days via an Alzet minipump (44 mg/ml delivered at 2.5 mul/h) along with a daily intraperitoneal administration of vehicle (saline) 10 mg/kg luteolin or 5 mg/kg galantamine (GAL) (a control drug for acetylcholinesterase (AChE) inhibitor) for 28 days. RESULTS: The administration of SCO significantly decreased the spatial alteration percentage in the Y-maze test compared to that in the vehicle (saline)-treated group. The administration of luteolin or GAL significantly improved the spatial alteration percentage compared to that in the SCO-treated group. Similarly, the administration of SCO significantly decreased the cell proliferation (Ki67-positive cells) and neuroblast differentiation (doubleocortin-positive cells) in the dentate gyrus. The administration of luteolin or GAL significantly mitigated the SCO-induced reduction of Ki67- and doublecortin-immunoreactive cells in the dentate gyrus. In addition, the administration of luteolin significantly decreased the lipid peroxidation (malondialdehyde (MDA) levels) and increased the brain-derived neurotrophic factor (BDNF) and AChE levels in the hippocampal homogenates compared to the SCO-treated group. CONCLUSION: These results suggest that the luteolin treatment improves the SCO-induced reduction of cell proliferation and neuroblast differentiation in the dentate gyrus. The mechanism underlying the amelioration of SCO-induced amnesia by luteolin may be associated with the increase in BDNF, acetylcholine, and the decrease in lipid peroxidation.
The action of beta-secretase (BACE1) is strongly correlated with the onset of Alzheimer's disease (AD). Aminochalcone derivatives were examined for their ability to inhibit BACE1. Parent aminochalcones showed two digit micromolar IC(50)s against BACE1. Potency was enhanced 10-fold or more by introducing benzenesulfonyl derivatives to the amino group: 1 (IC(50) = 48.2 muM) versus 4a (IC(50) = 1.44 muM) and 2 (IC(50) = 17.7 muM) versus 5a (IC(50) = 0.21 muM). The activity was significantly influenced by position and number of hydroxyl groups on the chalcone B-ring: 3,4-dihydroxy 5a (IC(50) = 0.21 muM) > 4-hydroxy 4a (IC(50) = 1.44 muM) > 2,4-dihydroxy 6 (IC(50) = 3.60 muM) > 2,5-dihydroxy 7 (IC(50) = 16.87 muM) > des hydroxy 4b (IC(50) = 168.7 muM). Lineweaver-Burk and Dixon plots and their secondary replots indicate that compound 5a was a mixed inhibitor with reversible and time-dependent behavior. Potent BACE1 inhibitors 4a,c,f, 5a-c showed moderate inhibition against two other enzymes implicated in AD pathogenesis, acetylcholinesterase (AChE) and butyrylcholinesterase (BChE), with IC(50)s ranging between 56.1 ~ 95.8 muM and 19.5 ~ 79.0 muM, respectively.
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.
Title: Novel metagenome-derived, cold-adapted alkaline phospholipase with superior lipase activity as an intermediate between phospholipase and lipase Lee MH, Oh KH, Kang CH, Kim JH, Oh TK, Ryu CM, Yoon JH Ref: Applied Environmental Microbiology, 78:4959, 2012 : PubMed
A novel lipolytic enzyme was isolated from a metagenomic library obtained from tidal flat sediments on the Korean west coast. Its putative functional domain, designated MPlaG, showed the highest similarity to phospholipase A from Grimontia hollisae CIP 101886, though it was screened from an emulsified tricaprylin plate. Phylogenetic analysis showed that MPlaG is far from family I.6 lipases, including Staphylococcus hyicus lipase, a unique lipase which can hydrolyze phospholipids, and is more evolutionarily related to the bacterial phospholipase A(1) family. The specific activities of MPlaG against olive oil and phosphatidylcholine were determined to be 2,957 +/- 144 and 1,735 +/- 147 U mg(-1), respectively, which means that MPlaG is a lipid-preferred phospholipase. Among different synthetic esters, triglycerides, and phosphatidylcholine, purified MPlaG exhibited the highest activity toward p-nitrophenyl palmitate (C(16)), tributyrin (C(4)), and 1,2-dihexanoyl-phosphatidylcholine (C(8)). Finally, MPlaG was identified as a phospholipase A(1) with lipase activity by cleavage of the sn-1 position of OPPC, interfacial activity, and triolein hydrolysis. These findings suggest that MPlaG is the first experimentally characterized phospholipase A(1) with lipase activity obtained from a metagenomic library. Our study provides an opportunity to improve our insight into the evolution of lipases and phospholipases.
The draft genome sequence of Lactobacillus salivarius GJ-24 isolated from the feces of healthy adults was determined. Its properties, including milk fermentation activity and bacteriocin production, suggest its potential uses as a probiotic lactic acid bacterium and start culture for dairy products.
Neurexin and neuroligin, which undergo heterophilic interactions with each other at the synapse, are mutated in some patients with autism spectrum disorder, a set of disorders characterized by deficits in social and emotional learning. We have explored the role of neurexin and neuroligin at sensory-to-motor neuron synapses of the gill-withdrawal reflex in Aplysia, which undergoes sensitization, a simple form of learned fear. We find that depleting neurexin in the presynaptic sensory neuron or neuroligin in the postsynaptic motor neuron abolishes both long-term facilitation and the associated presynaptic growth induced by repeated pulses of serotonin. Moreover, introduction into the motor neuron of the R451C mutation of neuroligin-3 linked to autism spectrum disorder blocks both intermediate-term and long-term facilitation. Our results suggest that activity-dependent regulation of the neurexin-neuroligin interaction may govern transsynaptic signaling required for the storage of long-term memory, including emotional memory that may be impaired in autism spectrum disorder.
Title: Development of a low-density DNA microarray for diagnosis of target-site mutations of pyrethroid and organophosphate resistance mutations in the whitefly Bemisia tabaci Chung IH, Kang S, Kim YR, Kim JH, Jung JW, Lee S, Lee SH, Hwang SY Ref: Pest Manag Sci, 67:1541, 2011 : PubMed
BACKGROUND: Rapid and accurate detection of mutations related to insecticide resistance is essential for development of resistance management strategies to support sustainable agriculture. The M918V, L925I and T929V mutations of the voltage-gated sodium channel gene (vgsc) and the F392W mutation of the acetylcholinesterase I gene (ace1) are reportedly associated with resistance to pyrethroids and organophosphates, respectively, in Bemisia tabaci. In order to detect known base substitutions in the ace1 and vgsc genes, a low-density microarray with an allele-specific probe was developed. RESULTS: Specific regions of the ace1 and vgsc gene mutations were amplified by multiplex asymmetrical PCR using Cy3-labelled primers, and then the PCR products were hybridised on the microarray. After analysing the probe signal data, the microarray containing 12 allele-specific probes produced a unique pattern of probe signals for field DNA samples of B. tabaci. To determine the optimal cut-off value of each probe, receiver operating characteristic (ROC) curve analysis was conducted using SPSS. Among 60 individual samples, microarray data for 57 samples were consistent with direct sequencing data. CONCLUSION: Although many molecular detection methods have been employed to monitor insecticide resistance, the present microarray provides rapid and accurate identification of target mutations in B. tabaci for resistance management.
Title: The effect of pyridostigmine on bispectral index during recovery from sevoflurane anesthesia Jeong SJ, Han JI, Baik HJ, Lee H, Lee GY, Kim JH Ref: Korean J Anesthesiol, 61:460, 2011 : PubMed
BACKGROUND: There have been some conflicting reports showing that muscle relaxants and anticholinesterases affect the level of the bispectral index (BIS). The purpose of this study was to investigate whether pyridostigmine affects the level of the BIS during recovery from sevoflurane anesthesia. METHODS: Fifty-two adult patients scheduled for laparoscopic cholecystectomy and laparoscopic appendectomy. Anesthesia was induced with thiopental 4 mg/kg and rocuronium 0.6 mg/kg. The lung was mechanically ventilated with 1-3 vol% sevoflurane, 50% oxygen and 50% nitrous oxide. After a specimen was removed, the sevoflurane concentration was maintained at 1.5 vol%. When skin closure began, sevoflurane was stopped; however, 50% oxygen and 50% nitrous oxide were maintained. The patients then received either (1) a group that received an injection of glycopyrrolate 0.04 mg/kg and pyridostigmine 0.2 mg/kg (reverse (R) group, n = 26) or (2) a group that received normal saline (control (C) group, n = 26). Group assignment was random. Pyridostigmine, a reversible cholinesterase inhibitor, is a parasympathomimetic. End-tidal sevoflurane concentration, train of four (TOF) ratio, bispectral index (BIS), blood pressure and heart rate were measured from the end of the operation to 15 min after inject of pyridostigmine or placebo. RESULTS: There were no significant between group differences in the time dependent decrease in end-tidal sevoflurane concentration (P = 0.0642). There were significant differences between the two groups for the time course for increases in the TOF value (P < 0.0001). There were significant differences between the two groups for the time course for increases in the BIS value (P = 0.0107). There were no significant differences in the mean BIS value up to 10 minutes after administering drug, but 15 minutes after administrating the reverse drug or the control drug, the BIS value showed significantly different BIS values: 68.2 +/- 6.2 (Group R) and 63.2 +/- 6.2 (Group C) (P = 0.0058). CONCLUSIONS: The finding that pyridostigmine increases TOF and BIS suggests that pyridostigmine may enhance recovery during recovery from sevoflurane anesthesia.
Title: Production of medium-chain-length polyhydroxyalkanoates by activated sludge enriched under periodic feeding with nonanoic acid Lee SH, Kim JH, Mishra D, Ni YY, Rhee YH Ref: Bioresour Technol, 102:6159, 2011 : PubMed
The potential use of activated sludge for the production of medium-chain-length polyhydroxyalkanoates (MCL-PHAs) was investigated. The enrichment of bacterial populations capable of producing MCL-PHAs was achieved by periodic feeding with nonanoic acid in a sequencing batch reactor (SBR). Denaturing gradient gel electrophoresis analysis revealed Pseudomonas aeruginosa strains to be predominant in the bacterial community during the SBR process. The composition of PHA synthesized by the enriched biomass from nonanoic acid consisted of a large concentration (>89 mol%) of MCL monomer units and a small amount of short-chain-length monomer units. Under fed-batch fermentation with continuous feeding of nonanoic acid at a flow rate of 0.225 g/L/h and a C/N ratio of 40, a maximum PHA content of 48.6% dry cell weight and a conversion yield (Y(p/s)) of 0.94 g/g were achieved. These results indicate that MCL-PHA production by activated sludge is a promising alternative to typical pure culture approaches.
There are ongoing events where aircraft engine lubricant containing tricresyl phosphates (TCPs) contaminates aircraft cabins. Some individuals have experienced tremors or other neurological symptoms that may last for many months following exposures. Mass spectrometric (MS) protocols are being developed to determine the percentage of "biomarker proteins" that are modified by such exposures, specifically on active site serines. Both plasma butyrylcholinesterase (BChE) and red cell acylpeptide hydrolase (APH) are readily inhibited by 2-(ortho-cresyl)-4H-1,3,2-benzodioxaphosphoran-2-one (CBDP) or phenyl saligenin cyclic phosphate (PSP) and have the potential to provide information about the level of exposure of an individual. We have developed immunomagnetic bead-based single-step purification protocols for both BChE and APH and have characterized the active site serine adducts of BChE by MS.
Despite considerable evidence for a critical role of neuroligin-1 in the specification of excitatory synapses, the cellular mechanisms and physiological roles of neuroligin-1 in mature neural circuits are poorly understood. In mutant mice deficient in neuroligin-1, or adult rats in which neuroligin-1 was depleted, we have found that neuroligin-1 stabilizes the NMDA receptors residing in the postsynaptic membrane of amygdala principal neurons, which allows for a normal range of NMDA receptor-mediated synaptic transmission. We observed marked decreases in NMDA receptor-mediated synaptic currents at afferent inputs to the amygdala of neuroligin-1 knockout mice. However, the knockout mice exhibited a significant impairment in spike-timing-dependent long-term potentiation (STD-LTP) at the thalamic but not the cortical inputs to the amygdala. Subsequent electrophysiological analyses indicated that STD-LTP in the cortical pathway is largely independent of activation of postsynaptic NMDA receptors. These findings suggest that neuroligin-1 can modulate, in a pathway-specific manner, synaptic plasticity in the amygdala circuits of adult animals, likely by regulating the abundance of postsynaptic NMDA receptors.
Over 1 billion pounds of organophosphorus (OP) chemicals are manufactured worldwide each year, including 70 million pounds of pesticides sprayed in the US. Current methods to monitor environmental and occupational exposures to OPs such as chlorpyrifos (CPS) have limitations, including low specificity and sensitivity, and short time windows for detection. Biomarkers for the OP tricresyl phosphate (TCP), which can contaminate bleed air from jet engines and cause an occupational exposure of commercial airline pilots, crewmembers and passengers, have not been identified. The aim of our work has been to identify, purify, and characterize new biomarkers of OP exposure. Butyrylcholinesterase (BChE) inhibition has been a standard for monitoring OP exposure. By identifying and characterizing molecular biomarkers with longer half-lives, we should be able to clinically detect TCP and OP insecticide exposure after longer durations of time than are currently possible. Acylpeptide hydrolase (APH) is a red blood cell (RBC) cytosolic serine proteinase that removes N-acetylated amino acids from peptides and cleaves oxidized proteins. Due to its properties, it is an excellent candidate for a biomarker of exposure. We have been able to purify APH and detect inhibition by both CPS and metabolites of TCP. The 120-day lifetime of the RBC offers a much longer window for detecting exposure. The OP-modified serine conjugate in the active site tryptic peptide has been characterized by mass spectrometry. This research uses functional proteomics and enzyme activities to identify and characterize useful biomarkers of neurotoxic environmental and occupational OP exposures.
Title: Cloning large natural product gene clusters from the environment: piecing environmental DNA gene clusters back together with TAR Kim JH, Feng Z, Bauer JD, Kallifidas D, Calle PY, Brady SF Ref: Biopolymers, 93:833, 2010 : PubMed
A single gram of soil can contain thousands of unique bacterial species, of which only a small fraction is regularly cultured in the laboratory. Although the fermentation of cultured microorganisms has provided access to numerous bioactive secondary metabolites, with these same methods it is not possible to characterize the natural products encoded by the uncultured majority. The heterologous expression of biosynthetic gene clusters cloned from DNA extracted directly from environmental samples (eDNA) has the potential to provide access to the chemical diversity encoded in the genomes of uncultured bacteria. One of the challenges facing this approach has been that many natural product biosynthetic gene clusters are too large to be readily captured on a single fragment of cloned eDNA. The reassembly of large eDNA-derived natural product gene clusters from collections of smaller overlapping clones represents one potential solution to this problem. Unfortunately, traditional methods for the assembly of large DNA sequences from multiple overlapping clones can be technically challenging. Here we present a general experimental framework that permits the recovery of large natural product biosynthetic gene clusters on overlapping soil-derived eDNA cosmid clones and the reassembly of these large gene clusters using transformation-associated recombination (TAR) in Saccharomyces cerevisiae. The development of practical methods for the rapid assembly of biosynthetic gene clusters from collections of overlapping eDNA clones is an important step toward being able to functionally study larger natural product gene clusters from uncultured bacteria.
Title: Imprinting and expression status of isoforms 1 and 2 of PEG1/MEST gene in uterine leiomyoma Moon YS, Park SK, Kim HT, Lee TS, Kim JH, Choi YS Ref: Gynecol Obstet Invest, 70:120, 2010 : PubMed
PEG1/MEST gene has been known to be an imprinting gene, which is associated with growth of mesodermal origin cells. Its expression was also reported to be increased in leiomyoma. Several reports showed that loss of imprinting is associated with carcinogenesis in some types of cancer. The purpose of this study was to investigate whether overexpression of PEG1/MEST gene in leiomyoma is associated with loss of imprinting of the gene (biallelic), or whether the overexpression occurs while maintaining the imprinting (monoallelic). We investigated the expression and the imprinting status of PEG1/MEST and its isoforms in samples from 25 patients with uterine leiomyomas as well as in matched normal myometrial tissue. The isoform 1 transcripts were found to be more increased in uterine leiomyomas, compared to myometrium. However, there was no difference in the mRNA levels of isoform 2 between normal myometrium and leiomyoma. All normal myometrial tissues and 19 of 20 leiomyomas showed monoallelic expression of PEG1/MEST. Thus, these data demonstrated that tumorigenesis of leiomyoma is associated with overexpression of isoform 1 of PEG1/MEST gene, but not with loss of imprinting of the gene.
Title: Cloning and expression analysis of the duplicated genes for carbon monoxide dehydrogenase of Mycobacterium sp. strain JC1 DSM 3803 Song T, Park SW, Park SJ, Kim JH, Yu JY, Oh JI, Kim YM Ref: Microbiology, 156:999, 2010 : PubMed
Carbon monoxide dehydrogenase (CO-DH) is an enzyme catalysing the oxidation of CO to carbon dioxide in Mycobacterium sp. strain JC1 DSM 3803. Cloning of the genes encoding CO-DH from the bacterium and sequencing of overlapping clones revealed the presence of duplicated sets of genes for three subunits of the enzyme, cutB1C1A1 and cutB2C2A2, in operons, and a cluster of genes encoding proteins that may be involved in CO metabolism, including a possible transcriptional regulator. Phylogenetic analysis based on the amino acid sequences of large subunits of CO-DH suggested that the CO-DHs of Mycobacterium sp. JC1 and other mycobacteria are distinct from those of other types of bacteria. The growth phenotype of mutant strains lacking cutA genes and of a corresponding complemented strain showed that both of the duplicated sets of CO-DH genes were functional in this bacterium. Transcriptional fusions of the cutB genes with lacZ revealed that the cutBCA operons were expressed regardless of the presence of CO and were further inducible by CO. Primer extension analysis indicated two promoters, one expressed in the absence of CO and the other induced in the presence of CO. This is believed to be the first report to show the presence of multiple copies of CO-DH genes with identical sequences and in close proximity in carboxydobacteria, and to present the genetic evidence for the function of the genes in mycobacteria.
The human intestine is densely populated by a microbial consortium whose metabolic activities are influenced by, among others, bifidobacteria. However, the genetic basis of adaptation of bifidobacteria to the human gut is poorly understood. Analysis of the 2,214,650-bp genome of Bifidobacterium bifidum PRL2010, a strain isolated from infant stool, revealed a nutrient-acquisition strategy that targets host-derived glycans, such as those present in mucin. Proteome and transcriptome profiling revealed a set of chromosomal loci responsible for mucin metabolism that appear to be under common transcriptional control and with predicted functions that allow degradation of various O-linked glycans in mucin. Conservation of the latter gene clusters in various B. bifidum strains supports the notion that host-derived glycan catabolism is an important colonization factor for B. bifidum with concomitant impact on intestinal microbiota ecology.
NDRG (N-Myc downstream-regulated gene)-2 is a member of the NDRG family. Although it has been suggested that NDRG2 is involved in cellular differentiation and tumor suppression, its intracellular signal and regulatory mechanism are not well known. Here, we show the differential expression of NDRG2 in human colon carcinoma cell lines and tissues by reverse transcription-polymerase chain reaction and immunohistochemical analyses with monoclonal antibody against NDRG2. NDRG2 was strongly expressed in normal colonic mucosa and colonic adenomatous tissues (25 of 25) but not in all invasive cancer tissues [44 of 99 (44%)]. Most distinctive results indicated that the high expression level of NDRG2 has a positive correlation with tumor differentiation and inverse correlation with tumor invasion depth and Dukes' stage of colon adenocarcinoma. To investigate the roles of NDRG2 in tumorigenesis, we used in vitro cell culture system. SW620 colon cancer cell line with a low level of intrinsic NDRG2 protein was transfected with NDRG2-expressing plasmid. TOPflash luciferase reporter assay showed that the transcriptional activity of T-cell factor (TCF)/lymphoid enhancer factor (LEF) was reduced by NDRG2 introduction, but not by the introduction of mutant NDRG2 generated by deletion or site-directed mutagenesis. Intracellular beta-catenin levels were slightly reduced in the NDRG2-transfected SW620 cells and this regulation of beta-catenin stability and TCF/LEF activity were mediated through the modulation of glycogen synthase kinase-3beta activity by NDRG2 function. Our results suggest that NDRG2 might play a pivotal role as a potent tumor suppressor by the attenuation of TCF/beta-catenin signaling for the maintenance of healthy colon tissues.
Title: Atromentin-induced apoptosis in human leukemia U937 cells Kim JH, Lee CH Ref: J Microbiol Biotechnol, 19:946, 2009 : PubMed
In the course of screening for apoptotic substances that induce apoptosis in human leukemia U937 cells, a fungal strain, F000487, which exhibits potent inducible activity, was selected. The active compound was purified from an ethyl acetate extract of the microorganism by Sep-pak C18 column chromatography and HPLC, and was identified as atromentin by spectroscopic methods. This compound induced caspase-3 processing in human leukemia U937 cells. The caspase-3 and poly (ADP-ribose) polymerase (PARP) were induced by atromentin in a dose-dependent manner. Furthermore, DNA fragmentation was also induced by this compound in a dose-dependent manner. These results show that atromentin potently induces apoptosis in U937 cells and that atromentin-induced apoptosis is related to the selective activation of caspases.
Title: Enzymatic characterization of class I DAD1-like acylhydrolase members targeted to chloroplast in Arabidopsis Seo YS, Kim EY, Kim JH, Kim WT Ref: FEBS Letters, 583:2301, 2009 : PubMed
In Arabidopsis, there are at least seven class I acylhydrolase members, which have a putative N-terminal chloroplast-targeting signal. Here, we show that all seven class I proteins are localized to the chloroplasts and hydrolyze phosphatidylcholine at the sn-1 position. However, based on their activities toward various lipids, Arabidopsis class I enzymes could be further divided into three sub-groups by substrate specificity, one with phospholipase-specific activity, another with phospholipase and galactolipase activities, and the other with broad lipolytic activity toward phosphatidylcholine, galactolipids, and triacylglycerol. These results suggest that the three sub-groups of class I acylhydrolases have specific roles in chloroplasts.
The M37 lipase from Photobacterium lipolyticum shows an extremely low activation energy and strong activity at low temperatures, with optimum activity seen at 298 K and more than 75% of the optimum activity retained down to 278 K. Though the M37 lipase is most closely related to the filamentous fungal lipase, Rhizomucor miehei lipase (RML) at the primary structure level, their activity characteristics are completely different. In an effort to identify structural components of cold adaptation in lipases, we determined the crystal structure of the M37 lipase at 2.2 A resolution and compared it to that of nonadapted RML. Structural analysis revealed that M37 lipase adopted a folding pattern similar to that observed for other lipase structures. However, comparison with RML revealed that the region beneath the lid of the M37 lipase included a significant and unique cavity that would be occupied by a lid helix upon substrate binding. In addition, the oxyanion hole was much wider in M37 lipase than RML. We propose that these distinct structural characteristics of M37 lipase may facilitate the lateral movement of the helical lid and subsequent substrate hydrolysis, which might explain its low activation energy and high activity at low temperatures.
Neuroligin-1 is a potent trigger for the de novo formation of synaptic connections, and it has recently been suggested that it is required for the maturation of functionally competent excitatory synapses. Despite evidence for the role of neuroligin-1 in specifying excitatory synapses, the underlying molecular mechanisms and physiological consequences that neuroligin-1 may have at mature synapses of normal adult animals remain unknown. By silencing endogenous neuroligin-1 acutely in the amygdala of live behaving animals, we have found that neuroligin-1 is required for the storage of associative fear memory. Subsequent cellular physiological studies showed that suppression of neuroligin-1 reduces NMDA receptor-mediated currents and prevents the expression of long-term potentiation without affecting basal synaptic connectivity at the thalamo-amygdala pathway. These results indicate that persistent expression of neuroligin-1 is required for the maintenance of NMDAR-mediated synaptic transmission, which enables normal development of synaptic plasticity and long-term memory in the amygdala of adult animals.
The time course of the requirement for local protein synthesis in the stabilization of learning-related synaptic growth and the persistence of long-term memory was examined using Aplysia bifurcated sensory neuron-motor neuron cultures. We find that, following repeated pulses of serotonin (5-HT), the local perfusion of emetine, an inhibitor of protein synthesis, or a TAT-AS oligonucleotide directed against ApCPEB blocks long-term facilitation (LTF) at either 24 or 48 hr and leads to a selective retraction of newly formed sensory neuron varicosities induced by 5-HT. By contrast, later inhibition of local protein synthesis, at 72 hr after 5-HT, has no effect on either synaptic growth or LTF. These results define a specific stabilization phase for the storage of long-term memory during which newly formed varicosities are labile and require sustained CPEB-dependent local protein synthesis to acquire the more stable properties of mature varicosities required for the persistence of LTF.
Following birth, the breast-fed infant gastrointestinal tract is rapidly colonized by a microbial consortium often dominated by bifidobacteria. Accordingly, the complete genome sequence of Bifidobacterium longum subsp. infantis ATCC15697 reflects a competitive nutrient-utilization strategy targeting milk-borne molecules which lack a nutritive value to the neonate. Several chromosomal loci reflect potential adaptation to the infant host including a 43 kbp cluster encoding catabolic genes, extracellular solute binding proteins and permeases predicted to be active on milk oligosaccharides. An examination of in vivo metabolism has detected the hallmarks of milk oligosaccharide utilization via the central fermentative pathway using metabolomic and proteomic approaches. Finally, conservation of gene clusters in multiple isolates corroborates the genomic mechanism underlying milk utilization for this infant-associated phylotype.
Scrub typhus is caused by the obligate intracellular rickettsia Orientia tsutsugamushi (previously called Rickettsia tsutsugamushi). The bacterium is maternally inherited in trombicuid mites and transmitted to humans by feeding larvae. We report here the 2,127,051-bp genome of the Boryong strain, which represents the most highly repeated bacterial genome sequenced to date. The repeat density of the scrub typhus pathogen is 200-fold higher than that of its close relative Rickettsia prowazekii, the agent of epidemic typhus. A total of 359 tra genes for components of conjugative type IV secretion systems were identified at 79 sites in the genome. Associated with these are >200 genes for signaling and host-cell interaction proteins, such as histidine kinases, ankyrin-repeat proteins, and tetratrico peptide-repeat proteins. Additionally, the O. tsutsugamushi genome contains >400 transposases, 60 phage integrases, and 70 reverse transcriptases. Deletions and rearrangements have yielded unique gene combinations as well as frequent pseudogenization in the tra clusters. A comparative analysis of the tra clusters within the genome and across strains indicates sequence homogenization by gene conversion, whereas complexity, diversity, and pseudogenization are acquired by duplications, deletions, and transposon integrations into the amplified segments. The results suggest intragenomic duplications or multiple integrations of a massively proliferating conjugative transfer system. Diversifying selection on host-cell interaction genes along with repeated population bottlenecks may drive rare genome variants to fixation, thereby short-circuiting selection for low complexity in bacterial genomes.
Title: Development of inhibitors against lipase and alpha-glucosidase from derivatives of monascus pigment Kim JH, Kim HJ, Park HW, Youn SH, Choi DY, Shin CS Ref: FEMS Microbiology Letters, 276:93, 2007 : PubMed
New derivatives of monascus pigment were produced during Monascus fermentation by the addition of unnatural amino acids, and the inhibitory activities of the derivatives against diet-related lipase and alpha-glucosidase were tested. Derivatives with penicillamine (H-Pen), cyclohexylalanine (H-Cha), butylglycine (L-t-Bg), and norleucine (H-Nle) showed relatively high inhibitory activities against lipase. The H-Pen derivative exhibited the highest inhibitory activity, with an IC(50) (50% inhibition) value of 24.0 microM. The four derivatives all showed noncompetitive inhibition patterns against lipase. The inhibition constant (K(i)) of the H-Pen derivative was estimated to be 20.7 microM. The H-Pen derivative also exhibited a relatively high inhibitory activity against alpha-glucosidase, with an IC(50) value of 50.9 microM. The inhibition pattern of the H-Pen derivative against alpha-glucosidase appeared to be of a mixed type. The inhibition constants K(i) and were estimated to be 25.9, and 98.9 microM, respectively.
Title: Platelet-activating factor-acetylhydrolase can monodeacylate and inactivate lipoteichoic acid Seo HS, Kim JH, Nahm MH Ref: Clin Vaccine Immunol, 13:452, 2006 : PubMed
Bacterial lipoteichoic acid (LTA) shares a structural motif with platelet-activating factor (PAF). Both molecules are strong inflammatory agents and have a glycerol backbone with two lipid chains at the sn-1 and sn-2 positions. PAF is normally inactivated by PAF-acetylhydrolase (PAF-AH), a phospholipase A2 (PLA2), which removes a short acyl group at the sn-2 position. To investigate whether PAF-AH can similarly degrade LTA, we studied the effects of porcine PLA2, bee venom PLA2, and recombinant human PAF-AH on pneumococcal LTA (PnLTA) and staphylococcal LTA (StLTA). After incubation with a porcine or bee venom PLA2, a large fraction of PnLTA lost 264 Da, which corresponds to the mass of the oleic acid group at the sn-2 position. After incubation with recombinant human PAF-AH, PnLTA lost 264 Da; the reduction did not occur when PAF-AH was exposed to Pefabloc SC, an irreversible inhibitor of the PAF-AH active site. Following PAF-AH treatment, PnLTA and StLTA were not able to stimulate mouse RAW 264.7 cells to produce tumor necrosis factor alpha but could stimulate CHO cells expressing human TLR2. This stimulation pattern has been observed with monoacyl PnLTA prepared by mild alkali hydrolysis (22). Taking these data together, we conclude that PAF-AH can remove one acyl chain at the sn-2 position of LTA and produce a monoacyl-LTA that is inactive against mouse cells.
AIM: To investigate the relationship between pancreatic amylase in bile duct and the clinico-pathological features in adult patients with choledochal cyst and anomalous pancreatico-biliary ductal union (APBDU). METHODS: From 39 patients who underwent surgery for choledochal cyst between March 1995 and March 2003, we selected 15 adult patients who had some symptoms and were radiologically diagnosed as APBDU, and their clinico-pathological features were subsequently evaluated retrospectively. However, we could not obtain biliary amylase in all the patients because of the surgeon's slip. Therefore, we measured the amylase level in gall bladder of 10 patients and in common bile duct of 11 patients. RESULTS: Levels of amylase in common bile duct and gall bladder ranged from 11,500 to 212,000 IU/L, and the younger the patients, the higher the biliary amylase level (r = -0.982, P<0.01). Pathologically, significant correlation was found between the size of choledochal cyst and the grade of inflammation (r = 0.798, P<0.01). And, significant correlation was found between the level of amylase in gall bladder and the grade of hyperplasia. On the other hand, there was no correlation to the age of symptomatic onset or inflammatory grade (r = 0.743, P<0.05). Level of lipase was elevated from 6,000 to 159,000 IU/L in bile duct and from 14,400 to 117,000 IU/L in the gall bladder; however, there was no significant correlation with age or clinico-pathological features. CONCLUSION: The results support the notion that amylase has a particular role in the onset of symptoms, and suggest that a large amount of biliary amylase induces early onset of symptom, thereby making early diagnosis possible.
The nucleotide sequence was determined for the genome of Xanthomonas oryzae pathovar oryzae (Xoo) KACC10331, a bacterium that causes bacterial blight in rice (Oryza sativa L.). The genome is comprised of a single, 4 941 439 bp, circular chromosome that is G + C rich (63.7%). The genome includes 4637 open reading frames (ORFs) of which 3340 (72.0%) could be assigned putative function. Orthologs for 80% of the predicted Xoo genes were found in the previously reported X.axonopodis pv. citri (Xac) and X.campestris pv. campestris (Xcc) genomes, but 245 genes apparently specific to Xoo were identified. Xoo genes likely to be associated with pathogenesis include eight with similarity to Xanthomonas avirulence (avr) genes, a set of hypersensitive reaction and pathogenicity (hrp) genes, genes for exopolysaccharide production, and genes encoding extracellular plant cell wall-degrading enzymes. The presence of these genes provides insights into the interactions of this pathogen with its gramineous host.
A presenilin 2 mutation is believed to be involved in the development of Alzheimer's disease. In addition, transgenic mice with a presenilin 2 mutation have been reported to have learning and memory impairments. In this study, exposing PC12 cells expressing mutant presenilin 2 to 50 microM AP25-35, 30 mM L-glutamate and 50 microM H2O2 caused a significant increase in acetylcholine esterase activity. An in vivo study revealed high levels of this enzyme activity in the mutant presenilin 2 transgenic brains compared with the wild type presenilin 2 transgenic and nontransgenic samples. These results suggest that a mutant presenilin 2-induced neurodegeneration in Alzheimer's disease might be involved in the increase in acetylcholinesterase activity. These findings might help in the development of an appropriate therapeutic intervention targeting mutant presenilin 2-induced Alzheimer's disease.
We report the complete genome sequence of Zymomonas mobilis ZM4 (ATCC31821), an ethanologenic microorganism of interest for the production of fuel ethanol. The genome consists of 2,056,416 base pairs forming a circular chromosome with 1,998 open reading frames (ORFs) and three ribosomal RNA transcription units. The genome lacks recognizable genes for 6-phosphofructokinase, an essential enzyme in the Embden-Meyerhof-Parnas pathway, and for two enzymes in the tricarboxylic acid cycle, the 2-oxoglutarate dehydrogenase complex and malate dehydrogenase, so glucose can be metabolized only by the Entner-Doudoroff pathway. Whole genome microarrays were used for genomic comparisons with the Z. mobilis type strain ZM1 (ATCC10988) revealing that 54 ORFs predicted to encode for transport and secretory proteins, transcriptional regulators and oxidoreductase in the ZM4 strain were absent from ZM1. Most of these ORFs were also found to be actively transcribed in association with ethanol production by ZM4.
Title: Serotonin-induced regulation of the actin network for learning-related synaptic growth requires Cdc42, N-WASP, and PAK in Aplysia sensory neurons Udo H, Jin I, Kim JH, Li HL, Youn T, Hawkins RD, Kandel ER, Bailey CH Ref: Neuron, 45:887, 2005 : PubMed
Application of Clostridium difficile toxin B, an inhibitor of the Rho family of GTPases, at the Aplysia sensory to motor neuron synapse blocks long-term facilitation and the associated growth of new sensory neuron varicosities induced by repeated pulses of serotonin (5-HT). We have isolated cDNAs encoding Aplysia Rho, Rac, and Cdc42 and found that Rho and Rac had no effect but that overexpression in sensory neurons of a dominant-negative mutant of ApCdc42 or the CRIB domains of its downstream effectors PAK and N-WASP selectively reduces the long-term changes in synaptic strength and structure. FRET analysis indicates that 5-HT activates ApCdc42 in a subset of varicosities contacting the postsynaptic motor neuron and that this activation is dependent on the PI3K and PLC signaling pathways. The 5-HT-induced activation of ApCdc42 initiates reorganization of the presynaptic actin network leading to the outgrowth of filopodia, some of which are morphological precursors for the learning-related formation of new sensory neuron varicosities.
Title: Effects of methanol extract of Uncariae Ramulus et Uncus on ibotenic acid-induced amnesia in the rat Kim JH, Chung JY, Lee YJ, Park S, Hahm DH, Lee HJ, Shim I Ref: J Pharmacol Sci, 96:314, 2004 : PubMed
In the present study, we investigated the effects of Uncariae Ramulus et Uncus (UR) on learning and memory in the Morris water maze task and the central cholinergic system of rats with excitotoxic medial septum (MS) lesion. In the water maze test, the animals were trained to find a platform in a fixed position during 6 days and then received a 60-s probe trial in which the platform was removed from the pool on the 7th day. Ibotenic lesion of the MS showed impaired performance of the maze test and severe cell losses in the septohippocampal cholinergic system (SHC), as indicated by decreased choline acetyltransferase-immunoreactivity and acetylcholinesterase-reactivity in the hippocampus. Daily administrations of UR (100 mg/kg, i.p.) for 21 consecutive days produced significant reversals of ibotenic acid-induced deficit in learning and memory. These treatments also reduced the loss of cholinergic immunoreactivity in the hippocampus induced by ibotenic acid. These results demonstrated that impairments of spatial learning and memory may be attributable to degeneration of SHC neurons and that UR ameliorated learning and memory deficits partly through neuroprotective effects on the central acetylcholine system. Our studies suggest that UR may be useful in the treatment of Alzheimer's disease.
Title: Pesticide poisoning events in wild birds in Korea from 1998 to 2002 Kwon YK, Wee SH, Kim JH Ref: J Wildl Dis, 40:737, 2004 : PubMed
We describe cases of pesticide poisoning of wild birds diagnosed at the National Veterinary Research and Quarantine Service (Kyunggi, Korea) from 1998 to 2002. Forty-one mortality events (759 birds) of 87 incidents (2,464 birds) investigated were associated with pesticide poisoning, and six organophosphates or carbamates were identified as being responsible for the poisoning. Phosphamidon was most frequently identified as the cause of poisoning, accounting for 23 mortality events. Other pesticides identified as poisons for birds were organophosphates monocrotophus, fenthion, parathion, EPN, and diazinon, and the carbamate carbofuran. Pesticide poisoning is a problem in wild birds in Korea.
Title: Construction and characterization of a recombinant esterase with high activity and enantioselectivity to (S)-ketoprofen ethyl ester Choi GS, Kim JY, Kim JH, Ryu YW, Kim GJ Ref: Protein Expr Purif, 29:85, 2003 : PubMed
The ester-hydrolyzing enzyme families, including lipase and esterase, mediated a broad range of reactions and, thus, were able to act on a variety of ester compounds that are found naturally or exploited industrially. With the increasing demand for pharmacological use, attempts to produce an enantiomer (S)-ketoprofen from the corresponding ethyl ester have recently been proliferating, but information about the structure and function of related enzymes has not been reported to date in detail. Here, we reported the construction, expression, and one-step purification of a potential esterase in Escherichia coli with a hexahistidine tag at its N-terminus. The expression level of the enzyme was more than 20% of the total protein in E. coli, resulting in approximately 1.2mg of the purified proteins by an affinity resin, Ni-NTA, from a 0.2L of bacterial culture in a single step. As typical properties, its innate traits that revealed favorable reactions at alkaline pH and high activity to the triglycerides composed of short chain fatty acids (99% ee(p)). The small-scale conversion using the recombinant enzyme strongly suggested the enzyme to be useful for enzyme-mediated chiral resolution of (S)-ketoprofen.
At Aplysia sensory-to-motor neuron synapses, the inhibitory neuropeptide Phe-Met-Arg-Phe-NH2 (FMRFa) produces depression, and serotonin (5-HT) produces facilitation. Short-term depression has been found to result from the activation of a phospholipase A2. The released arachidonate is metabolized by 12-lipoxygenase to active second messengers. We find that FMRFa leads to the phosphorylation and activation of p38 mitogen-activated protein (MAP) kinase. Short-term depression and the release of arachidonate are blocked by the specific p38 kinase inhibitor SB 203580. Both the inhibitor and an affinity-purified antibody raised against recombinant Aplysia p38 kinase injected into sensory neurons prevented long-term depression, which depends on the phosphorylation of translation factors cAMP response element-binding protein 2 (CREB2) and activating transcription factor 2. Facilitation produced by 5-HT, on the other hand, inactivates p38 kinase. Chromatin immunoprecipitation assays indicate that p38 kinase activates CREB2. p38 kinase also is pivotal in the bidirectional regulation of synaptic plasticity: when the kinase is inhibited, brief treatment with 5-HT that normally produces only short-term facilitation now results in long-term facilitation. Conversely, in sensory neurons injected with the activated kinase, long-term facilitation is blocked, and brief exposure to FMRFa, which normally results in short-term depression, results in long-term depression. We conclude that p38 kinase, which itself is bidirectionally regulated by FMRFa and 5-HT, acts as a modulator of synaptic plasticity by positively regulating depression and serving as an inhibitory constraint for facilitation.
Title: Presynaptic activation of silent synapses and growth of new synapses contribute to intermediate and long-term facilitation in Aplysia Kim JH, Udo H, Li HL, Youn TY, Chen M, Kandel ER, Bailey CH Ref: Neuron, 40:151, 2003 : PubMed
The time course and functional significance of the structural changes associated with long-term facilitation of Aplysia sensory to motor neuron synaptic connections in culture were examined by time-lapse confocal imaging of individual sensory neuron varicosities labeled with three different fluorescent markers: the whole-cell marker Alexa-594 and two presynaptic marker proteins-synaptophysin-eGFP to monitor changes in synaptic vesicle distribution and synapto-PHluorin to monitor active transmitter release sites. Repeated pulses of serotonin induce two temporally, morphologically, and molecularly distinct presynaptic changes: (1) a rapid activation of silent presynaptic terminals by filling of preexisting empty varicosities with synaptic vesicles, which parallels intermediate-term facilitation, is completed within 3-6 hr and requires translation but not transcription and (2) a slower generation of new functional varicosities which occurs between 12-18 hr and requires transcription and translation. Enrichment of empty varicosities with synaptophysin accounts for 32% of the newly activated synapses at 24 hr, whereas newly formed varicosities account for 68%.
The esterase-encoding gene, estA, was cloned from Acinetobacter lwoffii I6C-1 genomic DNA into Escherichia coli BL21(DE3) with plasmid vector pET-22b (pEM1). pEM1 has a 4.4-kb EcoRI insert that contained the complete estA gene. A 2.4-kb AvaI- SphI DNA fragment was subcloned (pEM3) and sequenced. estA gene encodes a protein of 366 amino acids (40,687 Da) with a pI of 9.17. The EstA signal peptide was 31 amino acids long, and the mature esterase sequence is 335 amino acids long (37.5 kDa). The conserved catalytic serine residue of EstA is in position 210. The EstA sequence was similar to that of the carboxylesterase from Acinetobacter calcoaceticus (75% identity, 85% similarity), Archaeoglobus fulgidus (37% identity, 59% similarity), and Mycobacterium tuberculosis (35% identity, 51% similarity). These enzymes contained the conserved motif G-X(1)-S-X(2)-G carrying the active-site serine of hydrolytic enzyme. The EstA activity in A. lwoffii I6C-1 remains constant throughout the stationary phase, and the activity in E. coil BL21 (DE3) with pEM1 was similar to A. lwoffii I6C-1.
Synapse-specific facilitation requires rapamycin-dependent local protein synthesis at the activated synapse. In Aplysia, rapamycin-dependent local protein synthesis serves two functions: (1) it provides a component of the mark at the activated synapse and thereby confers synapse specificity and (2) it stabilizes the synaptic growth associated with long-term facilitation. Here we report that a neuron-specific isoform of cytoplasmic polyadenylation element binding protein (CPEB) regulates this synaptic protein synthesis in an activity-dependent manner. Aplysia CPEB protein is upregulated locally at activated synapses, and it is needed not for the initiation but for the stable maintenance of long-term facilitation. We suggest that Aplysia CPEB is one of the stabilizing components of the synaptic mark.
Two hepatoprotective phenolic compounds, kaempferol (2) and salidroside (4), were isolated from the roots of Rhodiola sachalinensis together with two inactive compounds cinnamyl alcohol (1) and daucosterol (3) based on the hepatoprotective activity against tacrine-induced cytotoxicity in human liver-derived Hep G2 cells. The EC(50) values of compounds 2 and 4 were 33.5 and 51.3 micro m, respectively. Silybin as a positive control showed an EC(50) value of 68.4 micro m.
Excitatory and inhibitory inputs converge on single neurons and are integrated into a coherent output. Although much is known about short-term integration, little is known about how neurons sum opposing signals for long-term synaptic plasticity and memory storage. In Aplysia, we find that when a sensory neuron simultaneously receives inputs from the facilitatory transmitter 5-HT at one set of synapses and the inhibitory transmitter FMRFamide at another, long-term facilitation is blocked and synapse-specific long-term depression dominates. Chromatin immunoprecipitation assays show that 5-HT induces the downstream gene C/EBP by activating CREB1, which recruits CBP for histone acetylation, whereas FMRFa leads to CREB1 displacement by CREB2 and recruitment of HDAC5 to deacetylate histones. When the two transmitters are applied together, facilitation is blocked because CREB2 and HDAC5 displace CREB1-CBP, thereby deacetylating histones.
Title: Transdermal delivery of physostigmine: effects of enhancers and pressure-sensitive adhesives Kim JH, Lee CH, Choi HK Ref: Drug Dev Ind Pharm, 28:833, 2002 : PubMed
The purpose of this study was to investigate the effects of various pressure-sensitive adhesives (PSA) on the percutaneous absorption of physostigmine across hairless mouse skin. In addition, the influences of various vehicles and polyvinylpyrrolidone (PVP) on the percutaneous absorption of physostigmine from PSA matrix across hairless mouse skin were evaluated using a flow-through diffusion cell system at 37 degrees C. Physostigmine showed the highest permeability from silicone adhesive matrix, followed by polyisobutylene (PIB), styrene-isoprene-styrene (SIS), acrylic, and styrene-butadiene-styrene (SBS) matrix. Among acrylic adhesives, the permeability of physostigmine was the highest from grafted acrylic adhesive. Polyvinyl pyrrolidone inhibited the crystallization of physostigmine in the PIB adhesive matrix and enhanced the permeability of physostigmine from the PIB adhesive matrix. When esters of sorbitol and fatty acid, polyethylene glycol (PEG) alkyl esters, and caprylic/capric triglycerides were tested, the more lipophilic was a surfactant, the higher the permeation rate within the same group of surfactants. The enhancement effect of PEG derivatives was lower than that of non-PEG derivatives. Among non-linear fatty acid derivatives, linoleate derivatives showed higher permeability of physostigmine than oleate derivatives. This study showed that several non-ionic surfactants, including PEG-20 evening primrose glyceride, enhanced the permeation of physostigmine across hairless mouse skin better than oleic acid.
Title: Comparative Toxicities of Pyriproxyfen and Thiamethoxam against the Sweetpotato Whitefly, Bemisia tabaci (Homoptera: Aleyrodidae) Lee YS, Lee SY, Park EC, Kim JH, Kim GH Ref: Journal of Asia-Pacific Entomology, 5:117, 2002 : PubMed
This study was conducted to determine the comparative toxicities of pyriproxyfen and thiamethoxam on the sweetpotato whitefly, Bemisia tabaci biotype B. Ovicidal effect of pyriproxyfen (100 ppm) showed 94.5%, which was = 8 higher times than that of thiamethoxam (50 ppm) at the recommended concentration. The nymphal mortalities of both insecticides treated on the 3rd instar stage were over 85%. Thiamethoxam was very effective against adults, but the activity of pyriproxyfen was relatively low. Longevity and fecundity of newly emerged adults from the treated pupae of two chemicals were adveresely affected. Unlike pyriproxyfen, thiamethoxam highly showed root up-take systemic effect on nymphs and adults of B. tabaci. Pyriproxyfen and thiamethoxam showed residual effects against B. tabaci and particularly, thiamethoxam maintained high control effect with over 90% up to 7 days after treatment. In the control efficacy test on B. tabaci, the control values of pyriproxyfen and thiamethoxam at 9 days after treatment were 92.0 and 99.3%, respectively.
The responses to ionizing radiation (IR) in tumors are dependent on cellular context. We investigated radiation-related expression patterns in Jurkat T cells with nonsense mutation in p53 using cDNA microarray. Expression of 2400 genes in gamma-irradiated cells was distinct from other stimulations like anti-CD3, phetohemagglutinin (PHA) and concanavalin A (ConA) in unsupervised clustering analysis. Among them, 384 genes were selected for their IR-specific changes to make 'RadChip'. In spite of p53 status, every type of cells showed similar patterns in expression of these genes upon gamma-radiation. Moreover, radiation-induced responses were clearly separated from the responses to other genotoxic stress like UV radiation, cisplatin and doxorubicin. We focused on two IR-related genes, phospholipase Cgamma2 (PLCG2) and cytosolic epoxide hydrolase (EPHX2), which were increased at 12 h after gamma-radiation in RT-PCR. TPCK could suppress the induction of these two genes in either of Jurkat T cells and PBMCs, which might suggest the transcriptional regulation of PLCG2 and EPHX2 by NF-kappaB upon gamma-radiation. From these results, we could identify the IR-specific genes from expression profiling, which can be used as radiation biomarkers to screen radiation exposure as well as probing the mechanism of cellular responses to ionizing radiation.
In the course of screening natural products for anti-acetylcholinesterase (AChE) activity, we found that a total methanolic extract of the underground parts of Caragana chamlague (Leguminosae) had significant inhibition towards AChE. Bioactivity-guided fractionation of the total methanolic extract resulted in the isolation and identification of two active stilbene oligomers, (+)-alpha-viniferin (1) and kobophenol A (2). Both 1 and 2 inhibited AChE activity in a dose-dependent manner, and the IC50 values of 1 and 2 were 2.0 and 115.8 microM, respectively. The AChE inhibitory activity of 1 was specific, reversible and noncompetitive.
To investigate the hepatic metabolism of the new insecticide flupyrazofos [O,O-diethyl O-(1-phenyl-3-trifluoromethylpyrazol-5-yl) phosphorothioate], isolated rat liver was perfused with flupyrazofos under single-pass conditions. In outflow perfusate and bile, 1-phenyl-3-trifluoromethyl-5-hydroxyprazole (PTMHP), PTMHP-sulfate and PTMHP-glucuronide conjugates were identified as the metabolites of flupyrazofos. However, O,O-diethyl O-(1-phenyl-3-trifluoromethylpyrazol-5-yl) phosphate (flupyrazofos oxon) was not detected. A HPLC method with UV detection was used to investigate the hepatic disposition of flupyrazofos and its metabolite PTMHP. The concentrations of flupyrazofos, PTMHP and PTMHP conjugates in outflow perfusate reached steady-state levels within 20 min after commencing perfusion of 7.3 microM flupyrazofos. At steady state, the mean extraction ratio of flupyrazofos was 0.93 (+/- 0.01) and clearance was 26.1 (+/- 0.2) ml min-1 which nearly approached perfusate flow rate (28 ml min-1). PTMHP accounted for 55.7 (+/- 5.8)% of eliminated flupyrazofos and was recovered as unchanged PTMHP, PTMHP-sulfate and PTMHP-glucuronide in the bile as well as the outflow perfusate.
1. The effects of flupyrazofos on liver microsomal cytochrome P450 were investigated in the male Fischer 344 rat. When rats were treated intraperitoneally with flupyrazofos for 3 consecutive days, the activities of ethoxyresorufin O-deethylase and testosterone 2 beta-hydroxylase were significantly reduced, whereas the activities of pentoxyresorufin beta-depentylase and testosterone 6beta- and 7 alpha-hydroxylases were induced in liver microsomes. 2. Within 24 h after treatment with 50 m kg(-1) flupyrazofos, most enzyme activities were decreased, indicating the interaction of flupyrazofos with cytochrome P450. 3. In Western immunoblotting, cytochrome P4502B1/2 proteins were clearly induced by treatment with flupyrazofos, whereas P4501A1/2 and 2C6 proteins were reduced in liver microsomes. 4. The present results indicate that flupyrazofos modulates the expression of cytochrome P450 in rat.
Title: The infusion rate of mivacurium or atracurium for cesarean section compared with gynecological procedures Kim JH, Min KT, Ahn EK, Kim KH, Shin YS Ref: Yonsei Med J, 40:371, 1999 : PubMed
Mivacurium is mainly metabolized by plasma cholinesterase, whereas atracurium is removed by Hofman elimination. The purpose of this study was to compare the infusion rate of atracurium and mivacurium in maintaining surgical relaxation, and to compare their recovery indices between parturients and non-pregnant women. Muscle relaxation was maintained by the continuous infusion of relaxants to retain the first response of train-of-four (TOF) at 5% of control. When mivacurium was used, Bolus-T5 (duration from the end of mivacurium bolus injection to 5% single twitch recovery) was measured. After discontinuing the infusion, the recovery index was measured. The infusion rate of mivacurium, not atracurium, was significantly lower in parturients and Bolus-T5 of parturients was significantly longer than that of non-pregnant women. There was no significant difference in the recovery indices of both relaxants. The authors concluded that the infusion rate of mivacurium in maintaining muscle relaxation in parturients should be reduced compared to the rate in non-pregnant women and measuring Bolus-T5 may be helpful in determining the infusion rate to maintain muscle relaxation.
Title: Isolation and analysis of metA, a methionine biosynthetic gene encoding homoserine acetyltransferase in corynebacterium glutamicum Park SD, Lee JY, Kim Y, Kim JH, Lee HS Ref: Mol Cells, 8:286, 1998 : PubMed
The metA gene encoding homoserine acetyltransferase, the first enzyme of the methionine biosynthetic pathway, was isolated from a pMT1-based corynebacterium glutamicum gene library via complementation of an Escherichia coli metA mutant. A DNA-sequence analysis of the cloned DNA is identified an open-reading frame of 1,137 bp which encodes a protein with the molecular weight of 41,380 comprising 379 amino acids. The putative protein product showed good amino acid-sequence homology to its counterpart in other organisms. The internal fragment of the cloned DNA was successfully used to disrupt chromosomal metA, demonstrating the identity of the cloned gene. The C. glutamicum metA mutant lost the ability to grow on glucose minimal medium supplemented with homoserine. However, the mutant could grow on a minimal medium supplemented with cystathionine, demonstrating that C. glutamicum uses the cystathionine route to synthesize methionine. Introduction of a plasmid carrying cloned metA into C. glutamicum resulted in a 10-fold increase in enzyme activities and expression of a protein product of M(r) 41,000, which agrees with the sequence data and is similar in size to those of other homoserine acetyltransferases. Unlike E. coli whose metA product uses succinyl coenzyme A as a substrate, the cloned metA gene produced homoserine acetyltransferase which uses only acetyl coenzyme A as the acyl donor.
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.
Title: Cloning, nucleotide sequence and expression of gene coding for poly-3-hydroxybutyric acid (PHB) synthase of Rhodobacter sphaeroides 2.4.1. Kim JH, Lee JK Ref: J Microbiol Biotechnol, 7:229, 1997 : PubMed
1. The in vitro metabolism of the new insecticide flupyrazofos was studied using rat liver microsomes. Two metabolites were produced and identified as O,O-diethyl O-(1-phenyl-3-trifluoromethyl-5-pyrazoyl) phosphoric acid ester (flupyrazofos oxon) and 1-phenyl-3-trifluoromethyl-5-hydroxypyrazole (PTMHP) based on UV and mass spectral analysis. 2. Cytochrome P450 oxidatively converted flupyrazofos to flupyrazofos oxon, a major metabolite and phenobarbital-induced microsomes increased this desulphuration by 8-fold. 3. Flupyrazofos oxon was converted to PTMHP with a half-life of 47.8 min by chemical hydrolysis and this conversion also proceeded non-enzymatically under our microsomal incubation conditions.
Title: Cloning and nucleotide sequence of the beta-galactosidase gene from Lactococcus lactis ssp. lactis ATCC7962. Lee JM, Chung DK, Park JH, Lee WK, Chang HC, Kim JH, Lee HJ Ref: Biotechnol Lett, 19:179, 1997 : PubMed
