S-Inosyl-L-Homocysteine Hydrolase, a Novel Enzyme Involved in S-Adenosyl-L-Methionine Recycling.

Science.gov (United States)

Miller, Danielle; Xu, Huimin; White, Robert H

2015-07-01

S-Adenosyl-L-homocysteine, the product of S-adenosyl-L-methionine (SAM) methyltransferases, is known to be a strong feedback inhibitor of these enzymes. A hydrolase specific for S-adenosyl-L-homocysteine produces L-homocysteine, which is remethylated to methionine and can be used to regenerate SAM. Here, we show that the annotated S-adenosyl-L-homocysteine hydrolase in Methanocaldococcus jannaschii is specific for the hydrolysis and synthesis of S-inosyl-L-homocysteine, not S-adenosyl-L-homocysteine. This is the first report of an enzyme specific for S-inosyl-L-homocysteine. As with S-adenosyl-L-homocysteine hydrolase, which shares greater than 45% sequence identity with the M. jannaschii homologue, the M. jannaschii enzyme was found to copurify with bound NAD(+) and has Km values of 0.64 ± 0.4 mM, 0.0054 ± 0.006 mM, and 0.22 ± 0.11 mM for inosine, L-homocysteine, and S-inosyl-L-homocysteine, respectively. No enzymatic activity was detected with S-adenosyl-L-homocysteine as the substrate in either the synthesis or hydrolysis direction. These results prompted us to redesignate the M. jannaschii enzyme an S-inosyl-L-homocysteine hydrolase (SIHH). Identification of SIHH demonstrates a modified pathway in this methanogen for the regeneration of SAM from S-adenosyl-L-homocysteine that uses the deamination of S-adenosyl-L-homocysteine to form S-inosyl-L-homocysteine. In strictly anaerobic methanogenic archaea, such as Methanocaldococcus jannaschii, canonical metabolic pathways are often not present, and instead, unique pathways that are deeply rooted on the phylogenetic tree are utilized by the organisms. Here, we discuss the recycling pathway for S-adenosyl-L-homocysteine, produced from S-adenosyl-L-methionine (SAM)-dependent methylation reactions, which uses a hydrolase specific for S-inosyl-L-homocysteine, an uncommon metabolite. Identification of the pathways and the enzymes involved in the unique pathways in the methanogens will provide insight into the

Preparation, crystallization and preliminary X-ray crystallographic studies of diadenosine tetraphosphate hydrolase from Shigella flexneri 2a

Energy Technology Data Exchange (ETDEWEB)

Hu, Wenxin; Wang, Qihai; Bi, Ruchang, E-mail: rcbi@sun5.ibp.ac.cn [Institute of Biophysics, Chinese Academy of Sciences, 15 Datun Road, Chaoyang District, Beijing 100101 (China)

2005-12-01

The 31.3 kDa Ap{sub 4}A hydrolase from Shigella flexneri 2a has been cloned, expressed and purified using an Escherichia coli expression system. Crystals of Ap{sub 4}A hydrolase have been obtained by the hanging-drop technique at 291 K using PEG 550 MME as precipitant. Diadenosine tetraphosphate (Ap{sub 4}A) hydrolase (EC 3.6.1.41) hydrolyzes Ap{sub 4}A symmetrically in prokaryotes. It plays a potential role in organisms by regulating the concentration of Ap{sub 4}A in vivo. To date, no three-dimensional structures of proteins with significant sequence homology to this protein have been determined. The 31.3 kDa Ap{sub 4}A hydrolase from Shigella flexneri 2a has been cloned, expressed and purified using an Escherichia coli expression system. Crystals of Ap{sub 4}A hydrolase have been obtained by the hanging-drop technique at 291 K using PEG 550 MME as precipitant. Ap{sub 4}A hydrolase crystals diffract X-rays to 3.26 Å and belong to space group P2{sub 1}, with unit-cell parameters a = 118.9, b = 54.6, c = 128.5 Å, β = 95.7°.

In silico investigation of cycloartane triterpene derivatives from Cimicifuga dahurica (Turcz.) Maxim. roots for the development of potent soluble epoxide hydrolase inhibitors.

Science.gov (United States)

Thao, Nguyen Phuong; Kim, Jang Hoon; Thuy Luyen, Bui Thi; Dat, Nguyen Tien; Kim, Young Ho

2017-05-01

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 IC 50 values of about 5μM 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. Copyright © 2017 Elsevier B.V. All rights reserved.

Structure of HsaD, a steroid-degrading hydrolase, from Mycobacterium tuberculosis

International Nuclear Information System (INIS)

Lack, Nathan; Lowe, Edward D.; Liu, Jie; Eltis, Lindsay D.; Noble, Martin E. M.; Sim, Edith; Westwood, Isaac M.

2007-01-01

The structure of HsaD, a carbon–carbon bond serine hydrolase involved in steroid catabolism that is critical for the survival of M. tuberculosis inside human macrophages, has been solved by X-ray crystallography. Data were collected at the Diamond Light Source in Oxfordshire, England: this paper describes one of the first structures determined at the new synchrotron. Tuberculosis is a major cause of death worldwide. Understanding of the pathogenicity of Mycobacterium tuberculosis has been advanced by gene analysis and has led to the identification of genes that are important for intracellular survival in macrophages. One of these genes encodes HsaD, a meta-cleavage product (MCP) hydrolase that catalyzes the hydrolytic cleavage of a carbon–carbon bond in cholesterol metabolism. This paper describes the production of HsaD as a recombinant protein and, following crystallization, the determination of its three-dimensional structure to 2.35 Å resolution by X-ray crystallography at the Diamond Light Source in Oxfordshire, England. To the authors’ knowledge, this study constitutes the first report of a structure determined at the new synchrotron facility. The volume of the active-site cleft of the HsaD enzyme is more than double the corresponding active-site volumes of related MCP hydrolases involved in the catabolism of aromatic compounds, consistent with the specificity of HsaD for steroids such as cholesterol. Knowledge of the structure of the enzyme facilitates the design of inhibitors

Cellular localization of peptide hydrolases in chicken embryo tissues and influence of gamma irradiation on their activity

Energy Technology Data Exchange (ETDEWEB)

Khristov, D; Marinopolski, G

1975-01-01

Studied was the influence of chicken embryo irradiation at 600 R and 1000 R gamma rays on the activity of tissue peptide hydrolases in mitochondrial-lysosomal, microsomal and supernatant (cell hyaloplasm) cell fractions. The investigation was performed 50 to 168 hours post irradiation. The wole tissue (of the whole embryo) was examined following irradiation of 4-day-old embryos whose liver, muscle and brain tissues were post irradiation examined on day 12 and 16 of incubation. Prior to treatment, the tissues were threfold rinsed with sucrose solution to eliminate proeinase inhibitors. Lysosome membranes were destroyed by adding 0.5 % desoxycholate. It was found that: Peptide hydrolase activity of mitochondrial-lysosomal cell fractions of tissues of whole 6-day chicken embryos is 4-5 times as high as that of cell hyaloplasm. Peptide hydrolase activity of mitochondrial-lysosomal fractions of liver tissues decreases on day 18 and 19 post incubation, while the same fraction of muscle and brain tissues shows high activity. Peptide hydrolase activity of microsomal fraction and of cell hyaloplasm rises during embryonal development and exceeds the activity of liver tissue mitochondrial fraction. Peptide hydrolase activity of mitochondrial-lysosomal fraction of tissue of whole 6-day-old embryos 50 hours post irradiation is higher than the activity of non-irradiated embryos. Later the activity of this fraction diminishes and on the 168 hr post irradiation it drops below the normal. Microsomal fraction and cell hyaloplasm activity likewise show deviation from the norm. Peptide hydrolase activity of mitochondrial-lysosomal fraction of liver, muscle and brain tissue of 14 and 18-day-old embryos is higher than the control 50 hours post irradiation and then declines. The activity of mitochondrial-lysosomal fraction of embryo brain tissue changes most strikingly on irradiation, while other brain cell fractions change less compared with liver and muscle fractions.

Cellular localization of peptide hydrolases in chicken embryo tissues and influence of gamma irradiation on their activity

International Nuclear Information System (INIS)

Khristov, D.; Marinopolski, G.

1975-01-01

Studied was the influence of chicken embryo irradiation at 600 R and 1000 R gamma rays on the activity of tissue peptide hydrolases in mitochondrial-lysosomal, microsomal and supernatant (cell hyaloplasm) cell fractions. The investigation was performed 50 to 168 hours post irradiation. The wole tissue (of the whole embryo) was examined following irradiation of 4-day-old embryos whose liver, muscle and brain tissues were post irradiation examined on day 12 and 16 of incubation. Prior to treatment, the tissues were threfold rinsed with sucrose solution to eliminate proeinase inhibitors. Lysosome membranes were destroyed by adding 0.5 % desoxycholate. It was found that: Peptide hydrolase activity of mitochondrial-lysosomal cell fractions of tissues of whole 6-day chicken embryos is 4-5 times as high as that of cell hyaloplasm. Peptide hydrolase activity of mitochondrial-lysosomal fractions of liver tissues decreases on day 18 and 19 post incubation, while the same fraction of muscle and brain tissues shows high activity. Peptide hydrolase activity of microsomal fraction and of cell hyaloplasm rises during embryonal development and exceeds the activity of liver tissue mitochondrial fraction. Peptide hydrolase activity of mitochondrial-lysosomal fraction of tissue of whole 6-day-old embryos 50 hours post irradiation is higher than the activity of non-irradiated embryos. Later the activity of this fraction diminishes and on the 168 hr post irradiation it drops below the normal. Microsomal fraction and cell hyaloplasm activity likewise show deviation from the norm. Peptide hydrolase activity of mitochondrial-lysosomal fraction of liver, muscle and brain tissue of 14 and 18-day-old embryos is higher than the control 50 hours post irradiation and then declines. The activity of mitochondrial-lysosomal fraction of embryo brain tissue changes most strikingly on irradiation, while other brain cell fractions change less compared with liver and muscle fractions

4,3-α-Glucanotransferase, a novel reaction specificity in glycoside hydrolase family 70 and clan GH-H

NARCIS (Netherlands)

Gangoiti Muñecas, Joana; van Leeuwen, Sander S; Gerwig, Gerrit J; Duboux, Stéphane; Vafiadi, Christina; Pijning, Tjaard; Dijkhuizen, Lubbert

2017-01-01

Lactic acid bacteria possess a diversity of glucansucrase (GS) enzymes that belong to glycoside hydrolase family 70 (GH70) and convert sucrose into α-glucan polysaccharides with (α1 → 2)-, (α1 → 3)-, (α1 → 4)- and/or (α1 → 6)-glycosidic bonds. In recent years 3 novel subfamilies of GH70 enzymes,

Evaluation of fish models of soluble epoxide hydrolase inhibition.

OpenAIRE

Newman, J W; Denton, D L; Morisseau, C; Koger, C S; Wheelock, C E; Hinton, D E; Hammock, B D

2001-01-01

Substituted ureas and carbamates are mechanistic inhibitors of the soluble epoxide hydrolase (sEH). We screened a set of chemicals containing these functionalities in larval fathead minnow (Pimphales promelas) and embryo/larval golden medaka (Oryzias latipes) models to evaluate the utility of these systems for investigating sEH inhibition in vivo. Both fathead minnow and medaka sEHs were functionally similar to the tested mammalian orthologs (murine and human) with respect to substrate hydrol...

Regulation of calcium release from the endoplasmic reticulum by the serine hydrolase ABHD2.

Science.gov (United States)

Yun, Bogeon; Lee, HeeJung; Powell, Roger; Reisdorph, Nichole; Ewing, Heather; Gelb, Michael H; Hsu, Ku-Lung; Cravatt, Benjamin F; Leslie, Christina C

2017-09-02

The serine hydrolase inhibitors pyrrophenone and KT195 inhibit cell death induced by A23187 and H 2 O 2 by blocking the release of calcium from the endoplasmic reticulum and mitochondrial calcium uptake. The effect of pyrrophenone and KT195 on these processes is not due to inhibition of their known targets, cytosolic phospholipase A 2 and α/β-hydrolase domain-containing (ABHD) 6, respectively, but represent off-target effects. To identify targets of KT195, fibroblasts were treated with KT195-alkyne to covalently label protein targets followed by click chemistry with biotin azide, enrichment on streptavidin beads and tryptic peptide analysis by mass spectrometry. Although several serine hydrolases were identified, α/β-hydrolase domain-containing 2 (ABHD2) was the only target in which both KT195 and pyrrophenone competed for binding to KT195-alkyne. ABHD2 is a serine hydrolase with a predicted transmembrane domain consistent with its pull-down from the membrane proteome. Subcellular fractionation showed localization of ABHD2 to the endoplasmic reticulum but not to mitochondria or mitochondrial-associated membranes. Knockdown of ABHD2 with shRNA attenuated calcium release from the endoplasmic reticulum, mitochondrial calcium uptake and cell death in fibroblasts stimulated with A23187. The results describe a novel mechanism for regulating calcium transfer from the endoplasmic reticulum to mitochondria that involves the serine hydrolase ABHD2. Copyright © 2017 Elsevier Inc. All rights reserved.

Identification of N-ethylmethylamine as a novel scaffold for inhibitors of soluble epoxide hydrolase by crystallographic fragment screening.

Science.gov (United States)

Amano, Yasushi; Tanabe, Eiki; Yamaguchi, Tomohiko

2015-05-15

Soluble epoxide hydrolase (sEH) is a potential target for the treatment of inflammation and hypertension. X-ray crystallographic fragment screening was used to identify fragment hits and their binding modes. Eight fragment hits were identified via soaking of sEH crystals with fragment cocktails, and the co-crystal structures of these hits were determined via individual soaking. Based on the binding mode, N-ethylmethylamine was identified as a promising scaffold that forms hydrogen bonds with the catalytic residues of sEH, Asp335, Tyr383, and Tyr466. Compounds containing this scaffold were selected from an in-house chemical library and assayed. Although the starting fragment had a weak inhibitory activity (IC50: 800μM), we identified potent inhibitors including 2-({[2-(adamantan-1-yl)ethyl]amino}methyl)phenol exhibiting the highest inhibitory activity (IC50: 0.51μM). This corresponded to a more than 1500-fold increase in inhibitory activity compared to the starting fragment. Co-crystal structures of the hit compounds demonstrate that the binding of N-ethylmethylamine to catalytic residues is similar to that of the starting fragment. We therefore consider crystallographic fragment screening to be appropriate for the identification of weak but promising fragment hits. Copyright © 2015 Elsevier Ltd. All rights reserved.

Fluorometric determination of paraoxon in human serum using a gold nanoparticle-immobilized organophosphorus hydrolase and coumarin 1 as a competitive inhibitor

International Nuclear Information System (INIS)

Kamelipour, Nahid; Mohsenifar, Afshin; Rahmani-Cherati, Tavoos; Tabatabaei, Meisam; Khoshnevisan, Kamyar; Allameh, Abdolamir; Milani, Majid M.; Etemadikia, Batool; Najavand, Saeid

2014-01-01

A dimeric organophosphorus hydrolase (OPH; EC 3.1.8.1; 72 kDa) was isolated from wild-type bacteria, analyzed for its 16s rRNA sequence, purified, and immobilized on gold nanoparticles (AuNPs) to form the transducer part of a biosensor. The isolated strain was identified as Pseudomonas aeruginosa. The AuNPs were characterized by transmission electron microscopy and localized surface plasmon resonance. Covalent binding of OPH to the AuNPs was confirmed by spectrophotometry, enzymatic activity assays, and FTIR spectroscopy. Coumarin 1, a competitive inhibitor of OPH, was used as a fluorogenic probe. The bioconjugates quench the emission of coumarin 1 upon binding, but the addition of paraoxon results in an enhancement of fluorescence that is directly proportional to the concentration of paraoxon. The gold-OPH conjugates were then used to determine paraoxon in serum samples spiked with varying levels of paraoxon. The method works in the 50 to 1,050 nM concentration range, has a low standard deviation (with a CV of 5.7–11 %), and a detection limit as low as 5 × 10 −11 M. (author)

Evaluation of fish models of soluble epoxide hydrolase inhibition.

Science.gov (United States)

Newman, J W; Denton, D L; Morisseau, C; Koger, C S; Wheelock, C E; Hinton, D E; Hammock, B D

2001-01-01

Substituted ureas and carbamates are mechanistic inhibitors of the soluble epoxide hydrolase (sEH). We screened a set of chemicals containing these functionalities in larval fathead minnow (Pimphales promelas) and embryo/larval golden medaka (Oryzias latipes) models to evaluate the utility of these systems for investigating sEH inhibition in vivo. Both fathead minnow and medaka sEHs were functionally similar to the tested mammalian orthologs (murine and human) with respect to substrate hydrolysis and inhibitor susceptibility. Low lethality was observed in either larval or embryonic fish exposed to diuron [N-(3,4-dichlorophenyl), N'-dimethyl urea], desmethyl diuron [N-(3,4-dichlorophenyl), N'-methyl urea], or siduron [N-(1-methylcyclohexyl), N'-phenyl urea]. Dose-dependent inhibition of sEH was a sublethal effect of substituted urea exposure with the potency of siduron diuron = diuron, differing from the observed in vitro sEH inhibition potency of siduron > desmethyl diuron > diuron. Further, siduron exposure synergized the toxicity of trans-stilbene oxide in fathead minnows. Medaka embryos exposed to diuron, desmethyl diuron, or siduron displayed dose-dependent delays in hatch, and elevated concentrations of diuron and desmethyl diuron produced developmental toxicity. The dose-dependent toxicity and in vivo sEH inhibition correlated, suggesting a potential, albeit undefined, relationship between these factors. Additionally, the observed inversion of in vitro to in vivo potency suggests that these fish models may provide tools for investigating the in vivo stability of in vitro inhibitors while screening for untoward effects. PMID:11171526

Peptidoglycan Hydrolases of Escherichia coli

Science.gov (United States)

van Heijenoort, Jean

2011-01-01

Summary: The review summarizes the abundant information on the 35 identified peptidoglycan (PG) hydrolases of Escherichia coli classified into 12 distinct families, including mainly glycosidases, peptidases, and amidases. An attempt is also made to critically assess their functions in PG maturation, turnover, elongation, septation, and recycling as well as in cell autolysis. There is at least one hydrolytic activity for each bond linking PG components, and most hydrolase genes were identified. Few hydrolases appear to be individually essential. The crystal structures and reaction mechanisms of certain hydrolases having defined functions were investigated. However, our knowledge of the biochemical properties of most hydrolases still remains fragmentary, and that of their cellular functions remains elusive. Owing to redundancy, PG hydrolases far outnumber the enzymes of PG biosynthesis. The presence of the two sets of enzymes acting on the PG bonds raises the question of their functional correlations. It is difficult to understand why E. coli keeps such a large set of PG hydrolases. The subtle differences in substrate specificities between the isoenzymes of each family certainly reflect a variety of as-yet-unidentified physiological functions. Their study will be a far more difficult challenge than that of the steps of the PG biosynthesis pathway. PMID:22126997

Soluble epoxide hydrolase activity and pharmacologic inhibition in horses with chronic severe laminitis.

Science.gov (United States)

Guedes, A; Galuppo, L; Hood, D; Hwang, S H; Morisseau, C; Hammock, B D

2017-05-01

The roles of soluble epoxide hydrolase and lipid mediators in inflammatory and neuropathic pain could be relevant in laminitis pain management. To determine soluble epoxide hydrolase (sEH) activity in the digital laminae, sEH inhibitor potency in vitro, and efficacy of a sEH inhibitor as an adjunct analgesic therapy in chronic laminitic horses. In vitro experiments and clinical case series. sEH activity was measured in digital laminae from euthanised healthy and laminitic horses (n = 5-6/group). Potency of 7 synthetic sEH inhibitors was determined in vitro using equine liver cytosol. One of them (t-TUCB; 0.1 mg/kg bwt i.v. every 24 h) was selected based on potency and stability, and used as adjunct therapy in 10 horses with severe chronic laminitis (Obel grades 2, one horse; 3-4, nine horses). Daily assessments of forelimb lifts, pain scores, physiologic and laboratory examinations were performed before (baseline) and during t-TUCB treatment. Data are presented as mean ± s.d. and 95% confidence intervals (CI). sEH activity in the digital laminae from laminitic horses (0.9±0.6 nmol/min/mg; 95% CI 0.16-1.55 nmol/min/mg) was significantly greater (P = 0.01) than in healthy horses (0.17±0.09 nmol/min/mg; CI 0.07-0.26 nmol/min/mg). t-TUCB as an adjunct analgesic up to 10 days (4.3±3 days) in laminitic horses was associated with significant reduction in forelimb lifts (36±22%; 95% CI 9-64%) and in pain scores (18±23%; 95% CI 2-35%) compared with baseline (P = 0.04). One horse developed gas colic and another corneal vascularisation in a blind eye during treatment. No other significant changes were observed. Absence of control group and evaluator blinding in case series. sEH activity is significantly higher in the digital laminae of actively laminitic compared with healthy horses, and use of a potent inhibitor of equine sEH as adjunct analgesic therapy appears to decrease signs of pathologic pain in laminitic horses. © 2016 EVJ Ltd.

Lysophosphatidic acids are new substrates for the phosphatase domain of soluble epoxide hydrolase[S

Science.gov (United States)

Oguro, Ami; Imaoka, Susumu

2012-01-01

Soluble epoxide hydrolase (sEH) is a bifunctional enzyme that has a C-terminus epoxide hydrolase domain and an N-terminus phosphatase domain. The endogenous substrates of epoxide hydrolase are known to be epoxyeicosatrienoic acids, but the endogenous substrates of the phosphatase activity are not well understood. In this study, to explore the substrates of sEH, we investigated the inhibition of the phosphatase activity of sEH toward 4-methylumbelliferyl phosphate by using lecithin and its hydrolyzed products. Although lecithin itself did not inhibit the phosphatase activity, the hydrolyzed lecithin significantly inhibited it, suggesting that lysophospholipid or fatty acid can inhibit it. Next, we investigated the inhibition of phosphatase activity by lysophosphatidyl choline, palmitoyl lysophosphatidic acid, monopalmitoyl glycerol, and palmitic acid. Palmitoyl lysophosphatidic acid and fatty acid efficiently inhibited phosphatase activity, suggesting that lysophosphatidic acids (LPAs) are substrates for the phosphatase activity of sEH. As expected, palmitoyl, stearoyl, oleoyl, and arachidonoyl LPAs were efficiently dephosphorylated by sEH (Km, 3–7 μM; Vmax, 150–193 nmol/min/mg). These results suggest that LPAs are substrates of sEH, which may regulate physiological functions of cells via their metabolism. PMID:22217705

The Serine Hydrolase ABHD6 Is a Critical Regulator of the Metabolic Syndrome

Directory of Open Access Journals (Sweden)

Gwynneth Thomas

2013-10-01

Full Text Available The serine hydrolase α/β hydrolase domain 6 (ABHD6 has recently been implicated as a key lipase for the endocannabinoid 2-arachidonylglycerol (2-AG in the brain. However, the biochemical and physiological function for ABHD6 outside of the central nervous system has not been established. To address this, we utilized targeted antisense oligonucleotides (ASOs to selectively knock down ABHD6 in peripheral tissues in order to identify in vivo substrates and understand ABHD6’s role in energy metabolism. Here, we show that selective knockdown of ABHD6 in metabolic tissues protects mice from high-fat-diet-induced obesity, hepatic steatosis, and systemic insulin resistance. Using combined in vivo lipidomic identification and in vitro enzymology approaches, we show that ABHD6 can hydrolyze several lipid substrates, positioning ABHD6 at the interface of glycerophospholipid metabolism and lipid signal transduction. Collectively, these data suggest that ABHD6 inhibitors may serve as therapeutics for obesity, nonalcoholic fatty liver disease, and type II diabetes.

Fatty Acid Amide Hydrolase (FAAH) Inhibition Enhances Memory Acquisition through Activation of PPAR-alpha Nuclear Receptors

Science.gov (United States)

Mazzola, Carmen; Medalie, Julie; Scherma, Maria; Panlilio, Leigh V.; Solinas, Marcello; Tanda, Gianluigi; Drago, Filippo; Cadet, Jean Lud; Goldberg, Steven R.; Yasar, Sevil

2009-01-01

Inhibitors of fatty acid amide hydrolase (FAAH) increase endogenous levels of anandamide (a cannabinoid CB[subscript 1]-receptor ligand) and oleoylethanolamide and palmitoylethanolamide (OEA and PEA, ligands for alpha-type peroxisome proliferator-activated nuclear receptors, PPAR-alpha) when and where they are naturally released in the brain.…

Proteomic analysis of Oesophagostomum dentatum (Nematoda during larval transition, and the effects of hydrolase inhibitors on development.

Directory of Open Access Journals (Sweden)

Martina Ondrovics

Full Text Available In this study, in vitro drug testing was combined with proteomic and bioinformatic analyses to identify and characterize proteins involved in larval development of Oesophagostomum dentatum, an economically important parasitic nematode. Four hydrolase inhibitors ο-phenanthroline, sodium fluoride, iodoacetamide and 1,2-epoxy-3-(pnitrophenoxy-propane (EPNP significantly inhibited (≥90% larval development. Comparison of the proteomic profiles of the development-inhibited larvae with those of uninhibited control larvae using two-dimensional gel electrophoresis, and subsequent MALDI-TOF mass spectrometric analysis identified a down-regulation of 12 proteins inferred to be involved in various larval developmental processes, including post-embryonic development and growth. Furthermore, three proteins (i.e. intermediate filament protein B, tropomyosin and peptidyl-prolyl cis-trans isomerase inferred to be involved in the moulting process were down-regulated in moulting- and development-inhibited O. dentatum larvae. This first proteomic map of O. dentatum larvae provides insights in the protein profile of larval development in this parasitic nematode, and significantly improves our understanding of the fundamental biology of its development. The results and the approach used might assist in developing new interventions against parasitic nematodes by blocking or disrupting their key biological pathways.

Lysophosphatidylcholine hydrolases of human erythrocytes, lymphocytes, and brain: Sensitive targets of conserved specificity for organophosphorus delayed neurotoxicants

International Nuclear Information System (INIS)

Vose, Sarah C.; Holland, Nina T.; Eskenazi, Brenda; Casida, John E.

2007-01-01

Brain neuropathy target esterase (NTE), associated with organophosphorus (OP)-induced delayed neuropathy, has the same OP inhibitor sensitivity and specificity profiles assayed in the classical way (paraoxon-resistant, mipafox-sensitive hydrolysis of phenyl valerate) or with lysophosphatidylcholine (LysoPC) as the substrate. Extending our earlier observation with mice, we now examine human erythrocyte, lymphocyte, and brain LysoPC hydrolases as possible sensitive targets for OP delayed neurotoxicants and insecticides. Inhibitor profiling of human erythrocytes and lymphocytes gave the surprising result of essentially the same pattern as with brain. Human erythrocyte LysoPC hydrolases are highly sensitive to OP delayed neurotoxicants, with in vitro IC 50 values of 0.13-85 nM for longer alkyl analogs, and poorly sensitive to the current OP insecticides. In agricultural workers, erythrocyte LysoPC hydrolyzing activities are similar for newborn children and their mothers and do not vary with paraoxonase status but have high intersample variation that limits their use as a biomarker. Mouse erythrocyte LysoPC hydrolase activity is also of low sensitivity in vitro and in vivo to the OP insecticides whereas the delayed neurotoxicant ethyl n-octylphosphonyl fluoride inhibits activity in vivo at 1-3 mg/kg. Overall, inhibition of blood LysoPC hydrolases is as good as inhibition of brain NTE as a predictor of OP inducers of delayed neuropathy. NTE and lysophospholipases (LysoPLAs) both hydrolyze LysoPC, yet they are in distinct enzyme families with no sequence homology and very different catalytic sites. The relative contributions of NTE and LysoPLAs to LysoPC hydrolysis and clearance from erythrocytes, lymphocytes, and brain remain to be defined

4-alkyl-L-(Dehydro)proline biosynthesis in actinobacteria involves N-terminal nucleophile-hydrolase activity of γ-glutamyltranspeptidase homolog for C-C bond cleavage

Science.gov (United States)

Zhong, Guannan; Zhao, Qunfei; Zhang, Qinglin; Liu, Wen

2017-07-01

γ-Glutamyltranspeptidases (γ-GTs), ubiquitous in glutathione metabolism for γ-glutamyl transfer/hydrolysis, are N-terminal nucleophile (Ntn)-hydrolase fold proteins that share an autoproteolytic process for self-activation. γ-GT homologues are widely present in Gram-positive actinobacteria where their Ntn-hydrolase activities, however, are not involved in glutathione metabolism. Herein, we demonstrate that the formation of 4-Alkyl-L-(dehydro)proline (ALDP) residues, the non-proteinogenic α-amino acids that serve as vital components of many bioactive metabolites found in actinobacteria, involves unprecedented Ntn-hydrolase activity of γ-GT homologue for C-C bond cleavage. The related enzymes share a key Thr residue, which acts as an internal nucleophile for protein hydrolysis and then as a newly released N-terminal nucleophile for carboxylate side-chain processing likely through the generation of an oxalyl-Thr enzyme intermediate. These findings provide mechanistic insights into the biosynthesis of various ALDP residues/associated natural products, highlight the versatile functions of Ntn-hydrolase fold proteins, and particularly generate interest in thus far less-appreciated γ-GT homologues in actinobacteria.

Enzymatic synthesis of β-xylosyl-oligosaccharides by transxylosylation using two beta-xylosidases of glycoside hydrolase family 3 from Aspergillus nidulans FGSC A4

DEFF Research Database (Denmark)

Dilokpimol, Adiphol; Nakai, Hiroyuki; Gotfredsen, Charlotte Held

2011-01-01

Two beta-xylosidases of glycoside hydrolase family 3 (GH 3) from Aspergillus nidulans FGSC A4, BxlA and BxlB were produced recombinantly in Pichia pastoris and secreted to the culture supernatants in yields of 16 and 118 mg/L, respectively. BxlA showed about sixfold higher catalytic efficiency (k...

Soluble Epoxide Hydrolase Inhibitory Activity of Selaginellin Derivatives from Selaginella tamariscina

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Jang Hoon Kim

2015-12-01

Full Text Available Selaginellin derivatives 1–3 isolated from Selaginella tamariscina were evaluated for their inhibition of soluble epoxide hydrolase (sEH to demonstrate their potential for the treatment of cardiovascular disease. All selaginellin derivatives (1–3 inhibited sEH enzymatic activity and PHOME hydrolysis, in a dose-dependent manner, with IC50 values of 3.1 ± 0.1, 8.2 ± 2.2, and 4.2 ± 0.2 μM, respectively. We further determined that the derivatives function as non-competitive inhibitors. Moreover, the predicted that binding sites and interaction between 1–3 and sEH were solved by docking simulations. According to quantitative analysis, 1–3 were confirmed to have high content in the roots of S. tamariscina; among them, selaginellin 3 exhibited the highest content of 189.3 ± 0.0 μg/g.

Soluble epoxide hydrolase inhibitory activity of anthraquinone components from Aloe.

Science.gov (United States)

Sun, Ya Nan; Kim, Jang Hoon; Li, Wei; Jo, A Reum; Yan, Xi Tao; Yang, Seo Young; Kim, Young Ho

2015-10-15

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±4.2 μM. 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. Copyright © 2015 Elsevier Ltd. All rights reserved.

Structure of XC6422 from Xanthomonas campestris at 1.6 Å resolution: a small serine α/β-hydrolase

Energy Technology Data Exchange (ETDEWEB)

Yang, Chao-Yu; Chin, Ko-Hsin [Institute of Biochemistry, National Chung-Hsing University, Taichung 40227,Taiwan (China); Chou, Chia-Cheng; Wang, Andrew H.-J. [Institute of Biological Chemistry, Academia Sinica, Nankang, Taipei,Taiwan (China); Core Facility for Protein Crystallography, Academia Sinica, Nankang, Taipei,Taiwan (China); Chou, Shan-Ho, E-mail: shchou@nchu.edu.tw [Institute of Biochemistry, National Chung-Hsing University, Taichung 40227,Taiwan (China)

2006-06-01

The crystal structure of a conserved hypothetical protein from X. campestris has been determined to a resolution of 1.6 Å. The determined X. campestris structure shows that it belongs to the superfamily of serine α/β hydrolase, with an extra strand preceding the first β-strand to lead to extensive subunit interactions in the crystal. XC6422 is a conserved hypothetical protein from Xanthomonas campestris pathovar campestris (Xcc), a Gram-negative yellow-pigmented pathogenic bacterium that causes black rot, one of the major worldwide diseases of cruciferous crops. The protein consists of 220 amino acids and its structure has been determined to 1.6 Å resolution using the multi-wavelength anomalous dispersion (MAD) method. Although it has very low sequence identity to protein sequences in the PDB (less than 20%), the determined structure nevertheless shows that it belongs to the superfamily of serine α/β-hydrolases, with an active site that is fully accessible to solvent owing to the absence of a lid domain. Modelling studies with the serine esterase inhibitor E600 indicate that XC6422 adopts a conserved Ser-His-Asp catalytic triad common to this superfamily and has a preformed oxyanion hole for catalytic activation. These structural features suggest that XC6422 is most likely to be a hydrolase active on a soluble ester or a small lipid. An extra strand preceding the first β-strand in the canonical α/β-hydrolase fold leads to extensive subunit interactions between XC6422 monomers, which may explain why XC6422 crystals of good diffraction quality can grow to dimensions of up to 1.5 mm in a few days.

Characterization of Bile Salt Hydrolase from Lactobacillus gasseri FR4 and Demonstration of Its Substrate Specificity and Inhibitory Mechanism Using Molecular Docking Analysis

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Rizwana Parveen Rani

2017-05-01

Full Text Available Probiotic bacteria are beneficial to the health of poultry animals, thus are used as alternative candidates for antibiotics used as growth promoters (AGPs. However, they also reduce the body weight gain due to innate bile salt hydrolase (BSH activity. Hence, the addition of a suitable BSH inhibitor along with the probiotic feed can decrease the BSH activity. In this study, a BSH gene (981 bp encoding 326-amino acids was identified from the genome of Lactobacillus gasseri FR4 (LgBSH. The LgBSH-encoding gene was cloned and purified using an Escherichia coli BL21 (DE3 expression system, and its molecular weight (37 kDa was confirmed by SDS–PAGE and a Western blot analysis. LgBSH exhibited greater hydrolysis toward glyco-conjugated bile salts compared to tauro-conjugated bile salts. LgBSH displayed optimal activity at 52°C at a pH of 5.5, and activity was further increased by several reducing agents (DTT, surfactants (Triton X-100 and Tween 80, and organic solvents (isopropanol, butanol, and acetone. Riboflavin and penicillin V, respectively, inhibited LgBSH activity by 98.31 and 97.84%. A homology model of LgBSH was predicted using EfBSH (4WL3 as a template. Molecular docking analysis revealed that the glycocholic acid had lowest binding energy of -8.46 kcal/mol; on the other hand, inhibitors, i.e., riboflavin and penicillin V, had relatively higher binding energies of -6.25 and -7.38 kcal/mol, respectively. Our results suggest that L. gasseri FR4 along with riboflavin might be a potential alternative to AGPs for poultry animals.

Simultaneous Inhibition of Fatty Acid Amide Hydrolase and Monoacylglycerol Lipase Shares Discriminative Stimulus Effects with Δ9-Tetrahydrocannabinol in Mice

OpenAIRE

Hruba, Lenka; Seillier, Alexandre; Zaki, Armia; Cravatt, Benjamin F.; Lichtman, Aron H.; Giuffrida, Andrea; McMahon, Lance R.

2015-01-01

Monoacylglycerol lipase (MAGL) and fatty acid amide hydrolase (FAAH) inhibitors exert preclinical effects indicative of therapeutic potential (i.e., analgesia). However, the extent to which MAGL and FAAH inhibitors produce unwanted effects remains unclear. Here, FAAH and MAGL inhibition was examined separately and together in a Δ9-tetrahydrocannabinol (Δ9-THC; 5.6 mg/kg i.p.) discrimination assay predictive of subjective effects associated with cannabis use, and the relative contribution of N...

Variants of glycoside hydrolases

Science.gov (United States)

Teter, Sarah [Davis, CA; Ward, Connie [Hamilton, MT; Cherry, Joel [Davis, CA; Jones, Aubrey [Davis, CA; Harris, Paul [Carnation, WA; Yi, Jung [Sacramento, CA

2011-04-26

The present invention relates to variants of a parent glycoside hydrolase, comprising a substitution at one or more positions corresponding to positions 21, 94, 157, 205, 206, 247, 337, 350, 373, 383, 438, 455, 467, and 486 of amino acids 1 to 513 of SEQ ID NO: 2, and optionally further comprising a substitution at one or more positions corresponding to positions 8, 22, 41, 49, 57, 113, 193, 196, 226, 227, 246, 251, 255, 259, 301, 356, 371, 411, and 462 of amino acids 1 to 513 of SEQ ID NO: 2 a substitution at one or more positions corresponding to positions 8, 22, 41, 49, 57, 113, 193, 196, 226, 227, 246, 251, 255, 259, 301, 356, 371, 411, and 462 of amino acids 1 to 513 of SEQ ID NO: 2, wherein the variants have glycoside hydrolase activity. The present invention also relates to nucleotide sequences encoding the variant glycoside hydrolases and to nucleic acid constructs, vectors, and host cells comprising the nucleotide sequences.

The Soluble Epoxide Hydrolase Inhibitor AR9281 Decreases Blood Pressure, Ameliorates Renal Injury and Improves Vascular Function in Hypertension

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Sean Shaw

2009-12-01

Full Text Available Soluble epoxide hydrolase inhibitors (sEHIs are demonstrating promise as potential pharmaceutical agents for the treatment of cardiovascular disease, diabetes, inflammation, and kidney disease. The present study determined the ability of a first-inclass sEHI, AR9281, to decrease blood pressure, improve vascular function, and decrease renal inflammation and injury in angiotensin hypertension. Rats were infused with angiotensin and AR9281 was given orally during the 14-day infusion period. Systolic blood pressure averaged 180 ± 5 mmHg in vehicle treated and AR9281 treatment significantly lowered blood pressure to 142 ± 7 mmHg in angiotensin hypertension. Histological analysis demonstrated decreased injury to the juxtamedullary glomeruli. Renal expression of inflammatory genes was increased in angiotensin hypertension and two weeks of AR9281 treatment decreased this index of renal inflammation. Vascular function in angiotensin hypertension was also improved by AR9281 treatment. Decreased afferent arteriolar and mesenteric resistance endothelial dependent dilator responses were ameliorated by AR9281 treatment of angiotensin hypertensive rats. These data demonstrate that the first-in-class sEHI, AR9281, lowers blood pressure, improves vascular function and reduces renal damage in angiotensin hypertension.

The Vital Function of Fe3O4@Au nanocomposites for Hydrolase Biosensor Design and Its Application in Detection of Methyl Parathion

Energy Technology Data Exchange (ETDEWEB)

Zhao, Yuting; Zhang, Weiying; Lin, Yuehe; Du, Dan

2013-02-04

A nanocomposite of gold nanoparticles (AuNPs) decorating a magnetic Fe3O4 core was synthesized using cysteamine (SH–NH2) as linker, and characterized by TEM, XPS, UV and electrochemistry. Then a hydrolase biosensor, based on self-assembly of methyl parathion hydrolase (MPH) on the Fe3O4@Au nanocomposite, was developed for sensitive and selective detection of the organophosphorus pesticide (OP) methyl parathion. The magnetic nanocomposite provides an easy way to construct the enzyme biosensor by simply exerting an external magnetic field, and also provides a simple way to renew the electrode surface by removing the magnet. Unlike inhibition-based enzyme biosensors, the hydrolase is not poisoned by OPs and thus is reusable for continuous measurement. AuNPs not only provide a large surface area, high loading efficiency and fast electron transfer, but also stabilize the enzyme through electrostatic interactions. The MPH biosensor shows rapid response and high selectivity for detection of methyl parathion, with a linear range from 0.5 to 1000 ng/mL and a detection limit of 0.1 ng/mL. It also shows acceptable reproducibility and stability. The simplicity and ease of operation of the proposed method has great potential for on-site detection of P–S containing pesticides and provides a promising strategy to construct a robust biosensor.

Inhibition of soluble epoxide hydrolase attenuates hepatic fibrosis and endoplasmic reticulum stress induced by carbon tetrachloride in mice

International Nuclear Information System (INIS)

Harris, Todd R.; Bettaieb, Ahmed; Kodani, Sean; Dong, Hua; Myers, Richard; Chiamvimonvat, Nipavan; Haj, Fawaz G.; Hammock, Bruce D.

2015-01-01

Liver fibrosis is a pathological condition in which chronic inflammation and changes to the extracellular matrix lead to alterations in hepatic tissue architecture and functional degradation of the liver. Inhibitors of the enzyme soluble epoxide hydrolase (sEH) reduce fibrosis in the heart, pancreas and kidney in several disease models. In this study, we assess the effect of sEH inhibition on the development of fibrosis in a carbon tetrachloride (CCl 4 )-induced mouse model by monitoring changes in the inflammatory response, matrix remolding and endoplasmic reticulum stress. The sEH inhibitor 1-trifluoromethoxyphenyl-3-(1-propionylpiperidin-4-yl) urea (TPPU) was administered in drinking water. Collagen deposition in the liver was increased five-fold in the CCl 4 -treated group, and this was returned to control levels by TPPU treatment. Hepatic expression of Col1a2 and 3a1 mRNA was increased over fifteen-fold in the CCl 4 -treated group relative to the Control group, and this increase was reduced by 50% by TPPU treatment. Endoplasmic reticulum (ER) stress observed in the livers of CCl 4 -treated animals was attenuated by TPPU treatment. In order to support the hypothesis that TPPU is acting to reduce the hepatic fibrosis and ER stress through its action as a sEH inhibitor we used a second sEH inhibitor, trans-4-(4-[3-(4-trifluoromethoxy-phenyl)-ureido]-cyclohexyloxy)-benzoic acid (t-TUCB), and sEH null mice. Taken together, these data indicate that the sEH may play an important role in the development of hepatic fibrosis induced by CCl 4 , presumably by reducing endogenous fatty acid epoxide chemical mediators acting to reduce ER stress. - Highlights: • We administer an inhibitor of sEH in a CCl4 murine model. • sEH inhibition reduces liver collagen deposition and pro-fibrotic gene expression. • sEH inhibition induces MMP-1a activity

Inhibition of soluble epoxide hydrolase attenuates hepatic fibrosis and endoplasmic reticulum stress induced by carbon tetrachloride in mice

Energy Technology Data Exchange (ETDEWEB)

Harris, Todd R. [Department of Entomology and Comprehensive Cancer Center, University of California, Davis, CA 95616 (United States); Bettaieb, Ahmed [Department of Nutrition, University of California, Davis, CA 95616 (United States); Kodani, Sean; Dong, Hua [Department of Entomology and Comprehensive Cancer Center, University of California, Davis, CA 95616 (United States); Myers, Richard; Chiamvimonvat, Nipavan [Department of Internal Medicine: Cardiovascular, University of California, Davis, CA 95616 (United States); Haj, Fawaz G. [Department of Nutrition, University of California, Davis, CA 95616 (United States); Department of Internal Medicine: Endocrinology, Diabetes and Metabolism, University of California, Davis, CA 95616 (United States); Hammock, Bruce D., E-mail: bdhammock@ucdavis.edu [Department of Entomology and Comprehensive Cancer Center, University of California, Davis, CA 95616 (United States)

2015-07-15

Liver fibrosis is a pathological condition in which chronic inflammation and changes to the extracellular matrix lead to alterations in hepatic tissue architecture and functional degradation of the liver. Inhibitors of the enzyme soluble epoxide hydrolase (sEH) reduce fibrosis in the heart, pancreas and kidney in several disease models. In this study, we assess the effect of sEH inhibition on the development of fibrosis in a carbon tetrachloride (CCl{sub 4})-induced mouse model by monitoring changes in the inflammatory response, matrix remolding and endoplasmic reticulum stress. The sEH inhibitor 1-trifluoromethoxyphenyl-3-(1-propionylpiperidin-4-yl) urea (TPPU) was administered in drinking water. Collagen deposition in the liver was increased five-fold in the CCl{sub 4}-treated group, and this was returned to control levels by TPPU treatment. Hepatic expression of Col1a2 and 3a1 mRNA was increased over fifteen-fold in the CCl{sub 4}-treated group relative to the Control group, and this increase was reduced by 50% by TPPU treatment. Endoplasmic reticulum (ER) stress observed in the livers of CCl{sub 4}-treated animals was attenuated by TPPU treatment. In order to support the hypothesis that TPPU is acting to reduce the hepatic fibrosis and ER stress through its action as a sEH inhibitor we used a second sEH inhibitor, trans-4-(4-[3-(4-trifluoromethoxy-phenyl)-ureido]-cyclohexyloxy)-benzoic acid (t-TUCB), and sEH null mice. Taken together, these data indicate that the sEH may play an important role in the development of hepatic fibrosis induced by CCl{sub 4}, presumably by reducing endogenous fatty acid epoxide chemical mediators acting to reduce ER stress. - Highlights: • We administer an inhibitor of sEH in a CCl4 murine model. • sEH inhibition reduces liver collagen deposition and pro-fibrotic gene expression. • sEH inhibition induces MMP-1a activity.

[Discover potential inhibitors of 5-LOX and LTA4H from Rhei Radix et Rhizoma, Notopterygii Rhizoma et Radix and Genitana Macrophyllae Radix based on molecular simulation methods].

Science.gov (United States)

Gu, Yu; Zhang, Xu; Chen, Yan-Kun; Zhao, Bo-Wen; Zhang, Yan-Ling

2017-12-01

5-lipoxygenase (5-LOX) and leukotriene A4 hydrolase (LTA4H), as the major targets of 5-LOX branch in the arachidonic acid (AA) metabolic pathway, play an important role in the treatment of inflammation. Rhei Radix et Rhizoma, Notopterygii Rhizoma et Radix and Genitana Macrophyllae Radix have clear anti-inflammation activities. In this paper, the targets of 5-LOX and LTA4H were used as the research carrier, and Hiphop module in DS4.0 (Discovery studio) was used to construct ingredients database for preliminary screening of three traditional Chinese medicines based on target inhibitor pharmacophore, so as to obtain 5-LOX and LTA4H potential active ingredients. The ingredients obtained in initial pharmacophore screening were further screened by using CDOCKER module, and the screening rules were established based on the score of initial compound and the key amino acids to obtain 12 potential 5-LOX inhibitors and 7 potential LTA4H inhibitors. To be more specific, the potential 5-LOX inhibitors included 6 ingredients in Rhei Radix et Rhizoma, such as procyanidins B2-3,3'-O-double gallate and revandchinone 2; four ingredients in notopterygium, such as dodecanoic acid and so on. On the other hand, potential LTA4H inhibitors included revandchinone 1, revandchinone 4 in Rhei Radix et Rhizoma, tridecanoic acid, tetracosanoic acid and methyl eicosanoate in Notopterygii Rhizoma et Radix, montanic acid methyl ester and N-docosanoyl-O-aminobenzoate in Genitana Macrophyllae Radix and so on. The molecular simulation methods were highly efficient and time-saving to obtain the potential inhibitors of 5-LOX and LTA4H, which could provide assistance for discovering the chemical quality indicators of anti-inflammatory efficacy of three Chinese herbs, and may be helpful to promote the whole-process quality control of three Chinese herbs. Copyright© by the Chinese Pharmaceutical Association.

A comparison of effects of DPP-4 inhibitor and SGLT2 inhibitor on lipid profile in patients with type 2 diabetes.

Science.gov (United States)

Cha, Seon-Ah; Park, Yong-Moon; Yun, Jae-Seung; Lim, Tae-Seok; Song, Ki-Ho; Yoo, Ki-Dong; Ahn, Yu-Bae; Ko, Seung-Hyun

2017-04-13

Previous studies suggest that dipeptidyl peptidase-4 (DPP-4) inhibitors and sodium glucose cotransporter 2 (SGLT2) inhibitors have different effects on the lipid profile in patients with type 2 diabetes. We investigated the effects of DPP-4 inhibitors and SGLT2 inhibitors on the lipid profile in patients with type 2 diabetes. From January 2013 to December 2015, a total of 228 patients with type 2 diabetes who were receiving a DPP-4 inhibitor or SGLT2 inhibitor as add-on therapy to metformin and/or a sulfonylurea were consecutively enrolled. We compared the effects of DPP-4 inhibitors and SGLT2 inhibitors on the lipid profile at baseline and after 24 weeks of treatment. To compare lipid parameters between the two groups, we used the analysis of covariance (ANCOVA). A total of 184 patients completed follow-up (mean age: 53.1 ± 6.9 years, mean duration of diabetes: 7.1 ± 5.7 years). From baseline to 24 weeks, HDL-cholesterol (HDL-C) levels were increased by 0.5 (95% CI, -0.9 to 2.0) mg/dl with a DPP-4 inhibitor and by 5.1 (95% CI, 3.0 to 7.1) mg/dl with an SGLT2 inhibitor (p = 0.001). LDL-cholesterol (LDL-C) levels were reduced by 8.4 (95% CI, -14.0 to -2.8) mg/dl with a DPP-4 inhibitor, but increased by 1.3 (95% CI, -5.1 to 7.6) mg/dl with an SGLT2 inhibitor (p = 0.046). There was no significant difference in the mean hemoglobin A1c (8.3 ± 1.1 vs. 8.0 ± 0.9%, p = 0.110) and in the change of total cholesterol (TC) (p = 0.836), triglyceride (TG) (p = 0.867), apolipoprotein A (p = 0.726), apolipoprotein B (p = 0.660), and lipoprotein (a) (p = 0.991) between the DPP-4 inhibitor and the SGLT2 inhibitor. The SGLT2 inhibitor was associated with a significant increase in HDL-C and LDL-C after 24 weeks of SGLT2 inhibitor treatment in patients with type 2 diabetes compared with those with DPP-4 inhibitor treatment in this study. This study was conducted by retrospective medical record review.

Renal Effects of DPP-4 Inhibitors: A Focus on Microalbuminuria

Directory of Open Access Journals (Sweden)

Martin Haluzík

2013-01-01

Full Text Available Incretin-based therapies represent one of the most promising options in type 2 diabetes treatment owing to their good effectiveness with low risk of hypoglycemia and no weight gain. Other numerous potential beneficial effects of incretin-based therapies have been suggested based mostly on experimental and small clinical studies including its beta-cell- and vasculo-protective actions. One of the recently emerged interesting features of dipeptidyl peptidase-4 (DPP-4 inhibitors is its possible protective effect on the diabetic kidney disease. Here, we review the renal effects of DPP-4 inhibitors with special focus on its influence on the onset and progression of microalbuminuria, as presence of microalbuminuria represents an important early sign of kidney damage and is also associated with increased risk of hypoglycemia and cardiovascular complications. Mechanisms underlying possible nephroprotective properties of DPP-4 inhibitors include reduction of oxidative stress and inflammation and improvement of endothelial dysfunction. Effects of DPP-4 inhibitors may be both glucagon-like peptide-1 (GLP-1 dependent and independent. Ongoing prospective studies focused on the nephroprotective effects of DPP-4 inhibitors will further clarify its possible role in the prevention/attenuation of diabetic kidney disease beyond its glucose lowering properties.

DIPEPTIDYL PEPTIDASE 4 (DPP-4 INHIBITORS FOR THE TREATMENT OF TYPE 2 DIABETES MELLITUS

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Erna Kristin

2016-12-01

Diabetes mellitus (DM merupakan penyakit kronis yang menyebabkan sekitar 1,5 juta kematian pada tahun 2012 menurut Organisasi Kesehatan Dunia (WHO. DM tipe 2 (DMT2 banyaknya 90% dari keseluruhan DM di seluruh dunia. Prevalensi DMT2 meningkat karena obesitas. Pedoman klinis merekomendasikan penggunaan metformin sebagai pengobatan lini pertama kecuali ada kontraindikasi, maka bisa diikuti dengan penambahan 1 atau 2 OADs, seperti sulfonilurea (SU, inhibitor alpha-glucosidase, atau thiazolidinediones (TZD. Baru-baru ini, obat baru golongan dipeptidyl peptidase 4 (DPP-4 inhibitor telah ditambahkan ke algoritma pengobatan. Dipeptidyl peptidase 4 (DPP-4 inhibitor inhibitor adalah kelas obat antidiabetes oral yang menghambat DPP-4 enzim. Sitagliptin, saxagliptin, vildagliptin dan linagliptin yang merupakan golongan dipeptidyl peptidase-4 (DPP-4 inhibitor tersedia untuk pengobatan diabetes tipe 2 di Indonesia dan banyak negara lainnya. DPP-4 inhibitor memiliki khasiat glikemik yang setara. DPP-4 inhibitor menghasilkan peningkatan moderat hemoglobin terglikasi (A1C. Namun uji coba head-to-head jumlahnya terbatas, dan tidak ada data tentang penggunaan penggunaan jangka panjang (lebih dari dua tahun keamanan, kematian, komplikasi diabetes, atau kualitas-hidup pasien. Meskipun DPP-inhibitor tidak digunakan sebagai terapi awal untuk mayoritas pasien dengan diabetes tipe 2, DPP-4 inhibitor dapat digunakan sebagai terapi tambahan di tipe 2 pasien diabetes yang tidak toleran, ada kontraindikasi, atau tidak terkontrol dengan penggunaan metformin, sulfonilurea, atau thiazolidinediones. Peran sebenarnya dari DPP-4 inhibitor di antara beberapa obat lainnya untuk DMT2 tidak begitu jelas. Hanya ada sejumlah kecil studi jangka panjang pada DPP-4 inhibitor menilai penurunan glikemik, kemanjuran, kejadian kardiovaskular, kematian, atau keamanan. Pada pasien dengan gagal ginjal (perkiraan laju filtrasi glomerulus [eGFR] <30 mL / menit kronis dapat menggunakan DPP-4 inhibitor, linagliptin

Further characterization of intestinal lactase/phlorizin hydrolase

DEFF Research Database (Denmark)

Skovbjerg, H; Norén, O; Sjöström, H

1982-01-01

Pig intestinal lactase/phlorizin hydrolase (EC 3.2.1.23/62) was purified in its amphiphilic form by immunoadsorbent chromatography. The purified enzyme was free of other known brush border enzymes and appeared homogeneous in immunoelectrophoresis and polyacrylamide gel electrophoresis in the pres......Pig intestinal lactase/phlorizin hydrolase (EC 3.2.1.23/62) was purified in its amphiphilic form by immunoadsorbent chromatography. The purified enzyme was free of other known brush border enzymes and appeared homogeneous in immunoelectrophoresis and polyacrylamide gel electrophoresis...... in the presence of SDS. Pig lactase/phlorizin hydrolase was shown to have the same quaternary structure as the human enzyme, i.e., built up of two polypeptides of the same molecular weight (160000). In addition to hydrolyzing lactose, phlorizin and a number of synthetic substrates, both the human and the pig...... membranes (basolateral and intracellular membranes) exhibited in SDS-polyacrylamide gel electrophoresis the same size of constituent polypeptides and the same catalytic and immunological properties as a normal brush border lactase/phlorizin hydrolase....

Design of Selective Substrates and Activity-Based Probes for Hydrolase Important for Pathogenesis 1 (HIP1) from Mycobacterium tuberculosis.

Science.gov (United States)

Lentz, Christian S; Ordonez, Alvaro A; Kasperkiewicz, Paulina; La Greca, Florencia; O'Donoghue, Anthony J; Schulze, Christopher J; Powers, James C; Craik, Charles S; Drag, Marcin; Jain, Sanjay K; Bogyo, Matthew

2016-11-11

Although serine proteases are important mediators of Mycobacterium tuberculosis (Mtb) virulence, there are currently no tools to selectively block or visualize members of this family of enzymes. Selective reporter substrates or activity-based probes (ABPs) could provide a means to monitor infection and response to therapy using imaging methods. Here, we use a combination of substrate selectivity profiling and focused screening to identify optimized reporter substrates and ABPs for the Mtb "Hydrolase important for pathogenesis 1" (Hip1) serine protease. Hip1 is a cell-envelope-associated enzyme with minimal homology to host proteases, making it an ideal target for probe development. We identified substituted 7-amino-4-chloro-3-(2-bromoethoxy)isocoumarins as irreversible inhibitor scaffolds. Furthermore, we used specificity data to generate selective reporter substrates and to further optimize a selective chloroisocoumarin inhibitor. These new reagents are potentially useful in delineating the roles of Hip1 during pathogenesis or as diagnostic imaging tools for specifically monitoring Mtb infections.

The Nonglycemic Actions of Dipeptidyl Peptidase-4 Inhibitors

Directory of Open Access Journals (Sweden)

Na-Hyung Kim

2014-01-01

Full Text Available A cell surface serine protease, dipeptidyl peptidase 4 (DPP-4, cleaves dipeptide from peptides containing proline or alanine in the N-terminal penultimate position. Two important incretin hormones, glucagon-like peptide-1 (GLP-1 and glucose-dependent insulinotropic peptide (GIP, enhance meal-stimulated insulin secretion from pancreatic β-cells, but are inactivated by DPP-4. Diabetes and hyperglycemia increase the DPP-4 protein level and enzymatic activity in blood and tissues. In addition, multiple other functions of DPP-4 suggest that DPP-4 inhibitor, a new class of antidiabetic agents, may have pleiotropic effects. Studies have shown that DPP-4 itself is involved in the inflammatory signaling pathway, the stimulation of vascular smooth cell proliferation, and the stimulation of oxidative stress in various cells. DPP-4 inhibitor ameliorates these pathophysiologic processes and has been shown to have cardiovascular protective effects in both in vitro and in vivo experiments. However, in recent randomized clinical trials, DPP-4 inhibitor therapy in high risk patients with type 2 diabetes did not show cardiovascular protective effects. Some concerns on the actions of DPP-4 inhibitor include sympathetic activation and neuropeptide Y-mediated vascular responses. Further studies are required to fully characterize the cardiovascular effects of DPP-4 inhibitor.

S-Adenosyl-L-Homocysteine Hydrolase Inhibition by a Synthetic Nicotinamide Cofactor Biomimetic

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Lyn L. Kailing

2018-03-01

Full Text Available S-adenosyl-L-homocysteine (SAH hydrolases (SAHases are involved in the regulation of methylation reactions in many organisms and are thus crucial for numerous cellular functions. Consequently, their dysregulation is associated with severe health problems. The SAHase-catalyzed reaction is reversible and both directions depend on the redox activity of nicotinamide adenine dinucleotide (NAD+ as a cofactor. Therefore, nicotinamide cofactor biomimetics (NCB are a promising tool to modulate SAHase activity. In the present in vitro study, we investigated 10 synthetic truncated NAD+ analogs against a SAHase from the root-nodulating bacterium Bradyrhizobium elkanii. Among this set of analogs, one was identified to inhibit the SAHase in both directions. Isothermal titration calorimetry (ITC and crystallography experiments suggest that the inhibitory effect is not mediated by a direct interaction with the protein. Neither the apo-enzyme (i.e., deprived of the natural cofactor, nor the holo-enzyme (i.e., in the NAD+-bound state were found to bind the inhibitor. Yet, enzyme kinetics point to a non-competitive inhibition mechanism, where the inhibitor acts on both, the enzyme and enzyme-SAH complex. Based on our experimental results, we hypothesize that the NCB inhibits the enzyme via oxidation of the enzyme-bound NADH, which may be accessible through an open molecular gate, leaving the enzyme stalled in a configuration with oxidized cofactor, where the reaction intermediate can be neither converted nor released. Since the reaction mechanism of SAHase is quite uncommon, this kind of inhibition could be a viable pharmacological route, with a low risk of off-target effects. The NCB presented in this work could be used as a template for the development of more potent SAHase inhibitors.

Characterization of an epoxide hydrolase from the Florida red tide dinoflagellate, Karenia brevis.

Science.gov (United States)

Sun, Pengfei; Leeson, Cristian; Zhi, Xiaoduo; Leng, Fenfei; Pierce, Richard H; Henry, Michael S; Rein, Kathleen S

2016-02-01

Epoxide hydrolases (EH, EC 3.3.2.3) have been proposed to be key enzymes in the biosynthesis of polyether (PE) ladder compounds such as the brevetoxins which are produced by the dinoflagellate Karenia brevis. These enzymes have the potential to catalyze kinetically disfavored endo-tet cyclization reactions. Data mining of K. brevis transcriptome libraries revealed two classes of epoxide hydrolases: microsomal and leukotriene A4 (LTA4) hydrolases. A microsomal EH was cloned and expressed for characterization. The enzyme is a monomeric protein with molecular weight 44kDa. Kinetic parameters were evaluated using a variety of epoxide substrates to assess substrate selectivity and enantioselectivity, as well as its potential to catalyze the critical endo-tet cyclization of epoxy alcohols. Monitoring of EH activity in high and low toxin producing cultures of K. brevis over a three week period showed consistently higher activity in the high toxin producing culture implicating the involvement of one or more EH in brevetoxin biosynthesis. Copyright © 2015 Elsevier Ltd. All rights reserved.

Annotation and comparative analysis of the glycoside hydrolase genes in Brachypodium distachyon

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Wu Jiajie

2010-10-01

Full Text Available Abstract Background Glycoside hydrolases cleave the bond between a carbohydrate and another carbohydrate, a protein, lipid or other moiety. Genes encoding glycoside hydrolases are found in a wide range of organisms, from archea to animals, and are relatively abundant in plant genomes. In plants, these enzymes are involved in diverse processes, including starch metabolism, defense, and cell-wall remodeling. Glycoside hydrolase genes have been previously cataloged for Oryza sativa (rice, the model dicotyledonous plant Arabidopsis thaliana, and the fast-growing tree Populus trichocarpa (poplar. To improve our understanding of glycoside hydrolases in plants generally and in grasses specifically, we annotated the glycoside hydrolase genes in the grasses Brachypodium distachyon (an emerging monocotyledonous model and Sorghum bicolor (sorghum. We then compared the glycoside hydrolases across species, at the levels of the whole genome and individual glycoside hydrolase families. Results We identified 356 glycoside hydrolase genes in Brachypodium and 404 in sorghum. The corresponding proteins fell into the same 34 families that are represented in rice, Arabidopsis, and poplar, helping to define a glycoside hydrolase family profile which may be common to flowering plants. For several glycoside hydrolase familes (GH5, GH13, GH18, GH19, GH28, and GH51, we present a detailed literature review together with an examination of the family structures. This analysis of individual families revealed both similarities and distinctions between monocots and eudicots, as well as between species. Shared evolutionary histories appear to be modified by lineage-specific expansions or deletions. Within GH families, the Brachypodium and sorghum proteins generally cluster with those from other monocots. Conclusions This work provides the foundation for further comparative and functional analyses of plant glycoside hydrolases. Defining the Brachypodium glycoside hydrolases sets

Poly(aspartic acid) (PAA) hydrolases and PAA biodegradation: current knowledge and impact on applications.

Science.gov (United States)

Hiraishi, Tomohiro

2016-02-01

Thermally synthesized poly(aspartic acid) (tPAA) is a bio-based, biocompatible, biodegradable, and water-soluble polymer that has a high proportion of β-Asp units and equivalent moles of D- and L-Asp units. Poly(aspartic acid) (PAA) hydrolase-1 and hydrolase-2 are tPAA biodegradation enzymes purified from Gram-negative bacteria. PAA hydrolase-1 selectively cleaves amide bonds between β-Asp units via an endo-type process, whereas PAA hydrolase-2 catalyzes the exo-type hydrolysis of the products of tPAA hydrolysis by PAA hydrolase-1. The novel reactivity of PAA hydrolase-1 makes it a good candidate for a biocatalyst in β-peptide synthesis. This mini-review gives an overview of PAA hydrolases with emphasis on their biochemical and functional properties, in particular, PAA hydrolase-1. Functionally related enzymes, such as poly(R-3-hydroxybutyrate) depolymerases and β-aminopeptidases, are compared to PAA hydrolases. This mini-review also provides findings that offer an insight into the catalytic mechanisms of PAA hydrolase-1 from Pedobacter sp. KP-2.

Development and properties of a wax ester hydrolase in the cotyledons of jojoba seedlings.

Science.gov (United States)

Huang, A H; Moreau, R A; Liu, K D

1978-03-01

The activity of a wax ester hydrolase in the cotyledons of jojoba (Simmondsia chinensis) seedlings increased drastically during germination, parallel to the development of the gluconeogenic process. The enzyme at its peak of development was obtained in association with the wax body membrane, and its properties were studied. It had an optimal activity at alkaline pH (8.5-9). The apparent K(m) value for N-methylindoxylmyristate was 93 muM. It was stable at 40 C for 30 min but was inactivated at higher temperature. Various divalent cations and ethylenediaminetetraacetate had little effect on the activity. p-Chloromercuribenzoate was a strong inhibitor of the enzyme activity, and its effect was reversed by subsequent addition of dithiothreitol. It had a broad substrate specificity with highest activities on monoglycerides, wax esters, and the native substrate (jojoba wax).

Use of full recovery hydrolasing equipment for facility decommissioning - 16325

International Nuclear Information System (INIS)

Martin, Scott A.; Adams, Scott R.

2009-01-01

The removal of surface contamination is a major challenge for nearly all nuclear facilities undergoing, or awaiting, decommissioning. Conventional means of surface decontamination can expose workers to unnecessary hazards, and are often not fit-for-purpose due to size constraints or weight restrictions. Additionally, conventional methods are not always easily deployed remotely due to their complexity or required services. The use of ultra high pressure water for surface decontamination, known as hydrolasing, is recognized as a technology which can be used in various applications requiring surface removal. Hydrolasing is an advantageous technology for many reasons including its versatility, overall simplicity and relative ease of remote deployment. For the nuclear industry, one of the largest challenges with regards to the use of hydrolasing is the requirement for the full recovery of the injected water and removed solids. For nonnuclear applications, there is often no requirement for recovery of the liquid and solid waste, which has led to few system designs which will recover the waste in full. S.A. Robotics' experience with the deployment of ultra high pressure water systems for nuclear applications has shown that full recovery of injected water and removed solids is achievable in both underwater and in-air applications. Innovative equipment and system design have allowed S.A. Robotics' hydrolasing systems to achieve near 100% solid and liquid recovery during concrete hydrolasing. This technology has been deployed for Fluor Hanford at Hanford's K-Basins, as well as for UKAEA as part of the Windscale Piles decommissioning project. The purpose of this paper is to provide a short description of the hydrolasing process and the associated waste issues, describe the unique design features of S.A. Robotics' hydrolasing systems which combat these issues, and provide an overview of two of the hydrolasing projects that S.A. Robotics has completed. (authors)

Hydrolase activity in Jerusalem artichoke and chicory

Energy Technology Data Exchange (ETDEWEB)

Klaushofer, H.; Abraham, B.; Leichtfried, G.

1988-03-01

Post-harvest storage of chicory and Jerusalem artichoke and overwintering of Jerusalem artichoke in the soil cause a more or less pronounced shortening of the fructan chain, depending on the variety. The proportion of fructose in the total fructan thus shifts towards glucose. This reduction on the fructose/glucose ratio is undesirable if the intention is to obtain a sweetener of high fructose content. In this work an attempt was made, via the quantity of fructose formed after a 4(3)-hour reaction of a tuber (root) extract with inulin, to assign a characteristic value to the depolymerization tendency of the material in question. However, since the plant extract not only contains enzymes (hydrolase A and B) that shorten the fructan chains but the activity of fructosyltransferase (SST, FFT) and enzymes of microbial origin (inulinase II, invertase) must also be considered, the concept of 'hydrolase activity' used by the authors is essentially an expression of 'total activity'. The activity unit (EU) is defined as the ability to split of 1 ..mu..mol of fructose from (chicory) inulin per minute under experimental conditions. Values of 0.25 to 0.77 EU/g dry solids were found in Jerusalem artichoke. Considerable differences may occur between varieties from the same cultivated area and the same harvest period. With one and the same variety, the activity appears to be subject to marked yearly fluctuations, so that at present, because of hydrolase activity, nothing certain can be said about the depolymerization tendency of a variety.

Efficacy of S-adenosylhomocysteine hydrolase inhibitors, D-eritadenine and (S)-DHPA, against the growth of Cryptosporidium parvum in vitro

Czech Academy of Sciences Publication Activity Database

Čtrnáctá, Vlasta; Fritzler, J. M.; Šurínová, M.; Hrdý, I.; Zhu, G.; Stejskal, F.

2010-01-01

Roč. 126, č. 2 (2010), s. 113-116 ISSN 0014-4894 Institutional research plan: CEZ:AV0Z50520701 Keywords : S-adenosylhomocysteine hydrolase * D-eritadenine * (S)-DHPA Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 1.869, year: 2010

The dual FAAH/MAGL inhibitor JZL195 has enhanced effects on endocannabinoid transmission and motor behavior in rats as compared to those of the MAGL inhibitor JZL184

OpenAIRE

Seillier, Alexandre; Aguilar, David Dominguez; Giuffrida, Andrea

2014-01-01

The biological actions of the endocannabinoids anandamide and 2-arachidonoyl glycerol (2-AG) are terminated by enzymatic hydrolysis of these lipids via fatty acid amide hydrolase (FAAH ) and monoacylglycerol lipase (MAGL), respectively. While several selective FAAH inhibitors have been developed and characterized in vitro and in vivo, none of the initial MAGL blockers have shown adequate potency and specificity for in vivo applications. More recently, a selective MAGL inhibitor, JZL184, has b...

Lipoxin Generation Is Related to Soluble Epoxide Hydrolase Activity in Severe Asthma

Science.gov (United States)

Ono, Emiko; Dutile, Stefanie; Kazani, Shamsah; Wechsler, Michael E.; Yang, Jun; Hammock, Bruce D.; Douda, David Nobuhiro; Tabet, Yacine; Khaddaj-Mallat, Rayan; Sirois, Marco; Sirois, Chantal; Rizcallah, Edmond; Rousseau, Éric; Martin, Richard; Sutherland, E. Rand; Castro, Mario; N. Jarjour, Nizar; Israel, Elliot

2014-01-01

Rationale: Severe asthma is characterized by airway inflammatory responses associated with aberrant metabolism of arachidonic acid. Lipoxins (LX) are arachidonate-derived pro-resolving mediators that are decreased in severe asthma, yet mechanisms for defective LX biosynthesis and a means to increase LXs in severe asthma remain to be established. Objectives: To determine if oxidative stress and soluble epoxide hydrolase (sEH) activity are linked to decreased LX biosynthesis in severe asthma. Methods: Aliquots of blood, sputum, and bronchoalveolar lavage fluid were obtained from asthma subjects for mediator determination. Select samples were exposed to t-butyl-hydroperoxide or sEH inhibitor (sEHI) before activation. Peripheral blood leukocyte–platelet aggregates were monitored by flow cytometry, and bronchial contraction was determined with cytokine-treated human lung sections. Measurements and Main Results: 8-Isoprostane levels in sputum supernatants were inversely related to LXA4 in severe asthma (r = −0.55; P = 0.03) and t-butyl-hydroperoxide decreased LXA4 and 15-epi-LXA4 biosynthesis by peripheral blood leukocytes. LXA4 and 15-epi-LXA4 levels were inversely related to sEH activity in sputum supernatants and sEHIs significantly increased 14,15-epoxy-eicosatrienoic acid and 15-epi-LXA4 generation by severe asthma whole blood and bronchoalveolar lavage fluid cells. The abundance of peripheral blood leukocyte–platelet aggregates was related to asthma severity. In a concentration-dependent manner, LXs significantly inhibited platelet-activating factor–induced increases in leukocyte–platelet aggregates (70.8% inhibition [LXA4 100 nM], 78.3% inhibition [15-epi-LXA4 100 nM]) and 15-epi-LXA4 markedly inhibited tumor necrosis factor-α–induced increases in bronchial contraction. Conclusions: LX levels were decreased by oxidative stress and sEH activity. Inhibitors of sEH increased LXs that mediated antiphlogistic actions, suggesting a new therapeutic approach

Alogliptin: a new addition to the class of DPP-4 inhibitors

Directory of Open Access Journals (Sweden)

Radha Andukuri

2009-07-01

Full Text Available Radha Andukuri, Andjela Drincic, Marc RendellDivision of Endocrinology, Department of Medicine, Creighton University School of Medicine, Omaha, Nebraska, USABackground: Alogliptin is an oral antihyperglycemic agent that is a selective inhibitor of the enzyme dipeptidyl peptidase-4 (DPP-4. Inhibition of DPP-4 elevates levels of the incretin hormones glucagon-like peptide (GLP-1 and glucose-dependent insulinotropic polypeptide (GIP by preventing their degradation.Objective: To review the evolution of alogliptin and its pharmacokinetics, pharmacodynamics, clinical efficacy and adverse effects. In addition, we compared alogliptin to other DPP-4 inhibitors.Methods: A comprehensive literature search was performed using the term ‘alogliptin’. Original research articles and review articles as well as scientific abstracts were included. Results: Alogliptin raises postprandial levels of GLP-1. It has excellent bioavailability exhibiting a median Tmax ranging from 1 to 2 hours and a mean half-life of 12.4 to 21.4 hours across all doses. When given as monotherapy, mean hemoglobin A1c (HbA1c reductions achieved were 0.5% to 0.6%. Combination therapy yielded similar reductions (−0.5% with metformin, −0.6% with glyburide, −0.8% with pioglitazone and –0.6% with insulin. Administration of alogliptin does not promote weight loss but has not resulted in weight gain. The agent is relatively well tolerated with few adverse effects, the major finding being a marginally higher rate of skin events, primarily pruritus.Conclusions: Alogliptin causes significant reductions in HbA1c when used alone or in combination with other oral agents in patients with type 2 diabetes similar to other DPP-4 inhibitors in current clinical use. The side effect profile also does not differ from that of other DPP-4 inhibitors. However, long-term studies are necessary before the place of alogliptin in the management of type 2 diabetes can be established.Keywords: alogliptin, DPP

How to find soluble proteins: a comprehensive analysis of alpha/beta hydrolases for recombinant expression in E. coli

Directory of Open Access Journals (Sweden)

Barth Sandra

2005-04-01

Full Text Available Abstract Background In screening of libraries derived by expression cloning, expression of active proteins in E. coli can be limited by formation of inclusion bodies. In these cases it would be desirable to enrich gene libraries for coding sequences with soluble gene products in E. coli and thus to improve the efficiency of screening. Previously Wilkinson and Harrison showed that solubility can be predicted from amino acid composition (Biotechnology 1991, 9(5:443–448. We have applied this analysis to members of the alpha/beta hydrolase fold family to predict their solubility in E. coli. alpha/beta hydrolases are a highly diverse family with more than 1800 proteins which have been grouped into homologous families and superfamilies. Results The predicted solubility in E. coli depends on hydrolase size, phylogenetic origin of the host organism, the homologous family and the superfamily, to which the hydrolase belongs. In general small hydrolases are predicted to be more soluble than large hydrolases, and eukaryotic hydrolases are predicted to be less soluble in E. coli than prokaryotic ones. However, combining phylogenetic origin and size leads to more complex conclusions. Hydrolases from prokaryotic, fungal and metazoan origin are predicted to be most soluble if they are of small, medium and large size, respectively. We observed large variations of predicted solubility between hydrolases from different homologous families and from different taxa. Conclusion A comprehensive analysis of all alpha/beta hydrolase sequences allows more efficient screenings for new soluble alpha/beta hydrolases by the use of libraries which contain more soluble gene products. Screening of hydrolases from families whose members are hard to express as soluble proteins in E. coli should first be done in coding sequences of organisms from phylogenetic groups with the highest average of predicted solubility for proteins of this family. The tools developed here can be used

Glycoside hydrolase gene transcription by Alicyclobacillus acidocaldarius during growth on wheat arabinoxylan and monosaccharides: a proposed xylan hydrolysis mechanism

Energy Technology Data Exchange (ETDEWEB)

Lee, Brady D.; Apel, William A.; Sheridan, Peter P.; DeVeaux, Linda C.

2018-04-16

Background Metabolism of carbon bound in wheat arabinoxylan (WAX) polysaccharides by bacteria requires a number of glycoside hydrolases active toward different bonds between sugars and other molecules. Alicyclobacillus acidocaldarius is a Gram-positive thermoacidophilic bacterium capable of growth on a variety of mono-, di-, oligo-, and polysaccharides. Nineteen proposed glycoside hydrolases have been annotated in the A. acidocaldarius Type Strain ATCC27009/DSM 446 genome. Results Molecular analysis using high-density oligonucleotide microarrays was performed on A. acidocaldarius strain ATCC27009 when growing on WAX. When a culture growing exponentially at the expense of arabinoxylan saccharides was challenged with glucose or xylose, most glycoside hydrolases were down-regulated. Interestingly, regulation was more intense when xylose was added to the culture than when glucose was added, a clear departure from classical carbon catabolite repression demonstrated by many Gram-positive bacteria. In silico analyses of the regulated glycoside hydrolases, along with the results from the microarray analyses, yielded a potential mechanism for arabinoxylan metabolism by A. acidocaldarius. Glycoside hydrolases expressed by this strain may have broad substrate specificity, and initial hydrolysis is catalyzed by an extracellular xylanase, while subsequent steps are likely performed inside the growing cell. Conclusions Glycoside hydrolases, for the most part, appear to be found in clusters, throughout the A. acidocaldarius genome. Not all of the glycoside hydrolase genes found at loci within these clusters were regulated during the experiment, indicating that a specific subset of the 19 glycoside hydrolase genes found in A. acidocaldarius were used during metabolism of WAX. While specific functions of the glycoside hydrolases was not tested as part of the research discussed, many of the glycoside hydrolases found in the A. acidocaldarius Type Strain appear to have a broader

Development and Properties of a Wax Ester Hydrolase in the Cotyledons of Jojoba Seedlings 1

Science.gov (United States)

Huang, Anthony H. C.; Moreau, Robert A.; Liu, Kitty D. F.

1978-01-01

The activity of a wax ester hydrolase in the cotyledons of jojoba (Simmondsia chinensis) seedlings increased drastically during germination, parallel to the development of the gluconeogenic process. The enzyme at its peak of development was obtained in association with the wax body membrane, and its properties were studied. It had an optimal activity at alkaline pH (8.5-9). The apparent Km value for N-methylindoxylmyristate was 93 μM. It was stable at 40 C for 30 min but was inactivated at higher temperature. Various divalent cations and ethylenediaminetetraacetate had little effect on the activity. p-Chloromercuribenzoate was a strong inhibitor of the enzyme activity, and its effect was reversed by subsequent addition of dithiothreitol. It had a broad substrate specificity with highest activities on monoglycerides, wax esters, and the native substrate (jojoba wax). PMID:16660288

A possible usage of a CDK4 inhibitor for breast cancer stem cell-targeted therapy

International Nuclear Information System (INIS)

Han, Yu Kyeong; Lee, Jae Ho; Park, Ga-Young; Chun, Sung Hak; Han, Jeong Yun; Kim, Sung Dae; Lee, Janet; Lee, Chang-Woo; Yang, Kwangmo; Lee, Chang Geun

2013-01-01

Highlights: ► A CDK4 inhibitor may be used for breast cancer stem cell-targeted therapy. ► The CDK4 inhibitor differentiated the cancer stem cell population (CD24 − /CD44 + ) of MDA-MB-231. ► The differentiation of the cancer stem cells by the CDK4 inhibitor radiosensitized MDA-MB-231. -- Abstract: Cancer stem cells (CSCs) are one of the main reasons behind cancer recurrence due to their resistance to conventional anti-cancer therapies. Thus, many efforts are being devoted to developing CSC-targeted therapies to overcome the resistance of CSCs to conventional anti-cancer therapies and decrease cancer recurrence. Differentiation therapy is one potential approach to achieve CSC-targeted therapies. This method involves inducing immature cancer cells with stem cell characteristics into more mature or differentiated cancer cells. In this study, we found that a CDK4 inhibitor sensitized MDA-MB-231 cells but not MCF7 cells to irradiation. This difference appeared to be associated with the relative percentage of CSC-population between the two breast cancer cells. The CDK4 inhibitor induced differentiation and reduced the cancer stem cell activity of MDA-MB-231 cells, which are shown by multiple marker or phenotypes of CSCs. Thus, these results suggest that radiosensitization effects may be caused by reducing the CSC-population of MDA-MB-231 through the use of the CDK4 inhibitor. Thus, further investigations into the possible application of the CDK4 inhibitor for CSC-targeted therapy should be performed to enhance the efficacy of radiotherapy for breast cancer

ClbS Is a Cyclopropane Hydrolase That Confers Colibactin Resistance.

Science.gov (United States)

Tripathi, Prabhanshu; Shine, Emilee E; Healy, Alan R; Kim, Chung Sub; Herzon, Seth B; Bruner, Steven D; Crawford, Jason M

2017-12-13

Certain commensal Escherichia coli contain the clb biosynthetic gene cluster that codes for small molecule prodrugs known as precolibactins. Precolibactins are converted to colibactins by N-deacylation; the latter are postulated to be genotoxic and to contribute to colorectal cancer formation. Though advances toward elucidating (pre)colibactin biosynthesis have been made, the functions and mechanisms of several clb gene products remain poorly understood. Here we report the 2.1 Å X-ray structure and molecular function of ClbS, a gene product that confers resistance to colibactin toxicity in host bacteria and which has been shown to be important for bacterial viability. The structure harbors a potential colibactin binding site and shares similarity to known hydrolases. In vitro studies using a synthetic colibactin analog and ClbS or an active site residue mutant reveal cyclopropane hydrolase activity that converts the electrophilic cyclopropane of the colibactins into an innocuous hydrolysis product. As the cyclopropane has been shown to be essential for genotoxic effects in vitro, this ClbS-catalyzed ring-opening provides a means for the bacteria to circumvent self-induced genotoxicity. Our study provides a molecular-level view of the first reported cyclopropane hydrolase and support for a specific mechanistic role of this enzyme in colibactin resistance.

A remote but significant sequence homology between glycoside hydrolase clan GH-H and glycoside hydrolase family GH 31

DEFF Research Database (Denmark)

Janecek, S.; Svensson, Birte; MacGregor, E.A.

2007-01-01

Although both the α-amylase super-family, i.e. the glycoside hydrolase (GH) clan GH-H (the GH families 13, 70 and 77), and family GH31 share some characteristics, their different catalytic machinery prevents classification of GH31 in clan GH-H. A significant but remote evolutionary relatedness is...

Scaffold-hopping from xanthines to tricyclic guanines: A case study of dipeptidyl peptidase 4 (DPP4) inhibitors

Energy Technology Data Exchange (ETDEWEB)

Pissarnitski, Dmitri A.; Zhao, Zhiqiang; Cole, David; Wu, Wen-Lian; Domalski, Martin; Clader, John W.; Scapin, Giovanna; Voigt, Johannes; Soriano, Aileen; Kelly, Theresa; Powles, Mary Ann; Yao, Zuliang; Burnett, Duane A. (Merck)

2016-11-01

Molecular modeling of unbound tricyclic guanine scaffolds indicated that they can serve as effective bioisosteric replacements of xanthines. This notion was further confirmed by a combination of X-ray crystallography and SAR studies, indicating that tricyclic guanine DPP4 inhibitors mimic the binding mode of xanthine inhibitors, exemplified by linagliptin. Realization of the bioisosteric relationship between these scaffolds potentially will lead to a wider application of cyclic guanines as xanthine replacements in drug discovery programs for a variety of biological targets. Newly designed DPP4 inhibitors achieved sub-nanomolar potency range and demonstrated oral activity in vivo in mouse glucose tolerance test.

Inhibition of Xenobiotic-Degrading Hydrolases by Organophosphinates

Science.gov (United States)

1986-07-01

M 4 Q r 000 44 Table 11. Purification of arylester hydrolase Specific Total Total Activity Volume Activity Proteina (Umoles/ Purifi- Fraction (mL...did get re-adjusted after the sample was applied. After the sample was applied the column was washed with the above MES buffer an.+eluted with 100 ml...Lieske (94) and compared them to the reversed phase HPLC retention times we have previously reported (16). We get an excellent linear correlation

Identification of epoxybergamottin as a CYP3A4 inhibitor in grapefruit peel.

Science.gov (United States)

Wangensteen, H; Molden, E; Christensen, H; Malterud, K E

2003-02-01

The oral availability of many drugs metabolised by the enzyme cytochrome P(450) 3A4 (CYP3A4) is increased if co-administered with grapefruit juice. Extracts from grapefruit peel have also demonstrated inhibitory activity and, during commercial manufacturing of grapefruit juice, inhibitory components might be squeezed into the juice from the peel. Thus, the aim of this in vitro study was to identify CYP3A4 inhibitors in grapefruit peel. Grapefruit peel was extracted with diethyl ether, and the extract was further fractionated by normal-phase chromatography. Fractions demonstrating significant CYP3A4 inhibitory activity, as measured by the relative reduction in N-demethylation of diltiazem in transfected human liver epithelial cells, were subsequently separated by preparative thin-layer chromatography. Constituents of the fractions and isolated compounds were identified by nuclear magnetic resonance spectroscopy. Analysis of diltiazem and N-demethyl-diltiazem was performed using high-performance liquid chromatography. Of the identified components in grapefruit peel, only epoxybergamottin demonstrated a concentration-dependent inhibition of the CYP3A4-mediated N-demethylation of diltiazem. The IC(50) value was calculated to be 4.2+/-1.1 micro M. Coumarins without the furan ring and flavonoids isolated from grapefruit peel did not interfere with the metabolism of diltiazem. The results indicated the presence of other CYP3A4 inhibitors in grapefruit peel, but these agents were lost during the purification process excluding their identification. The furanocoumarin epoxybergamottin, present in grapefruit peel, is an inhibitor of CYP3A4. In commercial manufacturing of grapefruit juice, epoxybergamottin is possibly distributed into the juice. During manufacturing, however, epoxybergamottin may be hydrolysed to 6',7'-dihydroxybergamottin, which has been suggested as an important CYP3A4 inhibitor in grapefruit juice.

Compositional profile of α/β-hydrolase fold proteins in mangrove soil metagenomes: prevalence of epoxide hydrolases and haloalkane dehalogenases in oil-contaminated sites

Science.gov (United States)

Jiménez, Diego Javier; Dini-Andreote, Francisco; Ottoni, Júlia Ronzella; de Oliveira, Valéria Maia; van Elsas, Jan Dirk; Andreote, Fernando Dini

2015-01-01

The occurrence of genes encoding biotechnologically relevant α/β-hydrolases in mangrove soil microbial communities was assessed using data obtained by whole-metagenome sequencing of four mangroves areas, denoted BrMgv01 to BrMgv04, in São Paulo, Brazil. The sequences (215 Mb in total) were filtered based on local amino acid alignments against the Lipase Engineering Database. In total, 5923 unassembled sequences were affiliated with 30 different α/β-hydrolase fold superfamilies. The most abundant predicted proteins encompassed cytosolic hydrolases (abH08; ∼ 23%), microsomal hydrolases (abH09; ∼ 12%) and Moraxella lipase-like proteins (abH04 and abH01; mangroves BrMgv01-02-03. This suggested selection and putative involvement in local degradation/detoxification of the pollutants. Seven sequences that were annotated as genes for putative epoxide hydrolases and five for putative haloalkane dehalogenases were found in a fosmid library generated from BrMgv02 DNA. The latter enzymes were predicted to belong to Actinobacteria, Deinococcus-Thermus, Planctomycetes and Proteobacteria. Our integrated approach thus identified 12 genes (complete and/or partial) that may encode hitherto undescribed enzymes. The low amino acid identity (< 60%) with already-described genes opens perspectives for both production in an expression host and genetic screening of metagenomes. PMID:25171437

Enantioselectivity of a recombinant epoxide hydrolase from Agrobacterium radiobacter

NARCIS (Netherlands)

Lutje Spelberg, Jeffrey H.; Rink, Rick; Kellogg, Richard M.; Janssen, Dick B.

1998-01-01

The recombinant epoxide hydrolase from Agrobacterium radiobacter AD1 was used to obtain enantiomerically pure epoxides by means of a kinetic resolution. Epoxides such as styrene oxide and various derivatives thereof and phenyl glycidyl ether were obtained in high enantiomeric excess and in

Characterization of fatty acid amide hydrolase activity by a fluorescence-based assay.

Science.gov (United States)

Dato, Florian M; Maaßen, Andreas; Goldfuß, Bernd; Pietsch, Markus

2018-04-01

Fatty acid amide hydrolase (FAAH) is involved in many human diseases, particularly cancer, pain and inflammation as well as neurological, metabolic and cardiovascular disorders. Therefore, FAAH is an attractive target for the development of low-molecular-weight inhibitors as therapeutics, which requires robust assays that can be used for high-throughput screening (HTS) of compound libraries. Here, we report the development of a fluorometric assay based on FAAH's ability to effectively hydrolyze medium-chain fatty acid amides, introducing N-decanoyl-substituted 5-amino-2-methoxypyridine (D-MAP) as new amide substrate. D-MAP is cleaved by FAAH with an 8-fold larger specificity constant than the previously reported octanoyl-analog Oc-MAP (V max /K m of 1.09 and 0.134 mL min -1 mg -1 , respectively), with both MAP derivatives possessing superior substrate properties and much increased aqueous solubility compared to the respective p-nitroaniline compounds D-pNA and Oc-pNA. The new assay with D-MAP as substrate is highly sensitive using a lower enzyme concentration (1 μg mL -1 ) than literature-reported fluorimetric FAAH assays. In addition, D-MAP was validated in comparison to the substrate Oc-MAP for the characterization of FAAH inhibitors by means of the reference compounds URB597 and TC-F2 and was shown to be highly suitable for HTS in both kinetic and endpoint assays (Z' factors of 0.81 and 0.78, respectively). Copyright © 2018 Elsevier Inc. All rights reserved.

Structural analysis of Clostridium acetobutylicum ATCC 824 glycoside hydrolase from CAZy family GH105

International Nuclear Information System (INIS)

Germane, Katherine L.; Servinsky, Matthew D.; Gerlach, Elliot S.; Sund, Christian J.; Hurley, Margaret M.

2015-01-01

The crystal structure of the protein product of the C. acetobutylicum ATCC 824 gene CA-C0359 is structurally similar to YteR, an unsaturated rhamnogalacturonyl hydrolase from B. subtilis strain 168. Substrate modeling and electrostatic studies of the active site of the structure of CA-C0359 suggests that the protein can now be considered to be part of CAZy glycoside hydrolase family 105. Clostridium acetobutylicum ATCC 824 gene CA-C0359 encodes a putative unsaturated rhamnogalacturonyl hydrolase (URH) with distant amino-acid sequence homology to YteR of Bacillus subtilis strain 168. YteR, like other URHs, has core structural homology to unsaturated glucuronyl hydrolases, but hydrolyzes the unsaturated disaccharide derivative of rhamnogalacturonan I. The crystal structure of the recombinant CA-C0359 protein was solved to 1.6 Å resolution by molecular replacement using the phase information of the previously reported structure of YteR (PDB entry (http://scripts.iucr.org/cgi-bin/cr.cgi?rm)) from Bacillus subtilis strain 168. The YteR-like protein is a six-α-hairpin barrel with two β-sheet strands and a small helix overlaying the end of the hairpins next to the active site. The protein has low primary protein sequence identity to YteR but is structurally similar. The two tertiary structures align with a root-mean-square deviation of 1.4 Å and contain a highly conserved active pocket. There is a conserved aspartic acid residue in both structures, which has been shown to be important for hydration of the C=C bond during the release of unsaturated galacturonic acid by YteR. A surface electrostatic potential comparison of CA-C0359 and proteins from CAZy families GH88 and GH105 reveals the make-up of the active site to be a combination of the unsaturated rhamnogalacturonyl hydrolase and the unsaturated glucuronyl hydrolase from Bacillus subtilis strain 168. Structural and electrostatic comparisons suggests that the protein may have a slightly different substrate

Structural analysis of Clostridium acetobutylicum ATCC 824 glycoside hydrolase from CAZy family GH105

Energy Technology Data Exchange (ETDEWEB)

Germane, Katherine L., E-mail: katherine.germane.civ@mail.mil [Oak Ridge Associated Universities, 4692 Millennium Drive, Suite 101, Belcamp, MD 21017 (United States); Servinsky, Matthew D. [US Army Research Laboratory, 2800 Powder Mill Road, Adelphi, MD 20783 (United States); Gerlach, Elliot S. [Federal Staffing Resources, 2200 Somerville Road, Annapolis, MD 21401 (United States); Sund, Christian J. [US Army Research Laboratory, 2800 Powder Mill Road, Adelphi, MD 20783 (United States); Hurley, Margaret M., E-mail: katherine.germane.civ@mail.mil [US Army Research Laboratory, 4600 Deer Creek Loop, Aberdeen Proving Ground, MD 21005 (United States); Oak Ridge Associated Universities, 4692 Millennium Drive, Suite 101, Belcamp, MD 21017 (United States)

2015-07-29

The crystal structure of the protein product of the C. acetobutylicum ATCC 824 gene CA-C0359 is structurally similar to YteR, an unsaturated rhamnogalacturonyl hydrolase from B. subtilis strain 168. Substrate modeling and electrostatic studies of the active site of the structure of CA-C0359 suggests that the protein can now be considered to be part of CAZy glycoside hydrolase family 105. Clostridium acetobutylicum ATCC 824 gene CA-C0359 encodes a putative unsaturated rhamnogalacturonyl hydrolase (URH) with distant amino-acid sequence homology to YteR of Bacillus subtilis strain 168. YteR, like other URHs, has core structural homology to unsaturated glucuronyl hydrolases, but hydrolyzes the unsaturated disaccharide derivative of rhamnogalacturonan I. The crystal structure of the recombinant CA-C0359 protein was solved to 1.6 Å resolution by molecular replacement using the phase information of the previously reported structure of YteR (PDB entry (http://scripts.iucr.org/cgi-bin/cr.cgi?rm)) from Bacillus subtilis strain 168. The YteR-like protein is a six-α-hairpin barrel with two β-sheet strands and a small helix overlaying the end of the hairpins next to the active site. The protein has low primary protein sequence identity to YteR but is structurally similar. The two tertiary structures align with a root-mean-square deviation of 1.4 Å and contain a highly conserved active pocket. There is a conserved aspartic acid residue in both structures, which has been shown to be important for hydration of the C=C bond during the release of unsaturated galacturonic acid by YteR. A surface electrostatic potential comparison of CA-C0359 and proteins from CAZy families GH88 and GH105 reveals the make-up of the active site to be a combination of the unsaturated rhamnogalacturonyl hydrolase and the unsaturated glucuronyl hydrolase from Bacillus subtilis strain 168. Structural and electrostatic comparisons suggests that the protein may have a slightly different substrate

Selective inhibition of plant serine hydrolases by agrochemicals revealed by competitive ABPP.

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Kaschani, Farnusch; Nickel, Sabrina; Pandey, Bikram; Cravatt, Benjamin F; Kaiser, Markus; van der Hoorn, Renier A L

2012-01-15

Organophosphate and -phosphonates and their thio derivatives are often used in agroindustry as herbicides and insecticides, but their potential off-targets in the plant are poorly investigated. Here, we use competitive activity-based protein profiling (ABPP) of serine hydrolases (SHs) to detect targets of these agrochemicals and other compounds in Arabidopsis thaliana. Using broad-range and specific probes, and by overexpression of various SHs in planta, we are able to confirm eight SH-compound interactions, including selective inhibition of carboxylesterase CXE12, prolyloligopeptidase, methylesterase MES2 and tripeptidyl peptidase TPP2. These observations can be used for the design of novel probes and selective inhibitors and may help to assess physiological effects of agrochemicals on crop plants. Copyright © 2011 Elsevier Ltd. All rights reserved.

Comparison between SGLT2 inhibitors and DPP4 inhibitors added to insulin therapy in type 2 diabetes: a systematic review with indirect comparison meta-analysis.

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Min, Se Hee; Yoon, Jeong-Hwa; Hahn, Seokyung; Cho, Young Min

2017-01-01

Both sodium glucose cotransporter 2 (SGLT2) inhibitors and dipeptidyl peptidase-4 (DPP4) inhibitors can be used to treat patients with type 2 diabetes mellitus (T2DM) that is inadequately controlled with insulin therapy, and yet there has been no direct comparison of these two inhibitors. We searched MEDLINE, EMBASE, LILACS, the Cochrane Central Register of Controlled Trials and ClinicalTrials.gov through June 2015. Randomized controlled trials published in English that compare SGLT2 inhibitor plus insulin (SGLT2i/INS) with placebo plus insulin or DPP4 inhibitor plus insulin (DPP4i/INS) with placebo plus insulin in patients with T2DM were selected. Data on the study characteristics, efficacy and safety outcomes were extracted. We compared the efficacy and safety between SGLT2i/INS and DPP4i/INS indirectly with covariates adjustment. Risk of potential bias was assessed. Fourteen eligible randomized controlled trials comprising 6980 patients were included (five SGLT2 inhibitor studies and nine DPP4 inhibitor studies). Covariate-adjusted indirect comparison using meta-regression analyses revealed that SGLT2i/INS achieved greater reduction in HbA 1c [weighted mean difference (WMD) -0.24%, 95% confidence interval (CI) -0.43 to -0.05%], fasting plasma glucose (WMD -18.0 mg/dL, 95% CI -28.5 to -7.6 mg/dL) and body weight (WMD -2.38 kg, 95% CI -3.18 to -1.58 kg) from baseline than DPP4i/INS without increasing the risk of hypoglycaemia (relative risks 1.19, 95% CI 0.78 to 1.82). Sodium glucose cotransporter 2 inhibitors achieved better glycaemic control and greater weight reduction than DPP4 inhibitors without increasing the risk of hypoglycaemia in patients with T2DM that is inadequately controlled with insulin. There has been no direct comparison of SGLT2 inhibitors and DPP4 inhibitors in patients with T2DM inadequately controlled with insulin therapy. In this study, we performed indirect meta-analysis comparing SGLT2 inhibitors and DPP4 inhibitors added to insulin

V3-independent competitive resistance of a dual-X4 HIV-1 to the CXCR4 inhibitor AMD3100.

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Yosuke Maeda

Full Text Available A CXCR4 inhibitor-resistant HIV-1 was isolated from a dual-X4 HIV-1 in vitro. The resistant variant displayed competitive resistance to the CXCR4 inhibitor AMD3100, indicating that the resistant variant had a higher affinity for CXCR4 than that of the wild-type HIV-1. Amino acid sequence analyses revealed that the resistant variant harbored amino acid substitutions in the V2, C2, and C4 regions, but no remarkable changes in the V3 loop. Site-directed mutagenesis confirmed that the changes in the C2 and C4 regions were principally involved in the reduced sensitivity to AMD3100. Furthermore, the change in the C4 region was associated with increased sensitivity to soluble CD4, and profoundly enhanced the entry efficiency of the virus. Therefore, it is likely that the resistant variant acquired the higher affinity for CD4/CXCR4 by the changes in non-V3 regions. Taken together, a CXCR4 inhibitor-resistant HIV-1 can evolve using a non-V3 pathway.

Phosphodiesterase 4 inhibitors for chronic obstructive pulmonary disease.

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Chong, Jimmy; Leung, Bonnie; Poole, Phillippa

2017-09-19

Chronic obstructive pulmonary disease (COPD) is associated with cough, sputum production or dyspnoea and a reduction in lung function, quality of life and life expectancy. Apart from smoking cessation, there are no other treatments that slow lung function decline. Roflumilast and cilomilast are oral phosphodiesterase 4 (PDE 4 ) inhibitors proposed to reduce the airway inflammation and bronchoconstriction seen in COPD. This is an update of a Cochrane review first published in 2011 and updated in 2013. To evaluate the efficacy and safety of oral PDE 4 inhibitors in the management of stable COPD. We identified randomised controlled trials (RCTs) from the Cochrane Airways Trials Register (date of last search October 2016). We found other trials from web-based clinical trials registers. We included RCTs if they compared oral PDE 4 inhibitors with placebo in people with COPD. We allowed co-administration of standard COPD therapy. One review author extracted data and a second review author checked the data. We reported pooled data in Review Manager as mean differences (MD), standardised mean differences (SMD) or odds ratios (OR). We converted the odds ratios into absolute treatment effects in a 'Summary of findings' table. Thirty-four separate RCTs studying roflumilast (20 trials with 17,627 participants) or cilomilast (14 trials with 6457 participants) met the inclusion criteria, with a duration of between six weeks and one year. These included people across international study centres with moderate to very severe COPD (Global Initiative for Chronic Obstructive Lung Disease (GOLD) grades II-IV), with a mean age of 64 years.We considered that the methodological quality of the 34 published and unpublished trials was acceptable overall. Treatment with a PDE 4 inhibitor was associated with a significant improvement in forced expiratory volume in one second (FEV 1 ) over the trial period compared with placebo (MD 51.53 mL, 95% confidence interval (CI) 43.17 to 59.90, 27

Degradation of Polyester Polyurethane by Bacterial Polyester Hydrolases

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Juliane Schmidt

2017-02-01

Full Text Available Polyurethanes (PU are widely used synthetic polymers. The growing amount of PU used industrially has resulted in a worldwide increase of plastic wastes. The related environmental pollution as well as the limited availability of the raw materials based on petrochemicals requires novel solutions for their efficient degradation and recycling. The degradation of the polyester PU Impranil DLN by the polyester hydrolases LC cutinase (LCC, TfCut2, Tcur1278 and Tcur0390 was analyzed using a turbidimetric assay. The highest hydrolysis rates were obtained with TfCut2 and Tcur0390. TfCut2 also showed a significantly higher substrate affinity for Impranil DLN than the other three enzymes, indicated by a higher adsorption constant K. Significant weight losses of the solid thermoplastic polyester PU (TPU Elastollan B85A-10 and C85A-10 were detected as a result of the enzymatic degradation by all four polyester hydrolases. Within a reaction time of 200 h at 70 °C, LCC caused weight losses of up to 4.9% and 4.1% of Elastollan B85A-10 and C85A-10, respectively. Gel permeation chromatography confirmed a preferential degradation of the larger polymer chains. Scanning electron microscopy revealed cracks at the surface of the TPU cubes as a result of enzymatic surface erosion. Analysis by Fourier transform infrared spectroscopy indicated that the observed weight losses were a result of the cleavage of ester bonds of the polyester TPU.

Autolysis of dairy leuconostocs and detection of peptidoglycan hydrolases by renaturing SDS-PAGE.

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Cibik, R; Chapot-Chartier, M P

2000-11-01

The autolysis of lactic acid bacteria plays a major role during cheese ripening. The aim of this study was to evaluate the autolytic properties and peptidoglycan hydrolase content of dairy leuconostocs. Autolysis of 59 strains of dairy Leuconostoc was examined under starvation conditions in potassium phosphate buffer. The ability of dairy leuconostocs to lyse is strain dependant and not related to the species. The peptidoglycan hydrolase profile of Leuc. mesenteroides subsp. mesenteroides 10L was analysed by renaturing gel electrophoresis. Two major activity bands migrating at 41 and 52 kDa were observed. According to the specificity analysis, strain 10L seems to contain a glycosidase and an N-acetyl-muramyl-L-alanine amidase, or an endopeptidase. The peptidoglycan hydrolase profiles of various Leuconostoc species were also compared. Several peptidoglycan hydrolase activities could be detected in the different Leuconostoc species. Further characterization of the peptidoglycan hydrolases will help to control autolysis of leuconostocs in cheese.

Purification, crystallization and preliminary crystallographic studies of plant S-adenosyl-l-homocysteine hydrolase (Lupinus luteus)

Energy Technology Data Exchange (ETDEWEB)

Brzezinski, Krzysztof [Center for Biocrystallographic Research, Institute of Bioorganic Chemistry, Polish Academy of Sciences, Poznan (Poland); Department of Crystallography, Faculty of Chemistry, A. Mickiewicz University, Poznan (Poland); Bujacz, Grzegorz [Center for Biocrystallographic Research, Institute of Bioorganic Chemistry, Polish Academy of Sciences, Poznan (Poland); Faculty of Food Chemistry and Biotechnology, Technical University of Lodz (Poland); Jaskolski, Mariusz, E-mail: mariuszj@amu.edu.pl [Center for Biocrystallographic Research, Institute of Bioorganic Chemistry, Polish Academy of Sciences, Poznan (Poland); Department of Crystallography, Faculty of Chemistry, A. Mickiewicz University, Poznan (Poland)

2008-07-01

Single crystals of recombinant S-adenosyl-l-homocysteine hydrolase from L. luteus in complex with adenosine diffract X-rays to 1.17 Å resolution at 100 K. The crystals are tetragonal, space group P4{sub 3}2{sub 1}2, and contain one copy of the dimeric enzyme in the asymmetric unit. By degrading S-adenosyl-l-homocysteine, which is a byproduct of S-adenosyl-l-methionine-dependent methylation reactions, S-adenosyl-l-homocysteine hydrolase (SAHase) acts as a regulator of cellular methylation processes. S-Adenosyl-l-homocysteine hydrolase from the leguminose plant yellow lupin (Lupinus luteus), LlSAHase, which is composed of 485 amino acids and has a molecular weight of 55 kDa, has been cloned, expressed in Escherichia coli and purified. Crystals of LlSAHase in complex with adenosine were obtained by the hanging-drop vapour-diffusion method using 20%(w/v) PEG 4000 and 10%(v/v) 2-propanol as precipitants in 0.1 M Tris–HCl buffer pH 8.0. The crystals were tetragonal, space group P4{sub 3}2{sub 1}2, with unit-cell parameters a = 122.4, c = 126.5 Å and contained two protein molecules in the asymmetric unit, corresponding to the functional dimeric form of the enzyme. Atomic resolution (1.17 Å) X-ray diffraction data have been collected using synchrotron radiation.

Purification, crystallization and preliminary crystallographic studies of plant S-adenosyl-l-homocysteine hydrolase (Lupinus luteus)

International Nuclear Information System (INIS)

Brzezinski, Krzysztof; Bujacz, Grzegorz; Jaskolski, Mariusz

2008-01-01

Single crystals of recombinant S-adenosyl-l-homocysteine hydrolase from L. luteus in complex with adenosine diffract X-rays to 1.17 Å resolution at 100 K. The crystals are tetragonal, space group P4 3 2 1 2, and contain one copy of the dimeric enzyme in the asymmetric unit. By degrading S-adenosyl-l-homocysteine, which is a byproduct of S-adenosyl-l-methionine-dependent methylation reactions, S-adenosyl-l-homocysteine hydrolase (SAHase) acts as a regulator of cellular methylation processes. S-Adenosyl-l-homocysteine hydrolase from the leguminose plant yellow lupin (Lupinus luteus), LlSAHase, which is composed of 485 amino acids and has a molecular weight of 55 kDa, has been cloned, expressed in Escherichia coli and purified. Crystals of LlSAHase in complex with adenosine were obtained by the hanging-drop vapour-diffusion method using 20%(w/v) PEG 4000 and 10%(v/v) 2-propanol as precipitants in 0.1 M Tris–HCl buffer pH 8.0. The crystals were tetragonal, space group P4 3 2 1 2, with unit-cell parameters a = 122.4, c = 126.5 Å and contained two protein molecules in the asymmetric unit, corresponding to the functional dimeric form of the enzyme. Atomic resolution (1.17 Å) X-ray diffraction data have been collected using synchrotron radiation

Epoxide hydrolase-lasalocid a structure provides mechanistic insight into polyether natural product biosynthesis.

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Wong, Fong T; Hotta, Kinya; Chen, Xi; Fang, Minyi; Watanabe, Kenji; Kim, Chu-Young

2015-01-14

Biosynthesis of some polyether natural products involves a kinetically disfavored epoxide-opening cyclic ether formation, a reaction termed anti-Baldwin cyclization. One such example is the biosynthesis of lasalocid A, an ionophore antibiotic polyether. During lasalocid A biosynthesis, an epoxide hydrolase, Lsd19, converts the bisepoxy polyketide intermediate into the tetrahydrofuranyl-tetrahydropyran product. We report the crystal structure of Lsd19 in complex with lasalocid A. The structure unambiguously shows that the C-terminal domain of Lsd19 catalyzes the intriguing anti-Baldwin cyclization. We propose a general mechanism for epoxide selection by ionophore polyether epoxide hydrolases.

Activity-based protein profiling reveals off-target proteins of the FAAH inhibitor BIA 10-2474

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van Esbroeck, Annelot C M; Janssen, Antonius P A; Cognetta, Armand B; Ogasawara, Daisuke; Shpak, Guy; van der Kroeg, Mark; Kantae, Vasudev; Baggelaar, Marc P; de Vrij, Femke M S; Deng, Hui; Allarà, Marco; Fezza, Filomena; Lin, Zhanmin; van der Wel, Tom; Soethoudt, Marjolein; Mock, Elliot D; den Dulk, Hans; Baak, Ilse L; Florea, Bogdan I; Hendriks, Giel; De Petrocellis, Luciano; Overkleeft, Herman S; Hankemeier, Thomas; De Zeeuw, Chris I; Di Marzo, Vincenzo; Maccarrone, Mauro; Cravatt, Benjamin F; Kushner, Steven A; van der Stelt, Mario

2017-01-01

A recent phase 1 trial of the fatty acid amide hydrolase (FAAH) inhibitor BIA 10-2474 led to the death of one volunteer and produced mild-to-severe neurological symptoms in four others. Although the cause of the clinical neurotoxicity is unknown, it has been postulated, given the clinical safety

Activity of xyloglucan endotransglucosylases/hydrolases suggests a role during host invasion by the parasitic plant Cuscuta reflexa.

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Olsen, Stian; Krause, Kirsten

2017-01-01

The parasitic vines of the genus Cuscuta form haustoria that grow into other plants and connect with their vascular system, thus allowing the parasite to feed on its host. A major obstacle that meets the infection organ as it penetrates the host tissue is the rigid plant cell wall. In the present study, we examined the activity of xyloglucan endotransglucosylases/hydrolases (XTHs) during the host-invasive growth of the haustorium. The level of xyloglucan endotransglucosylation (XET) activity was found to peak at the penetrating stage of Cuscuta reflexa on its host Pelargonium zonale. In vivo colocalization of XET activity and donor substrate demonstrated XET activity at the border between host and parasite. A test for secretion of XET-active enzymes from haustoria of C. reflexa corroborated this and further indicated that the xyloglucan-modifying enzymes originated from the parasite. A known inhibitor of XET, Coomassie Brilliant Blue R250, was shown to reduce the level of XET in penetrating haustoria of C. reflexa. Moreover, the coating of P. zonale petioles with the inhibitor compound lowered the number of successful haustorial invasions of this otherwise compatible host plant. The presented data indicate that the activity of Cuscuta XTHs at the host-parasite interface is essential to penetration of host plant tissue.

Lysophosphatidic acids are new substrates for the phosphatase domain of soluble epoxide hydrolase.

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Oguro, Ami; Imaoka, Susumu

2012-03-01

Soluble epoxide hydrolase (sEH) is a bifunctional enzyme that has a C-terminus epoxide hydrolase domain and an N-terminus phosphatase domain. The endogenous substrates of epoxide hydrolase are known to be epoxyeicosatrienoic acids, but the endogenous substrates of the phosphatase activity are not well understood. In this study, to explore the substrates of sEH, we investigated the inhibition of the phosphatase activity of sEH toward 4-methylumbelliferyl phosphate by using lecithin and its hydrolyzed products. Although lecithin itself did not inhibit the phosphatase activity, the hydrolyzed lecithin significantly inhibited it, suggesting that lysophospholipid or fatty acid can inhibit it. Next, we investigated the inhibition of phosphatase activity by lysophosphatidyl choline, palmitoyl lysophosphatidic acid, monopalmitoyl glycerol, and palmitic acid. Palmitoyl lysophosphatidic acid and fatty acid efficiently inhibited phosphatase activity, suggesting that lysophosphatidic acids (LPAs) are substrates for the phosphatase activity of sEH. As expected, palmitoyl, stearoyl, oleoyl, and arachidonoyl LPAs were efficiently dephosphorylated by sEH (Km, 3-7 μM; Vmax, 150-193 nmol/min/mg). These results suggest that LPAs are substrates of sEH, which may regulate physiological functions of cells via their metabolism.

SVMDLF: A novel R-based Web application for prediction of dipeptidyl peptidase 4 inhibitors.

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Chandra, Sharat; Pandey, Jyotsana; Tamrakar, Akhilesh K; Siddiqi, Mohammad Imran

2017-12-01

Dipeptidyl peptidase 4 (DPP4) is a well-known target for the antidiabetic drugs. However, currently available DPP4 inhibitor screening assays are costly and labor-intensive. It is important to create a robust in silico method to predict the activity of DPP4 inhibitor for the new lead finding. Here, we introduce an R-based Web application SVMDLF (SVM-based DPP4 Lead Finder) to predict the inhibitor of DPP4, based on support vector machine (SVM) model, predictions of which are confirmed by in vitro biological evaluation. The best model generated by MACCS structure fingerprint gave the Matthews correlation coefficient of 0.87 for the test set and 0.883 for the external test set. We screened Maybridge database consisting approximately 53,000 compounds. For further bioactivity assay, six compounds were shortlisted, and of six hits, three compounds showed significant DPP4 inhibitory activities with IC 50 values ranging from 8.01 to 10.73 μm. This application is an OpenCPU server app which is a novel single-page R-based Web application for the DPP4 inhibitor prediction. The SVMDLF is freely available and open to all users at http://svmdlf.net/ocpu/library/dlfsvm/www/ and http://www.cdri.res.in/svmdlf/. © 2017 John Wiley & Sons A/S.

Identification, structure, and function of a novel type VI secretion peptidoglycan glycoside hydrolase effector-immunity pair.

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Whitney, John C; Chou, Seemay; Russell, Alistair B; Biboy, Jacob; Gardiner, Taylor E; Ferrin, Michael A; Brittnacher, Mitchell; Vollmer, Waldemar; Mougous, Joseph D

2013-09-13

Bacteria employ type VI secretion systems (T6SSs) to facilitate interactions with prokaryotic and eukaryotic cells. Despite the widespread identification of T6SSs among Gram-negative bacteria, the number of experimentally validated substrate effector proteins mediating these interactions remains small. Here, employing an informatics approach, we define novel families of T6S peptidoglycan glycoside hydrolase effectors. Consistent with the known intercellular self-intoxication exhibited by the T6S pathway, we observe that each effector gene is located adjacent to a hypothetical open reading frame encoding a putative periplasmically localized immunity determinant. To validate our sequence-based approach, we functionally investigate a representative family member from the soil-dwelling bacterium Pseudomonas protegens. We demonstrate that this protein is secreted in a T6SS-dependent manner and that it confers a fitness advantage in growth competition assays with Pseudomonas putida. In addition, we determined the 1.4 Å x-ray crystal structure of this effector in complex with its cognate immunity protein. The structure reveals the effector shares highest overall structural similarity to a glycoside hydrolase family associated with peptidoglycan N-acetylglucosaminidase activity, suggesting that T6S peptidoglycan glycoside hydrolase effector families may comprise significant enzymatic diversity. Our structural analyses also demonstrate that self-intoxication is prevented by the immunity protein through direct occlusion of the effector active site. This work significantly expands our current understanding of T6S effector diversity.

Identification, Structure, and Function of a Novel Type VI Secretion Peptidoglycan Glycoside Hydrolase Effector-Immunity Pair*

Science.gov (United States)

Whitney, John C.; Chou, Seemay; Russell, Alistair B.; Biboy, Jacob; Gardiner, Taylor E.; Ferrin, Michael A.; Brittnacher, Mitchell; Vollmer, Waldemar; Mougous, Joseph D.

2013-01-01

Bacteria employ type VI secretion systems (T6SSs) to facilitate interactions with prokaryotic and eukaryotic cells. Despite the widespread identification of T6SSs among Gram-negative bacteria, the number of experimentally validated substrate effector proteins mediating these interactions remains small. Here, employing an informatics approach, we define novel families of T6S peptidoglycan glycoside hydrolase effectors. Consistent with the known intercellular self-intoxication exhibited by the T6S pathway, we observe that each effector gene is located adjacent to a hypothetical open reading frame encoding a putative periplasmically localized immunity determinant. To validate our sequence-based approach, we functionally investigate a representative family member from the soil-dwelling bacterium Pseudomonas protegens. We demonstrate that this protein is secreted in a T6SS-dependent manner and that it confers a fitness advantage in growth competition assays with Pseudomonas putida. In addition, we determined the 1.4 Å x-ray crystal structure of this effector in complex with its cognate immunity protein. The structure reveals the effector shares highest overall structural similarity to a glycoside hydrolase family associated with peptidoglycan N-acetylglucosaminidase activity, suggesting that T6S peptidoglycan glycoside hydrolase effector families may comprise significant enzymatic diversity. Our structural analyses also demonstrate that self-intoxication is prevented by the immunity protein through direct occlusion of the effector active site. This work significantly expands our current understanding of T6S effector diversity. PMID:23878199

Compositional profile of α / β-hydrolase fold proteins in mangrove soil metagenomes: prevalence of epoxide hydrolases and haloalkane dehalogenases in oil-contaminated sites.

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Jiménez, Diego Javier; Dini-Andreote, Francisco; Ottoni, Júlia Ronzella; de Oliveira, Valéria Maia; van Elsas, Jan Dirk; Andreote, Fernando Dini

2015-05-01

The occurrence of genes encoding biotechnologically relevant α/β-hydrolases in mangrove soil microbial communities was assessed using data obtained by whole-metagenome sequencing of four mangroves areas, denoted BrMgv01 to BrMgv04, in São Paulo, Brazil. The sequences (215 Mb in total) were filtered based on local amino acid alignments against the Lipase Engineering Database. In total, 5923 unassembled sequences were affiliated with 30 different α/β-hydrolase fold superfamilies. The most abundant predicted proteins encompassed cytosolic hydrolases (abH08; ∼ 23%), microsomal hydrolases (abH09; ∼ 12%) and Moraxella lipase-like proteins (abH04 and abH01; mangroves BrMgv01-02-03. This suggested selection and putative involvement in local degradation/detoxification of the pollutants. Seven sequences that were annotated as genes for putative epoxide hydrolases and five for putative haloalkane dehalogenases were found in a fosmid library generated from BrMgv02 DNA. The latter enzymes were predicted to belong to Actinobacteria, Deinococcus-Thermus, Planctomycetes and Proteobacteria. Our integrated approach thus identified 12 genes (complete and/or partial) that may encode hitherto undescribed enzymes. The low amino acid identity (< 60%) with already-described genes opens perspectives for both production in an expression host and genetic screening of metagenomes. © 2014 The Authors. Microbial Biotechnology published by John Wiley & Sons Ltd and Society for Applied Microbiology.

A new insight into the physiological role of bile salt hydrolase among intestinal bacteria from the genus Bifidobacterium.

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Jarocki, Piotr; Podleśny, Marcin; Glibowski, Paweł; Targoński, Zdzisław

2014-01-01

This study analyzes the occurrence of bile salt hydrolase in fourteen strains belonging to the genus Bifidobacterium. Deconjugation activity was detected using a plate test, two-step enzymatic reaction and activity staining on a native polyacrylamide gel. Subsequently, bile salt hydrolases from B. pseudocatenulatum and B. longum subsp. suis were purified using a two-step chromatographic procedure. Biochemical characterization of the bile salt hydrolases showed that the purified enzymes hydrolyzed all of the six major human bile salts under the pH and temperature conditions commonly found in the human gastrointestinal tract. Next, the dynamic rheometry was applied to monitor the gelation process of deoxycholic acid under different conditions. The results showed that bile acids displayed aqueous media gelating properties. Finally, gel-forming abilities of bifidobacteria exhibiting bile salt hydrolase activity were analyzed. Our investigations have demonstrated that the release of deconjugated bile acids led to the gelation phenomenon of the enzymatic reaction solution containing purified BSH. The presented results suggest that bile salt hydrolase activity commonly found among intestinal microbiota increases hydrogel-forming abilities of certain bile salts. To our knowledge, this is the first report showing that bile salt hydrolase activity among Bifidobacterium is directly connected with the gelation process of bile salts. In our opinion, if such a phenomenon occurs in physiological conditions of human gut, it may improve bacterial ability to colonize the gastrointestinal tract and their survival in this specific ecological niche.

Luciferin Amides Enable in Vivo Bioluminescence Detection of Endogenous Fatty Acid Amide Hydrolase Activity.

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Mofford, David M; Adams, Spencer T; Reddy, G S Kiran Kumar; Reddy, Gadarla Randheer; Miller, Stephen C

2015-07-15

Firefly luciferase is homologous to fatty acyl-CoA synthetases. We hypothesized that the firefly luciferase substrate d-luciferin and its analogs are fatty acid mimics that are ideally suited to probe the chemistry of enzymes that release fatty acid products. Here, we synthesized luciferin amides and found that these molecules are hydrolyzed to substrates for firefly luciferase by the enzyme fatty acid amide hydrolase (FAAH). In the presence of luciferase, these molecules enable highly sensitive and selective bioluminescent detection of FAAH activity in vitro, in live cells, and in vivo. The potency and tissue distribution of FAAH inhibitors can be imaged in live mice, and luciferin amides serve as exemplary reagents for greatly improved bioluminescence imaging in FAAH-expressing tissues such as the brain.

Pharmacological inhibition of soluble epoxide hydrolase or genetic deletion reduces diclofenac-induced gastric ulcers.

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Goswami, Sumanta Kumar; Rand, Amelia Ann; Wan, Debin; Yang, Jun; Inceoglu, Bora; Thomas, Melany; Morisseau, Christophe; Yang, Guang-Yu; Hammock, Bruce D

2017-07-01

This research was conducted to evaluate the hypothesis that gastric ulcers caused by the NSAID diclofenac sodium (DCF) can be prevented by the soluble epoxide hydrolase inhibitor TPPU. Mice were administered a single dose of 10, 30 or 100mg/kg of DCF. Once an ulcerative dose of DCF was chosen, mice were pretreated with TPPU for 7days at 0.1mg/kg to evaluate anti-ulcer effects of the sEH inhibitor on anatomy, histopathology, pH, inflammatory markers and epithelial apoptosis of stomachs. Diclofenac caused ulceration of the stomach at a dose of 100mg/kg and a time post dose of 6h. Ulcers generated under these conditions were associated with a significant increase in the levels of TNF-α and IL-6 in serum and increased apoptosis compared to control mice. Pretreatment with TPPU resulted in a decrease of ulceration in mice treated with DCF with a significant decrease in the level of apoptosis, TNF-α and IL-6 in the serum in comparison to diclofenac-treated mice. TPPU did not affect the pH of the stomach, whereas omeprazole elevated the pH of the stomach as expected. A similar anti-ulcer effect was observed in sEH gene knockout mice treated with DCF. The sEH inhibitor TPPU decreases the NSAID-induced stomach ulcers. Copyright © 2017 Elsevier Inc. All rights reserved.

Teneligliptin: a DPP-4 inhibitor for the treatment of type 2 diabetes

Directory of Open Access Journals (Sweden)

Kishimoto M

2013-05-01

Full Text Available Miyako KishimotoDepartment of Diabetes and Metabolic Medicine, Center Hospital, Tokyo, Japan; Diabetes and Metabolism Information Center, Diabetes Research Center, National Center for Global Health and Medicine, Tokyo, JapanAbstract: Dipeptidyl peptidase-4 (DPP-4 inhibitors have recently emerged as a new class of antidiabetic that show favorable results in improving glycemic control with a minimal risk of hypoglycemia and weight gain. Teneligliptin, a novel DPP-4 inhibitor, exhibits a unique structure characterized by five consecutive rings, which produce a potent and long-lasting effect. Teneligliptin is currently used in cases showing insufficient improvement in glycemic control even after diet control and exercise or a combination of diet control, exercise, and sulfonylurea- or thiazolidine-class drugs. In adults, teneligliptin is orally administered at a dosage of 20 mg once daily, which can be increased up to 40 mg per day. Because the metabolites of this drug are eliminated via renal and hepatic excretion, no dose adjustment is necessary in patients with renal impairment. The safety profile of teneligliptin is similar to those of other available DPP-4 inhibitors. However, caution needs to be exercised when administering teneligliptin to patients who are prone to QT prolongation. One study has reported that the postprandial blood glucose-lowering effects of teneligliptin administered prior to breakfast were sustained throughout the day, and the effects observed after dinner were similar to those observed after breakfast or lunch. Thus, although clinical data for this new drug are limited, this drug shows promise in stabilizing glycemic fluctuations throughout the day and consequently suppressing the progression of diabetic complications. However, continued evaluation in long-term studies and clinical trials is required to evaluate the efficacy and safety of the drug as well as to identify additional indications for its clinical use

Les lipases sont des hydrolases atypiques : principales caractéristiques et applications

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Fickers P.

2008-01-01

Full Text Available ipases are atypical hydrolases: principal characteristics and applications. Due to their kinetic and substrate specificities, triacylglycerol acyl-hydrolases or lipases are atypical enzymes. In function of their microenvironment, lipases are able to act as hydrolases in aqueous solution or as biocatalysts in organic synthesis. As hydrolases, they are responsible of the triglycerids catabolism into fatty acids and glycerol. In many organisms, this reaction plays a major role in the fat and lipid metabolism. In addition, lipases are also able to hydrolyse phospholipids and cholesterol esters. In organic solvent, lipases could catalyse reactions such as esterifications, acidolysis or alcoolysis with enantio-, regio- and chimioselectivity. Lipases form a mixed class of enzyme due to their animal, vegetal or microbial origins. All those properties led to the development of many applications in the food and chemical industries but also in the medical and therapeutic field.

[11C]CURB: Evaluation of a novel radiotracer for imaging fatty acid amide hydrolase by positron emission tomography

International Nuclear Information System (INIS)

Wilson, Alan A.; Garcia, Armando; Parkes, Jun; Houle, Sylvain; Tong, Junchao; Vasdev, Neil

2011-01-01

Introduction: Fatty acid amide hydrolase (FAAH) is the enzyme responsible for metabolising the endogenous cannabinoid, anandamide, and thus represents an important target for molecular imaging. To date, no radiotracer has been shown to be useful for imaging of FAAH using either positron emission tomography (PET) or single photon emission computed tomography (SPECT). We here determine the suitability of a novel carbon-11-labeled inhibitor of FAAH via ex vivo biodistribution studies in rat brain in conjunction with pharmacological challenges. Methods: A potent irreversible inhibitor of FAAH, URB694, radiolabeled with carbon-11 in the carbonyl position ([ 11 C]CURB), was administered to male rats via tail-vein injection. Rats were sacrificed at various time points postinjection, and tissue samples were dissected, counted and weighed. Specific binding to FAAH was investigated by pretreatment of animals with URB694 or URB597. For metabolism and mechanism of binding studies, whole brains were excised post-radiotracer injection, homogenised and extracted exhaustively with 80% aq. acetonitrile to determine the time course and fraction of radioactivity that was irreversibly bound to brain parenchyma. Results: Upon intravenous injection into rats, [ 11 C]CURB showed high brain uptake [standard uptake value (SUV) of 1.6-2.4 at 5 min] with little washout over time, which is characteristic of irreversible binding. Highest uptake of radioactivity was seen in the cortex, intermediate in the cerebellum and lowest in the hypothalamus, reflecting the reported distribution of FAAH. Brain uptake of radioactivity was decreased in a dose-dependent manner by pretreatment with increasing amounts of URB694, demonstrating that binding was saturable. Pretreatment with the well-characterised FAAH inhibitor, URB597, reduced binding in all brain regions by 70-80%. Homogenised brain extraction experiments demonstrated unequivocally that [ 11 C]CURB was irreversibly bound to FAAH. Conclusions

Checkpoint inhibitors in cancer immunotherapy: Cross reactivity of a CTLA-4 antibody and IDO-inhibitor L-1MT in pigs

DEFF Research Database (Denmark)

Al-Shatrawi, Zina Adil; Frøsig, Thomas Mørch; Jungersen, Gregers

a non-specific activation of porcine T cells. This will be further investigated to provide the basis for in vivo studies investigating checkpoint inhibitor blockade in combination with other cancer immunotherapies. Eventually our goal is to establish pigs as an alternative large animal model......Blockade of checkpoint inhibitors has recently shown very convincing results in the treatment of cancer. One key target is CTLA-4, which has been demonstrated to be a potent negative regulator of lymphocyte activation. The treatment with the FDA-approved fully human CTLA-4 monoclonal antibody...... Ipilimumab increases anticancer T-cell reactivity and overall survival of metastatic cancer patients. Indole-amine 2,3-dioxygenase (IDO) is another checkpoint inhibitor which suppresses T-cell immunity by the depletion of tryptophan in the T-cell microenvironment, and also inhibition of IDO by L-1...

Sarcoid-like lung granulomas in a hemodialysis patient treated with a dipeptidyl peptidase-4 inhibitor.

Science.gov (United States)

Sada, Ken-Ei; Wada, Jun; Morinaga, Hiroshi; Tuchimochi, Shigeyuki; Uka, Mayu; Makino, Hirofumi

2014-04-01

It has been reported that the inhibition of dipeptidyl peptidase-4 (DPP-4)/CD26 on T-cells by DPP-4 enzymatic inhibitors suppresses lymphocyte proliferation and reduces the production of various cytokines, including tumor necrosis factor (TNF)-α. A 72-year-old female with diabetic nephropathy on hemodialysis developed multiple lung nodules following the administration of vildagliptin. A biopsy demonstrated the histology of granulomas without caseous necrosis. The discontinuation of vildagliptin resulted in the disappearance of the granulomas within 4 months. As granulomatosis often develops in patients under anti-TNF-α therapy, the accumulation of DPP-4 inhibitors or its metabolites is possibly linked to unrecognized complications, such as sarcoid-like lung granulomas.

Inhibition of the soluble epoxide hydrolase promotes albuminuria in mice with progressive renal disease.

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Oliver Jung

2010-08-01

Full Text Available Epoxyeicotrienoic acids (EETs are cytochrome P450-dependent anti-hypertensive and anti-inflammatory derivatives of arachidonic acid, which are highly abundant in the kidney and considered reno-protective. EETs are degraded by the enzyme soluble epoxide hydrolase (sEH and sEH inhibitors are considered treatment for chronic renal failure (CRF. We determined whether sEH inhibition attenuates the progression of CRF in the 5/6-nephrectomy model (5/6-Nx in mice. 5/6-Nx mice were treated with a placebo, an ACE-inhibitor (Ramipril, 40 mg/kg, the sEH-inhibitor cAUCB or the CYP-inhibitor fenbendazole for 8 weeks. 5/6-Nx induced hypertension, albuminuria, glomerulosclerosis and tubulo-interstitial damage and these effects were attenuated by Ramipril. In contrast, cAUCB failed to lower the blood pressure and albuminuria was more severe as compared to placebo. Plasma EET-levels were doubled in 5/6 Nx-mice as compared to sham mice receiving placebo. Renal sEH expression was attenuated in 5/6-Nx mice but cAUCB in these animals still further increased the EET-level. cAUCB also increased 5-HETE and 15-HETE, which derive from peroxidation or lipoxygenases. Similar to cAUCB, CYP450 inhibition increased HETEs and promoted albuminuria. Thus, sEH-inhibition failed to elicit protective effects in the 5/6-Nx model and showed a tendency to aggravate the disease. These effects might be consequence of a shift of arachidonic acid metabolism into the lipoxygenase pathway.

Biodegradation of phthalic acid esters (PAEs) and in silico structural characterization of mono-2-ethylhexyl phthalate (MEHP) hydrolase on the basis of close structural homolog.

Science.gov (United States)

Singh, Neha; Dalal, Vikram; Mahto, Jai Krishna; Kumar, Pravindra

2017-09-15

Three bacterial strains capable of degrading phthalates namely Pseudomonas sp. PKDM2, Pseudomonas sp. PKDE1 and Pseudomonas sp. PKDE2 were isolated and characterized for their degradative potential. These strains efficiently degraded 77.4%-84.4% of DMP, 75.0%-75.7% of DEP and 71.7%-74.7% of DEHP, initial amount of each phthalate is 500mgL -1 of each phthalate, after 44h of incubation. GC-MS results reveal the tentative DEHP degradation pathway, where hydrolases mediate the breakdown of DEHP to phthalic acid (PA) via an intermediate MEHP. MEHP hydrolase is a serine hydrolase which is involved in the reduction of the MEHP to PA. The predicted 3D model of MEHP hydrolase from Pseudomonas mosselii was docked with phthalate monoesters (PMEs) such as MEHP, mono-n-hexyl phthalate (MHP), mono-n-butyl phthalate (MBP) and mono-n-ethyl phthalate (MEP), respectively. Docking results show the distance between the carbonyl carbon of respective phthalate monoester and the hydroxyl group of catalytic serine lies in the range of 2.9 to 3.3Å, which is similar to the ES complex of other serine hydrolases. This structural study highlights the interaction and the role of catalytic residues of MEHP hydrolase involved in the biodegradation of PMEs to phthalate. Copyright © 2017 Elsevier B.V. All rights reserved.

Peptidoglycan Hydrolases of Local Lactic Acid Bacteria from Kazakh Traditional Food

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Serik Shaikhin

2014-01-01

Full Text Available Introduction: Peptidoglycan (PG is a major component of the cell wall of Gram-positive bacteria and is essential for maintaining the integrity of the bacterial cell and its shape. The bacteria synthesize PG hydrolases, which are capable of cleaving the covalent bonds of PG. They also play an important role in modeling PG, which is required for bacterial growth and division. In an era of increasing antibiotic-resistant pathogens, PG hydrolases that destroy these important structures of the cell wall act as a potential source of new antimicrobials. The aim of this study is to identify the main PG hydrolases of local lactic acid bacteria isolated from traditional foods that enhance probiotic activity of a biological preparation. Methods. Lactococcus lactis 17А and Lactococcus garvieae 19А were isolated from the traditional sausage-like meat product called kazy. They were isolated according to standards methods of microbiology. Genetic identification of the isolates were tested by determining the nucleotide sequences of 16S rDNA. The Republican collection of microorganisms took strains of Lactobacillus casei subsp. Rhamnosus 13-P, L. delbrueckii subsp. lactis CG-1 B-RKM 0044 from cheese, Lactobacillus casei subsp. casei B-RKM 0202 from homemade butter. They used the standard technique of renaturating polyacrylamide gel electrophoresis to detect PG hydrolases activity. Results. According to the profiles of PG hydrolase activity on zymograms, the enzymes of Lactococci 17A and 19A in kazy are similar in electrophoretic mobility to major autolysin AcmA, while the lactobacilli of industrial and home-made dairy products have enzymes similar to extracellular proteins p40 and p75, which have probiotic activity. Conclusions. Use of peptidoglycan hydrolases seems to be an interesting approach in the fight against multi-drug resistant strains of bacteria and could be a valuable tool for the treatment of diseases caused by these microorganisms in Kazakhstan.

Wound healing effects of dipeptidyl peptidase-4 inhibitors: An emerging concept in management of diabetic foot ulcer-A review.

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Saboo, Apoorva; Rathnayake, Ayeshmanthe; Vangaveti, Venkat N; Malabu, Usman H

2016-01-01

Dipeptidyl peptidase-4 (DPP-4) inhibitors have a well-known effect on glycaemic control in patients with diabetes but little is known on their wound healing role in this group of population. This paper reviews the effects of DPP-4 inhibitors on wound healing of diabetic foot ulcers. Published data on effects and mechanism of DDP-4 inhibitors on wound healing were derived from Medline, PubMed and Google Scholar search of English language literature from 1994 to 2014 using the key words such as "DPP-4 inhibitors", "endothelial healing" "diabetes" and "chronic ulcers". DPP-4 inhibitors show a potential benefit in processes of wound healing in diabetic chronic foot ulcers. The enzyme inhibitors promote recruitment of endothelial progenitor cells and allow the final scaffolding of wounds. Furthermore DPP-4 inhibitors augment angiogenesis and have widespread effects on optimising the immune response to persistent hypoxia in chronic diabetes wounds. DPP-4 inhibitors show promise in the local wound healing of diabetic foot ulcers in addition to its already established glycaemic control. In the light of high rate of amputations due to non-healing ulcers with profound psychological and economical liability, more investigations on the usefulness of DPP-4 inhibitors in the high risk diabetes population are needed. Copyright © 2015 Diabetes India. Published by Elsevier Ltd. All rights reserved.

[{sup 11}C]CURB: Evaluation of a novel radiotracer for imaging fatty acid amide hydrolase by positron emission tomography

Energy Technology Data Exchange (ETDEWEB)

Wilson, Alan A., E-mail: alan.wilson@camhpet.c [PET Centre, Centre for Addiction and Mental Health, Toronto, Ontario, M5T 1R8 (Canada); Human Neurochemical Pathology Laboratory, Centre for Addiction and Mental Health, Toronto, Ontario, M5T 1R8 (Canada); Garcia, Armando; Parkes, Jun [PET Centre, Centre for Addiction and Mental Health, Toronto, Ontario, M5T 1R8 (Canada); Houle, Sylvain [PET Centre, Centre for Addiction and Mental Health, Toronto, Ontario, M5T 1R8 (Canada); Human Neurochemical Pathology Laboratory, Centre for Addiction and Mental Health, Toronto, Ontario, M5T 1R8 (Canada); Tong, Junchao [Department of Psychiatry, University of Toronto, Toronto, Ontario, M5T 1R8 (Canada); Vasdev, Neil [PET Centre, Centre for Addiction and Mental Health, Toronto, Ontario, M5T 1R8 (Canada); Human Neurochemical Pathology Laboratory, Centre for Addiction and Mental Health, Toronto, Ontario, M5T 1R8 (Canada)

2011-02-15

Introduction: Fatty acid amide hydrolase (FAAH) is the enzyme responsible for metabolising the endogenous cannabinoid, anandamide, and thus represents an important target for molecular imaging. To date, no radiotracer has been shown to be useful for imaging of FAAH using either positron emission tomography (PET) or single photon emission computed tomography (SPECT). We here determine the suitability of a novel carbon-11-labeled inhibitor of FAAH via ex vivo biodistribution studies in rat brain in conjunction with pharmacological challenges. Methods: A potent irreversible inhibitor of FAAH, URB694, radiolabeled with carbon-11 in the carbonyl position ([{sup 11}C]CURB), was administered to male rats via tail-vein injection. Rats were sacrificed at various time points postinjection, and tissue samples were dissected, counted and weighed. Specific binding to FAAH was investigated by pretreatment of animals with URB694 or URB597. For metabolism and mechanism of binding studies, whole brains were excised post-radiotracer injection, homogenised and extracted exhaustively with 80% aq. acetonitrile to determine the time course and fraction of radioactivity that was irreversibly bound to brain parenchyma. Results: Upon intravenous injection into rats, [{sup 11}C]CURB showed high brain uptake [standard uptake value (SUV) of 1.6-2.4 at 5 min] with little washout over time, which is characteristic of irreversible binding. Highest uptake of radioactivity was seen in the cortex, intermediate in the cerebellum and lowest in the hypothalamus, reflecting the reported distribution of FAAH. Brain uptake of radioactivity was decreased in a dose-dependent manner by pretreatment with increasing amounts of URB694, demonstrating that binding was saturable. Pretreatment with the well-characterised FAAH inhibitor, URB597, reduced binding in all brain regions by 70-80%. Homogenised brain extraction experiments demonstrated unequivocally that [{sup 11}C]CURB was irreversibly bound to FAAH

Reduced Airway Hyperresponsiveness by Phosphodiesterase 3 and 4 Inhibitors in Guinea-Pigs

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Nöella Germain

1999-01-01

Full Text Available The aim of the present study was to compare the effects of selective phosphodiesterase (PDE 3, 4 and 5 inhibitors on antigen-induced airway hyperresponsiveness in sensitized guinea-pigs. When the sensitized guinea-pigs were orally pre-treated with the selective PDE4 inhibitor, Ro 20-1724 (30 mg/kg, and studied 48 h after OA, a significant reduction (p<0.01 of the leftward shift of the dose-response curve to ACh was noted, whereas it was ineffective at the lower dose (10 mg/kg. Administration of the selective PDE3 inhibitor, milrinone (30 mg/kg also elicited a significant reduction (p<0.01 of the airway hyperresponsiveness, whereas the PDE5 inhibitor zaprinast (30 mg/kg was ineffective. These results show that both PDE3 and PDE4 inhibitors are able to inhibit the antigen-induced airway hyperresponsiveness in sensitized guinea-pigs and support the potential utility of selective PDE inhibitors in the treatment of asthma.

Comparison of newly developed anti-bone morphogenetic protein 4 llama-derived antibodies with commercially available BMP4 inhibitors.

Science.gov (United States)

Calpe, Silvia; Correia, Ana C P; Sancho-Serra, Maria Del Carmen; Krishnadath, Kausilia K

2016-01-01

Due to improved understanding of the role of bone morphogenetic protein 4 (BMP4) in an increasing number of diseases, the development of selective inhibitors of BMP4 is an attractive therapeutic option. The currently available BMP4 inhibitors are not suitable as therapeutics because of their low specificity and low effectiveness. Here, we compared newly generated anti-BMP4 llama-derived antibodies (VHHs) with 3 different types of commercially available BMP4 inhibitors, natural antagonists, small molecule BMPR inhibitors and conventional anti-BMP4 monoclonal antibodies. We found that the anti-BMP4 VHHs were as effective as the natural antagonist or small molecule inhibitors, but had higher specificity. We also showed that commercial anti-BMP4 antibodies were inferior in terms of both specificity and effectiveness. These findings might result from the fact that the VHHs C4C4 and C8C8 target a small region within the BMPR1 epitope of BMP4, whereas the commercial antibodies target other areas of the BMP4 molecule. Our results show that the newly developed anti-BMP4 VHHs are promising antibodies with better specificity and effectivity for inhibition of BMP4, making them an attractive tool for research and for therapeutic applications.

High-throughput analysis of endogenous fruit glycosyl hydrolases using a novel chromogenic hydrogel substrate assay

DEFF Research Database (Denmark)

Schückel, Julia; Kracun, Stjepan Kresimir; Lausen, Thomas Frederik

2017-01-01

A broad range of enzyme activities can be found in a wide range of different fruits and fruiting bodies but there is a lack of methods where many samples can be handled in a high-throughput and efficient manner. In particular, plant polysaccharide degrading enzymes – glycosyl hydrolases (GHs) play...... led to a more profound understanding of the importance of GH activity and regulation, current methods for determining glycosyl hydrolase activity are lacking in throughput and fail to keep up with data output from transcriptome research. Here we present the use of a versatile, easy...

DPP4 inhibitors promote biological functions of human endothelial progenitor cells by targeting the SDF-1/CXCR4 signaling pathway

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Liu Feng

2016-01-01

Full Text Available Dipeptidyl peptidase 4 (DPP4 inhibitors(oral hypoglycemic agentshave beneficial effects during the early stages of diabetes. In this study, we evaluated the role of DPP4inhibitorsonthe biological functions of cultured human endothelial progenitor cells (EPCs. After treating EPCs with the DPP4 inhibitors sitagliptin and vildagliptin, we examined the mRNA expression of DPP4, vascular endothelial growth factor (VEGF,VEGF receptor 2 (VEGFR-2,endothelial nitric oxide synthase (eNOS, caspase-3,stromal cell-derived factor-1 (SDF-1, chemokine (C-X-C motif receptor 4 (CXCR4 were measured by RT-PCR. The protein expression of SDF-1 and CXCR4 was determined by Western blot; cell proliferation was tested by the MTT method, and DPP4 activity was determined by a DPP4 assay. Our results revealed that DPP4 expression and activity were inhibited following the treatment with various doses of DPP4 inhibitors. Cell proliferation and the expression of VEGF, VEGFR-2andeNOS were up regulated, while cell apoptosis was inhibited by DPP4 inhibitors in a dose-dependent manner. DPP4 inhibitors activated the SDF-1/CXCR4 signaling pathway, shown by the elevated expression of SDF-1/CXCR4. This further proved that after the SDF-1/CXCR4 signaling pathway was blocked by its inhibitor ADM3100, the effects of DPP4 inhibitors on the proliferation and apoptosis, and the expression of VEGF, VEGFR-2and eNOS of EPCs were significantly reduced. These findings suggest that DPP4 inhibitors promote the biological functions of human EPCs by up regulating the SDF-1/CXCR4 signaling pathway.

A screening method for β-glucan hydrolase employing Trypan Blue-coupled β-glucan agar plate and β-glucan zymography.

Science.gov (United States)

Park, Chang-Su; Yang, Hee-Jong; Kim, Dong-Ho; Kang, Dae-Ook; Kim, Min-Soo; Choi, Nack-Shick

2012-06-01

A new screening method for β-(1,3-1,6) glucan hydrolase was developed using a pure β-glucan from Aureobaisidum pullulans by zymography and an LB-agar plate. Paenibacillus sp. was screened as a producer a β-glucan hydrolase on the Trypan Blue-coupled β-glucan LB-agar plate and the activity of the enzyme was analyzed by SDS-β-glucan zymography. The β-glucan was not hydrolyzed by Bacillus spp. strains, which exhibit cellulolytic activity on CMC zymography. The gene, obtaining by shotgun cloning and encoding the β-glucan hydrolase of Paenibacillus sp. was sequenced.

Vildagliptin: the first innovative DDP-4 inhibitor

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Edvin Villkhauer

2010-09-01

Full Text Available A review of the main stages of investigation undertaken by Novartis Pharmaceuticals in search of a new molecule for the treatment of type 2 diabetesmellitus, dipeptidyl peptidase-4 (DPP-4 inhibitor (Vildaglyptin. The data on specificity and selectivity of the action of this molecule are presentedalong with the results of its comparison with another agent of this group (sitagliptin.

Discovery of Dengue Virus NS4B Inhibitors

Science.gov (United States)

Wang, Qing-Yin; Dong, Hongping; Zou, Bin; Karuna, Ratna; Wan, Kah Fei; Zou, Jing; Susila, Agatha; Yip, Andy; Shan, Chao; Yeo, Kim Long; Xu, Haoying; Ding, Mei; Chan, Wai Ling; Gu, Feng; Seah, Peck Gee; Liu, Wei; Lakshminarayana, Suresh B.; Kang, CongBao; Lescar, Julien; Blasco, Francesca; Smith, Paul W.

2015-01-01

ABSTRACT The four serotypes of dengue virus (DENV-1 to -4) represent the most prevalent mosquito-borne viral pathogens in humans. No clinically approved vaccine or antiviral is currently available for DENV. Here we report a spiropyrazolopyridone compound that potently inhibits DENV both in vitro and in vivo. The inhibitor was identified through screening of a 1.8-million-compound library by using a DENV-2 replicon assay. The compound selectively inhibits DENV-2 and -3 (50% effective concentration [EC50], 10 to 80 nM) but not DENV-1 and -4 (EC50, >20 μM). Resistance analysis showed that a mutation at amino acid 63 of DENV-2 NS4B (a nonenzymatic transmembrane protein and a component of the viral replication complex) could confer resistance to compound inhibition. Genetic studies demonstrate that variations at amino acid 63 of viral NS4B are responsible for the selective inhibition of DENV-2 and -3. Medicinal chemistry improved the physicochemical properties of the initial “hit” (compound 1), leading to compound 14a, which has good in vivo pharmacokinetics. Treatment of DENV-2-infected AG129 mice with compound 14a suppressed viremia, even when the treatment started after viral infection. The results have proven the concept that inhibitors of NS4B could potentially be developed for clinical treatment of DENV infection. Compound 14a represents a potential preclinical candidate for treatment of DENV-2- and -3-infected patients. IMPORTANCE Dengue virus (DENV) threatens up to 2.5 billion people and is now spreading in many regions in the world where it was not previously endemic. While there are several promising vaccine candidates in clinical trials, approved vaccines or antivirals are not yet available. Here we describe the identification and characterization of a spiropyrazolopyridone as a novel inhibitor of DENV by targeting the viral NS4B protein. The compound potently inhibits two of the four serotypes of DENV (DENV-2 and -3) both in vitro and in vivo. Our

Inhibitor of differentiation 4 (Id4) is a potential tumor suppressor in prostate cancer

International Nuclear Information System (INIS)

Carey, Jason PW; Asirvatham, Ananthi J; Galm, Oliver; Ghogomu, Tandeih A; Chaudhary, Jaideep

2009-01-01

Inhibitor of differentiation 4 (Id4), a member of the Id gene family is also a dominant negative regulator of basic helix loop helix (bHLH) transcription factors. Some of the functions of Id4 appear to be unique as compared to its other family members Id1, Id2 and Id3. Loss of Id4 gene expression in many cancers in association with promoter hypermethylation has led to the proposal that Id4 may act as a tumor suppressor. In this study we provide functional evidence that Id4 indeed acts as a tumor suppressor and is part of a cancer associated epigenetic re-programming. Data mining was used to demonstrate Id4 expression in prostate cancer. Methylation specific polymerase chain reaction (MSP) analysis was performed to understand molecular mechanisms associated with Id4 expression in prostate cancer cell lines. The effect of ectopic Id4 expression in DU145 cells was determined by cell cycle analysis (3H thymidine incorporation and FACS), expression of androgen receptor, p53 and cyclin dependent kinase inhibitors p27 and p21 by a combination of RT-PCR, real time-PCR, western blot and immuno-cytochemical analysis. Id4 expression was down-regulated in prostate cancer. Id4 expression was also down-regulated in prostate cancer line DU145 due to promoter hyper-methylation. Ectopic Id4 expression in DU145 prostate cancer cell line led to increased apoptosis and decreased cell proliferation due in part by an S-phase arrest. In addition to S-phase arrest, ectopic Id4 expression in PC3 cells also resulted in prolonged G2/M phase. At the molecular level these changes were associated with increased androgen receptor (AR), p21, p27 and p53 expression in DU145 cells. The results suggest that Id4 acts directly as a tumor suppressor by influencing a hierarchy of cellular processes at multiple levels that leads to a decreased cell proliferation and change in morphology that is possibly mediated through induction of previously silenced tumor suppressors

A comparison of the inhibitory activity of selective PDE4 inhibitors on eosinophil recruitment in guinea pig skin

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Mauro M Teixeira

1997-12-01

Full Text Available The elevation of intracellular cyclic AMP by phosphodiesterase (PDE4 inhibitors in eosinophils is associated with inhibition of the activation and recruitment of these cells. We have previously shown that systemic treatment with the PDE4 inhibitor rolipram effectively inhibt eosinophil migration in guinea pig skin. In the present study we compare the oral potency and efficacy of the PDE4 inhibitors rolipram, RP 73401 and CDP 840 on allergic and PAF-induced eosinophil recruitment. Rolipram and RP 73401 were equally effective and potent when given by the oral route and much more active than the PDE4 inhibitor CDP 840. We suggest that this guinea pig model of allergic and mediator-induced eosinophil recruitment is both a sensitive and simple tool to test the efficacy and potency of PDE4 inhibitors in vivo.

Effects of selective phosphodiesterases-4 inhibitors on learning and memory: a review of recent research.

Science.gov (United States)

Peng, Sheng; Sun, Haiyan; Zhang, Xiaoqing; Liu, Gongjian; Wang, Guanglei

2014-09-01

Phosphodiesterase-4 (PDE-4) regulates the intracellular level of cyclic adenosine monophosphate. Recent studies demonstrated that PDE-4 inhibitors can counteract deficits in long-term memory caused by aging or increased expression of mutant forms of human amyloid precursor proteins, and can influence the process of memory function and cognitive enhancement. Therapeutics, such as ketamine, a drug used in clinical anesthesia, can also cause memory deficits as adverse effects. Targeting PDE-4 with selective inhibitors may offer a novel therapeutic strategy to prevent, slow the progress, and, eventually, treat memory deficits.

Discovery and characterization of thermophilic limonene-1,2-epoxide hydrolases from hot spring metagenomic libraries

DEFF Research Database (Denmark)

Ferrandi, Erica Elisa; Sayer, Christopher; Isupov, Michail N.

2015-01-01

thermophilic sources, have higher optimal temperatures and apparent melting temperatures than Re-LEH. The new LEH enzymes have been crystallized and their structures solved to high resolution in the native form and in complex with the inhibitor valpromide for Tomsk-LEH and poly(ethylene glycol) for CH55-LEH......,2-epoxide hydrolase (LEH) family of enzymes. These two LEHs (Tomsk-LEH and CH55-LEH) show EH activities towards different epoxide substrates, differing in most cases from those previously identified for Rhodococcus erythropolis (Re-LEH) in terms of stereoselectivity. Tomsk-LEH and CH55-LEH, both from....... The structural analysis has provided insights into the LEH mechanism, substrate specificity and stereoselectivity of these new LEH enzymes, which has been supported by mutagenesis studies....

Mechanism-based fluorescent labeling of beta-galactosidases. An efficient method in proteomics for glycoside hydrolases.

Science.gov (United States)

Kurogochi, Masaki; Nishimura, Shin-Ichiro; Lee, Yuan Chuan

2004-10-22

(4-N-5-Dimethylaminonaphthalene-1-sulfonyl-2-difluoromethylphenyl)-beta-d-galactopyranoside was synthesized and successfully tested on beta-galactosidases from Xanthomonas manihotis (Wong-Madden, S. T., and Landry, D. Glycobiology (1995) 5, 19-28 and Taron, C. H., Benner, J. S., Hornstra, L. J., and Guthrie, E. P. (1995) Glycobiology 5, 603-610), Escherichia coli (Jacobson, R. H., Zhang, X. J., DuBose, R. F., and Matthews, B. W. (1994) Nature 369, 761-766), and Bacillus circulans (Fujimoto, H., Miyasato, M., Ito, Y., Sasaki, T., and Ajisaka, K. (1988) Glycoconj. J. 15, 155-160) for the rapid identification of the catalytic site. Reaction of the irreversible inhibitor with enzymes proceeded to afford a fluorescence-labeled protein suitable for further high throughput characterization by using antidansyl antibody and matrix-assisted laser desorption ionization time-of-flight/time-of-flight (MALDI-TOF/TOF). Specific probing by a fluorescent aglycon greatly facilitated identification of the labeled peptide fragments from beta-galactosidases. It was demonstrated by using X. manihotis beta-galactosidase that the Arg-58 residue, which is located within a sequence of 56IPRAYWKD63, was labeled by nucleophilic attack of the guanidinyl group. This sequence including Arg-58 (Leu-46 to Tyr-194) was similar to that (Met-1 to Tyr-151) of Thermus thermophilus A4, which is the first known structure of glycoside hydrolases family 42 (Hidaka, M., Fushinobu, S., Ohtsu, N., Motoshima, H., Matsuzawa, H., Shoun, H., and Wakagi, T. (2002) J. Mol. Biol. 322, 79-91). A catalytic glutamic acid (Glu-537) of E. coli beta-galactosidase was proved to be labeled by the same procedure, suggesting that the modification site with this irreversible substrate might depend both on the nucleophilicity of the amino acids and their spatial arrangement in the individual catalytic cavity. Similarly, a Glu-259 in 257TLEE260 was selectively labeled using B. circulans beta-galactosidase, indicating that Glu

4-Hydroxyphenylpyruvate Dioxygenase Inhibitors: From Chemical Biology to Agrochemicals.

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Ndikuryayo, Ferdinand; Moosavi, Behrooz; Yang, Wen-Chao; Yang, Guang-Fu

2017-10-04

The development of new herbicides is receiving considerable attention to control weed biotypes resistant to current herbicides. Consequently, new enzymes are always desired as targets for herbicide discovery. 4-Hydroxyphenylpyruvate dioxygenase (HPPD, EC 1.13.11.27) is an enzyme engaged in photosynthetic activity and catalyzes the transformation of 4-hydroxyphenylpyruvic acid (HPPA) into homogentisic acid (HGA). HPPD inhibitors constitute a promising area of discovery and development of innovative herbicides with some advantages, including excellent crop selectivity, low application rates, and broad-spectrum weed control. HPPD inhibitors have been investigated for agrochemical interests, and some of them have already been commercialized as herbicides. In this review, we mainly focus on the chemical biology of HPPD, discovery of new potential inhibitors, and strategies for engineering transgenic crops resistant to current HPPD-inhibiting herbicides. The conclusion raises some relevant gaps for future research directions.

Structure of a Trypanosoma brucei α/β-hydrolase fold protein with unknown function

International Nuclear Information System (INIS)

Merritt, Ethan A.; Holmes, Margaret; Buckner, Frederick S.; Van Voorhis, Wesley C.; Quartly, Erin; Phizicky, Eric M.; Lauricella, Angela; Luft, Joseph; DeTitta, George; Neely, Helen; Zucker, Frank; Hol, Wim G. J.

2008-01-01

T. brucei gene Tb10.6k15.0140 codes for an α/β-hydrolase fold protein of unknown function. The 2.2 Å crystal structure shows that members of this sequence family retain a conserved Ser residue at the expected site of a catalytic nucleophile, but that trypanosomatid sequences lack structural homologs for the other expected residues of the catalytic triad. The structure of a structural genomics target protein, Tbru020260AAA from Trypanosoma brucei, has been determined to a resolution of 2.2 Å using multiple-wavelength anomalous diffraction at the Se K edge. This protein belongs to Pfam sequence family PF08538 and is only distantly related to previously studied members of the α/β-hydrolase fold family. Structural superposition onto representative α/β-hydrolase fold proteins of known function indicates that a possible catalytic nucleophile, Ser116 in the T. brucei protein, lies at the expected location. However, the present structure and by extension the other trypanosomatid members of this sequence family have neither sequence nor structural similarity at the location of other active-site residues typical for proteins with this fold. Together with the presence of an additional domain between strands β6 and β7 that is conserved in trypanosomatid genomes, this suggests that the function of these homologs has diverged from other members of the fold family

Crystallization of mouse S-adenosyl-l-homocysteine hydrolase

International Nuclear Information System (INIS)

Ishihara, Masaaki; Kusakabe, Yoshio; Ohsumichi, Tsuyoshi; Tanaka, Nobutada; Nakanishi, Masayuki; Kitade, Yukio; Nakamura, Kazuo T.

2010-01-01

Mouse S-adenosyl-l-homocysteine hydrolase has been crystallized in the presence of the reaction product adenosine. Diffraction data to 1.55 Å resolution were collected using synchrotron radiation. S-Adenosyl-l-homocysteine hydrolase (SAHH; EC 3.3.1.1) catalyzes the reversible hydrolysis of S-adenosyl-l-homocysteine to adenosine and l-homocysteine. For crystallographic investigations, mouse SAHH (MmSAHH) was overexpressed in bacterial cells and crystallized using the hanging-drop vapour-diffusion method in the presence of the reaction product adenosine. X-ray diffraction data to 1.55 Å resolution were collected from an orthorhombic crystal form belonging to space group I222 with unit-cell parameters a = 100.64, b = 104.44, c = 177.31 Å. Structural analysis by molecular replacement is in progress

Design and synthesis of 4-heteroaryl 1,2,3,4-tetrahydroisoquinolines as triple reuptake inhibitors.

Science.gov (United States)

Liu, Shuang; Zha, Congxiang; Nacro, Kassoum; Hu, Min; Cui, Wenge; Yang, Yuh-Lin; Bhatt, Ulhas; Sambandam, Aruna; Isherwood, Matthew; Yet, Larry; Herr, Michael T; Ebeltoft, Sarah; Hassler, Carla; Fleming, Linda; Pechulis, Anthony D; Payen-Fornicola, Anne; Holman, Nicholas; Milanowski, Dennis; Cotterill, Ian; Mozhaev, Vadim; Khmelnitsky, Yuri; Guzzo, Peter R; Sargent, Bruce J; Molino, Bruce F; Olson, Richard; King, Dalton; Lelas, Snjezana; Li, Yu-Wen; Johnson, Kim; Molski, Thaddeus; Orie, Anitra; Ng, Alicia; Haskell, Roy; Clarke, Wendy; Bertekap, Robert; O'Connell, Jonathan; Lodge, Nicholas; Sinz, Michael; Adams, Stephen; Zaczek, Robert; Macor, John E

2014-07-10

A series of 4-bicyclic heteroaryl 1,2,3,4-tetrahydroisoquinoline inhibitors of the serotonin transporter (SERT), norepinephrine transporter (NET), and dopamine transporter (DAT) was discovered. The synthesis and structure-activity relationship (SAR) of these triple reuptake inhibitors (TRIs) will be discussed. Compound 10i (AMR-2), a very potent inhibitor of SERT, NET, and DAT, showed efficacy in the rat forced-swim and mouse tail suspension models with minimum effective doses of 0.3 and 1 mg/kg (po), respectively. At efficacious doses in these assays, 10i exhibited substantial occupancy levels at the three transporters in both rat and mouse brain. The study of the metabolism of 10i revealed the formation of a significant active metabolite, compound 13.

The apo structure of sucrose hydrolase from Xanthomonas campestris pv. campestris shows an open active-site groove

DEFF Research Database (Denmark)

Champion, Elise; Remaud-Simeon, Magali; Skov, Lars Kobberøe

2009-01-01

Glycoside hydrolase family 13 (GH-13) mainly contains starch-degrading or starch-modifying enzymes. Sucrose hydrolases utilize sucrose instead of amylose as the primary glucosyl donor. Here, the catalytic properties and X-ray structure of sucrose hydrolase from Xanthomonas campestris pv. campestris...... of GH-13. Comparisons with structures of the highly similar sucrose hydrolase from X. axonopodis pv. glycines most notably showed that residues Arg516 and Asp138, which form a salt bridge in the X. axonopodis sucrose complex and define part of the subsite -1 glucosyl-binding determinants...

Full Fatty Acid Amide Hydrolase Inhibition Combined with Partial Monoacylglycerol Lipase Inhibition: Augmented and Sustained Antinociceptive Effects with Reduced Cannabimimetic Side Effects in Mice.

Science.gov (United States)

Ghosh, Sudeshna; Kinsey, Steven G; Liu, Qing-Song; Hruba, Lenka; McMahon, Lance R; Grim, Travis W; Merritt, Christina R; Wise, Laura E; Abdullah, Rehab A; Selley, Dana E; Sim-Selley, Laura J; Cravatt, Benjamin F; Lichtman, Aron H

2015-08-01

Inhibition of fatty acid amide hydrolase (FAAH) or monoacylglycerol lipase (MAGL), the primary hydrolytic enzymes for the respective endocannabinoids N-arachidonoylethanolamine (AEA) and 2-arachidonylglycerol (2-AG), produces antinociception but with minimal cannabimimetic side effects. Although selective inhibitors of either enzyme often show partial efficacy in various nociceptive models, their combined blockade elicits augmented antinociceptive effects, but side effects emerge. Moreover, complete and prolonged MAGL blockade leads to cannabinoid receptor type 1 (CB1) receptor functional tolerance, which represents another challenge in this potential therapeutic strategy. Therefore, the present study tested whether full FAAH inhibition combined with partial MAGL inhibition would produce sustained antinociceptive effects with minimal cannabimimetic side effects. Accordingly, we tested a high dose of the FAAH inhibitor PF-3845 (N-​3-​pyridinyl-​4-​[[3-​[[5-​(trifluoromethyl)-​2-​pyridinyl]oxy]phenyl]methyl]-​1-​piperidinecarboxamide; 10 mg/kg) given in combination with a low dose of the MAGL inhibitor JZL184 [4-nitrophenyl 4-(dibenzo[d][1,3]dioxol-5-yl(hydroxy)methyl)piperidine-1-carboxylate] (4 mg/kg) in mouse models of inflammatory and neuropathic pain. This combination of inhibitors elicited profound increases in brain AEA levels (>10-fold) but only 2- to 3-fold increases in brain 2-AG levels. This combination produced significantly greater antinociceptive effects than single enzyme inhibition and did not elicit common cannabimimetic effects (e.g., catalepsy, hypomotility, hypothermia, and substitution for Δ(9)-tetrahydrocannabinol in the drug-discrimination assay), although these side effects emerged with high-dose JZL184 (i.e., 100 mg/kg). Finally, repeated administration of this combination did not lead to tolerance to its antiallodynic actions in the carrageenan assay or CB1 receptor functional tolerance. Thus, full FAAH inhibition

Biosynthesis of intestinal microvillar proteins. Dimerization of aminopeptidase N and lactase-phlorizin hydrolase

DEFF Research Database (Denmark)

Danielsen, E M

1990-01-01

of dimers of this enzyme therefore occurs prior to the Golgi-associated processing, and the slow rate of dimerization may be the rate-limiting step in the transport from the endoplasmic reticulum to the Golgi complex. For lactase-phlorizin hydrolase, the posttranslational processing includes a proteolytic......The pig intestinal brush border enzymes aminopeptidase N (EC 3.4.11.2) and lactase-phlorizin hydrolase (EC 3.2.1.23-62) are present in the microvillar membrane as homodimers. Dimethyl adipimidate was used to cross-link the two [35S]methionine-labeled brush border enzymes from cultured mucosal...... explants. For aminopeptidase N, dimerization did not begin until 5-10 min after synthesis, and maximal dimerization by cross-linking of the transient form of the enzyme required 1 h, whereas the mature form of aminopeptidase N cross-linked with unchanged efficiency from 45 min to 3 h of labeling. Formation...

Cloning, expression and mutation of a triazophos hydrolase gene from Burkholderia sp. SZL-1.

Science.gov (United States)

Zhang, Hao; Li, Qiang; Guo, Su-Hui; Cheng, Ming-Gen; Zhao, Meng-Jun; Hong, Qing; Huang, Xing

2016-06-01

Triazophos is a broad-spectrum and highly effective insecticide, and the residues of triazophos have been frequently detected in the environment. A triazophos-degrading bacterium, Burkholderia sp. SZL-1, was isolated from a long-term triazophos-polluted soil. Strain SZL-1 could hydrolyze triazophos to 1-phenyl-3-hydroxy-1,2,4-triazole, which was further utilized as the carbon sources for growth. The triazophos hydrolase gene trhA, cloned from strain SZL-1, was expressed and homogenously purified using Ni-nitrilotriacetic acid affinity chromatography. TrhA is 55 kDa and displays maximum activity at 25°C, pH 8.0. This enzyme still has nearly 60% activity at the range of 15°C-50°C for 30 min. TrhA was mutated by sequential error prone PCR and screened for improved activity for triazophos degradation. One purified variant protein (Val89-Gly89) named TrhA-M1 showed up to 3-fold improvement in specific activity against triazophos, and the specificity constants of Kcat and Kcat/Km for TrhA-M1 were improved up to 2.3- and 8.28-fold, respectively, compared to the wild-type enzyme. The results in this paper provided potential material for the contaminated soil remediation and hydrolase genetic structure research. © FEMS 2016. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

Inhibitor of differentiation 4 (Id4 is a potential tumor suppressor in prostate cancer

Directory of Open Access Journals (Sweden)

Carey Jason PW

2009-06-01

Full Text Available Abstract Background Inhibitor of differentiation 4 (Id4, a member of the Id gene family is also a dominant negative regulator of basic helix loop helix (bHLH transcription factors. Some of the functions of Id4 appear to be unique as compared to its other family members Id1, Id2 and Id3. Loss of Id4 gene expression in many cancers in association with promoter hypermethylation has led to the proposal that Id4 may act as a tumor suppressor. In this study we provide functional evidence that Id4 indeed acts as a tumor suppressor and is part of a cancer associated epigenetic re-programming. Methods Data mining was used to demonstrate Id4 expression in prostate cancer. Methylation specific polymerase chain reaction (MSP analysis was performed to understand molecular mechanisms associated with Id4 expression in prostate cancer cell lines. The effect of ectopic Id4 expression in DU145 cells was determined by cell cycle analysis (3H thymidine incorporation and FACS, expression of androgen receptor, p53 and cyclin dependent kinase inhibitors p27 and p21 by a combination of RT-PCR, real time-PCR, western blot and immuno-cytochemical analysis. Results Id4 expression was down-regulated in prostate cancer. Id4 expression was also down-regulated in prostate cancer line DU145 due to promoter hyper-methylation. Ectopic Id4 expression in DU145 prostate cancer cell line led to increased apoptosis and decreased cell proliferation due in part by an S-phase arrest. In addition to S-phase arrest, ectopic Id4 expression in PC3 cells also resulted in prolonged G2/M phase. At the molecular level these changes were associated with increased androgen receptor (AR, p21, p27 and p53 expression in DU145 cells. Conclusion The results suggest that Id4 acts directly as a tumor suppressor by influencing a hierarchy of cellular processes at multiple levels that leads to a decreased cell proliferation and change in morphology that is possibly mediated through induction of previously

Supplementing with non-glycoside hydrolase proteins enhances enzymatic deconstruction of plant biomass.

Science.gov (United States)

Su, Xiaoyun; Zhang, Jing; Mackie, Roderick I; Cann, Isaac K O

2012-01-01

The glycoside hydrolases (GH) of Caldicellulosiruptor bescii are thermophilic enzymes, and therefore they can hydrolyze plant cell wall polysaccharides at high temperatures. Analyses of two C. bescii glycoside hydrolases, CbCelA-TM1 and CbXyn10A with cellulase and endoxylanase activity, respectively, demonstrated that each enzyme is highly thermostable under static incubation at 70°C. Both enzymes, however, rapidly lost their enzymatic activities when incubated at 70°C with end-over-end shaking. Since crowding conditions, even at low protein concentrations, seem to influence enzymatic properties, three non-glycoside hydrolase proteins were tested for their capacity to stabilize the thermophilic proteins at high temperatures. The three proteins investigated were a small heat shock protein CbHsp18 from C. bescii, a histone MkHistone1 from Methanopyrus kandleri, and bovine RNase A, from a commercial source. Fascinatingly, each of these proteins increased the thermostability of the glycoside hydrolases at 70°C during end-over-end shaking incubation, and this property translated into increases in hydrolysis of several substrates including the bioenergy feedstock Miscanthus. Furthermore, MkHistone1 and RNase A also altered the initial products released from the cello-oligosaccharide cellopentaose during hydrolysis with the cellodextrinase CbCdx1A, which further demonstrated the capacity of the three non-GH proteins to influence hydrolysis of substrates by the thermophilic glycoside hydrolases. The non-GH proteins used in the present report were small proteins derived from each of the three lineages of life, and therefore expand the space from which different polypeptides can be tested for their influence on plant cell wall hydrolysis, a critical step in the emerging biofuel industry.

Supplementing with non-glycoside hydrolase proteins enhances enzymatic deconstruction of plant biomass.

Directory of Open Access Journals (Sweden)

Xiaoyun Su

Full Text Available The glycoside hydrolases (GH of Caldicellulosiruptor bescii are thermophilic enzymes, and therefore they can hydrolyze plant cell wall polysaccharides at high temperatures. Analyses of two C. bescii glycoside hydrolases, CbCelA-TM1 and CbXyn10A with cellulase and endoxylanase activity, respectively, demonstrated that each enzyme is highly thermostable under static incubation at 70°C. Both enzymes, however, rapidly lost their enzymatic activities when incubated at 70°C with end-over-end shaking. Since crowding conditions, even at low protein concentrations, seem to influence enzymatic properties, three non-glycoside hydrolase proteins were tested for their capacity to stabilize the thermophilic proteins at high temperatures. The three proteins investigated were a small heat shock protein CbHsp18 from C. bescii, a histone MkHistone1 from Methanopyrus kandleri, and bovine RNase A, from a commercial source. Fascinatingly, each of these proteins increased the thermostability of the glycoside hydrolases at 70°C during end-over-end shaking incubation, and this property translated into increases in hydrolysis of several substrates including the bioenergy feedstock Miscanthus. Furthermore, MkHistone1 and RNase A also altered the initial products released from the cello-oligosaccharide cellopentaose during hydrolysis with the cellodextrinase CbCdx1A, which further demonstrated the capacity of the three non-GH proteins to influence hydrolysis of substrates by the thermophilic glycoside hydrolases. The non-GH proteins used in the present report were small proteins derived from each of the three lineages of life, and therefore expand the space from which different polypeptides can be tested for their influence on plant cell wall hydrolysis, a critical step in the emerging biofuel industry.

The cardiovascular safety trials of DPP-4 inhibitors, GLP-1 agonists, and SGLT2 inhibitors.

Science.gov (United States)

Secrest, Matthew H; Udell, Jacob A; Filion, Kristian B

2017-04-01

In this paper, we review the results of large, double-blind, placebo-controlled randomized trials mandated by the US Food and Drug Administration to examine the cardiovascular safety of newly-approved antihyperglycemic agents in patients with type 2 diabetes. The cardiovascular effects of dipeptidyl peptidase-4 (DPP-4) inhibitors remain controversial: while these drugs did not reduce or increase the risk of primary, pre-specified composite cardiovascular outcomes, one DPP-4 inhibitor (saxagliptin) increased the risk of hospitalization for heart failure in the overall population; another (alogliptin) demonstrated inconsistent effects on heart failure hospitalization across subgroups of patients, and a third (sitagliptin) demonstrated no effect on heart failure. Evidence for cardiovascular benefits of glucagon-like peptide-1 (GLP-1) agonists has been similarly heterogeneous, with liraglutide and semaglutide reducing the risk of composite cardiovascular outcomes, but lixisenatide having no reduction or increase in cardiovascular risk. The effect of GLP-1 agonists on retinopathy remains a potential concern. In the only completed trial to date to assess a sodium-glucose cotransporter-2 (SGLT2) inhibitor, empagliflozin reduced the risk of composite cardiovascular endpoints, predominantly through its impact on cardiovascular mortality and heart failure hospitalization. Copyright © 2017 Elsevier Inc. All rights reserved.

Identification of the Gene Encoding Isoprimeverose-producing Oligoxyloglucan Hydrolase in Aspergillus oryzae*

Science.gov (United States)

Matsuzawa, Tomohiko; Mitsuishi, Yasushi; Kameyama, Akihiko

2016-01-01

Aspergillus oryzae produces a unique β-glucosidase, isoprimeverose-producing oligoxyloglucan hydrolase (IPase), that recognizes and releases isoprimeverose (α-d-xylopyranose-(1→6)-d-glucopyranose) units from the non-reducing ends of oligoxyloglucans. A gene encoding A. oryzae IPase, termed ipeA, was identified and expressed in Pichia pastoris. With the exception of cellobiose, IpeA hydrolyzes a variety of oligoxyloglucans and is a member of the glycoside hydrolase family 3. Xylopyranosyl branching at the non-reducing ends was vital for IPase activity, and galactosylation at a α-1,6-linked xylopyranosyl side chain completely abolished IpeA activity. Hepta-oligoxyloglucan saccharide (Xyl3Glc4) substrate was preferred over tri- (Xyl1Glc2) and tetra- (Xyl2Glc2) oligoxyloglucan saccharides substrates. IpeA transferred isoprimeverose units to other saccharides, indicating transglycosylation activity. The ipeA gene was expressed in xylose and xyloglucan media and was strongly induced in the presence of xyloglucan endo-xyloglucanase-hydrolyzed products. This is the first study to report the identification of a gene encoding IPase in eukaryotes. PMID:26755723

A Personal Retrospective: Elevating Anandamide (AEA by Targeting Fatty Acid Amide Hydrolase (FAAH and the Fatty Acid Binding Proteins (FABPs

Directory of Open Access Journals (Sweden)

Dale Deutsch

2016-10-01

Full Text Available This perspective was adapted from a Career Achievement Award talk given at the International Cannabinoid Research Society Symposium in Bukovina, Poland on June 27, 2016. As a biochemist working in the neurosciences, I was always fascinated with neurotransmitter inactivation. In 1993 we identified an enzyme activity that breaks down anandamide. We called the enzyme anandamide amidase, now called FAAH. We and other laboratories developed FAAH inhibitors that were useful reagents that also proved to have beneficial physiological effects and, until recently, new generations of inhibitors were in clinical trials. Nearly all neurotransmitters are water soluble and, as such, require a transmembrane protein transporter to pass through the lipid membrane for inactivation inside the cell. However, using model systems, we and others have shown that this is unnecessary for anandamide, an uncharged hydrophobic molecule that readily diffuses across the cellular membrane. Interestingly, its uptake is driven by the concentration gradient resulting from its breakdown mainly by FAAH localized in the endoplasmic reticulum. We identified the FABPs as intracellular carriers that solubilize anandamide, transporting anandamide to FAAH. Compounds that bind to FABPs block AEA breakdown, raising its level. The cannabinoids (THC and CBD also were discovered to bind FABPs and this may be one of the mechanisms by which CBD works in childhood epilepsy, raising anandamide levels. Targeting FABPs may be advantageous since they have some tissue specificity and do not require reactive serine hydrolase inhibitors, as does FAAH, with potential for off-target reactions.

Cycling Towards Progress: Ribociclib, CDK 4/6 inhibitor for Breast Cancer.

Science.gov (United States)

Spring, Laura; Bardia, Aditya

2018-04-23

Ribociclib is an orally active, highly selective inhibitor of cyclin-dependent kinase (CDK) 4 and 6. It is the second CDK 4/6 inhibitor approved for hormone receptor-positive breast cancer. The addition of ribociclib to an aromatase inhibitor has resulted in marked improvements in progression-free survival for patients with metastatic breast cancer. Copyright ©2018, American Association for Cancer Research.

Synthesis and preclinical evaluation of [11C-carbonyl]PF-04457845 for neuroimaging of fatty acid amide hydrolase

International Nuclear Information System (INIS)

Hicks, Justin W.; Parkes, Jun; Sadovski, Oleg; Tong, Junchao; Houle, Sylvain; Vasdev, Neil; Wilson, Alan A.

2013-01-01

Introduction: Fatty acid amide hydrolase (FAAH) has a significant role in regulating endocannabinoid signaling in the central nervous system. As such, FAAH inhibitors are being actively sought for pain, addiction, and other indications. This has led to the recent pursuit of positron emission tomography (PET) radiotracers targeting FAAH. We report herein the preparation and preclinical evaluation of [ 11 C-carbonyl]PF-04457845, an isotopologue of the potent irreversible FAAH inhibitor. Methods: PF-04457845 was radiolabeled at the carbonyl position via automated [ 11 C]CO 2 -fixation. Ex vivo brain biodistribution of [ 11 C-carbonyl]PF-04457845 was carried out in conscious rats. Specificity was determined by pre-administration of PF-04457845 or URB597 prior to [ 11 C-carbonyl]PF-04457845. In a separate experiment, rats injected with the title radiotracer had whole brains excised, homogenized and extracted to examine irreversible binding to brain parenchyma. Results: The title compound was prepared in 5 ± 1% (n = 4) isolated radiochemical yield based on starting [ 11 C]CO 2 (decay uncorrected) within 25 min from end-of-bombardment in > 98% radiochemical purity and a specific activity of 73.5 ± 8.2 GBq/μmol at end-of-synthesis. Uptake of [ 11 C-carbonyl]PF-04457845 into the rat brain was high (range of 1.2–4.4 SUV), heterogeneous, and in accordance with reported FAAH distribution. Saturable binding was demonstrated by a dose-dependent reduction in brain radioactivity uptake following pre-treatment with PF-04457845. Pre-treatment with the prototypical FAAH inhibitor, URB597, reduced the brain radiotracer uptake in all regions by 71–81%, demonstrating specificity for FAAH. The binding of [ 11 C-carbonyl]PF-04457845 to FAAH at 40 min post injection was irreversible as 98% of the radioactivity in the brain could not be extracted. Conclusions: [ 11 C-carbonyl]PF-04457845 was rapidly synthesized via an automated radiosynthesis. Ex vivo biodistribution studies in

HYDROLASING OF CONTAMINATED UNDERWATER BASIN SURFACES AT THE HANFORD K AREA

International Nuclear Information System (INIS)

CHRONISTER, G.B.

2005-01-01

This paper discusses selecting and implementing hydrolasing technology to reduce radioactive contamination in preparing to dispose of the K Basins; two highly contaminated concrete basins at the Hanford Site. A large collection of spent nuclear fuel stored for many years underwater at the K Basins has been removed to stable, dry, safe storage. Remediation activities have begun for the remaining highly contaminated water. sludge, and concrete basin structures. Hydrolasing will be used to decontaminate and prepare the basin structures for disposal

Fatty acid amide hydrolase inhibition heightens anandamide signaling without producing reinforcing effects in primates

Science.gov (United States)

Justinova, Zuzana; Mangieri, Regina A.; Bortolato, Marco; Chefer, Svetlana I.; Mukhin, Alexey G.; Clapper, Jason R.; King, Alvin R.; Redhi, Godfrey H.; Yasar, Sevil; Piomelli, Daniele; Goldberg, Steven R.

2008-01-01

Background CB1 cannabinoid receptors in the brain are known to participate in the regulation of reward-based behaviors, however, the contribution of each of the endocannabinoid transmitters, anandamide and 2-arachidonoylglycerol (2-AG), to these behaviors remains undefined. To address this question, we assessed the effects of URB597, a selective anandamide deactivation inhibitor, as a reinforcer of drug-seeking and drug-taking behavior in squirrel monkeys. Methods We investigated the reinforcing effects of the fatty acid amide hydrolase (FAAH) inhibitor URB597 in monkeys trained to intravenously self-administer Δ9-tetrahydrocannabinol (THC), anandamide or cocaine, and quantified brain endocannabinoid levels using liquid chromatography/mass spectrometry. We measured brain FAAH activity using an ex vivo enzyme assay. Results URB597 (0.3 mg/kg, intravenous) blocked FAAH activity and increased anandamide levels throughout the monkey brain. This effect was accompanied by a marked compensatory decrease in 2-AG levels. Monkeys did not self-administer URB597 and the drug did not promote reinstatement of extinguished drug-seeking behavior previously maintained by THC, anandamide, or cocaine. Pretreatment with URB597 did not modify self-administration of THC or cocaine even though, as expected, it significantly potentiated anandamide self-administration. Conclusions In the monkey brain, the FAAH inhibitor URB597 increases anandamide levels while causing a compensatory down-regulation in 2-AG levels. These effects are accompanied by a striking lack of reinforcing properties, which distinguishes URB597 from direct-acting cannabinoid agonists such as THC. Our results reveal an unexpected functional heterogeneity within the endocannabinoid signaling system, and suggest that FAAH inhibitors might be used therapeutically without risk of abuse or triggering of relapse to drug abuse. PMID:18814866

Factors Associated with Utilization of Dipeptidyl-4 Inhibitors in Patients with Type 2 Diabetes Mellitus: A Cross-Sectional Retrospective Study

Directory of Open Access Journals (Sweden)

Hasniza Zaman Huri

2014-01-01

Full Text Available Dipeptidyl-4 (DPP-4 inhibitors are oral antidiabetic agents recently introduced to Malaysia. Thus, limited data is available on their utilization patterns and factors associated with their use. This study aims to analyse the utilization patterns of DPP-4 inhibitors, factors that influenced the choice of agent, and the rationale for treatment with DPP-4 inhibitors in patients with type 2 diabetes mellitus. This retrospective study was conducted to address the utilization pattern of DPP-4 inhibitors and factors that influence choice in type 2 diabetes mellitus patients. 299 subjects taking either sitagliptin or vildagliptin from September 2008 to September 2012 were included in the study. Sitagliptin was more frequently prescribed than vildagliptin. Of the patients prescribed DPP-4 inhibitors, 95% received combinations of these and other agents, whereas only 5% were prescribed DPP-4 inhibitors as monotherapy. Factors affecting the utilization of DPP-4 inhibitors included age (P=0.049 and concomitant use of beta blockers (P=0.045 and aspirin (P=0.008. Early identification of factors associated with DPP-4 inhibitors is essential to enhance quality use of the drugs.

Comparative review of dipeptidyl peptidase-4 inhibitors and sulphonylureas

DEFF Research Database (Denmark)

Deacon, Carolyn F.; Lebovitz, HE

2016-01-01

compound being launched in 2006, but the class now globally encompasses at least 11 different compounds. DPP-4 inhibitors improve glycaemic control with similar efficacy to SUs, but do not usually provoke hypoglycaemia or weight gain, are relatively free from adverse side effects, and have recently been...... drugs (DPP-4 inhibitors and SUs), highlighting differences and similarities between members of the same class, as well as discussing the potential advantages and disadvantages of the two drug classes. While both classes have their merits, the choice of which to use depends on the characteristics of each...

Epoxide hydrolase affects estrogen production in the human ovary.

Science.gov (United States)

Hattori, N; Fujiwara, H; Maeda, M; Fujii, S; Ueda, M

2000-09-01

To investigate the mechanisms of ovarian cell differentiation, we raised a new monoclonal antibody, HCL-3, which reacted with human luteal cells. It also reacted with human and porcine hepatocytes. The immunoaffinity-purified HCL-3 antigen from human corpora lutea (CL) was shown to be a 46-kDa protein. The N-terminal 22 amino acids of the 46-kDa protein from porcine liver exhibited high homology (82%) to human microsomal epoxide hydrolase (mEH). The purified HCL-3 antigen from human CL or porcine liver showed EH enzyme activity, confirming that HCL-3 antigen is identical to mEH, which is reported to detoxify the toxic substrates in the liver. In human follicles, mEH was immunohistochemically detected on granulosa and theca interna cells. In the menstrual and pregnant CL, mEH was also expressed on large and small luteal cells. A competitive inhibitor of EH, 1,2-epoxy-3,3,3-trichloropropane, inhibited the conversion of estradiol from testosterone by granulosa cells cultured in vitro, indicating the involvement of mEH in ovarian estrogen production. Because anticonvulsant sodium valproate and its analogues were reported to inhibit EH enzyme activity, these findings provide a new insight into the etiology of endocrine disorders that are frequently observed among epileptic patients taking anticonvulsant drugs.

[Research progress of dipeptidyl peptidase 4 inhibitors on healing of chronic diabetic foot ulcers].

Science.gov (United States)

Gao, Yunyi; Liang, Yujie; Ran, Xingwu

2018-05-01

To review the effect of dipeptidyl peptidase 4 (DPP-4) inhibitors on the wound healing and its mechanisms in chronic diabetic foot ulcers. The latest literature concerning DPP-4 inhibitors for chronic diabetic foot ulcers was extensively reviewed, as well as the potential benefit and mechanism of DPP-4 inhibitors on wound healing of diabetic foot ulcers was analyzed thoroughly. DPP-4 inhibitors can accelerated the ulcer healing. The mechanisms probably include inhibiting the expression of the matrix metalloproteinase (MMP) and restoring the balance of the wound MMP and the tissue inhibitors of MMP; promoting recruitment of endothelial progenitor cells and augmenting angiogenesis; optimizing extracellular matrix construction and the immune response to persistent hypoxia in chronic diabetes wounds, and so on. At present, clinical researches show that DPP-4 inhibitors may be considered as an adjuvant treatment for chronic diabetic foot ulcers. DPP-4 inhibitors show promise in the local wound healing of chronic diabetic foot ulcers. However, more strictly designed, adequately powered, long-term follow-up, and high-quality randomized control trials are needed to further verify their efficacy and safety for chronic diabetic foot ulcers.

Purification and characterization of RihC, a xanthosine-inosine-uridine-adenosine-preferring hydrolase from Salmonella enterica serovar Typhimurium

DEFF Research Database (Denmark)

Hansen, Michael Riis; Dandanell, Gert

2005-01-01

as the sole carbon and energy source. By functional complementation, we have isolated a nucleoside hydrolase (rihC) that can complement a xapA deletion in E. coli and we have overexpressed, purified and characterized this hydrolase. RihC is a heat stable homotetrameric enzyme with a molecular weight of 135 k...... the neutral form of xanthosine....

Characterization of two novel bacterial type A exo-chitobiose hydrolases having C-terminal 5/12-type carbohydrate-binding modules

DEFF Research Database (Denmark)

Binti Jamek, Shariza; Nyffenegger, Christian; Muschiol, Jan

2017-01-01

"exo-chitobiose hydrolases." In this study, the chitinase type A from Serratia marcescens (SmaChiA) was used as a template for identifying two novel exo-chitobiose hydrolase type A enzymes, FbalChi18A and MvarChi18A, originating from the marine organisms Ferrimonas balearica and Microbulbifer...

Dipeptidyl peptidase-4 inhibitors for the treatment of type 2 diabetes

DEFF Research Database (Denmark)

Deacon, Carolyn F; Holst, Jens Juul

2013-01-01

INTRODUCTION: Dipeptidyl peptidase (DPP)-4 inhibitors belong to one class of drugs that have been approved for treatment of type 2 diabetes (T2D) based on the glucose-lowering actions of the gastrointestinal hormone glucagon-like peptide (GLP)-1. Several different compounds are now available...... of the different DPP-4 inhibitors, all are small orally active compounds with broadly similar HbA1c-lowering efficacy. They improve glycaemic control in T2D, without increasing the risk of hypoglycaemia or causing weight gain. They can be used as monotherapy or in combination with other anti-diabetic therapies......, and although their mechanism of action (inhibition of the catalytic activity of DPP-4) is the same, there are fundamental differences between them. AREAS COVERED: The authors discuss the differences between different DPP-4 inhibitors and review their therapeutic efficacy and key safety data. The literature...

A review of dipeptidyl peptidase-4 inhibitors. Hot topics from randomized controlled trials

DEFF Research Database (Denmark)

Deacon, Carolyn F

2018-01-01

The first clinical study to investigate effects of dipeptidyl peptidase-4 (DPP-4) inhibition was published in 2002, and since then, numerous randomized controlled trials (RCTs) have shown that DPP-4 inhibitors are efficacious, safe and well-tolerated. This review will focus upon RCTs which have i...

Post-exposure administration of diazepam combined with soluble epoxide hydrolase inhibition stops seizures and modulates neuroinflammation in a murine model of acute TETS intoxication

International Nuclear Information System (INIS)

Vito, Stephen T.; Austin, Adam T.; Banks, Christopher N.; Inceoglu, Bora; Bruun, Donald A.; Zolkowska, Dorota; Tancredi, Daniel J.; Rogawski, Michael A.; Hammock, Bruce D.; Lein, Pamela J.

2014-01-01

Tetramethylenedisulfotetramine (TETS) is a potent convulsant poison for which there is currently no approved antidote. The convulsant action of TETS is thought to be mediated by inhibition of type A gamma-aminobutyric acid receptor (GABA A R) function. We, therefore, investigated the effects of post-exposure administration of diazepam, a GABA A R positive allosteric modulator, on seizure activity, death and neuroinflammation in adult male Swiss mice injected with a lethal dose of TETS (0.15 mg/kg, ip). Administration of a high dose of diazepam (5 mg/kg, ip) immediately following the second clonic seizure (approximately 20 min post-TETS injection) effectively prevented progression to tonic seizures and death. However, this treatment did not prevent persistent reactive astrogliosis and microglial activation, as determined by GFAP and Iba-1 immunoreactivity and microglial cell morphology. Inhibition of soluble epoxide hydrolase (sEH) has been shown to exert potent anti-inflammatory effects and to increase survival in mice intoxicated with other GABA A R antagonists. The sEH inhibitor TUPS (1 mg/kg, ip) administered immediately after the second clonic seizure did not protect TETS-intoxicated animals from tonic seizures or death. Combined administration of diazepam (5 mg/kg, ip) and TUPS (1 mg/kg, ip, starting 1 h after diazepam and repeated every 24 h) prevented TETS-induced lethality and influenced signs of neuroinflammation in some brain regions. Significantly decreased microglial activation and enhanced reactive astrogliosis were observed in the hippocampus, with no changes in the cortex. Combining an agent that targets specific anti-inflammatory mechanisms with a traditional antiseizure drug may enhance treatment outcome in TETS intoxication. - Highlights: • Acute TETS intoxication causes delayed and persistent neuroinflammation. • Diazepam given post-TETS prevents lethal tonic seizures but not neuroinflammation. • A soluble epoxide hydrolase inhibitor alters

Post-exposure administration of diazepam combined with soluble epoxide hydrolase inhibition stops seizures and modulates neuroinflammation in a murine model of acute TETS intoxication

Energy Technology Data Exchange (ETDEWEB)

Vito, Stephen T., E-mail: stvito@ucdavis.edu [Department of Entomology, College of Agricultural and Environmental Sciences, University of California-Davis, Davis, CA 95616 (United States); Austin, Adam T., E-mail: aaustin@ucdavis.edu [Department of Pediatrics, School of Medicine, University of California-Davis Medical Center, Sacramento, CA 95817 (United States); Banks, Christopher N., E-mail: Christopher.Banks@oehha.ca.gov [Department of Molecular Biosciences, School of Veterinary Medicine, University of California-Davis, Davis, CA 95616 (United States); Inceoglu, Bora, E-mail: abinceoglu@ucdavis.edu [Department of Entomology, College of Agricultural and Environmental Sciences, University of California-Davis, Davis, CA 95616 (United States); Bruun, Donald A., E-mail: dabruun@ucdavis.edu [Department of Molecular Biosciences, School of Veterinary Medicine, University of California-Davis, Davis, CA 95616 (United States); Zolkowska, Dorota, E-mail: dzolkowska@gmail.com [Department of Neurology, School of Medicine, University of California-Davis, Sacramento, CA 95817 (United States); Tancredi, Daniel J., E-mail: djtancredi@ucdavis.edu [Department of Pediatrics, School of Medicine, University of California-Davis Medical Center, Sacramento, CA 95817 (United States); Rogawski, Michael A., E-mail: rogawski@ucdavis.edu [Department of Neurology, School of Medicine, University of California-Davis, Sacramento, CA 95817 (United States); Hammock, Bruce D., E-mail: bdhammock@ucdavis.edu [Department of Entomology, College of Agricultural and Environmental Sciences, University of California-Davis, Davis, CA 95616 (United States); Lein, Pamela J., E-mail: pjlein@ucdavis.edu [Department of Molecular Biosciences, School of Veterinary Medicine, University of California-Davis, Davis, CA 95616 (United States)

2014-12-01

Tetramethylenedisulfotetramine (TETS) is a potent convulsant poison for which there is currently no approved antidote. The convulsant action of TETS is thought to be mediated by inhibition of type A gamma-aminobutyric acid receptor (GABA{sub A}R) function. We, therefore, investigated the effects of post-exposure administration of diazepam, a GABA{sub A}R positive allosteric modulator, on seizure activity, death and neuroinflammation in adult male Swiss mice injected with a lethal dose of TETS (0.15 mg/kg, ip). Administration of a high dose of diazepam (5 mg/kg, ip) immediately following the second clonic seizure (approximately 20 min post-TETS injection) effectively prevented progression to tonic seizures and death. However, this treatment did not prevent persistent reactive astrogliosis and microglial activation, as determined by GFAP and Iba-1 immunoreactivity and microglial cell morphology. Inhibition of soluble epoxide hydrolase (sEH) has been shown to exert potent anti-inflammatory effects and to increase survival in mice intoxicated with other GABA{sub A}R antagonists. The sEH inhibitor TUPS (1 mg/kg, ip) administered immediately after the second clonic seizure did not protect TETS-intoxicated animals from tonic seizures or death. Combined administration of diazepam (5 mg/kg, ip) and TUPS (1 mg/kg, ip, starting 1 h after diazepam and repeated every 24 h) prevented TETS-induced lethality and influenced signs of neuroinflammation in some brain regions. Significantly decreased microglial activation and enhanced reactive astrogliosis were observed in the hippocampus, with no changes in the cortex. Combining an agent that targets specific anti-inflammatory mechanisms with a traditional antiseizure drug may enhance treatment outcome in TETS intoxication. - Highlights: • Acute TETS intoxication causes delayed and persistent neuroinflammation. • Diazepam given post-TETS prevents lethal tonic seizures but not neuroinflammation. • A soluble epoxide hydrolase

Oxidoreductases provide a more generic response to metallic stressors (Cu and Cd) than hydrolases in soil fungi: new ecotoxicological insights.

Science.gov (United States)

Lebrun, Jérémie D; Demont-Caulet, Nathalie; Cheviron, Nathalie; Laval, Karine; Trinsoutrot-Gattin, Isabelle; Mougin, Christian

2016-02-01

The present study investigates the effect of metals on the secretion of enzymes from 12 fungal strains maintained in liquid cultures. Hydrolases (acid phosphatase, β-glucosidase, β-galactosidase, and N-acetyl-β-glucosaminidase) and ligninolytic oxidoreductases (laccase, Mn, and lignin peroxidases) activities, as well as biomass production, were measured in culture fluids from fungi exposed to Cu or Cd. Our results showed that all fungi secreted most of the selected hydrolases and that about 50% of them produced a partial oxidative system in the absence of metals. Then, exposure of fungi to metals led to the decrease in biomass production. At the enzymatic level, Cu and Cd modified the secretion profiles of soil fungi. The response of hydrolases to metals was contrasted and complex and depended on metal, enzyme, and fungal strain considered. By contrast, the metals always stimulated the activity of ligninolytic oxidoreductases in fungal strains. In some of them, oxidoreductases were specifically produced following metal exposure. Fungal oxidoreductases provide a more generic response than hydrolases, constituting thus a physiological basis for their use as biomarkers of metal exposure in soils.

High-throughput screening identifies Ceefourin 1 and Ceefourin 2 as highly selective inhibitors of multidrug resistance protein 4 (MRP4).

Science.gov (United States)

Cheung, Leanna; Flemming, Claudia L; Watt, Fujiko; Masada, Nanako; Yu, Denise M T; Huynh, Tony; Conseil, Gwenaëlle; Tivnan, Amanda; Polinsky, Alexander; Gudkov, Andrei V; Munoz, Marcia A; Vishvanath, Anasuya; Cooper, Dermot M F; Henderson, Michelle J; Cole, Susan P C; Fletcher, Jamie I; Haber, Michelle; Norris, Murray D

2014-09-01

Multidrug resistance protein 4 (MRP4/ABCC4), a member of the ATP-binding cassette (ABC) transporter superfamily, is an organic anion transporter capable of effluxing a wide range of physiologically important signalling molecules and drugs. MRP4 has been proposed to contribute to numerous functions in both health and disease; however, in most cases these links remain to be unequivocally established. A major limitation to understanding the physiological and pharmacological roles of MRP4 has been the absence of specific small molecule inhibitors, with the majority of established inhibitors also targeting other ABC transporter family members, or inhibiting the production, function or degradation of important MRP4 substrates. We therefore set out to identify more selective and well tolerated inhibitors of MRP4 that might be used to study the many proposed functions of this transporter. Using high-throughput screening, we identified two chemically distinct small molecules, Ceefourin 1 and Ceefourin 2, that inhibit transport of a broad range of MRP4 substrates, yet are highly selective for MRP4 over other ABC transporters, including P-glycoprotein (P-gp), ABCG2 (Breast Cancer Resistance Protein; BCRP) and MRP1 (multidrug resistance protein 1; ABCC1). Both compounds are more potent MRP4 inhibitors in cellular assays than the most widely used inhibitor, MK-571, requiring lower concentrations to effect a comparable level of inhibition. Furthermore, Ceefourin 1 and Ceefourin 2 have low cellular toxicity, and high microsomal and acid stability. These newly identified inhibitors should be of great value for efforts to better understand the biological roles of MRP4, and may represent classes of compounds with therapeutic application. Copyright © 2014 Elsevier Inc. All rights reserved.

Screening and evaluation of the glucoside hydrolase activity in Saccharomyces and Brettanomyces brewing yeasts.

Science.gov (United States)

Daenen, L; Saison, D; Sterckx, F; Delvaux, F R; Verachtert, H; Derdelinckx, G

2008-02-01

The aim of this study was to select and examine Saccharomyces and Brettanomyces brewing yeasts for hydrolase activity towards glycosidically bound volatile compounds. A screening for glucoside hydrolase activity of 58 brewing yeasts belonging to the genera Saccharomyces and Brettanomyces was performed. The studied Saccharomyces brewing yeasts did not show 1,4-beta-glucosidase activity, but a strain dependent beta-glucanase activity was observed. Some Brettanomyces species did show 1,4-beta-glucosidase activity. The highest constitutive activity was found in Brettanomyces custersii. For the most interesting strains the substrate specificity was studied and their activity was evaluated in fermentation experiments with added hop glycosides. Fermentations with Br. custersii led to the highest release of aglycones. Pronounced exo-beta-glucanase activity in Saccharomyces brewing yeasts leads to a higher release of certain aglycones. Certain Brettanomyces brewing yeasts, however, are more interesting for hydrolysis of glycosidically bound volatiles of hops. The release of flavour active compounds from hop glycosides opens perspectives for the bioflavouring and product diversification of beverages like beer. The release can be enhanced by using Saccharomyces strains with high exo-beta-glucanase activity. Higher activities can be found in Brettanomyces species with beta-glucosidase activity.

Generation and characterization of epoxide hydrolase 3 (EPHX3-deficient mice.

Directory of Open Access Journals (Sweden)

Samantha L Hoopes

Full Text Available Cytochrome P450 (CYP epoxygenases metabolize arachidonic acid into epoxyeicosatrienoic acids (EETs, which play an important role in blood pressure regulation, protection against ischemia-reperfusion injury, angiogenesis, and inflammation. Epoxide hydrolases metabolize EETs to their corresponding diols (dihydroxyeicosatrienoic acids; DHETs which are biologically less active. Microsomal epoxide hydrolase (EPHX1, mEH and soluble epoxide hydrolase (EPHX2, sEH were identified >30 years ago and are capable of hydrolyzing EETs to DHETs. A novel epoxide hydrolase, EPHX3, was recently identified by sequence homology and also exhibits epoxide hydrolase activity in vitro with a substrate preference for 9,10-epoxyoctadecamonoenoic acid (EpOME and 11,12-EET. EPHX3 is highly expressed in the skin, lung, stomach, esophagus, and tongue; however, its endogenous function is unknown. Therefore, we investigated the impact of genetic disruption of Ephx3 on fatty acid epoxide hydrolysis and EET-related physiology in mice. Ephx3-/- mice were generated by excising the promoter and first four exons of the Ephx3 gene using Cre-LoxP methodology. LC-MS/MS analysis of Ephx3-/- heart, lung, and skin lysates revealed no differences in endogenous epoxide:diol ratios compared to wild type (WT. Ephx3-/- mice also exhibited no change in plasma levels of fatty acid epoxides and diols relative to WT. Incubations of cytosolic and microsomal fractions prepared from Ephx3-/- and WT stomach, lung, and skin with synthetic 8,9-EET, 11,12-EET, and 9,10-EpOME revealed no significant differences in rates of fatty acid diol formation between the genotypes. Ephx3-/- hearts had similar functional recovery compared to WT hearts following ischemia/reperfusion injury. Following intranasal lipopolysaccharide (LPS exposure, Ephx3-/- mice were not different from WT in terms of lung histology, bronchoalveolar lavage fluid cell counts, or fatty acid epoxide and diol levels. We conclude that genetic

Hepatitis C Virus NS3/4A Protease Inhibitors: A Light at the End of the Tunnel

Science.gov (United States)

Chatel-Chaix, Laurent; Baril, Martin; Lamarre, Daniel

2010-01-01

Hepatitis C virus (HCV) infection is a serious and growing threat to human health. The current treatment provides limited efficacy and is poorly tolerated, highlighting the urgent medical need for novel therapeutics. The membrane-targeted NS3 protein in complex with the NS4A comprises a serine protease domain (NS3/4A protease) that is essential for viral polyprotein maturation and contributes to the evasion of the host innate antiviral immunity by HCV. Therefore, the NS3/4A protease represents an attractive target for drug discovery, which is tied in with the challenge to develop selective small-molecule inhibitors. A rational drug design approach, based on the discovery of N-terminus product inhibition, led to the identification of potent and orally bioavailable NS3 inhibitors that target the highly conserved protease active site. This review summarizes the NS3 protease inhibitors currently challenged in clinical trials as one of the most promising antiviral drug class, and possibly among the first anti-HCV agents to be approved for the treatment of HCV infection. PMID:21994705

Hepatitis C Virus NS3/4A Protease Inhibitors: A Light at the End of the Tunnel

Directory of Open Access Journals (Sweden)

Laurent Chatel-Chaix

2010-08-01

Full Text Available Hepatitis C virus (HCV infection is a serious and growing threat to human health. The current treatment provides limited efficacy and is poorly tolerated, highlighting the urgent medical need for novel therapeutics. The membrane-targeted NS3 protein in complex with the NS4A comprises a serine protease domain (NS3/4A protease that is essential for viral polyprotein maturation and contributes to the evasion of the host innate antiviral immunity by HCV. Therefore, the NS3/4A protease represents an attractive target for drug discovery, which is tied in with the challenge to develop selective small-molecule inhibitors. A rational drug design approach, based on the discovery of N-terminus product inhibition, led to the identification of potent and orally bioavailable NS3 inhibitors that target the highly conserved protease active site. This review summarizes the NS3 protease inhibitors currently challenged in clinical trials as one of the most promising antiviral drug class, and possibly among the first anti-HCV agents to be approved for the treatment of HCV infection.

Dipeptidyl peptidase-4 inhibitor ameliorates early renal injury through its anti-inflammatory action in a rat model of type 1 diabetes

Energy Technology Data Exchange (ETDEWEB)

Kodera, Ryo, E-mail: kodera@cc.okayama-u.ac.jp [Center for Innovative Clinical Medicine, Okayama University Hospital, 2-5-1 Shikata-cho, Kita-ku, Okayama 700-8558 (Japan); Shikata, Kenichi [Center for Innovative Clinical Medicine, Okayama University Hospital, 2-5-1 Shikata-cho, Kita-ku, Okayama 700-8558 (Japan); Department of Medicine and Clinical Science, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, 2-5-1 Shikata-cho, Kita-ku, Okayama 700-8558 (Japan); Takatsuka, Tetsuharu; Oda, Kaori; Miyamoto, Satoshi; Kajitani, Nobuo; Hirota, Daisho; Ono, Tetsuichiro [Department of Medicine and Clinical Science, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, 2-5-1 Shikata-cho, Kita-ku, Okayama 700-8558 (Japan); Usui, Hitomi Kataoka [Department of Primary Care and Medical Education, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, 2-5-1 Shikata-cho, Kita-ku, Okayama 700-8558 (Japan); Makino, Hirofumi [Department of Medicine and Clinical Science, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, 2-5-1 Shikata-cho, Kita-ku, Okayama 700-8558 (Japan)

2014-01-17

Highlights: •DPP-4 inhibitor decreased urinary albumin excretion in a rat of type 1 diabetes. •DPP-4 inhibitor ameliorated histlogical changes of diabetic nephropathy. •DPP-4 inhibitor has reno-protective effects through anti-inflammatory action. •DPP-4 inhibitor is beneficial on diabetic nephropathy besides lowering blood glucose. -- Abstract: Introduction: Dipeptidyl peptidase-4 (DPP-4) inhibitors are incretin-based drugs in patients with type 2 diabetes. In our previous study, we showed that glucagon-like peptide-1 (GLP-1) receptor agonist has reno-protective effects through anti-inflammatory action. The mechanism of action of DPP-4 inhibitor is different from that of GLP-1 receptor agonists. It is not obvious whether DPP-4 inhibitor prevents the exacerbation of diabetic nephropathy through anti-inflammatory effects besides lowering blood glucose or not. The purpose of this study is to clarify the reno-protective effects of DPP-4 inhibitor through anti-inflammatory actions in the early diabetic nephropathy. Materials and methods: Five-week-old male Sprague–Dawley (SD) rats were divided into three groups; non-diabetes, diabetes and diabetes treated with DPP-4 inhibitor (PKF275-055; 3 mg/kg/day). PKF275-055 was administered orally for 8 weeks. Results: PKF275-055 increased the serum active GLP-1 concentration and the production of urinary cyclic AMP. PKF275-055 decreased urinary albumin excretion and ameliorated histological change of diabetic nephropathy. Macrophage infiltration was inhibited, and inflammatory molecules were down-regulated by PKF275-055 in the glomeruli. In addition, nuclear factor-κB (NF-κB) activity was suppressed in the kidney. Conclusions: These results indicate that DPP-4 inhibitor, PKF275-055, have reno-protective effects through anti-inflammatory action in the early stage of diabetic nephropathy. The endogenous biological active GLP-1 might be beneficial on diabetic nephropathy besides lowering blood glucose.

Dipeptidyl peptidase-4 inhibitor ameliorates early renal injury through its anti-inflammatory action in a rat model of type 1 diabetes

International Nuclear Information System (INIS)

Kodera, Ryo; Shikata, Kenichi; Takatsuka, Tetsuharu; Oda, Kaori; Miyamoto, Satoshi; Kajitani, Nobuo; Hirota, Daisho; Ono, Tetsuichiro; Usui, Hitomi Kataoka; Makino, Hirofumi

2014-01-01

Highlights: •DPP-4 inhibitor decreased urinary albumin excretion in a rat of type 1 diabetes. •DPP-4 inhibitor ameliorated histlogical changes of diabetic nephropathy. •DPP-4 inhibitor has reno-protective effects through anti-inflammatory action. •DPP-4 inhibitor is beneficial on diabetic nephropathy besides lowering blood glucose. -- Abstract: Introduction: Dipeptidyl peptidase-4 (DPP-4) inhibitors are incretin-based drugs in patients with type 2 diabetes. In our previous study, we showed that glucagon-like peptide-1 (GLP-1) receptor agonist has reno-protective effects through anti-inflammatory action. The mechanism of action of DPP-4 inhibitor is different from that of GLP-1 receptor agonists. It is not obvious whether DPP-4 inhibitor prevents the exacerbation of diabetic nephropathy through anti-inflammatory effects besides lowering blood glucose or not. The purpose of this study is to clarify the reno-protective effects of DPP-4 inhibitor through anti-inflammatory actions in the early diabetic nephropathy. Materials and methods: Five-week-old male Sprague–Dawley (SD) rats were divided into three groups; non-diabetes, diabetes and diabetes treated with DPP-4 inhibitor (PKF275-055; 3 mg/kg/day). PKF275-055 was administered orally for 8 weeks. Results: PKF275-055 increased the serum active GLP-1 concentration and the production of urinary cyclic AMP. PKF275-055 decreased urinary albumin excretion and ameliorated histological change of diabetic nephropathy. Macrophage infiltration was inhibited, and inflammatory molecules were down-regulated by PKF275-055 in the glomeruli. In addition, nuclear factor-κB (NF-κB) activity was suppressed in the kidney. Conclusions: These results indicate that DPP-4 inhibitor, PKF275-055, have reno-protective effects through anti-inflammatory action in the early stage of diabetic nephropathy. The endogenous biological active GLP-1 might be beneficial on diabetic nephropathy besides lowering blood glucose

Inhibition of soluble epoxide hydrolase lowers portal hypertension in cirrhotic rats by ameliorating endothelial dysfunction and liver fibrosis.

Science.gov (United States)

Deng, Wensheng; Zhu, Yiming; Lin, Jiayun; Zheng, Lei; Zhang, Chihao; Luo, Meng

2017-07-01

Epoxyeicostrienoic acids (EETs) are arachidonic acid derived meditators which are catalyzed by soluble epoxide hydrolase (sEH) to less active dihydroeicostrienoics acids (DHETS). The aim of our study is to investigate the effects of sEH inhibition on hepatic and systemic hemodynamics, hepatic endothelial dysfunction, and hepatic fibrosis in CCl4 cirrhotic rats. The sEH inhibitor,trans-4-{4-[3-(4-trifluoromethoxyphenyl)-ureido]cyclohexyloxy}benzoic acid (t-TUCB) was administered to stabilize hepatic EETs by gavage at a dose of 1mg/kg/d. Our results showed that hepatic sEH expression was markedly increased in portal hypertension, and led to a lower ratio of EETs/DHETs which was effectively reversed by t-TUCB administration. t-TUCB significantly decreased portal pressure without significant changes in systemic hemodynamics, which was associated with the attenuation of intrahepatic vascular resistance (IHVR) and liver fibrosis. t-TUCB ameliorated endothelial dysfunction, increased hepatic endothelial nitric oxide synthase (eNOS) phosphorylation and nitric oxide (NO) production. In addition, t-TUCB significantly reduced alpha-Smooth Muscle Actin (α-SMA) expression and liver fibrosis, which was associated with a decrease in NF-κB signaling. Taken together, inhibition of sEH reduces portal pressure, liver fibrosis and attenuates hepatic endothelial dysfunction in cirrhotic rats. Our results indicate that sEH inhbitors may be useful in the treatment of portal hypertension in patients with cirrhosis. Copyright © 2017 Elsevier Inc. All rights reserved.

α-Amylase: an enzyme specificity found in various families of glycoside hydrolases

DEFF Research Database (Denmark)

Janeček, Štefan; Svensson, Birte; MacGregor, E. Ann

2014-01-01

of all carbohydrate-active enzymes, it is one of the most frequently occurring glycoside hydrolases (GH). α-Amylase is the main representative of family GH13, but it is probably also present in the families GH57 and GH119, and possibly even in GH126. Family GH13, known generally as the main α...... investigation because of an obvious, but unexpected, homology with inverting β-glucan-active hydrolases....

Effects of URB597 as an inhibitor of fatty acid amide hydrolase on WIN55, 212-2-induced learning and memory deficits in rats.

Science.gov (United States)

Hasanein, Parisa; Teimuri Far, Massoud

2015-04-01

Cannabinoid and endocannabinoid systems have been implicated in several physiological functions including modulation of cognition. In this study we evaluated the effects and interaction between fatty-acid amide hydrolase (FAAH) inhibitor URB597 and CB1 receptor agonist WIN55, 212-2 on memory using object recognition and passive avoidance learning (PAL) tests. Learning and memory impairment was induced by WIN 55, 212-2 administration (1mg/kg, i.p.) 30min before the acquisition trial. URB597 (0.1, 0.3 and 1mg/kg, i.p.) or SR141716A (1mg/kg, i.p.) was injected to rats 10min before WIN 55, 212-2 or URB597 respectively. URB597 (0.3 and 1mg/kg) but not 0.1mg/kg induced higher discrimination index (DI) in object recognition test and enhanced memory acquisition in PAL test. The cognitive enhancing effect of URB597 was blocked by a CB1 receptor antagonist, SR141716A which at this dose alone had no effect on cognition. WIN55, 212-2 caused cognition deficits in both tests. URB597 (0.3 and 1mg/kg) treatment could alleviate the negative influence of WIN 55, 212-2 on cognition and memory. These results indicate URB597 potential to protect against memory deficits induced by cannabinoid. Therefore, in combination with URB597 beneficial effects, this study suggests that URB597 has recognition and acquisition memory enhancing effects. It may also constitute a novel approach for the treatment of cannabinoid induced memory deficits and lead to a better understanding of the brain mechanisms underlying cognition. Copyright © 2015 Elsevier Inc. All rights reserved.

Soluble epoxide hydrolase contamination of specific catalase preparations inhibits epoxyeicosatrienoic acid vasodilation of rat renal arterioles

Science.gov (United States)

Olson, Lauren; Harder, Adam; Isbell, Marilyn; Imig, John D.; Gutterman, David D.; Falck, J. R.; Campbell, William B.

2011-01-01

Cytochrome P-450 metabolites of arachidonic acid, the epoxyeicosatrienoic acids (EETs) and hydrogen peroxide (H2O2), are important signaling molecules in the kidney. In renal arteries, EETs cause vasodilation whereas H2O2 causes vasoconstriction. To determine the physiological contribution of H2O2, catalase is used to inactivate H2O2. However, the consequence of catalase action on EET vascular activity has not been determined. In rat renal afferent arterioles, 14,15-EET caused concentration-related dilations that were inhibited by Sigma bovine liver (SBL) catalase (1,000 U/ml) but not Calbiochem bovine liver (CBL) catalase (1,000 U/ml). SBL catalase inhibition was reversed by the soluble epoxide hydrolase (sEH) inhibitor tAUCB (1 μM). In 14,15-EET incubations, SBL catalase caused a concentration-related increase in a polar metabolite. Using mass spectrometry, the metabolite was identified as 14,15-dihydroxyeicosatrienoic acid (14,15-DHET), the inactive sEH metabolite. 14,15-EET hydrolysis was not altered by the catalase inhibitor 3-amino-1,2,4-triazole (3-ATZ; 10–50 mM), but was abolished by the sEH inhibitor BIRD-0826 (1–10 μM). SBL catalase EET hydrolysis showed a regioisomer preference with greatest hydrolysis of 14,15-EET followed by 11,12-, 8,9- and 5,6-EET (Vmax = 0.54 ± 0.07, 0.23 ± 0.06, 0.18 ± 0.01 and 0.08 ± 0.02 ng DHET·U catalase−1·min−1, respectively). Of five different catalase preparations assayed, EET hydrolysis was observed with two Sigma liver catalases. These preparations had low specific catalase activity and positive sEH expression. Mass spectrometric analysis of the SBL catalase identified peptide fragments matching bovine sEH. Collectively, these data indicate that catalase does not affect EET-mediated dilation of renal arterioles. However, some commercial catalase preparations are contaminated with sEH, and these contaminated preparations diminish the biological activity of H2O2 and EETs. PMID:21753077

Effects of Dipeptidyl Peptidase-4 Inhibitors on Hyperglycemia and Blood Cyclosporine Levels in Renal Transplant Patients with Diabetes: A Pilot Study

Directory of Open Access Journals (Sweden)

Jaehyun Bae

2016-03-01

Full Text Available BackgroundThe use of dipeptidyl peptidase-4 (DPP-4 inhibitors is increasing among renal transplant patients with diabetes. However, the glucose-lowering efficacies of various DPP-4 inhibitors and their effects on blood cyclosporine levels have not been fully investigated. We compared the glucose-lowering efficacies of DPP 4 inhibitors and evaluate their effects on the blood levels of cyclosporine in renal transplant recipients with diabetes.MethodsSixty-five renal allograft recipients who received treatment with DPP-4 inhibitors (vildagliptin, sitagliptin, or linagliptin following kidney transplant were enrolled. The glucose-lowering efficacies of the DPP-4 inhibitors were compared according to the changes in the hemoglobin A1c (HbA1c levels after 3 months of treatment. Changes in the trough levels of the cyclosporine were also assessed 2 months after treatment with each DPP-4 inhibitor.ResultsHbA1c significantly decreased in the linagliptin group in comparison with other DPP-4 inhibitors (vildagliptin –0.38%±1.03%, sitagliptin –0.53%±0.95%, and linagliptin –1.40±1.34; P=0.016. Cyclosporine trough levels were significantly increased in the sitagliptin group compared with vildagliptin group (30.62±81.70 ng/mL vs. –24.22±53.54 ng/mL, P=0.036. Cyclosporine trough levels were minimally changed in patients with linagliptin.ConclusionLinagliptin demonstrates superior glucose-lowering efficacy and minimal effect on cyclosporine trough levels in comparison with other DPP-4 inhibitors in kidney transplant patients with diabetes.

Structural Biology and Molecular Modeling in the Design of Novel DPP-4 Inhibitors

Science.gov (United States)

Scapin, Giovanna

Inhibition of dipeptidyl peptidase IV (DPP-4) is a promising new approach for the treatment of type 2 diabetes. DPP-4 is the enzyme responsible for inactivating the incretin hormones glucagon-like peptide 1 (GLP-1) and glucose dependent insulinotropic polypeptide (GIP), two hormones that play important roles in glucose homeostasis. The potent, orally bioavailable and highly selective small molecule DPP-4 inhibitor sitagliptin has been approved by the FDA as novel drug for the treatment of type 2 diabetes. The comparison between the binding mode of sitagliptin (a β-amino acid) and that of a second class of inhibitors (α-amino acid-based) initially led to the successful identification and design of structurally diverse and highly potent DPP-4 inhibitors. Further analysis of the crystal structure of sitagliptin bound to DPP-4 suggested that the central β-amino butanoyl moiety could be replaced by a rigid group. This was confirmed by molecular modeling, and the resulting cyclohexylamine analogs were synthesized and found to be potent DPP-4 inhibitors. However, the triazolopyrazine was predicted to be distorted in order to fit in the binding pocket, and the crystal structure showed that multiple conformations exist for this moiety. Additional molecular modeling studies were then used to improve potency of the cyclohexylamine series. In addition, a 3-D QSAR method was used to gain insight for reducing off-target DPP-8/9 activities. Novel compounds were thus synthesized and found to be potent DPP-4 inhibitors. Two compounds in particular were designed to be highly selective against off-target "DPP-4 Activity- and/or Structure Homologues" (DASH) enzymes while maintaining potency against DPP-4.

Purification and characterisation of a novel enantioselective epoxide hydrolase from Aspergillus niger M200

Czech Academy of Sciences Publication Activity Database

Kotík, Michael; Kyslík, Pavel

2006-01-01

Roč. 1760, - (2006), s. 245-252 ISSN 0006-3002 Institutional research plan: CEZ:AV0Z50200510 Keywords : epoxide hydrolase * enantioselectivity * aspergillus niger Subject RIV: EE - Microbiology, Virology

Mapping in vivo target interaction profiles of covalent inhibitors using chemical proteomics with label-free quantification.

Science.gov (United States)

van Rooden, Eva J; Florea, Bogdan I; Deng, Hui; Baggelaar, Marc P; van Esbroeck, Annelot C M; Zhou, Juan; Overkleeft, Herman S; van der Stelt, Mario

2018-04-01

Activity-based protein profiling (ABPP) has emerged as a valuable chemical proteomics method to guide the therapeutic development of covalent drugs by assessing their on-target engagement and off-target activity. We recently used ABPP to determine the serine hydrolase interaction landscape of the experimental drug BIA 10-2474, thereby providing a potential explanation for the adverse side effects observed with this compound. ABPP allows mapping of protein interaction landscapes of inhibitors in cells, tissues and animal models. Whereas our previous protocol described quantification of proteasome activity using stable-isotope labeling, this protocol describes the procedures for identifying the in vivo selectivity profile of covalent inhibitors with label-free quantitative proteomics. The optimization of our protocol for label-free quantification methods results in high proteome coverage and allows the comparison of multiple biological samples. We demonstrate our protocol by assessing the protein interaction landscape of the diacylglycerol lipase inhibitor DH376 in mouse brain, liver, kidney and testes. The stages of the protocol include tissue lysis, probe incubation, target enrichment, sample preparation, liquid chromatography-mass spectrometry (LC-MS) measurement, data processing and analysis. This approach can be used to study target engagement in a native proteome and to identify potential off targets for the inhibitor under investigation. The entire protocol takes at least 4 d, depending on the number of samples.

Changes in glucose-induced plasma active glucagon-like peptide-1 levels by co-administration of sodium–glucose cotransporter inhibitors with dipeptidyl peptidase-4 inhibitors in rodents

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Takahiro Oguma

2016-12-01

Full Text Available We investigated whether structurally different sodium–glucose cotransporter (SGLT 2 inhibitors, when co-administered with dipeptidyl peptidase-4 (DPP4 inhibitors, could enhance glucagon-like peptide-1 (GLP-1 secretion during oral glucose tolerance tests (OGTTs in rodents. Three different SGLT inhibitors—1-(β-d-Glucopyranosyl-4-chloro-3-[5-(6-fluoro-2-pyridyl-2-thienylmethyl]benzene (GTB, TA-1887, and canagliflozin—were examined to assess the effect of chemical structure. Oral treatment with GTB plus a DPP4 inhibitor enhanced glucose-induced plasma active GLP-1 (aGLP-1 elevation and suppressed glucose excursions in both normal and diabetic rodents. In DPP4-deficient rats, GTB enhanced glucose-induced aGLP-1 elevation without affecting the basal level, whereas metformin, previously reported to enhance GLP-1 secretion, increased both the basal level and glucose-induced elevation. Oral treatment with canagliflozin and TA-1887 also enhanced glucose-induced aGLP-1 elevation when co-administered with either teneligliptin or sitagliptin. These data suggest that structurally different SGLT2 inhibitors enhance plasma aGLP-1 elevation and suppress glucose excursions during OGTT when co-administered with DPP4 inhibitors, regardless of the difference in chemical structure. Combination treatment with DPP4 inhibitors and SGLT2 inhibitors having moderate SGLT1 inhibitory activity may be a promising therapeutic option for improving glycemic control in patients with type 2 diabetes mellitus.

Antiviral Activity and Resistance Analysis of NS3/4A Protease Inhibitor Grazoprevir and NS5A Inhibitor Elbasvir in Hepatitis C Virus GT4 Replicons.

Science.gov (United States)

Asante-Appiah, Ernest; Curry, Stephanie; McMonagle, Patricia; Ingravallo, Paul; Chase, Robert; Nickle, David; Qiu, Ping; Howe, Anita; Lahser, Frederick C

2017-07-01

Although genotype 4 (GT4)-infected patients represent a minor overall percentage of the global hepatitis C virus (HCV)-infected population, the high prevalence of the genotype in specific geographic regions coupled with substantial sequence diversity makes it an important genotype to study for antiviral drug discovery and development. We evaluated two direct-acting antiviral agents-grazoprevir, an HCV NS3/4A protease inhibitor, and elbasvir, an HCV NS5A inhibitor-in GT4 replicons prior to clinical studies in this genotype. Following a bioinformatics analysis of available GT4 sequences, a set of replicons bearing representative GT4 clinical isolates was generated. For grazoprevir, the 50% effective concentration (EC 50 ) against the replicon bearing the reference GT4a (ED43) NS3 protease and NS4A was 0.7 nM. The median EC 50 for grazoprevir against chimeric replicons encoding NS3/4A sequences from GT4 clinical isolates was 0.2 nM (range, 0.11 to 0.33 nM; n = 5). The difficulty in establishing replicons bearing NS3/4A resistance-associated substitutions was substantially overcome with the identification of a G162R adaptive substitution in NS3. Single NS3 substitutions D168A/V identified from de novo resistance selection studies reduced grazoprevir antiviral activity by 137- and 47-fold, respectively, in the background of the G162R replicon. For elbasvir, the EC 50 against the replicon bearing the reference full-length GT4a (ED43) NS5A gene was 0.0002 nM. The median EC 50 for elbasvir against chimeric replicons bearing clinical isolates from GT4 was 0.0007 nM (range, 0.0002 to 34 nM; n = 14). De novo resistance selection studies in GT4 demonstrated a high propensity to suppress the emergence of amino acid substitutions that confer high-potency reductions to elbasvir. Phenotypic characterization of the NS5A amino acid substitutions identified (L30F, L30S, M31V, and Y93H) indicated that they conferred 15-, 4-, 2.5-, and 7.5-fold potency losses, respectively, to elbasvir

The fatty acid amide hydrolase inhibitor URB597 exerts anti-inflammatory effects in hippocampus of aged rats and restores an age-related deficit in long-term potentiation

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Murphy Niamh

2012-04-01

Full Text Available Abstract Background Several factors contribute to the deterioration in synaptic plasticity which accompanies age and one of these is neuroinflammation. This is characterized by increased microglial activation associated with increased production of proinflammatory cytokines like interleukin-1β (IL-1β. In aged rats these neuroinflammatory changes are associated with a decreased ability of animals to sustain long-term potentiation (LTP in the dentate gyrus. Importantly, treatment of aged rats with agents which possess anti-inflammatory properties to decrease microglial activation, improves LTP. It is known that endocannabinoids, such as anandamide (AEA, have anti-inflammatory properties and therefore have the potential to decrease the age-related microglial activation. However, endocannabinoids are extremely labile and are hydrolyzed quickly after production. Here we investigated the possibility that inhibiting the degradation of endocannabinoids with the fatty acid amide hydrolase (FAAH inhibitor, URB597, could ameliorate age-related increases in microglial activation and the associated decrease in LTP. Methods Young and aged rats received subcutaneous injections of the FAAH inhibitor URB597 every second day and controls which received subcutaneous injections of 30% DMSO-saline every second day for 28 days. Long-term potentiation was recorded on day 28 and the animals were sacrificed. Brain tissue was analyzed for markers of microglial activation by PCR and for levels of endocannabinoids by liquid chromatography coupled to tandem mass spectrometry. Results The data indicate that expression of markers of microglial activation, MHCII, and CD68 mRNA, were increased in the hippocampus of aged, compared with young, rats and that these changes were associated with increased expression of the proinflammatory cytokines interleukin (IL-1β and tumor necrosis factor-α (TNFα which were attenuated by treatment with URB597. Coupled with these changes, we

Gastrointestinal Adverse Events of Dipeptidyl Peptidase 4 Inhibitors in Type 2 Diabetes: A Systematic Review and Network Meta-analysis.

Science.gov (United States)

Wu, Shanshan; Chai, Sanbao; Yang, Jun; Cai, Ting; Xu, Yang; Yang, Zhirong; Zhang, Yuan; Ji, Linong; Sun, Feng; Zhan, Siyan

2017-09-01

The purpose of this study was to systematically evaluate the effect of dipeptidyl peptidase 4 inhibitors on gastrointestinal adverse events in patients with type 2 diabetes. MEDLINE, Embase, the Cochrane Library, and ClinicalTrials.gov were searched from inception through April 28, 2016. Randomized controlled trials that compared dipeptidyl peptidase 4 inhibitor-based therapies with placebo and other hypoglycemic agents in type 2 diabetes were included. The duration of studies was at least 4 weeks. A total of 165 randomized controlled trials and 122,072 patients were included in the study. Dipeptidyl peptidase 4 inhibitors did not increase the incidence of gastrointestinal adverse events after the treatment with alogliptin (odds ratio [OR] = 0.83; 95% CI, 0.59-1.15), linagliptin (OR = 1.11; 95% CI, 0.92-1.35), saxagliptin (OR = 0.96; 95% CI, 0.80-1.15), sitagliptin (OR = 0.95; 95% CI, 0.64-1.14), teneligliptin (OR = 1.50; 95% CI, 0.81-2.77), and vildagliptin (OR = 0.80; 95% CI, 0.63-1.01) compared with placebo. Compared with glucagon-like peptide 1 receptor agonists, dipeptidyl peptidase 4 inhibitors significantly decreased the incidence of gastrointestinal adverse events with alogliptin (OR = 0.26; 95% CI, 0.15-0.44), linagliptin (OR = 0.43; 95% CI, 0.25-0.74), saxagliptin (OR = 0.28; 95% CI, 0.17-0.46), sitagliptin (OR = 0.24; 95% CI, 0.17-0.35), and vildagliptin (OR = 0.27; 95% CI, 0.18-0.41). Dipeptidyl peptidase 4 inhibitors were not associated with an increased risk of gastrointestinal adverse events relative to metformin and α-glucosidase inhibitors, respectively. The network meta-analysis found that compared with glucagon-like peptide 1 receptor agonists, metformin, and α-glucosidase inhibitor, dipeptidyl peptidase 4 inhibitors are associated with a lower incidence of gastrointestinal adverse events. Copyright © 2017 Elsevier HS Journals, Inc. All rights reserved.

A sensitive and specific radiochromatographic assay of fatty acid amide hydrolase activity.

Science.gov (United States)

Maccarrone, M; Bari, M; Agrò, A F

1999-02-15

A radiochromatographic method has been set up in order to determine fatty acid amide hydrolase (FAAH) activity, based on reversed-phase high-performance liquid chromatography and on-line scintillation counting. The reaction products were separated using a C18 column eluted with methanol-water-acetic acid and quantitated with an external standard. Baseline separation of the acid product from the substrate was completed in less than 4 min, with a detection limit of 2.5 fmol arachidonic acid at a signal to noise ratio of 4:1. The method enabled to determine the kinetic constants (i.e., apparent Km of 2.0 +/- 0.2 microM and Vmax of 800 +/- 75 pmol. min-1. mg protein-1 toward anandamide) and the substrate specificity of human brain FAAH, as well as the extent of enzyme inhibition by some anandamide congeners. The femtomole sensitivity and the accuracy of the method allow detection and characterization of the activity of FAAH in very minute tissue samples or in samples where the enzymatic activity is very low. Copyright 1999 Academic Press.

Dipeptidyl peptidase-4 (DPP-4) inhibitors are favourable to glucagon-like peptide-1 (GLP-1) agonists

DEFF Research Database (Denmark)

Madsbad, Sten

2012-01-01

Incretin-based therapies, which include the GLP-1 receptor agonists and DPP-4 inhibitors, use the antidiabetic properties of potentiating the GLP-1 receptor signalling via the regulation of insulin and glucagon secretion, inhibition of gastric emptying and suppression of appetite. Most physicians...... will start antidiabetic treatment with metformin, but adding a GLP-1 receptor agonist as the second drug seems to be optimal since more patients will reach an HbA1c below 7% than with a DPP-4 inhibitor or another oral antidiabetic agents and with minimal risk of hypoglycaemia. The GLP-1 receptor agonists...

Synthesis, pharmacological evaluation and molecular docking studies of pyrimidinedione based DPP-4 inhibitors as antidiabetic agents

Science.gov (United States)

Jha, Vibhu; Bhadoriya, Kamlendra Singh

2018-04-01

Dipeptidyl peptidase-4 (DPP-4) inhibitors are a class of newly developed antidiabetic drugs that bock DPP-4. DPP-4 is responsible for degradation of incretins harmones such as GLP-1 (Glucagon like Peptide) and GIP (Gastric inhibitory polypeptide) that maintain blood-glucose level. Pyrimidinedione based compounds were designed and synthesized for DPP-4 inhibitory activity. These heterocycles were designed by taking Alogliptin as a reference DPP-4 inhibitors and synthesized as N-methylated and N-benzylated pyrimidinediones. These compounds were subjected to DPP-4 assay, five out of nine synthesized compounds have shown in vitro DPP-4 inhibitory activity in significant range. Further, molecular docking studies of these compounds were performed on DPP-4 subunit and compared with natural DPP-4 inhibitors like Flavone, Resveratrol, Quercetin, Diprotin A. Docking studies have led to the conclusion that there are some identical amino acid interactions as Tyr 666 and Tyr 662, seen in both synthesized compounds and natural DPP-4 inhibitors. This study completely gives a good scope for further derivatisation and optimization of synthesized compounds to get clinical candidate as DPP-4 inhibitor for antidiabetic activity.

Angiotensin converting enzyme inhibitors and Alzheimer disease in the presence of the apolipoprotein E4 allele.

Science.gov (United States)

Qiu, Wendy Wei Qiao; Lai, Angela; Mon, Timothy; Mwamburi, Mkaya; Taylor, Warren; Rosenzweig, James; Kowall, Neil; Stern, Robert; Zhu, Haihao; Steffens, David C

2014-02-01

The effect of angiotensin converting enzyme (ACE) inhibitors on Alzheimer disease (AD) remains unclear, with conflicting results reported. We studied the interaction of the Apolipoprotein E (ApoE) genotype and ACE inhibitors on AD. This was a cross-sectional study of homebound elderly with an AD diagnosis and documentation of medications taken. ApoE genotype was determined. A total of 355 subjects with status on ApoE alleles and cognitive diagnoses were studied. The average age (mean ± SD) of this population was 73.3 ± 8.3 years old, and 73% were female. Cross-sectionally, there was no difference in the number of AD cases between ApoE4 carriers and ApoE4 non-carriers or between ACE inhibitor users and non-users in the homebound elderly. ApoE4 carriers treated with ACE inhibitors, however, had more diagnoses of AD compared with those who did not have the treatment (28% versus 6%, p = 0.01) or ApoE4 non-carriers treated with an ACE inhibitor (28% versus 10%, p = 0.03). ACE inhibitor use was associated with AD diagnosis only in the presence of an E4 allele. Using multivariate logistic regression analysis, we found that in diagnosed AD cases there was a significant interaction between ApoE4 and ACE inhibitor use (odds ratio: 20.85; 95% confidence interval: 3.08-140.95; p = 0.002) after adjusting for age, sex, ethnicity, and education. The effects of ACE inhibitors on AD may be different depending on ApoE genotype. A prospective study is needed to determine whether ACE inhibitor use accelerates or poorly delays AD development in ApoE4 carriers compared with ApoE4 non-carriers. Copyright © 2014. Published by Elsevier Inc.

Development of organophosphate hydrolase activity in a bacterial homolog of human cholinesterase

Science.gov (United States)

Legler, Patricia; Boisvert, Susanne; Compton, Jaimee; Millard, Charles

2014-07-01

We applied a combination of rational design and directed evolution (DE) to Bacillus subtilis p-nitrobenzyl esterase (pNBE) with the goal of enhancing organophosphorus acid anhydride hydrolase (OPAAH) activity. DE started with a designed variant, pNBE A107H, carrying a histidine homologous with human butyrylcholinesterase G117H to find complementary mutations that further enhance its OPAAH activity. Five sites were selected (G105, G106, A107, A190, and A400) within a 6.7 Å radius of the nucleophilic serine O?. All 95 variants were screened for esterase activity with a set of five substrates: pNP-acetate, pNP-butyrate, acetylthiocholine, butyrylthiocholine, or benzoylthiocholine. A microscale assay for OPAAH activity was developed for screening DE libraries. Reductions in esterase activity were generally concomitant with enhancements in OPAAH activity. One variant, A107K, showed an unexpected 7-fold increase in its kcat/Km for benzoylthiocholine, demonstrating that it is also possible to enhance the cholinesterase activity of pNBE. Moreover, DE resulted in at least three variants with modestly enhanced OPAAH activity compared to wild type pNBE. A107H/A190C showed a 50-fold increase in paraoxonase activity and underwent a slow time- and temperature-dependent change affecting the hydrolysis of OPAA and ester substrates. Structural analysis suggests that pNBE may represent a precursor leading to human cholinesterase and carboxylesterase 1 through extension of two vestigial specificity loops; a preliminary attempt to transfer the Ω-loop of BChE into pNBE is described. pNBE was tested as a surrogate scaffold for mammalian esterases. Unlike butyrylcholinesterase and pNBE, introducing a G143H mutation (equivalent to G117H) did not confer detectable OP hydrolase activity on human carboxylesterase 1. We discuss the importance of the oxyanion-hole residues for enhancing the OPAAH activity of selected serine hydrolases.

2-Butyne-1,4-diol as a novel corrosion inhibitor for API X65 steel pipeline in carbonate/bicarbonate solution

International Nuclear Information System (INIS)

Sadeghi Meresht, E.; Shahrabi Farahani, T.; Neshati, J.

2012-01-01

Highlights: ► Corrosion of API 5L X65 is effectively reduced by the addition of the inhibitor. ► The techniques include weight loss, potentiodynamic polarization, EIS and AFM. ► 2-Butyne-1,4-diol acts as a mixed-type inhibitor. ► The adsorption of 2-butyne-1,4-diol obeys Langmuir adsorption isotherm. - Abstract: The inhibition effects of 2-butyne-1,4-diol on the corrosion susceptibility of grade API 5L X65 steel pipeline in 2 M Na 2 CO 3 /1 M NaHCO 3 solution were studied by electrochemical techniques and weight loss measurements. The results indicated that this inhibitor was a mixed-type inhibitor, with a maximum percentage inhibition efficiency of approximately 92% in the presence of 5 mM inhibitor. Atomic force microscopy revealed that a protective film was formed on the surface of the inhibited sample. The adsorption of the inhibitor was found to conform to the Langmuir isotherm with the standard adsorption free energy of −21.08 kJ mol −1 .

Fluorescent Inhibitors as Tools To Characterize Enzymes: Case Study of the Lipid Kinase Phosphatidylinositol 4-Kinase IIIβ (PI4KB).

Science.gov (United States)

Humpolickova, Jana; Mejdrová, Ivana; Matousova, Marika; Nencka, Radim; Boura, Evzen

2017-01-12

The lipid kinase phosphatidylinositol 4-kinase IIIβ (PI4KB) is an essential host factor for many positive-sense single-stranded RNA (+RNA) viruses including human pathogens hepatitis C virus (HCV), Severe acute respiratory syndrome (SARS), coxsackie viruses, and rhinoviruses. Inhibitors of PI4KB are considered to be potential broad-spectrum virostatics, and it is therefore critical to develop a biochemical understanding of the kinase. Here, we present highly potent and selective fluorescent inhibitors that we show to be useful chemical biology tools especially in determination of dissociation constants. Moreover, we show that the coumarin-labeled inhibitor can be used to image PI4KB in cells using fluorescence-lifetime imaging microscopy (FLIM) microscopy.

Δ9-tetrahydrocannabinol and endocannabinoid degradative enzyme inhibitors attenuate intracranial self-stimulation in mice.

Science.gov (United States)

Wiebelhaus, Jason M; Grim, Travis W; Owens, Robert A; Lazenka, Matthew F; Sim-Selley, Laura J; Abdullah, Rehab A; Niphakis, Micah J; Vann, Robert E; Cravatt, Benjamin F; Wiley, Jenny L; Negus, S Stevens; Lichtman, Aron H

2015-02-01

A growing body of evidence implicates endogenous cannabinoids as modulators of the mesolimbic dopamine system and motivated behavior. Paradoxically, the reinforcing effects of Δ(9)-tetrahydrocannabinol (THC), the primary psychoactive constituent of cannabis, have been difficult to detect in preclinical rodent models. In this study, we investigated the impact of THC and inhibitors of the endocannabinoid hydrolytic enzymes fatty acid amide hydrolase (FAAH) and monoacylglycerol lipase (MAGL) on operant responding for electrical stimulation of the medial forebrain bundle [intracranial self-stimulation (ICSS)], which is known to activate the mesolimbic dopamine system. These drugs were also tested in assays of operant responding for food reinforcement and spontaneous locomotor activity. THC and the MAGL inhibitor JZL184 (4-[bis(1,3-benzodioxol-5-yl)hydroxymethyl]-1-piperidinecarboxylic acid 4-nitrophenyl ester) attenuated operant responding for ICSS and food, and also reduced spontaneous locomotor activity. In contrast, the FAAH inhibitor PF-3845 (N-3-pyridinyl-4-[[3-[[5-(trifluoromethyl)-2-pyridinyl]oxy]phenyl]methyl]-1-piperidinecarboxamide) was largely without effect in these assays. Consistent with previous studies showing that combined inhibition of FAAH and MAGL produces a substantially greater cannabimimetic profile than single enzyme inhibition, the dual FAAH-MAGL inhibitor SA-57 (4-[2-(4-chlorophenyl)ethyl]-1-piperidinecarboxylic acid 2-(methylamino)-2-oxoethyl ester) produced a similar magnitude of ICSS depression as that produced by THC. ICSS attenuation by JZL184 was associated with increased brain levels of 2-arachidonoylglycerol (2-AG), whereas peak effects of SA-57 were associated with increased levels of both N-arachidonoylethanolamine (anandamide) and 2-AG. The cannabinoid receptor type 1 receptor antagonist rimonabant, but not the cannabinoid receptor type 2 receptor antagonist SR144528, blocked the attenuating effects of THC, JZL184, and SA-57 on

Predictive Factors for Efficacy of Dipeptidyl Peptidase-4 Inhibitors in Patients with Type 2 Diabetes Mellitus

Directory of Open Access Journals (Sweden)

Shusuke Yagi

2015-08-01

Full Text Available BackgroundPredictive factors for the efficacy of dipeptidyl peptidase-4 (DPP-4 inhibitors for lowering glycosylated hemoglobin (HbA1c remain unclear in patients with type 2 diabetes mellitus. The aim of this study is therefore to clarify predictive factors of the efficacy of DPP-4 inhibitors for lowering HbA1c after 12 months of treatment.MethodsA total of 191 consecutive type 2 diabetic patients (male sex 55%, mean age, 68.3±35.8 years, who had been treated with DPP-4 inhibitors for 12 months, were enrolled in this study and evaluated retrospectively.ResultsAfter 12 months of DPP-4 inhibitor treatment, random blood glucose level, and HbA1c level, decreased from 167±63 to 151±49 mg/dL (P<0.01, and from 7.5%±1.3% to 6.9%±0.9% (P<0.01 respectively, without severe side effects. Multiple regression analysis showed that predictors of DPP-4 inhibitor treatment efficacy in lowering HbA1c level after 12 months were a decrease in HbA1c level after 3 months of treatment, a high baseline HbA1c level, a low baseline body mass index, and the absence of coronary artery disease.ConclusionMost suitable candidates for treatment with DPP-4 inhibitors are diabetics who are not obese and do not have coronary artery disease. In addition, long-term efficacy of DPP-4 inhibitors can be predicted by decrement of HbA1c after 3 months of treatment.

Mitochondrial NUDIX hydrolases: A metabolic link between NAD catabolism, GTP and mitochondrial dynamics.

Science.gov (United States)

Long, Aaron; Klimova, Nina; Kristian, Tibor

2017-10-01

NAD + catabolism and mitochondrial dynamics are important parts of normal mitochondrial function and are both reported to be disrupted in aging, neurodegenerative diseases, and acute brain injury. While both processes have been extensively studied there has been little reported on how the mechanisms of these two processes are linked. This review focuses on how downstream NAD + catabolism via NUDIX hydrolases affects mitochondrial dynamics under pathologic conditions. Additionally, several potential targets in mitochondrial dysfunction and fragmentation are discussed, including the roles of mitochondrial poly(ADP-ribose) polymerase 1(mtPARP1), AMPK, AMP, and intra-mitochondrial GTP metabolism. Mitochondrial and cytosolic NUDIX hydrolases (NUDT9α and NUDT9β) can affect mitochondrial and cellular AMP levels by hydrolyzing ADP- ribose (ADPr) and subsequently altering the levels of GTP and ATP. Poly (ADP-ribose) polymerase 1 (PARP1) is activated after DNA damage, which depletes NAD + pools and results in the PARylation of nuclear and mitochondrial proteins. In the mitochondria, ADP-ribosyl hydrolase-3 (ARH3) hydrolyzes PAR to ADPr, while NUDT9α metabolizes ADPr to AMP. Elevated AMP levels have been reported to reduce mitochondrial ATP production by inhibiting the adenine nucleotide translocase (ANT), allosterically activating AMPK by altering the cellular AMP: ATP ratio, and by depleting mitochondrial GTP pools by being phosphorylated by adenylate kinase 3 (AK3), which uses GTP as a phosphate donor. Recently, activated AMPK was reported to phosphorylate mitochondria fission factor (MFF), which increases Drp1 localization to the mitochondria and promotes mitochondrial fission. Moreover, the increased AK3 activity could deplete mitochondrial GTP pools and possibly inhibit normal activity of GTP-dependent fusion enzymes, thus altering mitochondrial dynamics. Published by Elsevier Ltd.

Identification and characterization of a bile salt hydrolase from Lactobacillus salivarius for development of novel alternatives to antibiotic growth promoters.

Science.gov (United States)

Wang, Zhong; Zeng, Ximin; Mo, Yiming; Smith, Katie; Guo, Yuming; Lin, Jun

2012-12-01

Antibiotic growth promoters (AGPs) have been used as feed additives to improve average body weight gain and feed efficiency in food animals for more than 5 decades. However, there is a worldwide trend to limit AGP use to protect food safety and public health, which raises an urgent need to discover effective alternatives to AGPs. The growth-promoting effect of AGPs has been shown to be highly correlated with the decreased activity of intestinal bile salt hydrolase (BSH), an enzyme that is produced by various gut microflora and involved in host lipid metabolism. Thus, BSH inhibitors are likely promising feed additives to AGPs to improve animal growth performance. In this study, the genome of Lactobacillus salivarius NRRL B-30514, a BSH-producing strain isolated from chicken, was sequenced by a 454 GS FLX sequencer. A BSH gene identified by genome analysis was cloned and expressed in an Escherichia coli expression system for enzymatic analyses. The BSH displayed efficient hydrolysis activity for both glycoconjugated and tauroconjugated bile salts, with slightly higher catalytic efficiencies (k(cat)/K(m)) on glycoconjugated bile salts. The optimal pH and temperature for the BSH activity were 5.5 and 41°C, respectively. Examination of a panel of dietary compounds using the purified BSH identified some potent BSH inhibitors, in which copper and zinc have been recently demonstrated to promote feed digestion and body weight gain in different food animals. In sum, this study identified and characterized a BSH with broad substrate specificity from a chicken L. salivarius strain and established a solid platform for us to discover novel BSH inhibitors, the promising feed additives to replace AGPs for enhancing the productivity and sustainability of food animals.

Novel microbial epoxide hydrolases for biohydrolysis of glycidyl derivatives

Czech Academy of Sciences Publication Activity Database

Kotík, Michael; Břicháč, Jiří; Kyslík, Pavel

2005-01-01

Roč. 120, - (2005), s. 364-375 ISSN 0168-1656 Institutional research plan: CEZ:AV0Z5020903 Keywords : screening * epoxide hydrolase * biotransformation Subject RIV: EE - Microbiology, Virology Impact factor: 2.687, year: 2005

INK4 proteins, a family of mammalian CDK inhibitors with novel biological functions.

Science.gov (United States)

Cánepa, Eduardo T; Scassa, María E; Ceruti, Julieta M; Marazita, Mariela C; Carcagno, Abel L; Sirkin, Pablo F; Ogara, María F

2007-07-01

The cyclin D-Cdk4-6/INK4/Rb/E2F pathway plays a key role in controlling cell growth by integrating multiple mitogenic and antimitogenic stimuli. The members of INK4 family, comprising p16(INK4a), p15(INK4b), p18(INK4c), and p19(INK4d), block the progression of the cell cycle by binding to either Cdk4 or Cdk6 and inhibiting the action of cyclin D. These INK4 proteins share a similar structure dominated by several ankyrin repeats. Although they appear to be structurally redundant and equally potent as inhibitors, the INK4 family members are differentially expressed during mouse development. The striking diversity in the pattern of expression of INK4 genes suggested that this family of cell cycle inhibitors might have cell lineage-specific or tissue-specific functions. The INK4 proteins are commonly lost or inactivated by mutations in diverse types of cancer, and they represent established or candidate tumor suppressors. Apart from their capacity to arrest cells in the G1-phase of the cell cycle they have been shown to participate in an increasing number of cellular processes. Given their emerging roles in fundamental physiological as well as pathological processes, it is interesting to explore the diverse roles for the individual INK4 family members in different functions other than cell cycle regulation. Extensive studies, over the past few years, uncover the involvement of INK4 proteins in senescence, apoptosis, DNA repair, and multistep oncogenesis. We will focus the discussion here on these unexpected issues.

CDK4/6 inhibitor PD0332991 in glioblastoma treatment: does it have a future?

Directory of Open Access Journals (Sweden)

Lisette eSchroder

2015-11-01

Full Text Available Glioblastoma is aggressive, highly infiltrating, and the most frequent malignant form of brain cancer. With a median survival time of only 14.6 months, when treated with the standard of care, it is essential to find new therapeutic options. A specific CDK4/6 inhibitor, PD0332991, obtained accelerated approval from the Food and Drug Administration for the treatment of patients with advanced estrogen receptor-positive and HER2-negative breast cancer. Common alterations in the cyclin D1-Cyclin Dependent Kinase 4/6-Retinoblastoma 1 pathway in glioblastoma make PD0332991 also an interesting drug for the treatment of glioblastoma. Promising results in in vitro studies, where patient derived glioblastoma cell lines showed sensitivity to PD0332991, gave motive to start in vivo studies. Outcomes of these studies have been contrasting in terms of PD0332991 efficacy within the brain: more research is necessary to conclude whether CDK4/6 inhibitor can be beneficial in the treatment of glioblastoma.

Cytosolic cholesterol ester hydrolase in adrenal cortex

OpenAIRE

Tocher, Douglas R.

1983-01-01

Cholesterol ester hydrolase (CEH) in adrenocortical cytosol was known to be phosphorylated and activated, in response to ACTH in a cAMPdependent protein kinase mediated process. The purification of CEH from bovine adrenocortical cytosol was attempted. The use of detergents to solubilise the enzyme from lipid-rich aggregates was investigated and sodium cholate was found to be effective. A purification procedure using cholate solubilised enzyme was developed. The detergent int...

Abemaciclib: a CDK4/6 inhibitor for the treatment of HR+/HER2– advanced breast cancer

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Corona SP

2018-02-01

Full Text Available Silvia Paola Corona,1 Daniele Generali2 1Radiation Oncology Department, Peter MacCallum Cancer Centre, Bentleigh East, VIC, Australia; 2Department of Medical, Surgery and Health Sciences, University of Trieste, Trieste, Italy Abstract: Although early breast cancer (BC is highly curable, advanced or metastatic disease poses numerous challenges in terms of medical management and treatment decisions and is associated with significantly worse prognosis. Among the new targeted agents, anticancer drugs exploiting the cell-cycle machinery have shown great potential in preclinical studies. CDK4/6 inhibitors target the cyclin D/CDK/retinoblastoma signaling pathway, inducing cell-cycle arrest, reduced cell viability and tumor shrinking. As the cyclin D/CDK complex is activated downstream of estrogen signaling, the combination of CDK4/6 inhibitors with standard endocrine therapies represents a rational approach to elicit synergic antitumor activity in hormone receptor-positive BC. The results of clinical trials have indeed confirmed the superiority of the combination of CDK4/6 inhibitors plus endocrine therapies over endocrine therapy alone. Currently approved are three compounds that exhibit similar structural characteristics as well as biological and clinical activities. Abemaciclib is the latest CDK4/6 inhibitor approved by the US Food and Drug Administration (FDA in view of the results of the MONARCH 1 and 2 trials. Further trials are ongoing as other important questions await response. In this review, we focus on abemaciclib to examine preclinical and clinical results, describing current therapeutic indications, open questions and ongoing clinical trials. Keywords: CDK4/6 inhibitor, abemaciclib, breast cancer, hormone receptor-positive BC, metastatic BC, mBC

Dipeptidyl peptidase-4 inhibitor induced angioedema - an overlooked and potentially lethal adverse drug reaction?

DEFF Research Database (Denmark)

Scott, Susanne Irene; Andersen, Michelle Fog; Aagaard, Lise

2018-01-01

to vasodilatation and increase in vascular permeability in the capillaries. Objective To assess the risk and pathomechanism of angioedema due to inhibition of dipeptidyl peptidase-4 inhibitors when used as monotherapy and in combination with angiotensin converting enzyme-inhibitors. Method PubMed, Embase......, the Cochrane Library, PubMed Central, Web of Science, Google Scholar and clinicaltrials.gov were searched using different combinations of keywords "angioedema", "dipeptidyl peptidase 4", "dipeptidyl peptidase 4 inhibitors", "gliptins", "bradykinin", "substance P" and "angiotensin converting enzyme...

Tetomilast: new promise for phosphodiesterase-4 inhibitors?

Science.gov (United States)

Bickston, Stephen J; Snider, Kenneth R; Kappus, Matthew R

2012-12-01

Tetomilast is a novel thiazole phosphodiesterase-4 (PDE-4) inhibitor, which may prove useful in both the treatment of inflammatory bowel disease (IBD) and chronic obstructive pulmonary disease (COPD). Here, the authors review the pharmacology of the drug, and offer critical review of the available data for use of tetomilast in the treatment of IBD. Peer-reviewed publications, including Phase I and II clinical trials, all other formats included. Tetomilast may be beneficial in IBD. Small differences in molecules and in recombinant proteins can translate into substantial differences in clinical effects and toxicity in IBD. This is a reasonable approach when exploring new options like tetomilast.

Structure of the Cyanuric Acid Hydrolase TrzD Reveals Product Exit Channel.

Science.gov (United States)

Bera, Asim K; Aukema, Kelly G; Elias, Mikael; Wackett, Lawrence P

2017-03-27

Cyanuric acid hydrolases are of industrial importance because of their use in aquatic recreational facilities to remove cyanuric acid, a stabilizer for the chlorine. Degradation of excess cyanuric acid is necessary to maintain chlorine disinfection in the waters. Cyanuric acid hydrolase opens the cyanuric acid ring hydrolytically and subsequent decarboxylation produces carbon dioxide and biuret. In the present study, we report the X-ray structure of TrzD, a cyanuric acid hydrolase from Acidovorax citrulli. The crystal structure at 2.19 Å resolution shows a large displacement of the catalytic lysine (Lys163) in domain 2 away from the active site core, whereas the two other active site lysines from the two other domains are not able to move. The lysine displacement is proposed here to open up a channel for product release. Consistent with that, the structure also showed two molecules of the co-product, carbon dioxide, one in the active site and another trapped in the proposed exit channel. Previous data indicated that the domain 2 lysine residue plays a role in activating an adjacent serine residue carrying out nucleophilic attack, opening the cyanuric acid ring, and the mobile lysine guides products through the exit channel.

Targeted discovery of glycoside hydrolases from a switchgrass-adapted compost community

Energy Technology Data Exchange (ETDEWEB)

Allgaier, M.; Reddy, A.; Park, J. I.; Ivanova, N.; D' haeseleer, P.; Lowry, S.; Sapra, R.; Hazen, T.C.; Simmons, B.A.; VanderGheynst, J. S.; Hugenholtz, P.

2009-11-15

Development of cellulosic biofuels from non-food crops is currently an area of intense research interest. Tailoring depolymerizing enzymes to particular feedstocks and pretreatment conditions is one promising avenue of research in this area. Here we added a green-waste compost inoculum to switchgrass (Panicum virgatum) and simulated thermophilic composting in a bioreactor to select for a switchgrass-adapted community and to facilitate targeted discovery of glycoside hydrolases. Small-subunit (SSU) rRNA-based community profiles revealed that the microbial community changed dramatically between the initial and switchgrass-adapted compost (SAC) with some bacterial populations being enriched over 20-fold. We obtained 225 Mbp of 454-titanium pyrosequence data from the SAC community and conservatively identified 800 genes encoding glycoside hydrolase domains that were biased toward depolymerizing grass cell wall components. Of these, {approx}10% were putative cellulases mostly belonging to families GH5 and GH9. We synthesized two SAC GH9 genes with codon optimization for heterologous expression in Escherichia coli and observed activity for one on carboxymethyl cellulose. The active GH9 enzyme has a temperature optimum of 50 C and pH range of 5.5 to 8 consistent with the composting conditions applied. We demonstrate that microbial communities adapt to switchgrass decomposition using simulated composting condition and that full-length genes can be identified from complex metagenomic sequence data, synthesized and expressed resulting in active enzyme.

Targeted Discovery of Glycoside Hydrolases from a Switchgrass-Adapted Compost Community

Energy Technology Data Exchange (ETDEWEB)

Reddy, Amitha; Allgaier, Martin; Park, Joshua I.; Ivanoval, Natalia; Dhaeseleer, Patrik; Lowry, Steve; Sapra, Rajat; Hazen, Terry C.; Simmons, Blake A.; VanderGheynst, Jean S.; Hugenholtz, Philip

2011-05-11

Development of cellulosic biofuels from non-food crops is currently an area of intense research interest. Tailoring depolymerizing enzymes to particular feedstocks and pretreatment conditions is one promising avenue of research in this area. Here we added a green-waste compost inoculum to switchgrass (Panicum virgatum) and simulated thermophilic composting in a bioreactor to select for a switchgrass-adapted community and to facilitate targeted discovery of glycoside hydrolases. Smallsubunit (SSU) rRNA-based community profiles revealed that the microbial community changed dramatically between the initial and switchgrass-adapted compost (SAC) with some bacterial populations being enriched over 20-fold. We obtained 225 Mbp of 454-titanium pyrosequence data from the SAC community and conservatively identified 800 genes encoding glycoside hydrolase domains that were biased toward depolymerizing grass cell wall components. Of these, ,10percent were putative cellulasesmostly belonging to families GH5 and GH9. We synthesized two SAC GH9 genes with codon optimization for heterologous expression in Escherichia coli and observed activity for one on carboxymethyl cellulose. The active GH9 enzyme has a temperature optimum of 50uC and pH range of 5.5 to 8 consistent with the composting conditions applied. We demonstrate that microbial communities adapt to switchgrass decomposition using simulated composting condition and that full-length genes can be identified from complex metagenomic sequence data, synthesized and expressed resulting in active enzyme.

Synergistic function of four novel thermostable glycoside hydrolases from a long-term enriched thermophilic methanogenic digester

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Meng eWang

2015-05-01

Full Text Available In biofuel production from lignocellulose, low thermostability and product inhibition strongly restrict the enzyme activities and production process. Application of multiple thermostable glycoside hydrolases, forming an enzyme cocktail, can result in a synergistic action and therefore improve production efficiency and reduce operational costs. Therefore, increasing enzyme thermostabilities and compatibility are important for the biofuel industry. In this study, we reported the screening, cloning and biochemical characterization of four novel thermostable lignocellulose hydrolases from a metagenomic library of a long-term dry thermophilic methanogenic digester community, which were highly compatible with optimal conditions and specific activities. The optimal temperatures of the four enzymes, β-xylosidase, xylanase, β-glucosidase, and cellulase ranged from 60°C to 75°C, and over 80% residual activities were observed after 2 h incubation at 50°C. Mixtures of these hydrolases retained high residual synergistic activities after incubation with cellulose, xylan, and steam-exploded corncob at 50°C for 72 h. In addition, about 55% dry weight of steam-exploded corncob was hydrolyzed to glucose and xylose by the synergistic action of the four enzymes at 50°C for 48 h. This work suggested that since different enzymes from a same ecosystem could be more compatible, screening enzymes from a long-term enriching community could be a favorable strategy.

A Personal Retrospective: Elevating Anandamide (AEA) by Targeting Fatty Acid Amide Hydrolase (FAAH) and the Fatty Acid Binding Proteins (FABPs).

Science.gov (United States)

Deutsch, Dale G

2016-01-01

This perspective was adapted from a Career Achievement Award talk given at the International Cannabinoid Research Society Symposium in Bukovina, Poland on June 27, 2016. As a biochemist working in the neurosciences, I was always fascinated with neurotransmitter inactivation. In 1993 we identified an enzyme activity that breaks down anandamide. We called the enzyme anandamide amidase, now called FAAH. We and other laboratories developed FAAH inhibitors that were useful reagents that also proved to have beneficial physiological effects and until recently, new generations of inhibitors were in clinical trials. Nearly all neurotransmitters are water soluble and as such, require a transmembrane protein transporter to pass through the lipid membrane for inactivation inside the cell. However, using model systems, we and others have shown that this is unnecessary for anandamide, an uncharged hydrophobic molecule that readily diffuses across the cellular membrane. Interestingly, its uptake is driven by the concentration gradient resulting from its breakdown mainly by FAAH localized in the endoplasmic reticulum. We identified the FABPs as intracellular carriers that "solubilize" anandamide, transporting anandamide to FAAH. Compounds that bind to FABPs block AEA breakdown, raising its level. The cannabinoids (THC and CBD) also were discovered to bind FABPs and this may be one of the mechanisms by which CBD works in childhood epilepsy, raising anandamide levels. Targeting FABPs may be advantageous since they have some tissue specificity and do not require reactive serine hydrolase inhibitors, as does FAAH, with potential for off-target reactions. At the International Cannabis Research Society Symposium in 1992, Raphe Mechoulam revealed that his laboratory isolated an endogenous lipid molecule that binds to the CB1 receptor (cannabinoid receptor type 1) and this became the milestone paper published in December of that year describing anandamide (AEA, Devane et al., 1992). As to

Sodium-glucose cotransporter 2 inhibitors combined with dipeptidyl peptidase-4 inhibitors in the management of type 2 diabetes: a review of current clinical evidence and rationale

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Yassin SA

2017-03-01

Full Text Available Sayf A Yassin,1 Vanita R Aroda2 1MedStar Union Memorial Hospital, Baltimore, 2MedStar Health Research Institute, Hyattsville, MD, USA Abstract: Type 2 diabetes mellitus (T2DM is a progressive and multifactorial cardiometabolic disorder. Almost half of adults with diabetes fail to achieve their recommended glucose control target. This has prompted some clinicians to advocate the use of more intensive initial therapy, including the use of combination therapy to target multiple physiologic defects in diabetes with the goal of achieving and sustaining glucose control. Numerous options exist for combining the various classes of glucose-lowering agents in the treatment of T2DM. This report reviews the mechanism, rationale, and evidence from clinical trials for combining two of the newer drug classes, namely, dipeptidyl peptidase-4 inhibitors and sodium-glucose cotransporter 2 inhibitors, and considers the possible role of such dual therapy in the management of T2DM. Keywords: sodium-glucose cotransporter 2 inhibitors, dipeptidyl peptidase-4 inhibitors, type 2 diabetes mellitus, combination therapy

2-Butyne-1,4-diol as a novel corrosion inhibitor for API X65 steel pipeline in carbonate/bicarbonate solution

Energy Technology Data Exchange (ETDEWEB)

Sadeghi Meresht, E. [Materials Engineering Department, Faculty of Engineering, Tarbiat Modares University, 1411713114, Tehran (Iran, Islamic Republic of); Shahrabi Farahani, T., E-mail: tshahrabi34@modares.ac.ir [Materials Engineering Department, Faculty of Engineering, Tarbiat Modares University, 1411713114, Tehran (Iran, Islamic Republic of); Neshati, J. [Research Institute of Petroleum Industry, RIPI, 1485733111, Tehran (Iran, Islamic Republic of)

2012-01-15

Highlights: Black-Right-Pointing-Pointer Corrosion of API 5L X65 is effectively reduced by the addition of the inhibitor. Black-Right-Pointing-Pointer The techniques include weight loss, potentiodynamic polarization, EIS and AFM. Black-Right-Pointing-Pointer 2-Butyne-1,4-diol acts as a mixed-type inhibitor. Black-Right-Pointing-Pointer The adsorption of 2-butyne-1,4-diol obeys Langmuir adsorption isotherm. - Abstract: The inhibition effects of 2-butyne-1,4-diol on the corrosion susceptibility of grade API 5L X65 steel pipeline in 2 M Na{sub 2}CO{sub 3}/1 M NaHCO{sub 3} solution were studied by electrochemical techniques and weight loss measurements. The results indicated that this inhibitor was a mixed-type inhibitor, with a maximum percentage inhibition efficiency of approximately 92% in the presence of 5 mM inhibitor. Atomic force microscopy revealed that a protective film was formed on the surface of the inhibited sample. The adsorption of the inhibitor was found to conform to the Langmuir isotherm with the standard adsorption free energy of -21.08 kJ mol{sup -1}.

Oxidoreductive Cellulose Depolymerization by the Enzymes Cellobiose Dehydrogenase and Glycoside Hydrolase 61▿†

Science.gov (United States)

Langston, James A.; Shaghasi, Tarana; Abbate, Eric; Xu, Feng; Vlasenko, Elena; Sweeney, Matt D.

2011-01-01

Several members of the glycoside hydrolase 61 (GH61) family of proteins have recently been shown to dramatically increase the breakdown of lignocellulosic biomass by microbial hydrolytic cellulases. However, purified GH61 proteins have neither demonstrable direct hydrolase activity on various polysaccharide or lignacious components of biomass nor an apparent hydrolase active site. Cellobiose dehydrogenase (CDH) is a secreted flavocytochrome produced by many cellulose-degrading fungi with no well-understood biological function. Here we demonstrate that the binary combination of Thermoascus aurantiacus GH61A (TaGH61A) and Humicola insolens CDH (HiCDH) cleaves cellulose into soluble, oxidized oligosaccharides. TaGH61A-HiCDH activity on cellulose is shown to be nonredundant with the activities of canonical endocellulase and exocellulase enzymes in microcrystalline cellulose cleavage, and while the combination of TaGH61A and HiCDH cleaves highly crystalline bacterial cellulose, it does not cleave soluble cellodextrins. GH61 and CDH proteins are coexpressed and secreted by the thermophilic ascomycete Thielavia terrestris in response to environmental cellulose, and the combined activities of T. terrestris GH61 and T. terrestris CDH are shown to synergize with T. terrestris cellulose hydrolases in the breakdown of cellulose. The action of GH61 and CDH on cellulose may constitute an important, but overlooked, biological oxidoreductive system that functions in microbial lignocellulose degradation and has applications in industrial biomass utilization. PMID:21821740

Discovery of Potent and Selective Inhibitors for ADAMTS-4 through DNA-Encoded Library Technology (ELT).

Science.gov (United States)

Ding, Yun; O'Keefe, Heather; DeLorey, Jennifer L; Israel, David I; Messer, Jeffrey A; Chiu, Cynthia H; Skinner, Steven R; Matico, Rosalie E; Murray-Thompson, Monique F; Li, Fan; Clark, Matthew A; Cuozzo, John W; Arico-Muendel, Christopher; Morgan, Barry A

2015-08-13

The aggrecan degrading metalloprotease ADAMTS-4 has been identified as a novel therapeutic target for osteoarthritis. Here, we use DNA-encoded Library Technology (ELT) to identify novel ADAMTS-4 inhibitors from a DNA-encoded triazine library by affinity selection. Structure-activity relationship studies based on the selection information led to the identification of potent and highly selective inhibitors. For example, 4-(((4-(6,7-dimethoxy-3,4-dihydroisoquinolin-2(1H)-yl)-6-(((4-methylpiperazin-1-yl)methyl)amino)-1,3,5-triazin-2-yl)amino)methyl)-N-ethyl-N-(m-tolyl)benzamide has IC50 of 10 nM against ADAMTS-4, with >1000-fold selectivity over ADAMT-5, MMP-13, TACE, and ADAMTS-13. These inhibitors have no obvious zinc ligand functionality.

Identification of [4-[4-(methylsulfonyl)phenyl]-6-(trifluoromethyl)-2-pyrimidinyl] amines and ethers as potent and selective cyclooxygenase-2 inhibitors.

Science.gov (United States)

Swarbrick, Martin E; Beswick, Paul J; Gleave, Robert J; Green, Richard H; Bingham, Sharon; Bountra, Chas; Carter, Malcolm C; Chambers, Laura J; Chessell, Iain P; Clayton, Nick M; Collins, Sue D; Corfield, John A; Hartley, C David; Kleanthous, Savvas; Lambeth, Paul F; Lucas, Fiona S; Mathews, Neil; Naylor, Alan; Page, Lee W; Payne, Jeremy J; Pegg, Neil A; Price, Helen S; Skidmore, John; Stevens, Alexander J; Stocker, Richard; Stratton, Sharon C; Stuart, Alastair J; Wiseman, Joanne O

2009-08-01

A novel series of [4-[4-(methylsulfonyl)phenyl]-6-(trifluoromethyl)-2-pyrimidine-based cyclooxygenase-2 (COX-2) inhibitors, which have a different arrangement of substituents compared to the more common 1,2-diarylheterocycle based molecules, have been discovered. For example, 2-(butyloxy)-4-[4-(methylsulfonyl)phenyl]-6-(trifluoromethyl)pyrimidine (47), a member of the 2-pyrimidinyl ether series, has been shown to be a potent and selective inhibitor with a favourable pharmacokinetic profile, high brain penetration and good efficacy in rat models of hypersensitivity.

The effects of GLP-1 analogues, DPP-4 inhibitors and SGLT2 inhibitors on the renal system.

Science.gov (United States)

Schernthaner, Guntram; Mogensen, Carl Erik; Schernthaner, Gerit-Holger

2014-09-01

Diabetic nephropathy (DN) affects an estimated 20%-40% of patients with type 2 diabetes mellitus (T2DM). Key modifiable risk factors for DN are albuminuria, anaemia, dyslipidaemia, hyperglycaemia and hypertension, together with lifestyle factors, such as smoking and obesity. Early detection and treatment of these risk factors can prevent DN or slow its progression, and may even induce remission in some patients. DN is generally preceded by albuminuria, which frequently remains elevated despite treatment in patients with T2DM. Optimal treatment and prevention of DN may require an early, intensive, multifactorial approach, tailored to simultaneously target all modifiable risk factors. Regular monitoring of renal function, including urinary albumin excretion, creatinine clearance and glomerular filtration rate, is critical for following any disease progression and making treatment adjustments. Dipeptidyl peptidase (DPP)-4 inhibitors and sodium-glucose cotransporter 2 (SGLT2) inhibitors lower blood glucose levels without additional risk of hypoglycaemia, and may also reduce albuminuria. Further investigation of the potential renal benefits of DPP-4 and SGLT2 inhibitors is underway. © The Author(s) 2014.

Emerging role of dipeptidyl peptidase-4 inhibitors in the management of type 2 diabetes

Directory of Open Access Journals (Sweden)

Bernd Richter

2008-08-01

Full Text Available Bernd Richter, Elizabeth Bandeira-Echtler, Karla Bergerhoff, Christian LerchCochrane Metabolic and Endocrine Disorders Group, Department of General Practice, Heinrich-Heine University Duesseldorf, Duesseldorf, GermanyBackground: In type 2 diabetes mellitus (T2DM there is a progressive loss of β-cell function. One new approach yielding promising results is the use of the orally active dipeptidyl peptidase-4 (DPP-4 inhibitors. However, every new compound for T2DM has to prove long-term safety especially on cardiovascular outcomes.Objectives: Systematic review and meta-analysis of the effects of sitagliptin and vildagliptin therapy on main efficacy parameters and safety.Selection criteria, data collection, and analysis: Randomized controlled clinical studies of at least 12 weeks’ duration in T2DM.Results: DPP-4 inhibitors versus placebo showed glycosylated hemoglobin A1c (A1c improvements of 0.7% versus placebo but not compared to monotherapy with other hypoglycemic agents (0.3% in favor of controls. The overall risk profile of DPP-4 inhibitors was low, however a 34% relative risk increase (95% confidence interval 10% to 64%, P = 0.004 was noted for all-cause infection associated with sitagliptin use. No data on immune function, health-related quality of life and diabetic complications could be extracted.Conclusions: DPP-4 inhibitors have some theoretical advantages over existing therapies with oral antidiabetic compounds but should currently be restricted to individual patients. Long-term data on cardiovascular outcomes and safety are needed before widespread use of these new agents.Keywords: DPP-4 inhibitors, sitagliptin, vildagliptin, systematic review, meta-analysis

Phosphodiesterase 4 inhibitor and phosphodiesterase 5 inhibitor combination therapy has antifibrotic and anti-inflammatory effects in mdx mice with Duchenne muscular dystrophy.

Science.gov (United States)

Nio, Yasunori; Tanaka, Masayuki; Hirozane, Yoshihiko; Muraki, Yo; Okawara, Mitsugi; Hazama, Masatoshi; Matsuo, Takanori

2017-12-01

Duchenne muscular dystrophy (DMD) is the most common inherited muscular dystrophy. Patients experience DMD in their 20s from cardiac or respiratory failure related to progressive muscle wasting. Currently, the only treatments for the symptoms of DMD are available. Muscle fibrosis, a DMD feature, leads to reduced muscle function and muscle mass, and hampers pharmaceutical therapeutic efficacy. Although antifibrotic agents may be useful, none is currently approved. Phosphodiesterase 4 (PDE4) inhibitors have exhibited antifibrotic effects in human and animal models. In this study, we showed beneficial effects of the PDE4 inhibitor piclamilast in the DMD mdx mouse. Piclamilast reduced the mRNA level of profibrotic genes, including collagen 1A1, in the gastrocnemius and diaphragm, in the mdx mouse, and significantly reduced the Sirius red staining area. The PDE5 inhibitors sildenafil and tadalafil ameliorated functional muscle ischemia in boys with DMD, and sildenafil reversed cardiac dysfunction in the mdx mouse. Single-treatment piclamilast or sildenafil showed similar antifibrotic effects on the gastrocnemius; combination therapy showed a potent antifibrotic effect, and piclamilast and combination therapy increased peroxisome proliferator-activated receptor γ coactivator-1α mRNA in mouse gastrocnemius. In summary, we confirmed that piclamilast has significant antifibrotic effects in mdx mouse muscle and is a potential treatment for muscle fibrosis in DMD.-Nio, Y., Tanaka, M., Hirozane, Y., Muraki, Y., Okawara, M., Hazama, M., Matsuo, T. Phosphodiesterase 4 inhibitor and phosphodiesterase 5 inhibitor combination therapy has antifibrotic and anti-inflammatory effects in mdx mice with Duchenne muscular dystrophy. © FASEB.

Structure-based drug design of a highly potent CDK1,2,4,6 inhibitor with novel macrocyclic quinoxalin-2-one structure.

Science.gov (United States)

Kawanishi, Nobuhiko; Sugimoto, Tetsuya; Shibata, Jun; Nakamura, Kaori; Masutani, Kouta; Ikuta, Mari; Hirai, Hiroshi

2006-10-01

The design of a novel series of cyclin-dependent kinase (CDK) inhibitors containing a macrocyclic quinoxaline-2-one is reported. Structure-based drug design and optimization from the starting point of diarylurea 2, which we previously reported as a moderate CDK1,2,4,6 inhibitor [J. Biol.Chem.2001, 276, 27548], led to the discovery of potent CDK1,2,4,6 inhibitor that were suitable for iv administration for in vivo study.

Computational study on the inhibitor binding mode and allosteric regulation mechanism in hepatitis C virus NS3/4A protein.

Directory of Open Access Journals (Sweden)

Weiwei Xue

Full Text Available HCV NS3/4A protein is an attractive therapeutic target responsible for harboring serine protease and RNA helicase activities during the viral replication. Small molecules binding at the interface between the protease and helicase domains can stabilize the closed conformation of the protein and thus block the catalytic function of HCV NS3/4A protein via an allosteric regulation mechanism. But the detailed mechanism remains elusive. Here, we aimed to provide some insight into the inhibitor binding mode and allosteric regulation mechanism of HCV NS3/4A protein by using computational methods. Four simulation systems were investigated. They include: apo state of HCV NS3/4A protein, HCV NS3/4A protein in complex with an allosteric inhibitor and the truncated form of the above two systems. The molecular dynamics simulation results indicate HCV NS3/4A protein in complex with the allosteric inhibitor 4VA adopts a closed conformation (inactive state, while the truncated apo protein adopts an open conformation (active state. Further residue interaction network analysis suggests the communication of the domain-domain interface play an important role in the transition from closed to open conformation of HCV NS3/4A protein. However, the inhibitor stabilizes the closed conformation through interaction with several key residues from both the protease and helicase domains, including His57, Asp79, Asp81, Asp168, Met485, Cys525 and Asp527, which blocks the information communication between the functional domains interface. Finally, a dynamic model about the allosteric regulation and conformational changes of HCV NS3/4A protein was proposed and could provide fundamental insights into the allosteric mechanism of HCV NS3/4A protein function regulation and design of new potent inhibitors.

Structural insight into catalytic mechanism of PET hydrolase

OpenAIRE

Han, Xu; Liu, Weidong; Huang, Jian-Wen; Ma, Jiantao; Zheng, Yingying; Ko, Tzu-Ping; Xu, Limin; Cheng, Ya-Shan; Chen, Chun-Chi; Guo, Rey-Ting

2017-01-01

PET hydrolase (PETase), which hydrolyzes polyethylene terephthalate (PET) into soluble building blocks, provides an attractive avenue for the bioconversion of plastics. Here we present the structures of a novel PETase from the PET-consuming microbe Ideonella sakaiensis in complex with substrate and product analogs. Through structural analyses, mutagenesis, and activity measurements, a substrate-binding mode is proposed, and several features critical for catalysis are elucidated.

Structural insight into catalytic mechanism of PET hydrolase.

Science.gov (United States)

Han, Xu; Liu, Weidong; Huang, Jian-Wen; Ma, Jiantao; Zheng, Yingying; Ko, Tzu-Ping; Xu, Limin; Cheng, Ya-Shan; Chen, Chun-Chi; Guo, Rey-Ting

2017-12-13

PET hydrolase (PETase), which hydrolyzes polyethylene terephthalate (PET) into soluble building blocks, provides an attractive avenue for the bioconversion of plastics. Here we present the structures of a novel PETase from the PET-consuming microbe Ideonella sakaiensis in complex with substrate and product analogs. Through structural analyses, mutagenesis, and activity measurements, a substrate-binding mode is proposed, and several features critical for catalysis are elucidated.

Identification and characterization of some Aspergillus pectinolytic glycoside hydrolases

NARCIS (Netherlands)

Zandleven, J.S.

2006-01-01

Keywords: Aspergillusniger , Arabidopsis thaliana , homogalacturonan, rhamnogalacturonan, xylogalacturonan, xylogalacturonan hydrolase, exo-polygalacturonasePectinases are used for many food

Linagliptin, a xanthine-based dipeptidyl peptidase-4 inhibitor with an unusual profile for the treatment of type 2 diabetes

DEFF Research Database (Denmark)

Deacon, Carolyn F; Holst, Jens Juul

2010-01-01

data presented at Scientific Meetings and peer-reviewed studies published since 2007. WHAT THE READER WILL GAIN: This article reviews pharmacokinetic and pharmacodynamic characteristics of linagliptin. Linagliptin belongs to a new chemical class of dipeptidyl pepidase-4 (DPP-4) inhibitors, which...... comprise xanthine-based compounds. It is a potent, long-acting inhibitor with high selectivity for DPP-4 versus the related enzymes DPP-8 and DPP-9. The drug has modest oral availability in humans, but is absorbed rapidly to inhibit plasma DPP-4 activity by > 80% over 24 h. It is not metabolized...

Targeted discovery of glycoside hydrolases from a switchgrass-adapted compost community.

Directory of Open Access Journals (Sweden)

Martin Allgaier

Full Text Available Development of cellulosic biofuels from non-food crops is currently an area of intense research interest. Tailoring depolymerizing enzymes to particular feedstocks and pretreatment conditions is one promising avenue of research in this area. Here we added a green-waste compost inoculum to switchgrass (Panicum virgatum and simulated thermophilic composting in a bioreactor to select for a switchgrass-adapted community and to facilitate targeted discovery of glycoside hydrolases. Small-subunit (SSU rRNA-based community profiles revealed that the microbial community changed dramatically between the initial and switchgrass-adapted compost (SAC with some bacterial populations being enriched over 20-fold. We obtained 225 Mbp of 454-titanium pyrosequence data from the SAC community and conservatively identified 800 genes encoding glycoside hydrolase domains that were biased toward depolymerizing grass cell wall components. Of these, approximately 10% were putative cellulases mostly belonging to families GH5 and GH9. We synthesized two SAC GH9 genes with codon optimization for heterologous expression in Escherichia coli and observed activity for one on carboxymethyl cellulose. The active GH9 enzyme has a temperature optimum of 50 degrees C and pH range of 5.5 to 8 consistent with the composting conditions applied. We demonstrate that microbial communities adapt to switchgrass decomposition using simulated composting condition and that full-length genes can be identified from complex metagenomic sequence data, synthesized and expressed resulting in active enzyme.

Method for enhancing amidohydrolase activity of fatty acid amide hydrolase

Science.gov (United States)

John, George; Nagarajan, Subbiah; Chapman, Kent; Faure, Lionel; Koulen, Peter

2017-12-26

A method for enhancing amidohydrolase activity of Fatty Acid Amide Hydrolase (FAAH) is disclosed. The method comprising administering a phenoxyacyl-ethanolamide that causes the enhanced activity. The enhanced activity can have numerous effects on biological organisms including, for example, enhancing the growth of certain seedlings.

Dipeptidyl Peptidase-4 Inhibitor Anagliptin Prevents Intracranial Aneurysm Growth by Suppressing Macrophage Infiltration and Activation.

Science.gov (United States)

Ikedo, Taichi; Minami, Manabu; Kataoka, Hiroharu; Hayashi, Kosuke; Nagata, Manabu; Fujikawa, Risako; Higuchi, Sei; Yasui, Mika; Aoki, Tomohiro; Fukuda, Miyuki; Yokode, Masayuki; Miyamoto, Susumu

2017-06-19

Chronic inflammation plays a key role in the pathogenesis of intracranial aneurysms (IAs). DPP-4 (dipeptidyl peptidase-4) inhibitors have anti-inflammatory effects, including suppressing macrophage infiltration, in various inflammatory models. We examined whether a DPP-4 inhibitor, anagliptin, could suppress the growth of IAs in a rodent aneurysm model. IAs were surgically induced in 7-week-old male Sprague Dawley rats, followed by oral administration of 300 mg/kg anagliptin. We measured the morphologic parameters of aneurysms over time and their local inflammatory responses. To investigate the molecular mechanisms, we used lipopolysaccharide-treated RAW264.7 macrophages. In the anagliptin-treated group, aneurysms were significantly smaller 2 to 4 weeks after IA induction. Anagliptin inhibited the accumulation of macrophages in IAs, reduced the expression of MCP-1 (monocyte chemotactic protein 1), and suppressed the phosphorylation of p65. In lipopolysaccharide-stimulated RAW264.7 cells, anagliptin treatment significantly reduced the production of tumor necrosis factor α, MCP-1, and IL-6 (interleukin 6) independent of GLP-1 (glucagon-like peptide 1), the key mediator in the antidiabetic effects of DPP-4 inhibitors. Notably, anagliptin activated ERK5 (extracellular signal-regulated kinase 5), which mediates the anti-inflammatory effects of statins, in RAW264.7 macrophages. Preadministration with an ERK5 inhibitor blocked the inhibitory effect of anagliptin on MCP-1 and IL-6 expression. Accordingly, the ERK5 inhibitor also counteracted the suppression of p65 phosphorylation in vitro. A DPP-4 inhibitor, anagliptin, prevents the growth of IAs via its anti-inflammatory effects on macrophages. © 2017 The Authors. Published on behalf of the American Heart Association, Inc., by Wiley.

N-3 Polyunsaturated Fatty Acids Decrease the Protein Expression of Soluble Epoxide Hydrolase via Oxidative Stress-Induced P38 Kinase in Rat Endothelial Cells.

Science.gov (United States)

Okada, Takashi; Morino, Katsutaro; Nakagawa, Fumiyuki; Tawa, Masashi; Kondo, Keiko; Sekine, Osamu; Imamura, Takeshi; Okamura, Tomio; Ugi, Satoshi; Maegawa, Hiroshi

2017-06-24

N -3 polyunsaturated fatty acids (PUFAs) improve endothelial function. The arachidonic acid-derived metabolites (epoxyeicosatrienoic acids (EETs)) are part of the endothelial hyperpolarization factor and are vasodilators independent of nitric oxide. However, little is known regarding the regulation of EET concentration by docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA) in blood vessels. Sprague-Dawley rats were fed either a control or fish oil diet for 3 weeks. Compared with the control, the fish oil diet improved acetylcholine-induced vasodilation and reduced the protein expression of soluble epoxide hydrolase (sEH), a key EET metabolic enzyme, in aortic strips. Both DHA and EPA suppressed sEH protein expression in rat aorta endothelial cells (RAECs). Furthermore, the concentration of 4-hydroxy hexenal (4-HHE), a lipid peroxidation product of n -3 PUFAs, increased in n -3 PUFA-treated RAECs. In addition, 4-HHE treatment suppressed sEH expression in RAECs, suggesting that 4-HHE (derived from n -3 PUFAs) is involved in this phenomenon. The suppression of sEH was attenuated by the p38 kinase inhibitor (SB203580) and by treatment with the antioxidant N-acetyl-L-cysteine. In conclusion, sEH expression decreased after n -3 PUFAs treatment, potentially through oxidative stress and p38 kinase. Mild oxidative stress induced by n -3 PUFAs may contribute to their cardio-protective effect.

Properties of epoxide hydrolase from the yeast Rhodotorula glutinis

NARCIS (Netherlands)

Ariës-Kronenburg, N.A.E.

2002-01-01

Epoxide hydrolases are ubiquitous enzymes that can be found in nearly all living organisms. Some of the enzymes play an important role in detoxifying xenobiotic and metabolic compounds. Others are important in the growth of organisms like