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Sample records for surface-expressed organophosphorus hydrolase

  1. Conformational Variability of Organophosphorus Hydrolase upon Soman and Paraoxon Binding

    Energy Technology Data Exchange (ETDEWEB)

    Gomes, Diego Eb; Lins, Roberto D.; Pascutti, Pedro G.; Lei, Chenghong; Soares, Thereza A.

    2011-12-31

    The bacterial enzyme organophosphorus hydrolase (OPH) exhibits both catalytic and substrate promiscuity. It hydrolyzes bonds in a variety of phosphotriester (P-O), phosphonothioate (P-S), phosphofluoridate (P-F) and phosphonocyanate (F-CN) compounds. However, its catalytic efficiency varies markedly for different substrates, limiting the broad-range application of OPH as catalyst in the bioremediation of pesticides and chemical war agents. In the present study, pK{sub a} calculations and multiple explicit-solvent molecular dynamics (MD) simulations were performed to characterize and contrast the structural dynamics of OPH bound to two substrates hydrolyzed with very distinct catalytic efficiencies: the nerve agent soman (O-pinacolyl-methyl-phosphonofluoridate) and the pesticide paraoxon (diethyl p-nitrophenyl phosphate). pK{sub a} calculations for the substrate-bound and unbound enzyme showed a significant pK{sub a} shift from standard values ({Delta}pK{sub a} = {+-} 3 units) for residues 254His and 275Arg. MD simulations of the doubly protonated 254His revealed a dynamic hydrogen bond network connecting the catalytic residue 301Asp via 254His to 232Asp, 233Asp, 275Arg and 235Asp, and is consistent with a previously postulated proton relay mechanism to ferry protons away from the active site with substrates that do not require activation of the leaving group. Hydrogen bonds between 301Asp and 254His were persistent in the OPH-paraoxon complex but not in the OPH-soman one, suggesting a potential role for such interaction in the more efficient hydrolysis of paraoxon over soman by OPH. These results are in line with previous mutational studies of residue 254His, which led to an increase of the catalytic efficiency of OPH over soman yet decreased its efficiency for paraoxon. In addition, comparative analysis of the molecular trajectories for OPH bound to soman and paraoxon suggests that binding of the latter facilitates the conformational transition of OPH from the

  2. Altering the Substrate Specificity of Organophosphorus Hydrolase for Enhanced Hydrolysis of Chlorpyrifos

    OpenAIRE

    Cho, Catherine Mee-Hie; Mulchandani, Ashok; Chen, Wilfred

    2004-01-01

    Chlorpyrifos is one of the most popular pesticides used for agriculture crop protection, and widespread contamination is a potential concern. However, chlorpyrifos is hydrolyzed almost 1,000-fold slower than the preferred substrate, paraoxon, by organophosphorus hydrolase (OPH), an enzyme that can degrade a broad range of organophosphate pesticides. We have recently demonstrated that directed evolution can be used to generate OPH variants with up to 25-fold improvement in hydrolysis of methyl...

  3. Detection of organophosphorus compound based on a sol-gel silica planar waveguide doped with a green fluorescent protein and an organophosphorus hydrolase

    Science.gov (United States)

    Enami, Y.; Tsuchiya, K.; Suye, S.

    2011-06-01

    In this letter, the authors report the real-time detection of an organophosphorus compound using a sol-gel silica planar waveguide doped with a green fluorescent protein and an organophosphorus hydrolase on a yeast-cell surface display. The waveguide was pumped at 488 nm, and it emitted green fluorescence at the far field. The green fluorescent light at 550 nm changed by 50% from the original power 1 min after application of the organophosphorus compound. The results enable the real-time detection of sarin and other biochemicals by using an in-line fiber sensor network.

  4. Assembly of the active center of organophosphorus hydrolase in metal-organic frameworks via rational combination of functional ligands.

    Science.gov (United States)

    Xia, Mengfan; Zhuo, Caixia; Ma, Xuejuan; Zhang, Xiaohong; Sun, Huaming; Zhai, Quanguo; Zhang, Yaodong

    2017-10-12

    Different from popular mimics of bimetallic nuclear centers bridged by a hydroxide, a total coordination sphere of the active center of organophosphorus hydrolase was assembled in metal-organic frameworks by rational design and combination of ligands, which resulted in efficient destruction of nerve agent stimulants without a base as a co-catalyst.

  5. Intensification of Organophosphorus Hydrolase Synthesis by Using Substances with Gas-Transport Function

    Directory of Open Access Journals (Sweden)

    Olga Senko

    2017-12-01

    Full Text Available We have performed studies and comparative analysis of the biosynthesis characteristics of intracellular recombinant enzyme, such as hexahistidine-containing organophosphorus hydrolase (His6-OPH in Escherichia coli SG13009[pREP4] cells when various perfluorocarbon compounds (PFC were introduced into the medium for cell cultivation. The PFC were found to facilitate the biosynthesis of His6-OPH: increased levels of the total OPH-activity (up to 37% were measured upon introduction of 1,1,1,2,2,3,3,4,4,5,5,6,6,6-tetradecafluorohexane (PFH and 4,7,10,13,16,19,22,25,28,31-decaoxaperfluoro-5,8,11,14,17,18,21,24,27,30-decamethyl tetratriacontane (Polyether II into culture medium. We have demonstrated the possibility of effective and multiple (at least five-fold use of PFH for biosynthesis of intracellular recombinant protein His6-OPH, which catalyzes the hydrolysis of organophosphorus pesticides (OP, is widely used in agriculture and can be applied as new antidote for OP-detoxification in vivo. The multiple use of PFH was achieved through recycling of this substance: sediment of Escherichia coli SG13009[pREP4] cell biomass was collected at the end of each culture growing step and disintegrated with ultrasound, and obtained residue containing almost all of the initially introduced PFC was then added to the medium at the start of the following culture growing step.

  6. Catalytic Characteristics of New Antibacterials Based on Hexahistidine-Containing Organophosphorus Hydrolase

    Directory of Open Access Journals (Sweden)

    Olga Maslova

    2017-09-01

    Full Text Available Catalytic characteristics of hexahistidine-containing organophosphorus hydrolase (His6-OPH and its enzyme-polyelectrolyte complexes with poly-l-glutamic acid or poly-l-aspartic acid (His6-OPH/PLD50, hydrolyzing organophosphorous compounds, and N-acyl homoserine lactones were studied in the presence of various antibiotics (ampicillin, gentamicin, kanamycin, and rifampicin. The antibiotics at concentrations below 1 g·L−1 had a negligible inhibiting effect on the His6-OPH activity. Mixed inhibition of His6-OPH was established for higher antibiotic concentrations, and rifampicin was the most potent inhibitor. Stabilization of the His6-OPH activity was observed in the presence of antibiotics at a concentration of 0.2 g·L−1 during exposure at 25–41 °C. Molecular docking of antibiotics to the surface of His6-OPH dimer revealed the antibiotics binding both to the area near active centers of the enzyme subunits and to the region of contact between subunits of the dimer. Such interactions between antibiotics and His6-OPH were verified with Fourier-transform infrared (FTIR spectroscopy. Considering all the results of the study, the combination of His6-OPH/PLD50 with β-lactam antibiotic ampicillin was established as the optimal one in terms of exhibition and persistence of maximal lactonase activity of the enzyme.

  7. Mimicking the Active Sites of Organophosphorus Hydrolase on the Backbone of Graphene Oxide to Destroy Nerve Agent Simulants.

    Science.gov (United States)

    Ma, Xuejuan; Zhang, Lin; Xia, Mengfan; Li, Shuangqin; Zhang, Xiaohong; Zhang, Yaodong

    2017-06-28

    Recent global military events, such as the conflict in Syria, have emphasized the need to find effective strategies to rapidly destroy organophosphorus-based nerve agents. In this work, we designed active site-engineered graphene oxide (GO) via polymerization (polymer bead-GOs) as organophosphorus hydrolase (OPH) mimetic hotspots for the rapid degradation of nerve agents. This hybrid catalyst has a high total turnover frequency value of 0.65 s -1 and good stability (94.8% activity maintained after 5 cycles). Mechanism investigations suggested that the high catalytic performance could be attributed to the synergistic effect among the clusters of imidazole and the presence of - COOH groups on the GO surface and Zn 2+ .

  8. Stabilization of organophosphorus hydrolase by entrapment in silk fibroin: formation of a robust enzymatic material suitable for surface coatings.

    Science.gov (United States)

    Dennis, Patrick B; Walker, Anne Y; Dickerson, Matthew B; Kaplan, David L; Naik, Rajesh R

    2012-07-09

    Organophosphates are some of the most acutely toxic compounds synthesized on an industrial scale, and organophosphorus hydrolase (OPH) has the ability to hydrolyze and inactivate a number of these chemicals. However, OPH activity is vulnerable to harsh environmental conditions that would accompany its practical utility in the field; a limitation that can also be extended to conditions required for incorporation of OPH into useful materials. Here we present evidence that entrapment of OPH in silk fibroin leads to stabilization of OPH activity under a variety of conditions that would otherwise reduce free enzyme activity, such as elevated temperature, UV light exposure and the presence of detergent. Silk fibroin entrapment of OPH also allowed for its dispersal into a polyurethane-based coating that retained organophosphate hydrolysis activity after formulation, application and drying. Together, the data presented here demonstrate the utility of silk fibroin entrapment for the protection of OPH activity under a variety of environmental conditions.

  9. Dramatic Differences in Organophosphorus Hydrolase Activity between Human and Chimeric Recombinant Mammalian Paraoxonase-1 Enzymes

    Science.gov (United States)

    2009-01-01

    1 s-1 (see Table 1)]. G2E6 PON1 had 13- and 6-fold greater catalytic efficiency for hydrolysis of paraoxon (the toxic metabolite of the pesticide ...Therapeutics (Hardman, J. G., Limbird, L. E., and Gilman, A. G., Eds.) pp 175-192, McGraw-Hill, New York. 3. Maynard, R. L., and Beswick, F. W. (1992...Organophosphorus compounds as chemical warfare agents. In Clinical and Experimental Toxicology of Organophosphates and Carbamates (Ballantyne, B., and Marrs, T

  10. Simultaneous degradation of organophosphate and organochlorine pesticides by Sphingobium japonicum UT26 with surface-displayed organophosphorus hydrolase.

    Science.gov (United States)

    Cao, Xiangyu; Yang, Chao; Liu, Ruihua; Li, Qiang; Zhang, Wei; Liu, Jianli; Song, Cunjiang; Qiao, Chuanling; Mulchandani, Ashok

    2013-04-01

    A genetically engineered microorganism (GEM) capable of simultaneously degrading organophosphate and organochlorine pesticides was constructed for the first time by display of organophosphorus hydrolase (OPH) on the cell surface of a hexachlorocyclohexane (HCH)-degrading Sphingobium japonicum UT26. The GEM could potentially be used for removing the two classes of pesticides that may be present in mixtures at contaminated sites. A surface anchor system derived from the truncated ice nucleation protein (INPNC) from Pseudomonas syringae was used to target OPH onto the cell surface of UT26, reducing the potential substrate uptake limitation. The surface localization of INPNC-OPH fusion was verified by cell fractionation, western blot, proteinase accessibility, and immunofluorescence microscopy. Furthermore, the functionality of the surface-exposed OPH was demonstrated by OPH activity assays. Surface display of INPNC-OPH fusion (82 kDa) neither inhibited cell growth nor affected cell viability. The engineered UT26 could degrade parathion as well as γ-HCH rapidly in minimal salt medium. The removal of parathion and γ-HCH by engineered UT26 in sterile and non-sterile soil was also studied. In both soil samples, a mixture of parathion (100 mg kg(-1)) and γ-HCH (10 mg kg(-1)) could be degraded completely within 15 days. Soil treatment results indicated that the engineered UT26 is a promising multifunctional bacterium that could be used for the bioremediation of multiple pesticide-contaminated environments.

  11. Optical Detection of Paraoxon Using Single-Walled Carbon Nanotube Films with Attached Organophosphorus Hydrolase-Expressed Escherichia coli

    Directory of Open Access Journals (Sweden)

    Intae Kim

    2015-05-01

    Full Text Available In whole-cell based biosensors, spectrophotometry is one of the most commonly used methods for detecting organophosphates due to its simplicity and reliability. The sensor performance is directly affected by the cell immobilization method because it determines the amount of cells, the mass transfer rate, and the stability. In this study, we demonstrated that our previously-reported microbe immobilization method, a microbe-attached single-walled carbon nanotube film, can be applied to whole-cell-based organophosphate sensors. This method has many advantages over other whole-cell organophosphate sensors, including high specific activity, quick cell immobilization, and excellent stability. A device with circular electrodes was fabricated for an enlarged cell-immobilization area. Escherichia coli expressing organophosphorus hydrolase in the periplasmic space and single-walled carbon nanotubes were attached to the device by our method. Paraoxon was hydrolyzed using this device, and detected by measuring the concentration of the enzymatic reaction product, p-nitrophenol. The specific activity of our device was calculated, and was shown to be over 2.5 times that reported previously for other whole-cell organophosphate sensors. Thus, this method for generation of whole-cell-based OP biosensors might be optimal, as it overcomes many of the caveats that prevent the widespread use of other such devices.

  12. Isolation of the opdE gene that encodes for a new hydrolase of Enterobacter sp. capable of degrading organophosphorus pesticides.

    Science.gov (United States)

    Chino-Flores, Concepción; Dantán-González, Edgar; Vázquez-Ramos, Alejandra; Tinoco-Valencia, Raunel; Díaz-Méndez, Rafael; Sánchez-Salinas, Enrique; Castrejón-Godínez, Maria Luisa; Ramos-Quintana, Fernando; Ortiz-Hernández, Maria Laura

    2012-06-01

    Microbial enzymes that can hydrolyze organophosphorus compounds have been isolated, identified and characterized from different microbial species in order to use them in biodegradation of organophosphorus compounds. We isolated a bacterial strain Cons002 from an agricultural soil bacterial consortium, which can hydrolyze methyl-parathion (MP) and other organophosphate pesticides. HPLC analysis showed that strain Cons002 is capable of degrading pesticides MP, parathion and phorate. Pulsed-field gel electrophoresis and 16S rRNA amplification were performed for strain characterization and identification, respectively, showing that the strain Cons002 is related to the genus Enterobacter sp. which has a single chromosome of 4.6 Mb and has no plasmids. Genomic library was constructed from DNA of Enterobacter sp. Cons002. A gene called opdE (Organophosphate Degradation from Enterobacter) consists of 753 bp and encodes a protein of 25 kDa, which was isolated using activity methods. This gene opdE had no similarity to any genes reported to degrade organophosphates. When kanamycin-resistance cassette was placed in the gene opdE, hydrolase activity was suppressed and Enterobacter sp. Cons002 had no growth with MP as a nutrients source.

  13. Efficient Degradation of Malathion in the Presence of Detergents Using an Engineered Organophosphorus Hydrolase Highly Expressed by Pichia pastoris without Methanol Induction.

    Science.gov (United States)

    Bai, Yun-Peng; Luo, Xiao-Jing; Zhao, Yu-Lian; Li, Chun-Xiu; Xu, Dian-Sheng; Xu, Jian-He

    2017-10-18

    The biodegradation of pesticides by organophosphorus hydrolases (OPHs) requires an efficient enzyme production technology in industry. Herein, a Pichia pastoris strain was constructed for the extracellular expression of PoOPH M9 , an engineered malathion-degrading enzyme. After optimization, the maximum titer and yield of fermentation reached 50.8 kU/L and 4.1 g protein /L after 3 days, with the highest space-time yield (STY) reported so far, 640 U L -1 h -1 . PoOPH M9 displayed its high activity and stability in the presence of 0.1% (w/w) plant-derived detergent. Only 0.04 mg/mL enzyme could completely remove 0.15 mM malathion in aqueous solution within 20 min. Furthermore, 12 μmol malathion on apples and cucumbers surfaces was completely removed by 0.05 mg/mL PoOPH M9 in tap water after 35 min washing. The efficient production of the highly active PoOPH M9 has cleared a major barrier to biodegradation of pesticide residues in food industry.

  14. BIOSENSOR FOR DIRECT DETERMINATION OF ORGANOPHOSPHATE NERVE AGENTS USING RECOMBINANT ESCHERICHIA COLI WITH SURFACE-EXPRESSED ORGANOPHOSPHORUS HYDROLASE. 1. POTENTIOMETRIC MICROBIAL ELECTRODE. (R823663)

    Science.gov (United States)

    The perspectives, information and conclusions conveyed in research project abstracts, progress reports, final reports, journal abstracts and journal publications convey the viewpoints of the principal investigator and may not represent the views and policies of ORD and EPA. Concl...

  15. AMPEROMETRIC MICROBIAL BIOSENSOR FOR DIRECT DETERMINATION OF ORGANOPHOSPHATE NERVE AGENTS USING RECOMBINANT MORAXELLA SP. WITH SURFACE EXPRESSED ORGANOPHOSPHORUS HYDROLASE. (R828160)

    Science.gov (United States)

    The perspectives, information and conclusions conveyed in research project abstracts, progress reports, final reports, journal abstracts and journal publications convey the viewpoints of the principal investigator and may not represent the views and policies of ORD and EPA. Concl...

  16. MICROBIAL BIOSENSOR FOR DIRECT DETERMINATION OF ORGANOPHOSPHATE NERVE AGENTS USING RECOMBINANT ESCHERICHIA COLI WITH SURFACE-EXPRESSED ORGANOPHOSPHORUS HYDROLASE. 2. FIBER-OPTIC MICROBIAL BIOSENSOR. (R823663)

    Science.gov (United States)

    The perspectives, information and conclusions conveyed in research project abstracts, progress reports, final reports, journal abstracts and journal publications convey the viewpoints of the principal investigator and may not represent the views and policies of ORD and EPA. Concl...

  17. Green fluorescent protein-doped sol-gel silica planar waveguide to detect organophosphorus compound

    Science.gov (United States)

    Enami, Y.; Suye, S.

    2012-02-01

    We report novel living protein-doped planar waveguide, and real-time detection of an organophosphorus compound using a sol-gel silica planar waveguide doped with a green fluorescent protein and an organophosphorus hydrolase on a yeast-cell surface. The waveguide was pumped at 488 nm, and emitted green fluorescence at the far field. The green fluorescent light at 550 nm changed by 50% from the original power 1 min after application of the organophosphorus compound. The results enable the real-time detection of biochemical weapon and insecticide harmful for human body by using an in-line fiber sensor network.

  18. Rapid biodegradation of organophosphorus pesticides by Stenotrophomonas sp. G1

    International Nuclear Information System (INIS)

    Deng, Shuyan; Chen, Yao; Wang, Daosheng; Shi, Taozhong; Wu, Xiangwei; Ma, Xin; Li, Xiangqiong; Hua, Rimao; Tang, Xinyun; Li, Qing X.

    2015-01-01

    Highlights: • Stenotrophomonas sp. G1 was isolated from chlorpyrifos contaminated sludge. • Strain G1 is closest to Stenotrophomonas acidaminiphila. • Strain G1 can efficiently degrade 8 organophosphorus pesticides (OPs). • Intracellular methyl parathion hydrolase is responsible for the OP degradation. • Three factors were orthogonally optimized for degradation of methyl parathion. - Abstract: Organophosphorus insecticides have been widely used, which are highly poisonous and cause serious concerns over food safety and environmental pollution. A bacterial strain being capable of degrading O,O-dialkyl phosphorothioate and O,O-dialkyl phosphate insecticides, designated as G1, was isolated from sludge collected at the drain outlet of a chlorpyrifos manufacture plant. Physiological and biochemical characteristics and 16S rDNA gene sequence analysis suggested that strain G1 belongs to the genus Stenotrophomonas. At an initial concentration of 50 mg/L, strain G1 degraded 100% of methyl parathion, methyl paraoxon, diazinon, and phoxim, 95% of parathion, 63% of chlorpyrifos, 38% of profenofos, and 34% of triazophos in 24 h. Orthogonal experiments showed that the optimum conditions were an inoculum volume of 20% (v/v), a substrate concentration of 50 mg/L, and an incubation temperature in 40 °C. p-Nitrophenol was detected as the metabolite of methyl parathion, for which intracellular methyl parathion hydrolase was responsible. Strain G1 can efficiently degrade eight organophosphorus pesticides (OPs) and is a very excellent candidate for applications in OP pollution remediation

  19. Rapid biodegradation of organophosphorus pesticides by Stenotrophomonas sp. G1

    Energy Technology Data Exchange (ETDEWEB)

    Deng, Shuyan; Chen, Yao [Key Laboratory of Agri-food Safety of Anhui Province, Lab of Quality & Safety and Risk Assessment for Agro-products on Storage and Preservation (Hefei), Ministry of Agriculture, School of Resource and Environment, Anhui Agricultural University, Hefei 230036 (China); Wang, Daosheng [School of Life Science, Anhui Agricultural University, Hefei 230036 (China); Shi, Taozhong; Wu, Xiangwei; Ma, Xin; Li, Xiangqiong [Key Laboratory of Agri-food Safety of Anhui Province, Lab of Quality & Safety and Risk Assessment for Agro-products on Storage and Preservation (Hefei), Ministry of Agriculture, School of Resource and Environment, Anhui Agricultural University, Hefei 230036 (China); Hua, Rimao, E-mail: rimaohua@ahau.edu.cn [Key Laboratory of Agri-food Safety of Anhui Province, Lab of Quality & Safety and Risk Assessment for Agro-products on Storage and Preservation (Hefei), Ministry of Agriculture, School of Resource and Environment, Anhui Agricultural University, Hefei 230036 (China); Tang, Xinyun [School of Life Science, Anhui Agricultural University, Hefei 230036 (China); Li, Qing X. [Department of Molecular Biosciences and Bioengineering, University of Hawaii at Manoa, 1955 East–West Road, Honolulu, HI 957822 (United States)

    2015-10-30

    Highlights: • Stenotrophomonas sp. G1 was isolated from chlorpyrifos contaminated sludge. • Strain G1 is closest to Stenotrophomonas acidaminiphila. • Strain G1 can efficiently degrade 8 organophosphorus pesticides (OPs). • Intracellular methyl parathion hydrolase is responsible for the OP degradation. • Three factors were orthogonally optimized for degradation of methyl parathion. - Abstract: Organophosphorus insecticides have been widely used, which are highly poisonous and cause serious concerns over food safety and environmental pollution. A bacterial strain being capable of degrading O,O-dialkyl phosphorothioate and O,O-dialkyl phosphate insecticides, designated as G1, was isolated from sludge collected at the drain outlet of a chlorpyrifos manufacture plant. Physiological and biochemical characteristics and 16S rDNA gene sequence analysis suggested that strain G1 belongs to the genus Stenotrophomonas. At an initial concentration of 50 mg/L, strain G1 degraded 100% of methyl parathion, methyl paraoxon, diazinon, and phoxim, 95% of parathion, 63% of chlorpyrifos, 38% of profenofos, and 34% of triazophos in 24 h. Orthogonal experiments showed that the optimum conditions were an inoculum volume of 20% (v/v), a substrate concentration of 50 mg/L, and an incubation temperature in 40 °C. p-Nitrophenol was detected as the metabolite of methyl parathion, for which intracellular methyl parathion hydrolase was responsible. Strain G1 can efficiently degrade eight organophosphorus pesticides (OPs) and is a very excellent candidate for applications in OP pollution remediation.

  20. The organophosphorus extractants

    International Nuclear Information System (INIS)

    Zaoul, B.; Attou, M.; Azzouz, A.

    1989-07-01

    This work consists in a bibliographic review dealing with phosphorus and organophosphorus compounds chemistry and especially with the main extracting agents used in uranium ore treatment. In this context, a special interest is devoted to TBP, D 2 EHPA and TOPO. The content of this work is based on a large bibliography including cca. One hundred references related to many aspects concerning as well the nomenclature, the classification and the chemical structures of the organophosphorus compounds as synthesis methods, purification and analysis of the main extracting agents used in uranium extraction

  1. Glycoside hydrolases having multiple hydrolase activities

    Energy Technology Data Exchange (ETDEWEB)

    Chen, Zhiwei; Friedland, Gregory D.; Chhabra, Swapnil R.; Chivian, Dylan C.; Simmons, Blake A

    2017-08-08

    Glycoside hydrolases having at least two different hydrolytic activities are provided. In one embodiment, an isolated recombinant hydrolase having at least two activities selected from a group including asparagine derivatives, glutamine derivatives, and histidine derivatives is provided. Further, a method of generating free sugars from a mixture comprising asparagine derivatives, glutamine derivatives, and histidine derivatives is provided.

  2. Poisoning by organophosphorus insecticides

    OpenAIRE

    Martínez Parra, Pedro P.

    2014-01-01

    The agricultural and industrial development that is reaching our country has conditioned the emergence of numerous types of occupational diseases, among which stand out the poison in the work environment, and within poisoning organophosphorus insecticides. Substances acting on harmful insects transmit diseases to both the man and the vegetable kingdom. The recent and ever-increasing use of new insecticides, raises the need to know the physiological actions of these products so that their bene...

  3. Organophosphorus Insecticide Pharmacokinetics

    Energy Technology Data Exchange (ETDEWEB)

    Timchalk, Charles

    2010-01-01

    This chapter highlights a number of current and future applications of pharmacokinetics to assess organophosphate (OP) insecticide dosimetry, biological response and risk in humans exposed to these agents. Organophosphates represent a large family of pesticides where insecticidal as well as toxicological mode of action is associated with their ability to target and inhibit acetylcholinesterase (AChE). Pharmacokinetics entails the quantitative integration of physiological and metabolic processes associated with the absorption, distribution, metabolism and excretion (ADME) of drugs and xenobiotics. Pharmacokinetic studies provide important data on the amount of toxicant delivered to a target site as well as species-, age-, gender-specific and dose-dependent differences in biological response. These studies have been conducted with organophosphorus insecticides in multiple species, at various dose levels, and across different routes of exposure to understand their in vivo pharmacokinetics and how they contribute to the observed toxicological response. To access human exposure to organophosphorus insecticides, human pharmacokinetic studies have been conducted and used to develop biological monitoring strategies based on the quantitation of key metabolites in biological fluids. Pharmacokinetic studies with these insecticides are also useful to facilitate extrapolation of dosimetry and biological response from animals to humans and for the assessment of human health risk. In this regard, physiologically based pharmacokinetic and pharmacodynamic (PBPK/PD) models are being utilized to assess risk and understand the toxicological implications of known or suspected exposures to various insecticides. In this chapter a number of examples are presented that illustrate the utility and limitation of pharmacokinetic studies to address human health concerns associated with organophosphorus insecticides.

  4. Enzymatic synthesis of organophosphorus compounds

    International Nuclear Information System (INIS)

    Kolodiazhnyi, Oleg I

    2011-01-01

    Data on biocatalytic methods for the preparation of chiral organophosphorus compounds are generalized and described systematically. Various examples of enzymatic and microbiological synthesis of hydroxyphosphonates, aminophosphonates, phosphinites, phosphine oxides and tertiary phosphines are discussed. The bibliography includes 154 references.

  5. Enzymatic synthesis of organophosphorus compounds

    Science.gov (United States)

    Kolodiazhnyi, Oleg I.

    2011-09-01

    Data on biocatalytic methods for the preparation of chiral organophosphorus compounds are generalized and described systematically. Various examples of enzymatic and microbiological synthesis of hydroxyphosphonates, aminophosphonates, phosphinites, phosphine oxides and tertiary phosphines are discussed. The bibliography includes 154 references.

  6. Construction of genetically engineered bacteria that degrades organophosphorus pesticide residues and can be easily detected by the fluorescence.

    Science.gov (United States)

    Li, Qin; Wang, Pan; Chen, Rui; Li, Wei; Wu, Yi-Jun

    2014-01-01

    Organophosphorus compounds (OPs) are widely used in agriculture and industry and there is increased concern about their toxicological effects in the environment. Bioremediation can offer an efficient and cost-effective option for the removal of OPs. Herein, we describe the construction of a genetically engineered microorganism (GEM) that can degrade OPs and be directly detected and monitored in the environment using an enhanced green fluorescent protein (EGFP) fusion strategy. The coding regions of EGFP, a reporter protein that can fluoresce by itself, and organophosphorus hydrolase (OPH), which has a broad substrate specificity and is able to hydrolyse a number of organophosphorus pesticides, were cloned into the expression vector pET-28b. The fusion protein of EGFP-OPH was expressed in E. coli BL21 (DE3) and the protein expression reached the highest level at 11 h after isopropyl beta-D-thiogalactopyranoside induction. The fluorescence of the GEM was detected by fluorescence spectrophotometry and microscopy, and its ability to degrade OPs was determined by OPH activity assay. Those GEM that express the fusion protein (EGFP and OPH) exhibited strong fluorescence intensity and also potent hydrolase activity, which could be used to degrade organophosphorus pesticide residues in the environment and can also be directly monitored by fluorescence.

  7. Variants of glycoside hydrolases

    Energy Technology Data Exchange (ETDEWEB)

    Teter, Sarah; Ward, Connie; Cherry, Joel; Jones, Aubrey; Harris, Paul; Yi, Jung

    2017-07-11

    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.

  8. Variants of glycoside hydrolases

    Science.gov (United States)

    Teter, Sarah; Ward, Connie; Cherry, Joel; Jones, Aubrey; Harris, Paul; Yi, Jung

    2013-02-26

    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.

  9. Crystal Structures of Brain Group-VIII Phospholipase A2 in Non-aged Complexes with the Organophosphorus Nerve Agents Soman and Sarin†‡

    OpenAIRE

    Epstein, Todd M.; Samanta, Uttamkumar; Kirby, Stephen D.; Cerasoli, Douglas M.; Bahnson, Brian J.

    2009-01-01

    Insecticide and nerve agent organophosphorus compounds are potent inhibitors of the serine hydrolase superfamily of enzymes. Nerve agents, such as sarin, soman, tabun and VX exert their toxicity by inhibiting human acetycholinesterase at nerve synapses. Following the initial phosphonylation of the active site serine, the enzyme may reactivate spontaneously or through reaction with an appropriate nucleophilic oxime. Alternatively, the enzyme-nerve agent complex can undergo a secondary process,...

  10. Zebrafish Models for Human Acute Organophosphorus Poisoning.

    Science.gov (United States)

    Faria, Melissa; Garcia-Reyero, Natàlia; Padrós, Francesc; Babin, Patrick J; Sebastián, David; Cachot, Jérôme; Prats, Eva; Arick Ii, Mark; Rial, Eduardo; Knoll-Gellida, Anja; Mathieu, Guilaine; Le Bihanic, Florane; Escalon, B Lynn; Zorzano, Antonio; Soares, Amadeu M V M; Raldúa, Demetrio

    2015-10-22

    Terrorist use of organophosphorus-based nerve agents and toxic industrial chemicals against civilian populations constitutes a real threat, as demonstrated by the terrorist attacks in Japan in the 1990 s or, even more recently, in the Syrian civil war. Thus, development of more effective countermeasures against acute organophosphorus poisoning is urgently needed. Here, we have generated and validated zebrafish models for mild, moderate and severe acute organophosphorus poisoning by exposing zebrafish larvae to different concentrations of the prototypic organophosphorus compound chlorpyrifos-oxon. Our results show that zebrafish models mimic most of the pathophysiological mechanisms behind this toxidrome in humans, including acetylcholinesterase inhibition, N-methyl-D-aspartate receptor activation, and calcium dysregulation as well as inflammatory and immune responses. The suitability of the zebrafish larvae to in vivo high-throughput screenings of small molecule libraries makes these models a valuable tool for identifying new drugs for multifunctional drug therapy against acute organophosphorus poisoning.

  11. 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

  12. Glycemic Status in Organophosphorus Poisoning.

    Science.gov (United States)

    Panda, S; Nanda, R; Mangaraj, M; Rathod, P K; Mishra, P K

    2015-01-01

    Organophosphorus(OP) poisoning, in addition to its cholinergic manifestations shows metabolic derangements leading to hyperglycemia. Apart from inhibiting acetylcholinesterase it also induces oxidative stress to exhibit this manifestation. The present study aims to assess the glycemic status of OP poisoned patients and its association with various factors in OP poisoning like oxidative stress and dose of atropine. This is a prospective study which recruited 102 patients above 18 years of age with history of OP poisoning. They were categorized into 3 grades-mild, moderate and severe based on the Peradeniya Organophosphorus Poisining Scale. The routine biochemical parameters along with serum malondialdehyde (MDA) and cholinesterase were estimated in the study group. Hyperglycemia and glycosuria were observed, with majority cases of hyperglycemia (57%) noticed in the severe group. There was a rise in the random plasma glucose (RPG), serum malondialdehyde (MDA), total dose of atropine across the groups along with a fall in the serum cholinesterase with increase in severity of poisoning. The fall in plasma glucose at the time of discharge was significant in all three groups when compared to the admission random plasma glucose(RPG) level. This transient hyperglycemia exhibited a significant positive association with serum MDA and dose of atropine administered during treatment (p<0.05). Glycemic status in OP poisoning may play a role in identifying the severity of poisoning at the time of admission.

  13. Organophosphorus pesticide poisoning : cases and developments

    NARCIS (Netherlands)

    Aardema, H.; Ligtenberg, J. J. M.; Peters-Polman, O. M.; Tulleken, J. E.; Zijlstra, J. G.; Meertens, John H. J. M.

    Self-poisoning with organophosphate pesticides is a major health problem world-wide. Through the inhibition of acetylcholinesterase, organophosphorus poisoning is characterised by the clinical picture of acute cholinergic crisis. Other manifestations are the intermediate neurotoxic syndrome and

  14. Preparation of Electrochemical Biosensor for Detection of Organophosphorus Pesticides

    Science.gov (United States)

    Gothwal, Ashish; Beniwal, Puneet; Dhull, Vikas

    2014-01-01

    Polyvinyl chloride (PVC) can be used to develop reaction beaker which acts as electrochemical cell for the measurement of OP pesticides. Being chemically inert, corrosion resistant, and easy in molding to various shapes and size, PVC can be used for the immobilization of enzyme. Organophosphorus hydrolase was immobilized covalently onto the chemically activated inner surface of PVC beaker by using glutaraldehyde as a coupling agent. The carbon nanotubes paste working electrode was constructed for amperometric measurement at a potential of +0.8 V. The biosensor showed optimum response at pH 8.0 with incubation temperature of 40°C. K m and I max for substrate (methyl parathion) were 322.58 µM and 1.1 µA, respectively. Evaluation study showed a correlation of 0.985, which was in agreement with the standard method. The OPH biosensor lost 50% of its initial activity after its regular use for 25 times over a period of 50 days when stored in 0.1 M sodium phosphate buffer, pH 8.0 at 4°C. No interference was observed by interfering species. PMID:25667593

  15. Carborane-containing organophosphorus compounds. Synthesis and properties

    Science.gov (United States)

    Godovikov, Nikolai N.; Balema, Viktor P.; Rys, Evgenii G.

    1997-12-01

    Published data on the synthesis, chemical properties and practical use of carborane-containing organophosphorus compounds are surveyed. Their properties are compared with those of similar organophosphorus derivatives devoid of carboranyl groups. The bibliography includes 89 references.

  16. of Several Organophosphorus Insecticide Metabolites

    Directory of Open Access Journals (Sweden)

    Russell L. Carr

    2015-01-01

    Full Text Available Paraoxonase (PON1 is a calcium dependent enzyme that is capable of hydrolyzing organophosphate anticholinesterases. PON1 activity is present in most mammals and previous research established that PON1 activity differs depending on the species. These studies mainly used the organophosphate substrate paraoxon, the active metabolite of the insecticide parathion. Using serum PON1 from different mammalian species, we compared the hydrolysis of paraoxon with the hydrolysis of the active metabolites (oxons of two additional organophosphorus insecticides, methyl parathion and chlorpyrifos. Paraoxon hydrolysis was greater than that of methyl paraoxon, but the level of activity between species displayed a similar pattern. Regardless of the species tested, the hydrolysis of chlorpyrifos-oxon was significantly greater than that of paraoxon or methyl paraoxon. These data indicate that chlorpyrifos-oxon is a better substrate for PON1 regardless of the species. The pattern of species differences in PON1 activity varied with the change in substrate to chlorpyrifos-oxon from paraoxon or methyl paraoxon. For example, the sex difference observed here and reported elsewhere in the literature for rat PON1 hydrolysis of paraoxon was not present when chlorpyrifos-oxon was the substrate.

  17. Organophosphorus ester-induced chronic neurotoxicity.

    Science.gov (United States)

    Abou-Donia, Mohamed B

    2003-08-01

    Organophosphorus compounds are potent neurotoxic chemicals that are widely used in medicine, industry, and agriculture. The neurotoxicity of these chemicals has been documented in accidental human poisoning, epidemiological studies, and animal models. Organophosphorus compounds have 3 distinct neurotoxic actions. The primary action is the irreversible inhibition of acetylcholinesterase, resulting in the accumulation of acetylcholine and subsequent overstimulation of the nicotinic and muscarinic acetylcholine receptors, resulting in cholinergic effects. Another action of some of these compounds, arising from single or repeated exposure, is a delayed onset of ataxia, accompanied by a Wallerian-type degeneration of the axon and myelin in the most distal portion of the longest tracts in both the central and peripheral nervous systems, and is known as organophosphorus ester-induced delayed neurotoxicity (OPIDN). In addition, since the introduction and extensive use of synthetic organophosphorus compounds in agriculture and industry half a century ago, many studies have reported long-term, persistent, chronic neurotoxicity symptoms in individuals as a result of acute exposure to high doses that cause acute cholinergic toxicity, or from long-term, low-level, subclinical doses of these chemicals. The author attempts to define the neuronal disorder that results from organophosphorus ester-induced chronic neurotoxicity (OPICN), which leads to long-term neurological and neurobehavioral deficits. Although the mechanisms of this neurodegenerative disorder have yet to be established, the sparse available data suggest that large toxic doses of organophosphorus compounds cause acute necrotic neuronal cell death in the brain, whereas sublethal or subclinical doses produce apoptotic neuronal cell death and involve oxidative stress.

  18. Organophosphorus and carbamate insecticide poisoning.

    Science.gov (United States)

    Vale, Allister; Lotti, Marcello

    2015-01-01

    Both organophosphorus (OP) and carbamate insecticides inhibit acetylcholinesterase (AChE), which results in accumulation of acetylcholine (ACh) at autonomic and some central synapses and at autonomic postganglionic and neuromuscular junctions. As a consequence, ACh binds to, and stimulates, muscarinic and nicotinic receptors, thereby producing characteristic features. With OP insecticides (but not carbamates), "aging" may also occur by partial dealkylation of the serine group at the active site of AChE; recovery of AChE activity requires synthesis of new enzyme in the liver. Relapse after apparent resolution of cholinergic symptoms has been reported with OP insecticides and is termed the intermediate syndrome. This involves the onset of muscle paralysis affecting particularly upper-limb muscles, neck flexors, and cranial nerves some 24-96 hours after OP exposure and is often associated with the development of respiratory failure. OP-induced delayed neuropathy results from phosphorylation and subsequent aging of at least 70% of neuropathy target esterase. Cramping muscle pain in the lower limbs, distal numbness, and paresthesiae are followed by progressive weakness, depression of deep tendon reflexes in the lower limbs and, in severe cases, in the upper limbs. The therapeutic combination of oxime, atropine, and diazepam is well established experimentally in the treatment of OP pesticide poisoning. However, there has been controversy as to whether oximes improve morbidity and mortality in human poisoning. The explanation may be that the solvents in many formulations are primarily responsible for the high morbidity and mortality; oximes would not be expected to reduce toxicity in these circumstances. even if given in appropriate dose. © 2015 Elsevier B.V. All rights reserved.

  19. A retrospective analysis of acute organophosphorus poisoning ...

    African Journals Online (AJOL)

    A retrospective analysis of acute organophosphorus poisoning cases admitted to the tertiary care teaching hospital in South India. ... Young adult males were more commonly involved than females (M:F 2.5:1). The mean age of the patients was 28 years (range 2-72 years, SD ± 14.3 years). Mean time to receive treatment ...

  20. Determination of selected organophosphorus pesticides using GC ...

    African Journals Online (AJOL)

    A reproducible analytical method utilising GC-MS is presented for the analysis of thirteen organophosphorus insecticides (OPs) often found in environmental aqueous samples. Extraction of filtered river and canal water (100 mL), fortified with the OPs, was conducted using solid-phase extraction and eluted with a variety of ...

  1. Efficacious Oxime for Organophosphorus Poisoning: A Minireview ...

    African Journals Online (AJOL)

    Oximes are well known as acetylcholinesterase reactivators and are used in organophosphorus poisoning to reactivate inhibited acetylcholinesterase. Therapeutically available oximes, namely, pralidoxime (2-PAM), obidoxime, trimedoxime and Hagedorn oxime (HI-6), have no broad-spectrum activity against structurally ...

  2. Biochemical Markers for Exposure to Low Doses of Organophosphorus Insecticides

    National Research Council Canada - National Science Library

    Lockridge, Oksana

    2003-01-01

    Though acetylcholinesterase is the primary target of organophosphorus toxicants, our finding that acetylcholinesterase knockout mice are supersensitive to the lethal effects of VX, DFP, chlorpyrifos...

  3. Biochemical Markers for Exposure to Low Doses of Organophosphorus Insecticides

    National Research Council Canada - National Science Library

    Lockridge, Oksana

    2004-01-01

    Though acetylcholinesterase is the primary target of organophosphorus toxicants, our finding that acetylcholinesterase knockout mice are supersensitive to the lethal effects of VX, DFP, chlorpyrifos...

  4. Biochemical Markers for Exposure to Low Doses of Organophosphorus Insecticides

    National Research Council Canada - National Science Library

    Lockridge, Oksana

    2002-01-01

    Though acetyicholinesterase is the primary target of organophosphorus toxicants, our finding that acetyicholinesterase knockout mice are supersensitive to the lethal effects of VX, DFP, chlorpyrifos...

  5. Evaluation of organophosphorus pesticide biodegradation by halophilic bacteria

    Directory of Open Access Journals (Sweden)

    Shokufeh Rafieyan

    2018-03-01

    Discussion and conclusion: Halophilic bacteria due to compatibility with the salty condition, can be a good option for the removal of organophosphorus pesticides in the contaminated salty environments.

  6. Development of a versatile organophosphorous-hydrolase-based assay for organophosphate pesticides

    Science.gov (United States)

    Rogers, Kim R.; Wang, Yi; Mulchandani, Ashok; Mulchandani, P.; Chen, Wilfred

    1999-02-01

    We report a rapid and versatile organophosphorus hydrolase (OPH)-based method for measurement of organophosphate pesticides. This assay is based on a substrate-dependant change in pH near the active site of the enzyme. The pH change is monitored using fluorescein isothiocyanate (FITC) which is covalently immobilized to the enzyme. This method employs FITC-labeled enzyme adsorbed to polymethylmethacrylate beads. Analytes were measured using a microbead fluorescence analyzer. The dynamic concentration range for the assay extends from 25 (mu) M to 400 (mu) M for paraoxon with a detection limit of 8 (mu) M. This assay compared favorably to an HPLC method for monitoring the concentration of coumaphos in bioremediation filtrate samples.

  7. [Today's threat of use of organophosphorus compounds].

    Science.gov (United States)

    Sokołowski, Rafał; Płusa, Tadeusz

    2015-09-01

    Organophosphates are stable cholinesterases inhibitors (AChE). Inhibition of AChE activity leads to the accumulation of large amounts of acetylcholine and hyperactivity of the cholinergic system by stimulating acetylcholine receptors - muscarinic and nicotinic. This group included tabun, sarin, soman and VX gases. Exposure to gaseous form causes symptoms within a few seconds of exposure. This depends on the gas concentration in the atmosphere. The most sensitive organ is the eyes and the respiratory system. Severe poisoning are characterized by the immediate loss of consciousness with convulsions. Therapeutic management of acute poisoning organophosphorus compounds boils down to treating symptomatic and supportive vital functions. Monitoring of cardiovascular, respiratory and renal failure in intensive care gives only guarantee the effective treatment of poisoning. Properties toxic organophosphorus compounds also are of interest to terrorist groups. © 2015 MEDPRESS.

  8. Towards a Capacitive Enzyme Sensor for Direct Determination of Organophosphorus Pesticides: Fundamental Studies and Aspects of Development

    Directory of Open Access Journals (Sweden)

    Ashok Mulchandani

    2003-06-01

    Full Text Available The realisation of a miniaturised potentiometric enzyme biosensor is presented. The biosensor chip utilises the enzyme organophosphorus hydrolase (OPH for the direct determination of pesticides. The transducer structure of the sensors chip consists of a pH-sensitive capacitive electrolyte-insulator-semiconductor (EIS structure that reacts towards pH changes caused by the OPH-catalised hydrolysis of the organophosphate compounds. The biosensor is operated versus a conventional Ag/AgCl reference electrode. Measurements were performed in the capacitance/voltage (C/V and the constant capacitance (ConCap mode for the two different pesticides paraoxon and parathion. For the development of this new type of biosensor, different immobilisation strategies, influence of buffer composition and concentration, transducer material, detection limit, long-term stability and selectivity have been studied.

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

    Directory of Open Access Journals (Sweden)

    Patricia Marie Legler

    2014-07-01

    Full Text Available 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.

  10. Chlorination of organophosphorus pesticides in natural waters

    Energy Technology Data Exchange (ETDEWEB)

    Acero, Juan L. [Departamento de Ingenieria Quimica y Energetica, Universidad de Extremadura, 06071 Badajoz (Spain)], E-mail: jlacero@unex.es; Benitez, F. Javier; Real, Francisco J.; Gonzalez, Manuel [Departamento de Ingenieria Quimica y Energetica, Universidad de Extremadura, 06071 Badajoz (Spain)

    2008-05-01

    Unknown second-order rate constants for the reactions of three organophosphorus pesticides (chlorpyrifos, chlorfenvinfos and diazinon) with chlorine were determined in the present study, and the influence of pH and temperature was established. It was found that an increase in the pH provides a negative effect on the pesticides degradation rates. Apparent second-order rate constants at 20 {sup o}C and pH 7 were determined to be 110.9, 0.004 and 191.6 M{sup -1} s{sup -1} for chlorpyrifos, chlorfenvinfos and diazinon, respectively. A higher reactivity of chlorine with the phosphorothioate group (chlorpyrifos and diazinon) than with the phosphate moiety (chlorfenvinfos) could explain these results. Intrinsic rate constant for the elementary reactions of chlorine species with chlorpyrifos and diazinon were also calculated, leading to the conclusion that the reaction between hypochlorous acid and the pesticide is predominant at neutral pH. The elimination of these pesticides in surface waters was also investigated. A chlorine dose of 2.5 mg L{sup -1} was enough to oxidize chlorpyrifos and diazinon almost completely, with a formation of trihalomethanes below the EU standard for drinking water. However, the removal of chlorfenvinfos was not appreciable. Therefore, chlorination is a feasible option for the removal of organophosphorus pesticides with phosphorothioate group during oxidation and disinfection processes, but not for the elimination of pesticides with phosphate moiety.

  11. QSAR for cholinesterase inhibition by organophosphorus esters and CNDO/2 calculations for organophosphorus ester hydrolysis

    Science.gov (United States)

    Johnson, H.; Kenley, R. A.; Rynard, C.; Golub, M. A.

    1985-01-01

    Quantitative structure-activity relationships were derived for acetyl- and butyrylcholinesterase inhibition by various organophosphorus esters. Bimolecular inhibition rate constants correlate well with hydrophobic substituent constants, and with the presence or absence of catonic groups on the inhibitor, but not with steric substituent constants. CNDO/2 calculations were performed on a separate set of organophosphorus esters, RR'P(O)X, where R and R' are alkyl and/or alkoxy groups and X is fluorine, chlorine or a phenoxy group. For each subset with the same X, the CNDO-derived net atomic charge at the central phosphorus atom in the ester correlates well with the alkaline hydrolysis rate constant. For the whole set of esters with different X, two equations were derived that relate either charge and leaving group steric bulk, or orbital energy and bond order to the hydrogen hydrolysis rate constant.

  12. Downregulation of transferrin receptor surface expression by intracellular antibody

    International Nuclear Information System (INIS)

    Peng Jilin; Wu Sha; Zhao Xiaoping; Wang Min; Li Wenhan; Shen Xin; Liu Jing; Lei Ping; Zhu Huifen; Shen Guanxin

    2007-01-01

    To deplete cellular iron uptake, and consequently inhibit the proliferation of tumor cells, we attempt to block surface expression of transferrin receptor (TfR) by intracellular antibody technology. We constructed two expression plasmids (scFv-HAK and scFv-HA) coding for intracellular single-chain antibody against TfR with or without endoplasmic reticulum (ER) retention signal, respectively. Then they were transfected tumor cells MCF-7 by liposome. Applying RT-PCR, Western blotting, immunofluorescence microscopy and immunoelectron microscope experiments, we insure that scFv-HAK intrabody was successfully expressed and retained in ER contrasted to the secreted expression of scFv-HA. Flow cytometric analysis confirmed that the TfR surface expression was markedly decreased approximately 83.4 ± 2.5% in scFv-HAK transfected cells, while there was not significantly decrease in scFv-HA transfected cells. Further cell growth and apoptosis characteristics were evaluated by cell cycle analysis, nuclei staining and MTT assay. Results indicated that expression of scFv-HAK can dramatically induce cell cycle G1 phase arrest and apoptosis of tumor cells, and consequently significantly suppress proliferation of tumor cells compared with other control groups. For First time this study demonstrates the potential usage of anti-TfR scFv-intrabody as a growth inhibitor of TfR overexpressing tumors

  13. Neurotoxic disorders of organophosphorus compounds and their managements.

    Science.gov (United States)

    Balali-Mood, Mahdi; Balali-Mood, Kia

    2008-01-01

    Organophosphorus compounds have been used as pesticides and as chemical warfare nerve agents. The mechanism of toxicity of organophosphorus compounds is the inhibition of acetylcholinesterase, which results in accumulation of acetylcholine and the continued stimulation of acetylcholine receptors. Therefore, they are also called anticholinesterase agents. Organophosphorus pesticides have largely been used worldwide, and poisoning by these agents, particularly in developing countries, is a serious health problem. Organophosphorus nerve agents were used by Iraqi army against Iranian combatants and even civilian population in 1983 - 1988. They were also used for chemical terrorism in Japan in 1994 - 1995. Their use is still a constant threat to the population. Therefore, medical and health professionals should be aware and learn more about the toxicology and proper management of organophosphorus poisoning. Determination of acetylcholinesterase and butyrylcholinesterase activity in blood remains a mainstay for the fast initial screening of organophosphorus compounds but lacks sensitivity and specificity. Quantitative analysis of organophosphorus compounds and their degradation products in plasma and urine by mass spectrometric methods may prove exposure but is expensive and is limited to specialized laboratories. However, history of exposure to organophosphorous compounds and clinical manifestations of a cholinergic syndrome are sufficient for management of the affected patients. The standard management of poisoning with organophosphorous compounds consists of decontamination, and injection of atropine sulfate with an oxime. Recent advances on treatment of organophosphorus pesticides poisoning revealed that blood alkalinization with sodium bicarbonate and also magnesium sulfate as adjunctive therapies are promising. Patients who receive prompt proper treatment usually recover from acute toxicity but may suffer from neurologic complications.

  14. Competitive reactions of organophosphorus radicals on coke surfaces.

    Science.gov (United States)

    Catak, Saron; Hemelsoet, Karen; Hermosilla, Laura; Waroquier, Michel; Van Speybroeck, Veronique

    2011-10-17

    The efficacy of organophosphorus radicals as anticoking agents was subjected to a computational study in which a representative set of radicals derived from industrially relevant organophosphorus additives was used to explore competitive reaction pathways on the graphene-like coke surface formed during thermal cracking. The aim was to investigate the nature of the competing reactions of different organophosphorus radicals on coke surfaces, and elucidate their mode of attack and inhibiting effect on the forming coke layer by use of contemporary computational methods. Density functional calculations on benzene and a larger polyaromatic hydrocarbon, namely, ovalene, showed that organophosphorus radicals have a high propensity to add to the periphery of the coke surface, inhibiting methyl radical induced hydrogen abstraction, which is known to be a key step in coke growth. Low addition barriers reported for a phosphatidyl radical suggest competitive aptitude against coke formation. Moreover, organophosphorus additives bearing aromatic substituents, which were shown to interact with the coke surface through dispersive π-π stacking interactions, are suggested to play a nontrivial role in hindering further stacking among coke surfaces. This may be the underlying rationale behind experimental observation of softer coke in the presence of organophosphorus radicals. The ultimate goal is to provide information that will be useful in building single-event microkinetic models. This study presents pertinent information on potential reactions that could be taken up in these models. Copyright © 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  15. [Decontamination of organophosphorus compounds: Towards new alternatives].

    Science.gov (United States)

    Poirier, L; Jacquet, P; Elias, M; Daudé, D; Chabrière, E

    2017-05-01

    Organophosphorus coumpounds (OP) are toxic chemicals mainly used for agricultural purpose such as insecticides and were also developed and used as warfare nerve agents. OP are inhibitors of acetylcholinesterase, a key enzyme involved in the regulation of the central nervous system. Chemical, physical and biological approaches have been considered to decontaminate OP. This review summarizes the current and emerging strategies that are investigated to tackle this issue with a special emphasis on enzymatic remediation methods. During the last decade, many studies have been dedicated to the development of biocatalysts for OP removal. Among these, recent reports have pointed out the promising enzyme SsoPox isolated from the archaea Sulfolobus solfataricus. Considering both its intrinsic stability and activity, this hyperthermostable enzyme is highly appealing for the decontamination of OP. Copyright © 2017 Académie Nationale de Pharmacie. All rights reserved.

  16. Are Titan's radial Labyrinth terrains surface expressions of large laccoliths?

    Science.gov (United States)

    Schurmeier, L.; Dombard, A. J.; Malaska, M.; Radebaugh, J.

    2017-12-01

    The Labyrinth terrain unit may be the one of the best examples of the surface expression of Titan's complicated history. They are characterized as highly eroded, dissected, and elevated plateaus and remnant ridges, with an assumed composition that is likely organic-rich based on radar emissivity. How these features accumulated organic-rich sediments and formed topographic highs by either locally uplifting or surviving pervasive regional deflation or erosion is an important question for understanding the history of Titan. There are several subsets of Labyrinth terrains, presumably with differing evolutionary histories and formation processes. We aim to explain the formation of a subset of Labyrinth terrain units informally referred to as "radial Labyrinth terrains." They are elevated and appear dome-like, circular in planform, have a strong radial dissection pattern, are bordered by Undifferentiated Plains units, and are found in the mid-latitudes. Based on their shape, clustering, and dimensions, we suggest that they may be the surface expression of large subsurface laccoliths. A recent study by Manga and Michaut (Icarus, 2017) explained Europa's lenticulae (pits, domes, spots) with the formation of saucer-shaped sills that form laccoliths around the brittle-ductile transition depth within the ice shell (1-5 km). Here, we apply the same scaling relationships and find that the larger size of radial labyrinth terrains with Titan's higher gravity implies deeper intrusion depths of around 20-40 km. This intrusion depth matches the expected brittle-ductile transition on Titan based on our finite element simulations and yield strength envelope analyses. We hypothesize that Titan's radial labyrinth terrains formed as cryovolcanic (water) intrusions that rose to the brittle-ductile transition within the ice shell where they spread horizontally, and uplifted the overlying ice. The organic-rich sedimentary cover also uplifted, becoming more susceptible to pluvial and fluvial

  17. THE ALPHA/BETA-HYDROLASE FOLD

    NARCIS (Netherlands)

    OLLIS, DL; CHEAH, E; CYGLER, M; FROLOW, F; FRANKEN, SM; HAREL, M; REMINGTON, SJ; SILMAN, [No Value; SCHRAG, J; SUSSMAN, JL; VERSCHUEREN, KHG; GOLDMAN, A

    We have identified a new protein fold-the alpha/beta-hydrolase fold-that is common to several hydrolytic enzymes of widely differing phylogenetic origin and catalytic function. The core of each enzyme is similar: an alpha/beta-sheet, not barrel, of eight beta-sheets connected by alpha-helices. These

  18. Sorption of Organophosphorus Flame Retardants (OPFRs) on ...

    Science.gov (United States)

    Organophosphorus flame retardants (OPFRs) are widely used as additives in industrial and consumer products such as electrical and electronic products, furniture, plastics, textiles, and building/construction materials. Due to human exposure and potential health effects, OPFRs including tris(2-chloroethyl) phosphate (TCEP), tris(1-chloro-2-propyl) phosphate (TCPP), and tris(1,3-dichloro-2-propyl) phosphate (TDCPP) are EPA Action Plan chemicals for chemical assessments under the Toxic Substances Control Act (TSCA). This work investigated the sorption of these three compounds from the air to settled Arizona Test Dust (ATD) and house dust (HD) in a dual small chamber system. The OPFR exposed dust was analyzed to determine the sorption concentration and sorption rate of OPFRs on the dust. The effect of the composition of the dust on OPFR sorption was evaluated. The results showed that ATD and HD have varied sorption capacity for OPFRs from air. This work explores the relationship between OPFR concentrations in settled dust and air. The data can be used to determine partitioning of OPFRs between the gas phase and settled dust indoors and to inform strategies to reduce exposure and risk.

  19. Critical evaluation of some equilibrium constants involving organophosphorus extractants

    CERN Document Server

    Marcus, Y

    2013-01-01

    Critical Evaluation of Some Equilibrium Constants Involving Organophosphorus Extractants is a supplementary text to the compilation ""Equilibrium Constants of Liquid-Liquid Distribution Reactions (Introduction, and Part I: Organophosphorus Extractants).The book contains a number of well documented chemical reactions that are critically evaluated. The reactions evaluated comprise those from List 1 for which data are available. There are, however, a great variety of reactions that cannot be critically evaluated due to lack of experimental data and unverifiable results.Chemists will find this com

  20. Biodegradation of organophosphorus pesticides by soil bacteria

    Science.gov (United States)

    de Pasquale, C.; Fodale, R.; Lo Piccolo, L.; Palazzolo, E.; Alonzo, G.; Quatrini, P.

    2009-04-01

    A number of studies in the 1980s and 1990s showed that crop-protection products, applied to drained fields, could move downwards through the soil profile and to the groundwater. Organophosphorus insecticides (OPs) are used all over the world for crop protection, for other agricultural practices such as sheep dipping and, in aquaculture, for the control of sea lice. Ops besides showing a specific neurotoxicity and have also been related to various modern diseases, including Creutzfeldt-Jakob (CJD) and the Gulf War syndrome. Although OPs are less persistent than Organoclorine pesticides (OCs), they still constitute an environmental risks thus increasing the social concern about their levels in soils, surface waters, and ground waters. Degradation of OPs by microorganisms has been assessed for a few bacterial strains. In the present study the OPs degrading potential of indigenous soil microorganisms was investigated. Using enrichment cultures in which parathion was the only C and energy sources many bacterial strains were isolated from OPs contaminated and pristine agricultural soils characterized by different physico-chemical properties. More than 40 potential OPs degraders were isolated and grouped in operational taxonomic units (OTU) using analysis of polymorphism showed by the ribosomal internal transcribed spacer (ITS). Partial sequencing of 16S rRNA gene of representative isolates of each OTU revealed that most of them belong to Proteobacteria and Actinobacteria. All the analyzed soils showed the presence of putative OPs degraders: the highest diversity was found in organic cultivated soils, the lowest in chemically cultivated soils. Degradation of different OPs, characterized by different physical and chemical properties, was obtained by different selected representative strains using SPME GC-MS analysis on water and soil microcosms. The results showed that, after the incubation period, the amount of pesticide residues were in the range 20-80%. Some of the

  1. The European Continent : Surface Expression of Upper Mantle Dynamics

    Science.gov (United States)

    Tondi, M. R.; Schivardi, R.; Molinari, I.; Morelli, A.

    2012-12-01

    images of the European upper mantle isotropic shear-wave speeds and mass densities, recently recovered by combined inversion of surface-wave information and GRACE satellite gravity data (Tondi et al., 2012) are used to select the regions where the residual topography and the residual mantle gravity anomalies are strongly correlated (correlation coefficient is equal to 1). We assume surface uplift processes with negative density anomalies and downward pull with positive anomalies. Our work shows a strong correlation among the areas where, on the basis of our assumptions, the mantle dynamics have surface expression and the areas of low values of radial anisotropy: (1) the southern margins of the East European Craton, (2) the North-Eastern edges of the Arabian Plateau, (3) the northern edge of the CEVP (Central European Volcanic Province), (4) the North-Eastern part of the Atlantic Ocean, between Greenland and Iceland.

  2. FLOW INJECTION AMPEROMETRIC DETECTION OF OP NERVE AGENTS BASED ON AN ORGANOPHOSPHORUS-HYDROLASE BIOSENSOR DETECTOR. (R828160)

    Science.gov (United States)

    The perspectives, information and conclusions conveyed in research project abstracts, progress reports, final reports, journal abstracts and journal publications convey the viewpoints of the principal investigator and may not represent the views and policies of ORD and EPA. Concl...

  3. A 1000-L Scale-Up Fermentation of Escherichia Coli Containing PVSEOP7 for Production of Organophosphorus Hydrolase

    National Research Council Canada - National Science Library

    Lukens, Dennis C; Rastogi, Vipin K; Cheng, Tu-c; DeFrank, Joseph J; Valdes, James J; Kim, Michael H

    2003-01-01

    .... coli XL1 containing pVSEOP7. The specific OPH activity was monitored during each 20-L batch fermentation culture by assaying several times between the IPTG induction point (OD of 0.4 to 0.5 at 600 nm...

  4. Production of Self-Purifying Proteins in a Variety of Expression Hosts with Focus on Organophosphorus Hydrolase

    Science.gov (United States)

    2012-08-17

    beta - lactamase , OPH, a second CA and an antimicrobial peptide), and we have generated non-cleaving control fusions of different sized ELP tags with...CA) and beta -Galactosidase (Bgal). The most critical results for CA are summarized in Figure 8. In particular, we observed that the overall yield

  5. Solvolysis of organophosphorus pesticide parathion with simple and ...

    Indian Academy of Sciences (India)

    CHANDAN SAHU

    P 2007 Comparison of photocatalysis and photolysis of malathion, isomalathion, malaoxon, and commercial malathion–products and toxicity studies Water Res. 41. 4504. 8. Bravcon Kralj M, Franko M and Trebse P 2007. Photodegradation of organophosphorus insecticides –. Investigations of products and their toxicity ...

  6. Acute organophosphorus compound poisoning in cattle: A case report

    African Journals Online (AJOL)

    A case of an acute organophosphorus compound, GOLDFLEECE poisoning involving 39 cattle at Abubakar Tafawa Balewa University, Bauchi is reported. The animals were overexposed to the compound following routine tick spray. Within 15 minutes after the spray of the compound, 26 animals were recumbent showing ...

  7. Acute organo-phosphorus pesticide poisoning in North Karnataka ...

    African Journals Online (AJOL)

    Background: Pesticide poisoning is an important cause of morbidity and mortality in India. Objectives: To assess the oxidative damage, hemoglobin level and leukocyte count in acute organophosphorus pesticide poisoning. Methods: Plasma cholinesterase was assessed as a toxicity marker. Oxidative damage was ...

  8. Study of Serum Amylase and Serum Cholinesterase in Organophosphorus Poisoning

    Directory of Open Access Journals (Sweden)

    Sharan Badiger

    2016-04-01

    Full Text Available Background: Poisoning due to organophosphorus compounds is most commonly seen. Earlier plasma cholinesterase level was used to assess the severity of poisoning. Presently serum amylase is being recommended as a better indicator of severity. Aims and Objectives: To study plasma cholinesterase and serum amylase levels in acute organophosphorus and to correlate serum amylase levels with clinical severity and outcome. Material and Methods: A total of 80 patients in the study admitted to a tertiary care centre within 24 hours with a history of organophosphorus poisoning were included in study. Estimation of plasma cholinesterase and serum rd amylase was done at the time of admission, and on 3 th day and on 5 day. Results: Occurrence of organophosphorus poisoning was more common among age group 21-30 years and among males (57.5%. They were 25 (31.2% farmers, 23 (28.8% st u d e n ts, a n d 2 2 ( 2 7 . 5% h o u s ewi v e s. Monocrotophos (45.0% was commonly used compound. Mean value of plasma cholinesterase and serum amylase at admission are 3693 U/L, and 185.4 U/L. There was significant inhibition of plasma cholinesterase and elevation of serum amylase at th admission with return to normal values on 5 day. Conclusion: Plasma cholinesterase inhibition 200 U/L has been associated with poor prognosis and proneness to respiratory failure.

  9. Crystal structures of human group-VIIA phospholipase A2 inhibited by organophosphorus nerve agents exhibit non-aged complexes.

    Science.gov (United States)

    Samanta, Uttamkumar; Kirby, Stephen D; Srinivasan, Prabhavathi; Cerasoli, Douglas M; Bahnson, Brian J

    2009-08-15

    The enzyme group-VIIA phospholipase A2 (gVIIA-PLA2) is bound to lipoproteins in human blood and hydrolyzes the ester bond at the sn-2 position of phospholipid substrates with a short sn-2 chain. The enzyme belongs to a serine hydrolase superfamily of enzymes, which react with organophosphorus (OP) nerve agents. OPs ultimately exert their toxicity by inhibiting human acetycholinesterase at nerve synapses, but may additionally have detrimental effects through inhibition of other serine hydrolases. We have solved the crystal structures of gVIIA-PLA2 following inhibition with the OPs diisopropylfluorophosphate, sarin, soman and tabun. The sarin and soman complexes displayed a racemic mix of P(R) and P(S) stereoisomers at the P-chiral center. The tabun complex displayed only the P(R) stereoisomer in the crystal. In all cases, the crystal structures contained intact OP adducts that had not aged. Aging refers to a secondary process OP complexes can go through, which dealkylates the nerve agent adduct and results in a form that is highly resistant to either spontaneous or oxime-mediated reactivation. Non-aged OP complexes of the enzyme were corroborated by trypsin digest and matrix-assisted laser desorption ionization mass spectrometry of OP-enzyme complexes. The lack of stereoselectivity of sarin reaction was confirmed by gas chromatography/mass spectrometry using a chiral column to separate and quantitate the unbound stereoisomers of sarin following incubation with enzyme. The structural details and characterization of nascent reactivity of several toxic nerve agents is discussed with a long-term goal of developing gVIIA-PLA2 as a catalytic bioscavenger of OP nerve agents.

  10. Cell-surface expression of Hsp70 on hematopoietic cancer cells after inhibition of HDAC activity

    DEFF Research Database (Denmark)

    Jensen, Helle; Andresen, Lars; Hansen, Karen Aagaard

    or normal ER/Golgi transport did not affect Hsp70 surface expression. Intracellular Calcium and the transcription factor Sp1, that has previously been shown to be important for the intracellular stress mediated by HDAC-inhibitors, were not involved in Hsp70 surface expression. We also found that HDAC...

  11. Cell-surface expression of Hsp70 on hematopoietic cancer cells after inhibition of HDAC activity

    DEFF Research Database (Denmark)

    Jensen, Helle; Andresen, Lars; Hansen, Karen Aagaard

    clinically applied reagents, such as alkyl-lysophospholipides, chemotherapeutic agents, and anti-inflammatory reagents, have been found to enhance Hsp70 surface expression on cancer cells. We have found that inhibition of histone deacetylase (HDAC) activity leads to surface expression of Hsp70 on various...

  12. Vesicular Stomatitis Virus Infection Promotes Immune Evasion by Preventing NKG2D-Ligand Surface Expression

    DEFF Research Database (Denmark)

    Jensen, Helle; Andresen, Lars; Nielsen, Jens

    2011-01-01

    leads to a robust induction of MICA mRNA expression, however the subsequent surface expression is potently hindered. Thus, VSV lines up with human cytomegalovirus (HCMV) and adenovirus, which actively subvert the immune system by negatively affecting NKG2D-ligand surface expression. VSV infection caused...

  13. Photodegradation of organophosphorus pesticides in honey medium.

    Science.gov (United States)

    Yuan, Zhimin; Yao, Jun; Liu, Haijun; Han, Jun; Trebše, Polonca

    2014-10-01

    Honey can be polluted due to environmental pollution and misuse of beekeeping practices. In the present study, photodegradation experiments of organophosphorus pesticides (coumaphos, methyl parathion and fenitrothion) in honey medium were conducted using Atlas Suntest simulator CPS+ as a sunlight producer. Photodegradation experiments were conducted under three different intensities as 250W/m(2), 500W/m(2) and 750W/m(2) to evaluate the impact of sunlight intensity on removal of OPs in honey medium. Significant decreases of three OPs' concentrations were observed. Coumaphos showed the highest degradability, reaching a degradation percentage of 90 percent within 15min. After 1h irradiation, residual percentages of coumaphos were 6.62 percent for 250W/m(2), 3.48 percent for 500W/m(2) and 2.98 percent for 750W/m(2), respectively. Methyl parathion and fenitrothion also could be removed through photodegradation efficiently. After 1h irradiation, the residual percentages of methyl parathion and fenitrothion under 750W/m(2) sunlight irradiation were 26.89 percent and 16.70 percent, respectively. Intensity of sunlight showed a positive impact on removal of OPs in honey medium. The higher intensity, the lower residual percentage. Photodegradation of three OPs fitted well with pseudo-first order kinetics. Half-lives calculated from pseudo-first order kinetics were 17.61min (250W/m(2)), 16.67min (500W/m(2)) and 17.58min (750W/m(2)) for coumaphos, 57.62min (250W/m(2)), 34.13min (500W/m(2)) and 31.69min (750W/m(2)) for methyl parathion and 144.70min (250W/m(2)), 95.47min (500W/m(2)) and 22.57min (750W/m(2)) for fenitrothion, respectively. Most of the three OPs could dissipate in a short time under sunlight irradiation. Photodegradation could be accepted as an appropriate method for the removal of OPs in honey medium. Copyright © 2014 Elsevier Inc. All rights reserved.

  14. OH initiated heterogeneous degradation of organophosphorus compounds

    Science.gov (United States)

    Liggio, J.; Liu, Y.; Harner, T.; Jantunen, L.; Shoeib, M.; Li, S.

    2013-12-01

    Organophosphorus compounds (OPs) have been extensively used worldwide as flame retardants, plasticizers, antifoaming agents, and additives because of their favorable physicochemical characteristics. The global consumption of OPs is likely to greatly increase due to the phasing out of bromine-containing flame retardants (BFRs) with OPs identified as possible substitutes. In most applications, OPs easily leach out of the material into the environment via volatilization, abrasion, and dissolution and have been observed widely in atmospheric particles even in polar regions. However, little is known about their atmospheric fate. The Canadian Chemicals Management Plan (CMP) has targeted OP FRs for risk assessment, including assessing stability and atmospheric transport potential of OP FRs and other priority chemicals that are associated primarily with particles. In the current study, OH initiated heterogeneous reaction kinetics of tris(1,3-dichloro-2-propyl) phosphate (TDCPP), tris-2-ethylhexyl-phosphate (TEHP), tris-2-butoxyethyl-phosphate (TBEP), and tri-phenyl phosphate (TPhP) coated on (NH4)2SO4 were investigated using a photo-chemical flow tube which was coupled to an Aerosol Mass Spectrometer (AMS) and Proton Transfer Reaction Mass Spectrometer (PTR-MS). second-order rate constants (k2) for the heterogeneous loss of TPhP, TEHP and TDCPP were (2.07×0.19)×10-12, (2.69×0.63)×10-12 and (9.22×0.92)×10-13 cm3 molecule-1 s-1, respectively, from which approximate atmospheric lifetimes were estimated to be 5.6 (5.2-6.0), 4.3 (3.5-5.6), and 12.6 (11.4-14.0) days. These results represent the first reported estimates of heterogeneous rate constants for these species, and suggest that particle bound OPEs will be highly persistent in the atmosphere, supporting the assumption that OPEs can undergo medium or long-range transport, as proposed on the basis of field measurements.

  15. Cooperative degradation of chitin by extracellular and cell surface-expressed chitinases from Paenibacillus sp. strain FPU-7.

    Science.gov (United States)

    Itoh, Takafumi; Hibi, Takao; Fujii, Yutaka; Sugimoto, Ikumi; Fujiwara, Akihiro; Suzuki, Fumiko; Iwasaki, Yukimoto; Kim, Jin-Kyung; Taketo, Akira; Kimoto, Hisashi

    2013-12-01

    Chitin, a major component of fungal cell walls and invertebrate cuticles, is an exceedingly abundant polysaccharide, ranking next to cellulose. Industrial demand for chitin and its degradation products as raw materials for fine chemical products is increasing. A bacterium with high chitin-decomposing activity, Paenibacillus sp. strain FPU-7, was isolated from soil by using a screening medium containing α-chitin powder. Although FPU-7 secreted several extracellular chitinases and thoroughly digested the powder, the extracellular fluid alone broke them down incompletely. Based on expression cloning and phylogenetic analysis, at least seven family 18 chitinase genes were found in the FPU-7 genome. Interestingly, the product of only one gene (chiW) was identified as possessing three S-layer homology (SLH) domains and two glycosyl hydrolase family 18 catalytic domains. Since SLH domains are known to function as anchors to the Gram-positive bacterial cell surface, ChiW was suggested to be a novel multimodular surface-expressed enzyme and to play an important role in the complete degradation of chitin. Indeed, the ChiW protein was localized on the cell surface. Each of the seven chitinase genes (chiA to chiF and chiW) was cloned and expressed in Escherichia coli cells for biochemical characterization of their products. In particular, ChiE and ChiW showed high activity for insoluble chitin. The high chitinolytic activity of strain FPU-7 and the chitinases may be useful for environmentally friendly processing of chitin in the manufacture of food and/or medicine.

  16. Organophosphorus pentavalent compounds: history, synthetic methods of preparation and application as insecticides and antitumor agents

    International Nuclear Information System (INIS)

    Santos, Viviane Martins Rebello dos; Donnici, Claudio Luis; DaCosta, Joao Batista Neves; Caixeiro, Janaina Marques Rodrigues

    2007-01-01

    This paper is a review of the history, synthesis and application of organophosphorus compounds, especially of those of pentavalent phosphorus, such as phosphoramidates, phosphorothioates, phosphonates and phosphonic acids with insecticide and anticancer activities. The organophosphorus compounds with agrochemical applications show great structural variety, They include not only insecticides, but also fungicides, herbicides, and others. The large variety of commercially available organophosphorus pesticides is remarkable. Even more interesting is the high efficiency of some organophosphorus compounds as anticancer agents such as cyclophosphamide and its derivatives. (author)

  17. Atypical Organophosphorus Toxicology of the Herbicides Glufosinate and Ethephon

    OpenAIRE

    Lantz, Stephen R.

    2013-01-01

    Organophosphorus (OP) compounds have found tremendous use as industrial chemicals, pharmaceuticals and chemical warfare agents. The intended toxicity of warfare agents and the unwanted toxicity of industrial and agrochemicals results primarily from inhibition of cholinesterase enzymes, predominantly acetylcholinesterase (AChE). Functionalization of the OP class has led to development of compounds with selective action at various primary protein targets and the identification of numerous sec...

  18. Importance and toxicological effects of organophosphorus pesticides: A comprehensive review

    OpenAIRE

    M. Kazemi; A. M. Tahmasbi; R. Valizadeh; A. A. Naserian; M. M. Moheghi; A. Soni

    2012-01-01

    Environmental risk assessment is an estimate of the likelihood or probability of an adverse impact on the environment resulting from human activities such as applying of pesticides against of target pests (insects, plant pathogens, weeds, nematodes, microbes, mollusks, birds, mammals, fish and so on) in the agriculture. Since the Withdrawal of organochlorine pesticides from use, organophosphorus pesticides (OPs) have become the most widely used pesticides available today in the entire world e...

  19. The application of green chemistry methods in organophosphorus synthesis

    International Nuclear Information System (INIS)

    Odinets, Irina L; Matveeva, E V

    2012-01-01

    Data concerning the synthesis of organophosphorus compounds in ionic liquids, in water and under solvent-free conditions are considered and summarized. It is shown that this strategy, which complies with the definition of green chemistry, has advantages in terms of the rate of the process and the yields of target products as compared with syntheses in common organic solvents. The Wittig, Horner–Wadsworth–Emmons, Kabachnik–Fields, Arbuzov and Michaelis reactions are considered as examples. The bibliography includes 178 references.

  20. Lanthanide sorbent based on magnetite nanoparticles functionalized with organophosphorus extractants

    OpenAIRE

    Basualto, C.; Gaete, J.; Molina, L.; Valenzuela, F.; Yáñez, C.; Marco, J.F.

    2015-01-01

    © 2015 National Institute for Materials Science. In this work, an adsorbent was prepared based on the attachment of organophosphorus acid extractants, namely, D2EHPA, CYANEX 272, and CYANEX 301, to the surface of superparamagnetic magnetite (Fe3O4) nanoparticles. The synthesized nanoparticles were coated with oleic acid, first by a chemisorption mechanism and later by the respective extractant via physical adsorption. The obtained core-shell functionalized magnetite nanoparticle composites we...

  1. Novel organophosphorus compounds; synthesis, spectroscopy and X-ray crystallography

    Czech Academy of Sciences Publication Activity Database

    Shariatinia, Z.; Sohrabi, M.; Yousefi, M.; Kovaľ, Tomáš; Dušek, Michal

    2012-01-01

    Roč. 11, č. 2 (2012), s. 125-133 ISSN 1024-1221 Grant - others:AV ČR(CZ) AP0701 Program:Akademická prémie - Praemium Academiae Institutional research plan: CEZ:AV0Z10100521 Keywords : organophosphorus compounds * NMR * X-ray crystallography * hydrogen bond Subject RIV: BM - Solid Matter Physics ; Magnetism Impact factor: 0.686, year: 2012

  2. The application of green chemistry methods in organophosphorus synthesis

    Science.gov (United States)

    Odinets, Irina L.; Matveeva, E. V.

    2012-03-01

    Data concerning the synthesis of organophosphorus compounds in ionic liquids, in water and under solvent-free conditions are considered and summarized. It is shown that this strategy, which complies with the definition of green chemistry, has advantages in terms of the rate of the process and the yields of target products as compared with syntheses in common organic solvents. The Wittig, Horner-Wadsworth-Emmons, Kabachnik-Fields, Arbuzov and Michaelis reactions are considered as examples. The bibliography includes 178 references.

  3. Third International Meeting on Esterases Reacting with Organophosphorus Compounds

    Science.gov (United States)

    1998-01-01

    ESTHER database (for esterases, oc/ß hydrolase enzymes and relatives) since 1994. This internet server is dedicated to several aspects of biology...Neurociencias University of Zagreb Facultad de Medicina Strossmayerov trg 14 Campus Universitario de San Juan 10000 Zagreb, Croatia 03050 Alicante, Spain

  4. 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

  5. The brain imaging study of the organophosphorus pesticides poisoning

    International Nuclear Information System (INIS)

    Yang Yanmei; Liu Huaijun; Li Shuling; Wang Yongsheng; Huang Boyuan; Chi Cen; Shi Zhenyang; Cui Caixia; Zhou Lixia; Liu Runtian

    2004-01-01

    Objective: To summarize the CT and MR imaging findings in acute organophosphorus pesticides poisoning patients, and to improve the early diagnostic ability. Methods: The imaging of 34 patients of organophosphorus pesticides poisoning was analyzed, the poisons were all taken orally. The pesticides included methamidophos (12 cases), omethoate (15 cases), DDV (3 cases), and methylparathion (4 cases). According to the diagnosis and classification diagnosis criterion of acute organophosphorus pesticides poisoning, the patients were divided into two groups: mild or moderate grade group (24 cases) and severe grade group (10 cases). The relationship between the clinic grade and CT and MRI findings was studied. Results: in the severe grade group, 4 patients showed brain edema, presenting as sulcus and fissure flattened or disappeared, and ventricles and cisterns narrowed or closed 2-3 days after poisoning. In 3 patients 3 days to 3 months after poisoning, bilateral basal ganglion and cerebral cortex showed prolonged T 1 and T 2 signals, and high signal intensity was detected on FLAIR, and bilateral basal ganglion low density was revealed on CT. T 1 relaxation was shortened, T 2 WI and FLAIR imaging showed high signal intensity in 1 patient. The imaging of 1 patient 6 months after poisoning showed the cerebral sulcus, fissure and ventricle were enlarged. CT and MRI in the mild or moderate group were normal. By the Fisher's exact probabilities test, the imaging exhibition difference between the severe grade and mild or moderate grade patients was significant. Conclusion: The CT and MRI can reflect the brain injury after poisoning, and the imaging exhibitions were various. The imaging information can provide credible foundation for the therapy for lightening the brain edema and nourishing the brain cell

  6. Surface expression of NMDA receptor changes during memory consolidation in the crab Neohelice granulata

    Science.gov (United States)

    Hepp, Yanil; Salles, Angeles; Carbo-Tano, Martin

    2016-01-01

    The aim of the present study was to analyze the surface expression of the NMDA-like receptors during the consolidation of contextual learning in the crab Neohelice granulata. Memory storage is based on alterations in the strength of synaptic connections between neurons. The glutamatergic synapses undergo various forms of N-methyl-D aspartate receptor (NMDAR)-dependent changes in strength, a process that affects the abundance of other receptors at the synapse and underlies some forms of learning and memory. Here we propose a direct regulation of the NMDAR. Changes in NMDAR's functionality might be induced by the modification of the subunit's expression or cellular trafficking. This trafficking does not only include NMDAR's movement between synaptic and extra-synaptic localizations but also the cycling between intracellular compartments and the plasma membrane, a process called surface expression. Consolidation of contextual learning affects the surface expression of the receptor without affecting its general expression. The surface expression of the GluN1 subunit of the NMDAR is down-regulated immediately after training, up-regulated 3 h after training and returns to naïve and control levels 24 h after training. The changes in NMDAR surface expression observed in the central brain are not seen in the thoracic ganglion. A similar increment in surface expression of GluN1 in the central brain is observed 3 h after administration of the competitive GABAA receptor antagonist, bicuculline. These consolidation changes are part of a plasticity event that first, during the down-regulation, stabilizes the trace and later, at 3-h post-training, changes the threshold for synapse activation. PMID:27421895

  7. Photodegradation of organophosphorus pesticides in water, ice and snow

    Science.gov (United States)

    Kurkova, R.; Weber, J.; Klanova, J.; Klan, P.; Halsall, C.

    2009-04-01

    Organophosphorus pesticides (OPs) represent more polar compounds with better water solubility and higher degradation rates than organochlorine pesticides, which makes them attractive for a wide application in the agriculture. A fact, that they have been detected in the polar regions, suggests that even though they can be degraded, they still belong to the group of compounds capable of long-range transport. In the cold regions, snow and ice affect the processes of OPs deposition, accumulation and transformation and they can significantly alter their life-times, degradation and elimination rates. However, our knowledge on photodegradation processes of OPs in the matrices of ice and snow is still very limited. This study is focused on the photochemical behaviour of fenitrothion and methyl-parathion as two currently used organophosphorus pesticides. Methyl-parathion is one of the most extensively applied pesticides and both, methyl-parathion and fenitrothion are susceptible to a direct photolysis. Photodegradation is considered to be an important transformation process for both, but the mechanism of degradation in the cold environments has not been studied yet. Filling this gap is crucial for an enhancement of our understanding to the fate of the organic compounds in pristine arctic environments.

  8. Toward antibody-catalyzed hydrolysis of organophosphorus poisons

    Science.gov (United States)

    Vayron, Philippe; Renard, Pierre-Yves; Taran, Frédéric; Créminon, Christophe; Frobert, Yveline; Grassi, Jacques; Mioskowski, Charles

    2000-01-01

    We report here our preliminary results on the use of catalytic antibodies as an approach to neutralizing organophosphorus chemical weapons. A first-generation hapten, methyl-α-hydroxyphosphinate Ha, was designed to mimic the approach of an incoming water molecule for the hydrolysis of exceedingly toxic methylphosphonothioate VX (1a). A moderate protective activity was first observed on polyclonal antibodies raised against Ha. The results were further confirmed by using a mAb PAR 15 raised against phenyl-α-hydroxyphosphinate Hb, which catalyzes the hydrolysis of PhX (1b), a less toxic phenylphosphonothioate analog of VX with a rate constant of 0.36 M−1⋅min−1 at pH 7.4 and 25°C, which corresponds to a catalytic proficiency of 14,400 M−1 toward the rate constant for the uncatalyzed hydrolysis of 1b. This is a demonstration on the organophosphorus poisons themselves that mAbs can catalytically hydrolyze nerve agents, and a significant step toward the production of therapeutically active abzymes to treat poisoning by warfare agents. PMID:10860971

  9. Cell-surface expression of Hsp70 on hematopoietic cancer cells after inhibition of HDAC activity

    DEFF Research Database (Denmark)

    Jensen, Helle; Andresen, Lars; Hansen, Karen Aagaard

    2009-01-01

    surface-negative despite effective induction of apoptosis. Interestingly, inhibition of endolysosomes or normal ER/Golgi transport did not affect Hsp70 surface expression. Intracellular calcium and the transcription factor Sp1, which has been shown previously to be important for the intracellular stress...

  10. SRC Inhibition Reduces NR2B Surface Expression and Synaptic Plasticity in the Amygdala

    Science.gov (United States)

    Sinai, Laleh; Duffy, Steven; Roder, John C.

    2010-01-01

    The Src protein tyrosine kinase plays a central role in the regulation of N-methyl-d-aspartate receptor (NMDAR) activity by regulating NMDAR subunit 2B (NR2B) surface expression. In the amygdala, NMDA-dependent synaptic plasticity resulting from convergent somatosensory and auditory inputs contributes to emotional memory; however, the role of Src…

  11. Basement to surface expressions and critical factors in the genesis of unconformity-related deposits

    International Nuclear Information System (INIS)

    Potter, Eric

    2014-01-01

    Two subprojects: 1) Basement to surface expressions of deep mineralization and refinement of critical factors leading to the genesis of unconformity-related uranium deposits; and 2) Recognition of uranium ore system alteration signatures in complex terranes: IOCG vs albite-hosted uranium vs volcanic-hosted uranium.

  12. Surface Expression of NMDA Receptor Changes during Memory Consolidation in the Crab "Neohelice granulata"

    Science.gov (United States)

    Hepp, Yanil; Salles, Angeles; Carbo-Tano, Martin; Pedreira, Maria Eugenia; Freudenthal, Ramiro

    2016-01-01

    The aim of the present study was to analyze the surface expression of the NMDA-like receptors during the consolidation of contextual learning in the crab "Neohelice granulata". Memory storage is based on alterations in the strength of synaptic connections between neurons. The glutamatergic synapses undergo various forms of…

  13. Vesicular stomatitis virus infection promotes immune evasion by preventing NKG2D-ligand surface expression.

    Science.gov (United States)

    Jensen, Helle; Andresen, Lars; Nielsen, Jens; Christensen, Jan Pravsgaard; Skov, Søren

    2011-01-01

    Vesicular stomatitis virus (VSV) has recently gained attention for its oncolytic ability in cancer treatment. Initially, we hypothesized that VSV infection could increase immune recognition of cancer cells through induction of the immune stimulatory NKG2D-ligands. Here we show that VSV infection leads to a robust induction of MICA mRNA expression, however the subsequent surface expression is potently hindered. Thus, VSV lines up with human cytomegalovirus (HCMV) and adenovirus, which actively subvert the immune system by negatively affecting NKG2D-ligand surface expression. VSV infection caused an active suppression of NKG2D-ligand surface expression, affecting both endogenous and histone deacetylase (HDAC)-inhibitor induced MICA, MICB and ULBP-2 expression. The classical immune escape mechanism of VSV (i.e., the M protein blockade of nucleocytoplasmic mRNA transport) was not involved, as the VSV mutant strain, VSV(ΔM51), which possess a defective M protein, prevented MICA surface expression similarly to wild-type VSV. The VSV mediated down modulation of NKG2D-ligand expression did not involve apoptosis. Constitutive expression of MICA bypassed the escape mechanism, suggesting that VSV affect NKG2D-ligand expression at an early post-transcriptional level. Our results show that VSV possess an escape mechanism, which could affect the immune recognition of VSV infected cancer cells. This may also have implications for immune recognition of cancer cells after combined treatment with VSV and chemotherapeutic drugs.

  14. Crystal Structures of Human Group-VIIA Phospholipase A2 Inhibited by Organophosphorus Nerve Agents Exhibit Non-aged Complexes ☆,☆☆

    Science.gov (United States)

    Samanta, Uttamkumar; Kirby, Stephen D.; Srinivasan, Prabhavathi; Cerasoli, Douglas M.; Bahnson, Brian J.

    2009-01-01

    The enzyme group-VIIA phospholipase A2 (gVIIA-PLA2) is bound to lipoproteins in human blood and hydrolyzes the ester bond at the sn-2 position of phospholipid substrates with a short sn-2 chain. The enzyme belongs to a serine hydrolase superfamily of enzymes, which react with organophosphorus (OP) nerve agents. OPs ultimately exert their toxicity by inhibiting human acetycholinesterase at nerve synapses, but may additionally have detrimental effects through inhibition of other serine hydrolases. We have solved the crystal structures of gVIIA-PLA2 following inhibition with the OPs diisopropylfluorophosphate, sarin, soman and tabun. The sarin and soman complexes displayed a racemic mix of PR and PS stereoisomers at the P-chiral center. The tabun complex displayed only the PR stereoisomer in the crystal. In all cases, the crystal structures contained intact OP adducts that had not aged. Aging refers to a secondary process OP complexes can go through, which dealkylates the nerve agent adduct and results in a form that is highly resistant to either spontaneous or oxime-mediated reactivation. Non-aged OP complexes of the enzyme were corroborated by trypsin digest and matrix assisted laser desorption ionization mass spectrometry of OP-enzyme complexes. The lack of stereoselectivity of sarin reaction was confirmed by gas chromatography/mass spectrometry using a chiral column to separate and quantitate the unbound stereoisomers of sarin following incubation with enzyme. The structural details and characterization of nascent reactivity of several toxic nerve agents is discussed with a long term goal of developing gVIIA-PLA2 as a catalytic bioscavenger of OP nerve agents. PMID:19394314

  15. Crystal Structures of Brain Group-VIII Phospholipase A2 in Non-aged Complexes with the Organophosphorus Nerve Agents Soman and Sarin†‡

    Science.gov (United States)

    Epstein, Todd M.; Samanta, Uttamkumar; Kirby, Stephen D.; Cerasoli, Douglas M.; Bahnson, Brian J.

    2009-01-01

    Insecticide and nerve agent organophosphorus compounds are potent inhibitors of the serine hydrolase superfamily of enzymes. Nerve agents, such as sarin, soman, tabun and VX exert their toxicity by inhibiting human acetycholinesterase at nerve synapses. Following the initial phosphonylation of the active site serine, the enzyme may reactivate spontaneously or through reaction with an appropriate nucleophilic oxime. Alternatively, the enzyme-nerve agent complex can undergo a secondary process, called “aging”, which dealkylates the nerve agent adduct and results in a product that is highly resistant to reactivation by any known means. Here we report the structures of paraoxon, soman and sarin complexes of group-VIII phospholipase A2 from bovine brain. In each case, the crystal structures indicate a non-aged adduct; a stereoselective preference for binding of the PSCS isomer of soman and the PS isomer of sarin was also noted. The stability of the non-aged complexes was corroborated by trypsin digest and electrospray ionization mass spectrometry, which indicates non-aged complexes are formed with diisopropylfluorophosphate, soman and sarin. The PS stereoselectivity for reaction with sarin was confirmed by reaction of racemic sarin, followed by gas chromatography/mass spectrometry using a chiral column to separate and quantitate each stereoisomer. The PS stereoisomers of soman and sarin are known to be the more toxic stereoisomers, as they react preferentially to inhibit human acetylcholinesterase. The results obtained for non-aged complexes of group-VIII phospholipase A2 are compared to those obtained for other serine hydrolases and discussed to partly explain determinants of OP aging. Furthermore, structural insights can now be exploited to engineer variant versions of this enzyme with enhanced nerve agent binding and hydrolysis functions. PMID:19271773

  16. Crystal structures of brain group-VIII phospholipase A2 in nonaged complexes with the organophosphorus nerve agents soman and sarin.

    Science.gov (United States)

    Epstein, Todd M; Samanta, Uttamkumar; Kirby, Stephen D; Cerasoli, Douglas M; Bahnson, Brian J

    2009-04-21

    Insecticide and nerve agent organophosphorus (OP) compounds are potent inhibitors of the serine hydrolase superfamily of enzymes. Nerve agents, such as sarin, soman, tabun, and VX exert their toxicity by inhibiting human acetycholinesterase at nerve synapses. Following the initial phosphonylation of the active site serine, the enzyme may reactivate spontaneously or through reaction with an appropriate nucleophilic oxime. Alternatively, the enzyme-nerve agent complex can undergo a secondary process, called "aging", which dealkylates the nerve agent adduct and results in a product that is highly resistant to reactivation by any known means. Here we report the structures of paraoxon, soman, and sarin complexes of group-VIII phospholipase A2 from bovine brain. In each case, the crystal structures indicate a nonaged adduct; a stereoselective preference for binding of the P(S)C(S) isomer of soman and the P(S) isomer of sarin was also noted. The stability of the nonaged complexes was corroborated by trypsin digest and electrospray ionization mass spectrometry, which indicates nonaged complexes are formed with diisopropylfluorophosphate, soman, and sarin. The P(S) stereoselectivity for reaction with sarin was confirmed by reaction of racemic sarin, followed by gas chromatography/mass spectrometry using a chiral column to separate and quantitate each stereoisomer. The P(S) stereoisomers of soman and sarin are known to be the more toxic stereoisomers, as they react preferentially to inhibit human acetylcholinesterase. The results obtained for nonaged complexes of group-VIII phospholipase A2 are compared to those obtained for other serine hydrolases and discussed to partly explain determinants of OP aging. Furthermore, structural insights can now be exploited to engineer variant versions of this enzyme with enhanced nerve agent binding and hydrolysis functions.

  17. Human valacyclovir hydrolase/biphenyl hydrolase-like protein is a highly efficient homocysteine thiolactonase.

    Directory of Open Access Journals (Sweden)

    Judit Marsillach

    Full Text Available Homocysteinylation of lysine residues by homocysteine thiolactone (HCTL, a reactive homocysteine metabolite, results in protein aggregation and malfunction, and is a well-known risk factor for cardiovascular, autoimmune and neurological diseases. Human plasma paraoxonase-1 (PON1 and bleomycin hydrolase (Blmh have been reported as the physiological HCTL detoxifying enzymes. However, the catalytic efficiency of HCTL hydrolysis by Blmh is low and not saturated at 20 mM HCTL. The catalytic efficiency of PON1 for HCTL hydrolysis is 100-fold lower than that of Blmh. A homocysteine thiolactonase (HCTLase was purified from human liver and identified by mass spectrometry (MS as the previously described human biphenyl hydrolase-like protein (BPHL. To further characterize this newly described HCTLase activity, BPHL was expressed in Escherichia coli and purified. The sequence of the recombinant BPHL (rBPHL and hydrolytic products of the substrates HCTL and valacyclovir were verified by MS. We found that the catalytic efficiency (kcat/Km of rBPHL for HCTL hydrolysis was 7.7 × 10(4 M(-1s(-1, orders of magnitude higher than that of PON1 or Blmh, indicating a more significant physiological role for BPHL in detoxifying HCTL.

  18. Surface Chemistry of Enzymes for Detection and Decontamination of Organophosphorus Compounds

    National Research Council Canada - National Science Library

    Leblanc, Roger

    2003-01-01

    The principal objective of this project was to understand the molecular interactions between organophosphorus compounds and the enzymes that catalyze the hydrolysis of these compounds at air water-interface (Langmuir films...

  19. UPTAKE AND PHYTOTRANSFORMATION OF ORGANOPHOSPHORUS PESTICIDES BY AXENICALLY CULTIVATED AQUATIC PLANTS

    Science.gov (United States)

    The uptake and phytotransformation of organophosphorus (OP) pesticides (malathion, demeton-S-methyl, and crufomate) was investigated in vitro using the axenically aquatic cultivated plants parrot feather (Myriophyllum aquaticum), duckweed (Spirodela oligorrhiza L.), and elodea (E...

  20. HIGH PERFORMANCE LIQUID CHROMATOGRAPHIC SEPARATION OF THE ENANTIOMERS OF ORGANOPHOSPHORUS PESTICIDES ON POLYSACCHARIDE CHIRAL STATIONARY PHASES

    Science.gov (United States)

    High-performance liquid chromatographic separation of the individual enantiomers of 12 organophosphorus pesticides (OPs) was obtained on polysaccharide enantioselective HPLC columns using alkane-alcohol mobile phase. The OP pesticides were crotoxyphos, dialifor, fonofos, fenamiph...

  1. HPLC SEPARATION OF CHIRAL ORGANOPHOSPHORUS PESTICIDES ON POLYSACCHARIDE CHIRAL STATIONARY PHASES

    Science.gov (United States)

    High-performance liquid chromatographic separation of the individual enantiomers of 12 organophosphorus pesticides (OPs) were obtained on polysaccharide chiral HPLC columns using an alkane-alcohol mobile phase. The OP pesticides were crotoxyphos, dialifor, dyfonate, fenamiphos, ...

  2. Levels of organophosphorus pesticides in medicinal plants commonly consumed in Iran

    Directory of Open Access Journals (Sweden)

    Sarkhail Parisa

    2012-08-01

    Full Text Available Abstract The frequent occurrence of pesticide residues in herbal materials was indicated by previous studies. In this study, the concentration of some of the organophosphorus pesticides including parathion, malathion, diazinon and pirimiphos methyl in different kinds of medicinal plants were determined. The samples were collected randomly from ten local markets of different areas of Iran. At the detection limit of 0.5 ng g-1, parathion and pirimiphos methyl were not detected in any of the samples. Some amounts of malathion and diazinon were found in Zataria, Matricaria chamomile, Spearmint and Cumin Seed samples while, the concentrations of target organophosphorus pesticides in Borage samples were below the detection limits of the methods which could be a result of intensive transformation of organophosphorus pesticides by Borage. In addition the organophosphorus pesticides were detected in all of the samples below the maximum residue levels (MRLs proposed by the international organizations.

  3. Degradation of atrazine and several organophosphorus pesticides in oranges

    Energy Technology Data Exchange (ETDEWEB)

    Bouaid, A.; Martin-Esteban, A.; Fernandez, P.; Camara, C. [Madrid Univ., Madrid (Spain). Facultad de Ciencias Quimicas, Dept. de Quimica Analitica

    2001-02-01

    The degradation of atrazine and four organophosphorus pesticides (chloropyriphos, fenamiphos, methidathion and methyl-parathion) in oranges was studied. Oranges were immersed in a Milli-Q water solution spiked with 10 mg litre{sup -}1 of each pesticide for one day, allowing their adsorption on the orange peel. Then, the oranges were rinsed with Milli-Q water and left outdoors to expose them to natural ambient conditions for two weeks. In parallel, an aqueous solution containing 1 mg litre{sup -}1 of each pesticide was placed in a Pyrex flask, which was tightly closed, and exposed to the same ambient conditions. Both samples (orange peel and Milli-Q water) were analyzed periodically by gas chromatography coupled to a nitrogen phosphorus detector. The pesticide degradation in both samples could be described using a first-order degradation curve. Half-lives varied from 14.5 to more than 30 days in aqueous solution and from 2.3 to 4.1 days in oranges for organophosphorus pesticides, while those for atrazine were 3.1 days and 14.2 days, respectively. The presence of some organophosphorus degradation products in water samples after storage under the above conditions was confirmed by gas chromatography-mass spectrometry. [Italian] E' stata studiata la degradazione dell'atrazina e quattro pesticidi organofosforati (chlorpyriphos, fenamiphos, methidathion and methyl-parathion) nelle arance. Le arance sono state immerse, per un giorno, in una soluzione acquosa (Milli-Q) addizionata di 10 mg L di ciascun pesticida al fine di ottenerne l'assorbimento sulla biccia. Le arance sono state successivamente lavate con acqua Milli-Q e collocate all'esterno per esporle alle condizioni ambientali per due settimane. In parallelo si e' collocata, una soluzione acquosa contenente 1 mg L, in un matraccio di pirex chiuso ed esposto alle stesse condizioni ambientali. Entrambi i campioni (buccia d'arancia ed acqua Milli-Q) sono stati analizzati periodicamente

  4. DL-endopeptidases function as both cell wall hydrolases and poly-γ-glutamic acid hydrolases.

    Science.gov (United States)

    Fukushima, Tatsuya; Uchida, Natsuki; Ide, Masatoshi; Kodama, Takeko; Sekiguchi, Junichi

    2018-03-01

    Biopolymers on the cell surface are very important for protecting microorganisms from environmental stresses, as well as storing nutrients and minerals. Synthesis of biopolymers is well studied, while studies on the modification and degradation processes of biopolymers are limited. One of these biopolymers, poly-γ-glutamic acid (γ-PGA), is produced by Bacillus species. Bacillus subtilis PgdS, possessing three NlpC/P60 domains, hydrolyses γ-PGA. Here, we have demonstrated that several dl-endopeptidases with an NlpC/P60 domain (LytE, LytF, CwlS, CwlO, and CwlT) in B. subtilis digest not only an amide bond of d-γ-glutamyl-diaminopimelic acid in peptidoglycans but also linkages of γ-PGA produced by B. subtilis. The hydrolase activity of dl-endopeptidases towards γ-PGA was inhibited by IseA, which also inhibits their hydrolase activity towards peptidoglycans, while the hydrolysis of PgdS towards γ-PGA was not inhibited. PgdS hydrolysed only the d-/l-Glu‒d-Glu linkages of d-Glu-rich γ-PGA (d-Glu:l-Glu=7 : 3) and l-Glu-rich γ-PGA (d-Glu:l-Glu=1 : 9), indicating that PgdS can hydrolyse only restricted substrates. On the other hand, the dl-endopeptidases in B. subtilis cleaved d-/l-Glu‒d-/l-Glu linkages of d-Glu-rich γ-PGA (d-Glu:l-Glu=7 : 3), indicating that these enzymes show different substrate specificities. Thus, the dl-endopeptidases digest γ-PGA more flexibly than PgdS, even though they are annotated as "dl-endopeptidase, digesting the d-γ-glutamyl-diaminopimelic acid linkage (d‒l amino acid bond)".

  5. Determination of organophosphorus pesticide residues in tomatoes by gas chromatography

    International Nuclear Information System (INIS)

    Souissi, Sihem

    2010-01-01

    Chloropyriphos and malathion are two organophosphorus pesticides from many others pesticides widely used by famers in agriculture .Because of their bad effect on human health, officials standards are set by the international organisations and communities to ensure safer food for consumer .In the same way, scientists over the world are working hard to develop new detection techniques responding to the international requirements. In this study, an ' IAEA-ethylacetate method ', an adaptation of the popular QuEChERS multi residue method, was optimized to analyse chloropyriphos and Malathion residues in tomatoes .Ethyl-Acetate was used as an extraction solvent the PSA was kept for the clean up procedure. GC-NPD is used for samples analysis .The method optimized is specifique, selective with a recovery averaged more than 70 pour cent. A complete validation of the method is necessary to be used for routine analysis.

  6. Fluorescent Chemosensors for Toxic Organophosphorus Pesticides: A Review

    Directory of Open Access Journals (Sweden)

    Kenneth Fletcher

    2010-07-01

    Full Text Available Many organophosphorus (OP based compounds are highly toxic and powerful inhibitors of cholinesterases that generate serious environmental and human health concerns. Organothiophosphates with a thiophosphoryl (P=S functional group constitute a broad class of these widely used pesticides. They are related to the more reactive phosphoryl (P=O organophosphates, which include very lethal nerve agents and chemical warfare agents, such as, VX, Soman and Sarin. Unfortunately, widespread and frequent commercial use of OP-based compounds in agricultural lands has resulted in their presence as residues in crops, livestock, and poultry products and also led to their migration into aquifers. Thus, the design of new sensors with improved analyte selectivity and sensitivity is of paramount importance in this area. Herein, we review recent advances in the development of fluorescent chemosensors for toxic OP pesticides and related compounds. We also discuss challenges and progress towards the design of future chemosensors with dual modes for signal transduction.

  7. Fluorescent chemosensors for toxic organophosphorus pesticides: a review.

    Science.gov (United States)

    Obare, Sherine O; De, Chandrima; Guo, Wen; Haywood, Tajay L; Samuels, Tova A; Adams, Clara P; Masika, Noah O; Murray, Desmond H; Anderson, Ginger A; Campbell, Keith; Fletcher, Kenneth

    2010-01-01

    Many organophosphorus (OP) based compounds are highly toxic and powerful inhibitors of cholinesterases that generate serious environmental and human health concerns. Organothiophosphates with a thiophosphoryl (P=S) functional group constitute a broad class of these widely used pesticides. They are related to the more reactive phosphoryl (P=O) organophosphates, which include very lethal nerve agents and chemical warfare agents, such as, VX, Soman and Sarin. Unfortunately, widespread and frequent commercial use of OP-based compounds in agricultural lands has resulted in their presence as residues in crops, livestock, and poultry products and also led to their migration into aquifers. Thus, the design of new sensors with improved analyte selectivity and sensitivity is of paramount importance in this area. Herein, we review recent advances in the development of fluorescent chemosensors for toxic OP pesticides and related compounds. We also discuss challenges and progress towards the design of future chemosensors with dual modes for signal transduction.

  8. Radiodiagnosis of pulmonary alterations induced by organophosphorus insecticides poisoning

    International Nuclear Information System (INIS)

    Kishkovskij, A.N.; Tyutin, L.A.; Ivanov, N.V.

    1982-01-01

    In order to assess possibilities of radiodiagnosis of pulmonary complications, developing as a result of organophosphorus insecticides poisoning, clinical and X-ray examination of 400 patients, aged 17 to 73 years, has been carried out. The lung plethora was manifested by a symmetric emlargement of the lung roots and vessels. A diffuse cleaness of the lung pattern, lung root widening and structural disorder, as well as an appearance of the small focal shadows in the hilar zones, were all characteristic of interstitial edema. X-ray signs of alveolar edema were confined to the coalescing shadows of different size and intensity, and to a massive infiltration of both the lung roots and emphysematous zones. The pneumonic infiltration foci of the lung parenchyma were usually distributed in the peripheral segments. Routine radiography in lateral projection played a decisive role in atelectasis diagnosis

  9. 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...... enzyme were shown to have a considerable activity against cellotriose and cellotetraose, and a low but significant activity against cellulose. The lactase/phlorizin hydrolase isolated from pigs in which the pancreatic ducts had been disconnected 3 days before death and from Ca2+-precipitated enterocyte...

  10. Method for enhancing amidohydrolase activity of fatty acid amide hydrolase

    Energy Technology Data Exchange (ETDEWEB)

    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.

  11. 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

  12. ENGINEERING OF PEPTIDOGLYCAN HYDROLASES FOR CONTROL OF PATHOGENIC BACTERIA

    Science.gov (United States)

    Bacteriophages are viruses exclusively infecting bacteria and therefore offer suitable tools for their detection and control. At the end of their multiplication cycle, most phages lyse their hosts from within by means of an endolysin (peptidoglycan hydrolase), thereby enabling release of the phage p...

  13. Carboxylic ester hydrolases in mitochondria from rat skeletal muscle

    DEFF Research Database (Denmark)

    Kirkeby, S; Moe, D; Zelander, T

    1990-01-01

    A mitochondrial pellet, prepared from rat skeletal muscle, contained a number of carboxylic ester hydrolase isoenzymes. The esterases which split alpha-naphthyl acetate were organophosphate sensitive, whereas two out of three indoxyl acetate hydrolysing enzymes were resistant to both organophosph...

  14. α/β Hydrolase fold enzymes: the family keeps growing

    NARCIS (Netherlands)

    Nardini, Marco; Dijkstra, B W

    1999-01-01

    The alpha/beta hydrolase fold is a typical example of a tertiary fold adopted by proteins that have no obvious sequence similarity, but nevertheless, in the course of evolution, diverged from a common ancestor. Recently solved structures demonstrate a considerably increased variability in fold

  15. 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...

  16. Consolidation of glycosyl hydrolase family 30 : a dual domain 4/7 hydrolase family consisting of two structurally distinct groups

    Science.gov (United States)

    Franz J. St John; Javier M. Gonzalez; Edwin Pozharski

    2010-01-01

    In this work glycosyl hydrolase (GH) family 30 (GH30) is analyzed and shown to consist of its currently classified member sequences as well as several homologous sequence groups currently assigned within family GH5. A large scale amino acid sequence alignment and a phylogenetic tree were generated and GH30 groups and subgroups were designated. A partial rearrangement...

  17. Regulation of NKG2D-ligand cell surface expression by intracellular calcium after HDAC-inhibitor treatment

    DEFF Research Database (Denmark)

    Jensen, Helle; Hagemann-Jensen, Michael Henrik; Lauridsen, Felicia Kathrine Bratt

    2013-01-01

    cell surface expression on melanoma cells and Jurkat T-cells. A NKG2D-dependent cytolytic assay and staining with a recombinant NKG2D-Fc fusion protein showed that calcium chelation impaired the functional ability of NKG2D-ligands induced by HDAC-inhibitor treatment. The HDAC-inhibitor induced cell...... surface expression of ULBP2, but not MICA/B, was sensitive to treatment calmidazolium and trifluoperazine, two agents known to block calcium signaling. siRNA-mediated knock-down of the calcium-regulated proteins calmodulin or calpain did however not affect NKG2D-ligand cell surface expression on Jurkat T...

  18. Repeated exposure to morphine alters surface expression of AMPA receptors in the rat medial prefrontal cortex.

    Science.gov (United States)

    Mickiewicz, Amanda L; Napier, T Celeste

    2011-01-01

    Behavioral sensitization describes the intensification of motor activity that results from repeated exposure to drugs of misuse, and the underlying neuronal adaptations are hypothesized to model aspects of the brain changes that occur in humans misusing such drugs. The α-amino-3-hydroxyl-5-methyl-4-isoxazole-propionate (AMPA) receptor is an ionotropic glutamate receptor involved in the neuroplasticity that accompanies acute and repeated drug administration. Changing surface expression is one means to regulate AMPA receptor function, and the present study tested the hypothesis that behavioral sensitization to the μ-opioid receptor agonist morphine is accompanied by changes in the subcellular distribution of AMPA receptors in limbic brain regions. To test this hypothesis, we used a protein cross-linking assay to assess cell surface and intracellular levels of GluA1 and GluA2 subunits in the nucleus accumbens, medial prefrontal cortex and ventral pallidum. Repeated morphine treatment decreased surface expression of GluA1 in the medial prefrontal cortex without affecting levels of GluA2. In contrast, surface levels of GluA1 or GluA2 were unchanged in the nucleus accumbens and ventral pallidum, demonstrating that although AMPA receptors in accumbal and pallidal regions are critical mediators of behaviors induced by repeated opiate exposure, these effects are not accompanied by changes in surface expression. The findings reveal that the involvement of AMPA receptor trafficking in opiate-induced behavioral sensitization is relegated to selective regions and that AMPA receptors in the medial prefrontal cortex may be particularly sensitive to these actions. © 2010 The Authors. European Journal of Neuroscience © 2010 Federation of European Neuroscience Societies and Blackwell Publishing Ltd.

  19. AMP-activated protein kinase downregulates Kv7.1 cell surface expression

    DEFF Research Database (Denmark)

    Andersen, Martin N; Krzystanek, Katarzyna; Jespersen, Thomas

    2012-01-01

    The potassium channel Kv7.1 is expressed in the heart, where it contributes to the repolarization of the cardiac action potential. Additionally, Kv7.1 is expressed in epithelial tissues playing a role in salt and water transport. We recently demonstrated that surface-expressed Kv7.1 is internalized...... in response to polarization of the epithelial Madin-Darby canine kidney (MDCK) cell line and that this was mediated by activation of protein kinase C (PKC). In this study, the pathway downstream of PKC, which leads to internalization of Kv7.1 upon cell polarization, is elucidated. We show by confocal...

  20. Effect of Bile Salt Hydrolase Inhibitors on a Bile Salt Hydrolase from Lactobacillus acidophilus

    Directory of Open Access Journals (Sweden)

    Jun Lin

    2014-12-01

    Full Text Available Bile salt hydrolase (BSH, a widely distributed function of the gut microbiota, has a profound impact on host lipid metabolism and energy harvest. Recent studies suggest that BSH inhibitors are promising alternatives to antibiotic growth promoters (AGP for enhanced animal growth performance and food safety. Using a high-purity BSH from Lactobacillus salivarius strain, we have identified a panel of BSH inhibitors. However, it is still unknown if these inhibitors also effectively inhibit the function of the BSH enzymes from other bacterial species with different sequence and substrate spectrum. In this study, we performed bioinformatics analysis and determined the inhibitory effect of identified BSH inhibitors on a BSH from L. acidophilus. Although the L. acidophilus BSH is phylogenetically distant from the L. salivarius BSH, sequence analysis and structure modeling indicated the two BSH enzymes contain conserved, catalytically important amino residues and domain. His-tagged recombinant BSH from L. acidophilus was further purified and used to determine inhibitory effect of specific compounds. Previously identified BSH inhibitors also exhibited potent inhibitory effects on the L. acidophilus BSH. In conclusion, this study demonstrated that the BSH from L. salivarius is an ideal candidate for screening BSH inhibitors, the promising alternatives to AGP for enhanced feed efficiency, growth performance and profitability of food animals.

  1. Orf virus interferes with MHC class I surface expression by targeting vesicular transport and Golgi

    Directory of Open Access Journals (Sweden)

    Rohde Jörg

    2012-07-01

    Full Text Available Abstract Background The Orf virus (ORFV, a zoonotic Parapoxvirus, causes pustular skin lesions in small ruminants (goat and sheep. Intriguingly, ORFV can repeatedly infect its host, despite the induction of a specific immunity. These immune modulating and immune evading properties are still unexplained. Results Here, we describe that ORFV infection of permissive cells impairs the intracellular transport of MHC class I molecules (MHC I as a result of structural disruption and fragmentation of the Golgi apparatus. Depending on the duration of infection, we observed a pronounced co-localization of MHC I and COP-I vesicular structures as well as a reduction of MHC I surface expression of up to 50%. These subversion processes are associated with early ORFV gene expression and are accompanied by disturbed carbohydrate trimming of post-ER MHC I. The MHC I population remaining on the cell surface shows an extended half-life, an effect that might be partially controlled also by late ORFV genes. Conclusions The presented data demonstrate that ORFV down-regulates MHC I surface expression in infected cells by targeting the late vesicular export machinery and the structure and function of the Golgi apparatus, which might aid to escape cellular immune recognition.

  2. Subchronic organophosphorus ester-induced delayed neurotoxicity in mallards

    Science.gov (United States)

    Hoffman, D.J.; Sileo, L.; Murray, H.C.

    1984-01-01

    Eighteen-week-old mallard hens received 0, 10, 30, 90, or 270 ppm technical grade EPN (phenylphosphonothioic acid O-ethyl-O-4-nitrophenyl ester) in the diet for 90 days. Ataxia was first observed in the 270-ppm group after 16 days, in the 90-ppm group after 20 days, in the 30-ppm group after 38 days; 10 ppm failed to produce ataxia. By the end of 90 days all 6 birds in the 270-ppm group exhibited ataxia or paralysis whereas 5 of 6 birds in the 90-ppm group and 2 of 6 birds in the 30-ppm group were visibly affected. Treatment with 30 ppm or more resulted in a significant reduction in body weight. Brain neurotoxic esterase activity was inhibited by averages of 16, 69, 73, and 74% in the 10-, 30-, 90-, and 270-ppm groups, respectively. Brain acetylcholinesterase, plasma cholinesterase, and plasma alkaline phosphatase were significantly inhibited as well. Distinct histopathological effects were seen in the 30-, 90-, and 270-ppm groups which included demyelination and degeneration of axons of the spinal cord. Additional ducks were exposed in a similar manner to 60-, 270-, or 540-ppm leptophos (phosphonothioic acid O-4-bromo-2,5-dichlorophenyl-O-methylphenyl ester) which resulted in similar behavioral, biochemical, and histopathological alterations. These findings indicate that adult mallards are probably somewhat less sensitive than chickens to subchronic dietary exposure to organophosphorus insecticides that induce delayed neurotoxicity.

  3. Microsomal transformation of organophosphorus pesticides by white rot fungi.

    Science.gov (United States)

    Jauregui, Juan; Valderrama, Brenda; Albores, Arnulfo; Vazquez-Duhalt, Rafael

    2003-12-01

    The enzymatic mechanism for the transformation of organophosphorus pesticides (OPPs) by different white-rot fungi strains was studied. With the exception of Ganoderma applanatum 8168, all strains from a collection of 17 different fungi cultures were able to deplete parathion. Three strains showing the highest activities were selected for further studies: Bjerkandera adusta 8258, Pleurotus ostreatus 7989 and Phanerochaete chrysosporium 3641. These strains depleted 50 to 96% of terbufos, azinphos-methyl, phosmet and tribufos after four-days exposure to the pesticides. In order to identify the cellular localization of the transformation activity, the extracellular and microsomal fractions of Pleuronts ostreatus 7989 were evaluated in vitro. While the activities of ligninolytic enzymes (lignin peroxidase, manganese peroxidase and laccase) were detected in the extracellular fraction, no enzymatic modification of any of the five pesticides tested could be found, suggesting the intracellular origin of the transformation activity. In accordance with this observation the microsomal fraction was found able to transform three OPPs with the following rates: 10 micromol mg prot(-1) h(-1) for phosmet, 5.7 micromol mg prot(-1) h(-1) for terbufos, and 2.2 micromol mg prot(-1) h(-1) for azinphos-methyl. The products from these reactions and from the transformation of trichlorfon and malathion, were identified by mass-spectrometry. These results, supported by specific inhibition experiments and the stringent requirement for NADPH during the in vitro assays suggest the involvement of a cytochrome P450.

  4. Lanthanide sorbent based on magnetite nanoparticles functionalized with organophosphorus extractants.

    Science.gov (United States)

    Basualto, Carlos; Gaete, José; Molina, Lorena; Valenzuela, Fernando; Yañez, Claudia; Marco, Jose F

    2015-06-01

    In this work, an adsorbent was prepared based on the attachment of organophosphorus acid extractants, namely, D2EHPA, CYANEX 272, and CYANEX 301, to the surface of superparamagnetic magnetite (Fe 3 O 4 ) nanoparticles. The synthesized nanoparticles were coated with oleic acid, first by a chemisorption mechanism and later by the respective extractant via physical adsorption. The obtained core-shell functionalized magnetite nanoparticle composites were characterized by dynamic light scattering, scanning electron microscopy, transmission electron microscopy, thermogravimetry, infrared absorption and vibrating sample magnetometry. All the prepared nanoparticles exhibited a high saturation magnetization capacity that varied between 72 and 46 emu g -1 and decreased as the magnetite nanoparticle was coated with oleic acid and functionalized. The scope of this study also included adsorption tests for lanthanum, cerium, praseodymium, and neodymium and the corresponding analysis of their results. Sorption tests indicated that the functionalized nanoparticles were able to extract the four studied lanthanide metal ions, although the best extraction performance was observed when the sorbent was functionalized with CYANEX 272, which resulted in a loading capacity of approximately 12-14 mg La /g MNP . The magnetization of the synthesized nanoparticles was verified during the separation of the lanthanide-loaded sorbent from the raffinate by using a conventional magnet.

  5. Ecological risk estimation of organophosphorus pesticides in riverine ecosystems.

    Science.gov (United States)

    Wee, Sze Yee; Aris, Ahmad Zaharin

    2017-12-01

    Pesticides are of great concern because of their existence in ecosystems at trace concentrations. Worldwide pesticide use and its ecological impacts (i.e., altered environmental distribution and toxicity of pesticides) have increased over time. Exposure and toxicity studies are vital for reducing the extent of pesticide exposure and risk to the environment and humans. Regional regulatory actions may be less relevant in some regions because the contamination and distribution of pesticides vary across regions and countries. The risk quotient (RQ) method was applied to assess the potential risk of organophosphorus pesticides (OPPs), primarily focusing on riverine ecosystems. Using the available ecotoxicity data, aquatic risks from OPPs (diazinon and chlorpyrifos) in the surface water of the Langat River, Selangor, Malaysia were evaluated based on general (RQ m ) and worst-case (RQ ex ) scenarios. Since the ecotoxicity of quinalphos has not been well established, quinalphos was excluded from the risk assessment. The calculated RQs indicate medium risk (RQ m  = 0.17 and RQ ex  = 0.66; 0.1 ≤ RQ  1 (high risk) was observed for both the general and worst cases of chlorpyrifos, but only for the worst cases of diazinon at all sites from downstream to upstream regions. Thus, chlorpyrifos posed a higher risk than diazinon along the Langat River, suggesting that organisms and humans could be exposed to potentially high levels of OPPs. Copyright © 2017 Elsevier Ltd. All rights reserved.

  6. Medical treatment of acute poisoning with organophosphorus and carbamate pesticides.

    Science.gov (United States)

    Jokanović, Milan

    2009-10-28

    Organophosphorus compounds (OPs) are used as pesticides and developed as warfare nerve agents such as tabun, soman, sarin, VX and others. Exposure to even small amounts of an OP can be fatal and death is usually caused by respiratory failure. The mechanism of OP poisoning involves inhibition of acetylcholinesterase (AChE) leading to inactivation of the enzyme which has an important role in neurotransmission. AChE inhibition results in the accumulation of acetylcholine at cholinergic receptor sites, producing continuous stimulation of cholinergic fibers throughout the nervous systems. During more than five decades, pyridinium oximes have been developed as therapeutic agents used in the medical treatment of poisoning with OP. They act by reactivation of AChE inhibited by OP. However, they differ in their activity in poisoning with pesticides and warfare nerve agents and there is still no universal broad-spectrum oxime capable of protecting against all known OP. In spite of enormous efforts devoted to development of new pyridinium oximes as potential antidotes against poisoning with OP only four compounds so far have found its application in human medicine. Presently, a combination of an antimuscarinic agent, e.g. atropine, AChE reactivator such as one of the recommended pyridinium oximes (pralidoxime, trimedoxime, obidoxime and HI-6) and diazepam are used for the treatment of OP poisoning in humans. In this article the available data related to medical treatment of poisoning with OP pesticides are reviewed and the current recommendations are presented.

  7. Sorption of Organophosphorus Flame-Retardants on Settled ...

    Science.gov (United States)

    Dust is an important sink for indoor air pollutants, such as organophosphorus flame-retardants (OPFRs) that are used as additives in industrial and consumer products including electrical and electronic products, furniture, plastics, textile, and building/construction materials. This research investigated the sorption of OPFRs, tris(2-chloroethyl) phosphate (TCEP), tris(1-chlor-2-propyl) phosphate (TCPP), and tris(1,3-dichloro-2-propyl) phosphate (TDCPP) on settled Arizona Test Dust (ATD) using a dual small chamber system. During the test, seven free film release paper dust trays covered with ATD were placed in the sink test chamber. Constant gas phase OPFRs from the source chamber were dosed into the test chamber. The dust evenly spread on each dust tray was removed from the test chamber at different exposure times to determine the amount of OPFRs absorbed by the dust. The ATD has been characterized for a nominal particle size and surface area. The mass of dust on each of seven dust trays was weighed before the dust was placed inside the sink chamber. OPFRs concentrations at the inlet and faceplate of the test chamber were monitored by collecting polyurethane foam (PUF) samples. The OPFR exposed dust and PUF samples were extracted by 1:1 ethyl acetate/methylene chloride and analyzed on GC/MS. The data were used to calculate the OPFR sorption concentration on the dust through dust/air partition. Settled dust can adsorb OPFR from air. The sorption concentration wa

  8. Lanthanide sorbent based on magnetite nanoparticles functionalized with organophosphorus extractants

    Science.gov (United States)

    Basualto, Carlos; Gaete, José; Molina, Lorena; Valenzuela, Fernando; Yañez, Claudia; Marco, Jose F.

    2015-06-01

    In this work, an adsorbent was prepared based on the attachment of organophosphorus acid extractants, namely, D2EHPA, CYANEX 272, and CYANEX 301, to the surface of superparamagnetic magnetite (Fe3O4) nanoparticles. The synthesized nanoparticles were coated with oleic acid, first by a chemisorption mechanism and later by the respective extractant via physical adsorption. The obtained core-shell functionalized magnetite nanoparticle composites were characterized by dynamic light scattering, scanning electron microscopy, transmission electron microscopy, thermogravimetry, infrared absorption and vibrating sample magnetometry. All the prepared nanoparticles exhibited a high saturation magnetization capacity that varied between 72 and 46 emu g-1 and decreased as the magnetite nanoparticle was coated with oleic acid and functionalized. The scope of this study also included adsorption tests for lanthanum, cerium, praseodymium, and neodymium and the corresponding analysis of their results. Sorption tests indicated that the functionalized nanoparticles were able to extract the four studied lanthanide metal ions, although the best extraction performance was observed when the sorbent was functionalized with CYANEX 272, which resulted in a loading capacity of approximately 12-14 mgLa/gMNP. The magnetization of the synthesized nanoparticles was verified during the separation of the lanthanide-loaded sorbent from the raffinate by using a conventional magnet.

  9. Development of a Novel Optical Biosensor for Detection of Organophoshorus Pesticides Based on Methyl Parathion Hydrolase Immobilized by Metal-Chelate Affinity

    Science.gov (United States)

    Lan, Wensheng; Chen, Guoping; Cui, Feng; Tan, Feng; Liu, Ran; Yushupujiang, Maolidan

    2012-01-01

    We have developed a novel optical biosensor device using recombinant methyl parathion hydrolase (MPH) enzyme immobilized on agarose by metal-chelate affinity to detect organophosphorus (OP) compounds with a nitrophenyl group. The biosensor principle is based on the optical measurement of the product of OP catalysis by MPH (p-nitrophenol). Briefly, MPH containing six sequential histidines (6× His tag) at its N-terminal was bound to nitrilotriacetic acid (NTA) agarose with Ni ions, resulting in the flexible immobilization of the bio-reaction platform. The optical biosensing system consisted of two light-emitting diodes (LEDs) and one photodiode. The LED that emitted light at the wavelength of the maximum absorption for p-nitrophenol served as the signal light, while the other LED that showed no absorbance served as the reference light. The optical sensing system detected absorbance that was linearly correlated to methyl parathion (MP) concentration and the detection limit was estimated to be 4 μM. Sensor hysteresis was investigated and the results showed that at lower concentration range of MP the difference got from the opposite process curves was very small. With its easy immobilization of enzymes and simple design in structure, the system has the potential for development into a practical portable detector for field applications. PMID:23012501

  10. Organophosphorus reagents in actinide separations: Unique tools for production, cleanup and disposal

    Energy Technology Data Exchange (ETDEWEB)

    Nash, K. L.

    2000-01-12

    Interactions of actinide ions with phosphate and organophosphorus reagents have figured prominently in nuclear science and technology, particularly in the hydrometallurgical processing of irradiated nuclear fuel. Actinide interactions with phosphorus-containing species impact all aspects from the stability of naturally occurring actinides in phosphate mineral phases through the application of the bismuth phosphate and PUREX processes for large-scale production of transuranic elements to the development of analytical separation and environment restoration processes based on new organophosphorus reagents. In this report, an overview of the unique role of organophosphorus compounds in actinide production, disposal, and environment restoration is presented. The broad utility of these reagents and their unique chemical properties is emphasized.

  11. Organophosphorus reagents in actinide separations: Unique tools for production, cleanup and disposal

    International Nuclear Information System (INIS)

    Nash, K. L.

    2000-01-01

    Interactions of actinide ions with phosphate and organophosphorus reagents have figured prominently in nuclear science and technology, particularly in the hydrometallurgical processing of irradiated nuclear fuel. Actinide interactions with phosphorus-containing species impact all aspects from the stability of naturally occurring actinides in phosphate mineral phases through the application of the bismuth phosphate and PUREX processes for large-scale production of transuranic elements to the development of analytical separation and environment restoration processes based on new organophosphorus reagents. In this report, an overview of the unique role of organophosphorus compounds in actinide production, disposal, and environment restoration is presented. The broad utility of these reagents and their unique chemical properties is emphasized

  12. 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...

  13. Marine Extremophiles: A Source of Hydrolases for Biotechnological Applications

    OpenAIRE

    Dalmaso, Gabriel Zamith Leal; Ferreira, Davis; Vermelho, Alane Beatriz

    2015-01-01

    The marine environment covers almost three quarters of the planet and is where evolution took its first steps. Extremophile microorganisms are found in several extreme marine environments, such as hydrothermal vents, hot springs, salty lakes and deep-sea floors. The ability of these microorganisms to support extremes of temperature, salinity and pressure demonstrates their great potential for biotechnological processes. Hydrolases including amylases, cellulases, peptidases and lipases from hy...

  14. 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.

  15. 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.

  16. A dual tag system for facilitated detection of surface expressed proteins in Escherichia coli

    Directory of Open Access Journals (Sweden)

    Jarmander Johan

    2012-09-01

    Full Text Available Abstract Background The discovery of the autotransporter family has provided a mechanism for surface expression of proteins in laboratory strains of Escherichia coli. We have previously reported the use of the AIDA-I autotransport system to express the Salmonella enterica serovar Enteritidis proteins SefA and H:gm. The SefA protein was successfully exposed to the medium, but the orientation of H:gm in the outer membrane could not be determined due to proteolytic cleavage of the N-terminal detection-tag. The goal of the present work was therefore to construct a vector containing elements that facilitates analysis of surface expression, especially for proteins that are sensitive to proteolysis or otherwise difficult to express. Results The surface expression system pAIDA1 was created with two detection tags flanking the passenger protein. Successful expression of SefA and H:gm on the surface of E. coli was confirmed with fluorescently labeled antibodies specific for the N-terminal His6-tag and the C-terminal Myc-tag. While both tags were detected during SefA expression, only the Myc-tag could be detected for H:gm. The negative signal indicates a proteolytic cleavage of this protein that removes the His6-tag facing the medium. Conclusions Expression levels from pAIDA1 were comparable to or higher than those achieved with the formerly used vector. The presence of the Myc- but not of the His6-tag on the cell surface during H:gm expression allowed us to confirm the hypothesis that this fusion protein was present on the surface and oriented towards the cell exterior. Western blot analysis revealed degradation products of the same molecular weight for SefA and H:gm. The size of these fragments suggests that both fusion proteins have been cleaved at a specific site close to the C-terminal end of the passenger. This proteolysis was concluded to take place either in the outer membrane or in the periplasm. Since H:gm was cleaved to a much greater extent

  17. Simultaneous hydrolysis of carbaryl and chlorpyrifos by Stenotrophomonas sp. strain YC-1 with surface-displayed carbaryl hydrolase.

    Science.gov (United States)

    Yang, Chao; Xu, Xiaoqing; Liu, Yanping; Jiang, Hong; Wu, Yunbo; Xu, Ping; Liu, Ruihua

    2017-10-17

    Many sites are often co-contaminated with multiple pesticides. To date, there are no reports on simultaneous degradation of different classes of pesticides by a natural microorganism. In this work, we aim at constructing a live biocatalyst able to simultaneously hydrolyze carbaryl and chlorpyrifos. For this purpose, carbaryl hydrolase (CH) was displayed on the cell surface of a chlorpyrifos-degrading bacterium Stenotrophomonas sp. strain YC-1 using N- and C-terminal domain of ice nucleation protein (INPNC) from Pseudomonas syringae INA5 as an anchoring motif. The localization of INPNC-CH fusion protein in the outer membrane fraction was demonstrated by cell fractionation followed by Western blot analysis. Surface display of INPNC-CH was further confirmed by proteinase accessibility experiment and immunofluorescence microscope. CH was present in an active form on cell surface without causing any growth inhibition, suggesting that the INP-based display system is a useful tool for surface expression of macromolecular heterologous proteins on the bacterial cell surface. Because surface-displayed CH has free access to pesticides, this bacterium can be used as a whole-cell biocatalyst for efficient hydrolysis of pesticides.

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

    Energy Technology Data Exchange (ETDEWEB)

    Tyler, Ludmila [United States Department of Agriculture (USDA), Western Regional Research Center (WRRC), Albany; Bragg, Jennifer [United States Department of Agriculture (USDA), Western Regional Research Center (WRRC), Albany; Wu, Jiajie [United States Department of Agriculture (USDA), Western Regional Research Center (WRRC), Albany; Yang, Xiaohan [ORNL; Tuskan, Gerald A [ORNL; Vogel, John [United States Department of Agriculture (USDA), Western Regional Research Center (WRRC), Albany

    2010-01-01

    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, both at the whole-genome level and at the level of 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. Examination of individual glycoside hydrolase familes (GH5, GH13, GH18, GH19, GH28, and GH51) revealed both similarities and distinctions between monocots and dicots, as well as between species. Shared evolutionary histories appear to be modified by lineage-specific expansions or deletions. Within 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 the stage for Brachypodium to be a monocot model for investigations of these enzymes and their diverse roles in planta. Insights

  19. Kv7.1 surface expression is regulated by epithelial cell polarization

    DEFF Research Database (Denmark)

    Andersen, Martin N; Olesen, Søren-Peter; Rasmussen, Hanne Borger

    2011-01-01

    The potassium channel K(V)7.1 is expressed in the heart where it contributes to the repolarization of the cardiac action potential. In addition, K(V)7.1 is expressed in epithelial tissues where it plays a role in salt and water transport. Mutations in the kcnq1 gene can lead to long QT syndrome...... and deafness, and several mutations have been described as trafficking mutations. To learn more about the basic mechanisms that regulate K(V)7.1 surface expression, we have investigated the trafficking of K(V)7.1 during the polarization process of the epithelial cell line Madin-Darby Canine Kidney (MDCK) using...... is regulated by signaling mechanisms involved in epithelial cell polarization in particular signaling cascades involving protein kinase C and PI3K....

  20. The surface expression of Inertial Oscillations in the south shore of Oahu, Hawaii

    Science.gov (United States)

    Castillo-Trujillo, A. C.; Flament, P. J.

    2016-12-01

    HF radars are used to analyze the surface expression of Near Inertial Oscillations (NIO) on the south shore of Oahu, Hawaii over a two year period. The spatial coverage and high resolution of HF radars allows us to focus on the spatial structure of the NIOs in relation to the wind forcing and background circulation. Preliminary results show larger NIOs amplitudes in spring months when sporadic southwest wind events are found. These NIOs mostly display a red frequency shift, often associated with anticylonic background vorticity. The characteristics of the observed NIOs will be compared to simulations of the classic slab layer model. We will also present the energy exchange between these NIOs and the background flow.

  1. Surface expression of protein A on magnetosomes and capture of pathogenic bacteria by magnetosome/ antibody complexes

    Directory of Open Access Journals (Sweden)

    Jun eXu

    2014-04-01

    Full Text Available Magnetosomes are membrane-enclosed magnetite nanocrystals synthesized by magnetotactic bacteria (MTB. They display chemical purity, narrow size ranges, and species-specific crystal morphologies. Specific transmembrane proteins are sorted to the magnetosome membrane (MM. MamC is the most abundant MM protein of Magnetospirillum gryphiswaldense strain MSR-1. MamF is the second most abundant MM protein of MSR-1 and forms stable oligomers. We expressed staphylococcal protein A (SPA, an immunoglobulin-binding protein from the cell wall of Staphylococcus aureus, on MSR-1 magnetosomes by fusion with MamC or MamF. The resulting recombinant magnetosomes were capable of self-assembly with the Fc region of mammalian antibodies (Abs and were therefore useful for functionalization of magnetosomes. Recombinant plasmids pBBR-mamC-spa and pBBR-mamF-spa were constructed by fusing spa (the gene that encodes SPA with mamC and mamF, respectively. Recombinant magnetosomes with surface expression of SPA were generated by introduction of these fusion genes into wild-type MSR-1 or a mamF mutant strain. Studies with a Zeta Potential Analyzer showed that the recombinant magnetosomes had hydrated radii significantly smaller than those of WT magnetosomes and zeta potentials less than -30 mV, indicating that the magnetosome colloids were relatively stable. Observed conjugation efficiencies were as high as 71.24 µg Ab per mg recombinant magnetosomes, and the conjugated Abs retained most of their activity. Numbers of Vibrio parahaemolyticus (a common pathogenic bacterium in seafood captured by recombinant magnetosome/ Ab complexes were measured by real-time fluorescence-based quantitative PCR. One mg of complex was capable of capturing as many as 1.74×107 Vibrio cells. The surface expression system described here will be useful for design of functionalized magnetosomes from MSR-1 and other MTB.

  2. Inflammation promotes oral squamous carcinoma immune evasion via induced programmed death ligand-1 surface expression.

    Science.gov (United States)

    Lu, Wanlu; Lu, Libing; Feng, Yun; Chen, Jiao; Li, Yan; Kong, Xiangli; Chen, Sixiu; Li, Xiaoyu; Chen, Qianming; Zhang, Ping

    2013-05-01

    The association between inflammation and cancer provides a new target for tumor biotherapy. The inflammatory cells and molecules within the tumor microenvironment have decisive dual roles in antitumor immunity and immune evasion. In the present study, phytohemagglutinin (PHA) was used to stimulate peripheral blood mononuclear cells (PBMCs) to simulate the tumor inflammatory microenvironment. The effect of immune cells and inflammatory cytokines on the surface expression of programmed cell death-1 ligand 1 (PD-L1) and tumor immune evasion was investigated using flow cytometry (FCM) and an in vivo xenotransplantation model. Based on the data, PHA-activated, but not resting, immune cells were able to promote the surface expression of PD-L1 in Tca8113 oral squamous carcinoma cells via the secretion of inflammatory cytokines, but not by cell-cell contact. The majority of the inflammatory cytokines had no significant effect on the proliferation, cell cycle progression and apoptosis of the Tca8113 cells, although they each induced the expression of PD-L1 in a dose-dependent manner. In total, 99% of the Tca8113 cells expressed PD-L1 following treatment with the supernatant of PHA-stimulated PBMCs. The PHA-supernatant pretreated Tca8113 cells unusually induced Tca8113 antigen-specific CD8 + T cell apoptosis in vitro and the evasion of antigen-specific T cell attraction in a nude mouse tumor-bearing model. These results indicate a new mechanism for the promotion of tumor immune evasion by the tumor inflammatory microenvironment.

  3. The surface expression of HLA-F on decidual trophoblasts increases from mid to term gestation.

    Science.gov (United States)

    Shobu, Takanori; Sageshima, Noriko; Tokui, Hiroshi; Omura, Motoko; Saito, Keigo; Nagatsuka, Yuka; Nakanishi, Mari; Hayashi, Yukio; Hatake, Katsuhiko; Ishitani, Akiko

    2006-12-01

    HLA-F has recently only begun to be studied in earnest, and has been thought not to be expressed on the cell surface. However, in our previous report, we demonstrated surface expression of HLA-F on extravillous trophoblasts (EVTs) invading the decidua in term placental tissues. To better understand its function, we attempted to determine when surface expression of HLA-F begins during normal pregnancy, and whether there is a difference in expression between normal and preeclamptic placentas, by comparing the expression of HLA-G and -E by immunohistochemical staining with anti-HLA-E, -F and -G antibodies (3D12, 3D11 and 87G, respectively). In EVTs, HLA-F was expressed only in the cytoplasm weakly during the first trimester, after which expression increased and moved to the cell surface with the progression of pregnancy from the second trimester, which was confirmed by the results of double-labeled immunofluorescence staining with anti-HLA-F and anti-HLA-G antibodies, and by flow cytometry using trophoblasts isolated from the decidua. HLA-E showed similar expression as HLA-F, though it was expressed on the cell surface from the first trimester, while HLA-G was expressed strongly in the cytoplasm and on the cell surface during all stages of pregnancy. The expressions of HLA-E, -F and -G in preeclamptic placentas were not different from those in normal placentas, though there were a greater number of necrotic EVTs in preeclampsia. The increase in expression of HLA-E and HLA-F from the second trimester to full term was coincident with the timing of rapid growth of the fetus. Our results suggest that these may function together to prepare an environment that supports fetal growth.

  4. In Vivo Delivery and Activation of Masked Fluorogenic Hydrolase Substrates by Endogenous Hydrolases in C. elegans.

    Science.gov (United States)

    Dube, Shataakshi; Dube, Hitesh; Green, Nicole B; Larsen, Erik M; White, Alex; Johnson, R Jeremy; Kowalski, Jennifer R

    2017-09-19

    Protein expression and localization are often studied in vivo by tagging molecules with green fluorescent protein (GFP), yet subtle changes in protein levels are not easily detected. To develop a sensitive in vivo method to amplify fluorescence signals and allow cell-specific quantification of protein abundance changes, we sought to apply an enzyme-activated cellular fluorescence system in vivo by delivering ester-masked fluorophores to Caenorhabditis elegans neurons expressing porcine liver esterase (PLE). To aid uptake into sensory neuron membranes, we synthesized two novel fluorogenic hydrolase substrates with long hydrocarbon tails. Recombinant PLE activated these fluorophores in vitro. In vivo activation occurred in sensory neurons, along with potent activation in intestinal lysosomes quantifiable by imaging and microplate and partially attributable to gut esterase 1 (GES-1) activity. These data demonstrate the promise of biorthogonal hydrolases and their fluorogenic substrates as in vivo neuronal imaging tools and for characterizing endogenous C. elegans hydrolase substrate specificities. © 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

  5. QSAR for cholinesterase inhibition by organophosphorus esters and CNDO/2 calculations for organophosphorus ester hydrolysis. [quantitative structure-activity relationship, complete neglect of differential overlap

    Science.gov (United States)

    Johnson, H.; Kenley, R. A.; Rynard, C.; Golub, M. A.

    1985-01-01

    Quantitative structure-activity relationships were derived for acetyl- and butyrylcholinesterase inhibition by various organophosphorus esters. Bimolecular inhibition rate constants correlate well with hydrophobic substituent constants, and with the presence or absence of cationic groups on the inhibitor, but not with steric substituent constants. CNDO/2 calculations were performed on a separate set of organophosphorus esters, RR-primeP(O)X, where R and R-prime are alkyl and/or alkoxy groups and X is fluorine, chlorine or a phenoxy group. For each subset with the same X, the CNDO-derived net atomic charge at the central phosphorus atom in the ester correlates well with the alkaline hydrolysis rate constant. For the whole set of esters with different X, two equations were derived that relate either charge and leaving group steric bulk, or orbital energy and bond order to the hydrolysis rate constant.

  6. Presence of organophosphorus pesticide oxygen analogs in air samples

    Science.gov (United States)

    Armstrong, Jenna L.; Fenske, Richard A.; Yost, Michael G.; Galvin, Kit; Tchong-French, Maria; Yu, Jianbo

    2013-02-01

    A number of recent toxicity studies have highlighted the increased potency of oxygen analogs (oxons) of several organophosphorus (OP) pesticides. These findings were a major concern after environmental oxons were identified in environmental samples from air and surfaces following agricultural spray applications in California and Washington State. This paper reports on the validity of oxygen analog measurements in air samples for the OP pesticide, chlorpyrifos. Controlled environmental and laboratory experiments were used to examine artificial formation of chlorpyrifos-oxon using OSHA Versatile Sampling (OVS) tubes as recommended by NIOSH method 5600. Additionally, we compared expected chlorpyrifos-oxon attributable to artificial transformation to observed chlorpyrifos-oxon in field samples from a 2008 Washington State Department of Health air monitoring study using non-parametric statistical methods. The amount of artificially transformed oxon was then modeled to determine the amount of oxon present in the environment. Toxicity equivalency factors (TEFs) for chlorpyrifos-oxon were used to calculate chlorpyrifos-equivalent air concentrations. The results demonstrate that the NIOSH-recommended sampling matrix (OVS tubes with XAD-2 resin) was found to artificially transform up to 30% of chlorpyrifos to chlorpyrifos-oxon, with higher percentages at lower concentrations (<30 ng m-3) typical of ambient or residential levels. Overall, the 2008 study data had significantly greater oxon than expected by artificial transformation, but the exact amount of environmental oxon in air remains difficult to quantify with the current sampling method. Failure to conduct laboratory analysis for chlorpyrifos-oxon may result in underestimation of total pesticide concentration when using XAD-2 resin matrices for occupational or residential sampling. Alternative methods that can accurately measure both OP pesticides and their oxygen analogs should be used for air sampling, and a toxicity

  7. Degradation of organophosphorus compounds by X-ray irradiation

    International Nuclear Information System (INIS)

    Trebse, P.; Arcon, I.

    2002-01-01

    Complete text of publication follows. Our research presented in this contribution aims to elucidate the degradation mechanisms of organophosphate pesticides as organic pollutants by X-ray irradiation. Diazinon, one of the most widely used pesticides in Slovenia, has been chosen as a model compound for the study of radiation induced degradation of organophosphorus pesticides. Recent studies have shown that the degradation of the pesticide at normal conditions (room temperature, exposure to daylight) is strongly enhanced when the pesticide is exposed to UV light (Hg lamp, λ=254 nm, XeCl excimer laser, λ=308 nm). In our study we irradiated the pesticide in aqueous media with a white x-ray beam from a conventional x-ray source. Mo X-ray tube operating at the high voltage of 55 kV and a current of 45 mA was used. The flux of the continuous X-ray beam was stabilized within 1%. Saturated water solution of the pesticide (volume: 4mL, conc. of 40 mg L -1 ) was inserted in 1 cm long lucite cell with 1 mm thick lucite windows. The whole volume of the solution in the cell was exposed to the unfiltered X-ray beam. The dose rate on the sample was about 1 mGy/s. Different irradiation times between 30 min to 120 min were chosen to study the dependence of the pesticide decomposition with the absorbed dose. Solid phase extraction was employed for sample extraction from the solution, and gas chromatography was used for the identification and quantification of the compounds. The results show that the concentration of the pesticide in the solution decreases exponentially with the exposure time, i.e. with the absorbed dose. At irradiation conditions described above, the time constant of the exponential decrease was 74 min

  8. Organophosphorus flame retardants – Toxicity and influence on human health

    Directory of Open Access Journals (Sweden)

    Elżbieta Bruchajzer

    2015-06-01

    Full Text Available Organophosphorus flame retardants (flame retardants, FRs have been used for several decades in many industries, including the production of dyes, varnishes, adhesives, synthetic resins, polyvinyl chloride, hydraulic fluids, plastics and textiles. Their importance in recent times has increased due to i.a., significantly reduced use of polybrominated diphenyl ethers (PBDEs – persistent organic pollutants, dangerous for the environment. The aim of this study was to review the available literature data concerning phosphorous FRs primarily for neurotoxic, fertility, reproductive and carcinogenic effects. The analysis concerned the following most commonly used substances: tris(2-ethylhexylphosphate (TEHP, tris(2-butoxyethylphosphate (TBEP, triphenyl phosphate (TPP, tris(2-chloroethylphosphate (TCEP, tetrakis(hydroxymethyl-phosphonium chloride (THPC, tributyl phosphate (TBP, tricresyl phosphate (TCP, tris(2-chloroisopropylphosphate (TCPP, tris(1,3-dichloroisopropylphosphate (TDCP and tetrakis(hydroxymethyl phosphonium sulphate (THPS. In animal studies neurotoxic effects were found after exposure to TBEP, THPC, TBP and TCP, while in humans they were observed only after exposure to TCP. TCEP, THPS, TBP, TCP and TDCP caused disorders in fertility and/or fetal development of animals. Adverse effects on reproduction in humans may be caused by TPP, TCP, and TDCP. In laboratory animals the development of tumors was observed after high doses of TEHP, TCEP, TBP and TDCP. None of these compounds is classified as a human carcinogen. The environmental toxicity of phosphate FRs is low (except for TPP, TCEP and TBEP. They are not stable compounds, in living organisms they are metabolised and quickly excreted. Therefore, they can be used as an alternative to PBDEs. Med. Pr. 2015;66(2:235–264

  9. Effects of Organophosphorus Flame Retardants on Spontaneous Activity in Neuronal Networks Grown on Microelectrode Arrays

    Science.gov (United States)

    EFFECTS OF ORGANOPHOSPHORUS FLAME RETARDANTS ON SPONTANEOUS ACTIVITY IN NEURONAL NETWORKS GROWN ON MICROELECTRODE ARRAYS TJ Shafer1, K Wallace1, WR Mundy1, M Behl2,. 1Integrated Systems Toxicology Division, NHEERL, USEPA, RTP, NC, USA, 2National Toxicology Program, NIEHS, RTP, NC...

  10. Determination of organophosphorus acids by thermo-spray liquid chromatography-mass spectrometry

    NARCIS (Netherlands)

    Wils, E.R.J.; Hulst, A.G.

    1988-01-01

    The determination of thirteen organophosphorus acids, hydrolysis products of nerve agents and pesticides, by a combination of ion-pair liquid chromatography on a reversed-phase C18 column and thermospray mass spectrometry was investigated. Ammonium acetate and three tetraalkylammonium salts with

  11. Assessing the Developmental Neurotoxicity of 27 Organophosphorus Pesticides Using a Zebrafish Behavioral Assay

    Science.gov (United States)

    Assessing the Developmental Neurotoxicity of 27 Organophosphorus Pesticides Using a Zebrafish Behavioral Assay, Waalkes, M., Hunter, D.L., Jarema, K., Mundy, W., and S. Padilla. The U.S. Environmental Protection Agency is evaluating methods to screen and prioritize organophosphor...

  12. Mineralisation and primary biodegradation of aromatic organophosphorus flame retardants in activated sludge

    NARCIS (Netherlands)

    Jurgens, S.S.; Helmus, R.; Waaijers, S.L.; Uittenbogaard, D.; Dunnebier, D.; Vleugel, M.; Kraak, M.H.S.; de Voogt, P.; Parsons, J.R.

    2014-01-01

    Halogen-free flame retardants (HFFRs), such as the aromatic organophosphorus flame retardants (OPFRs) triphenyl phosphate (TPHP), resorcinol bis(diphenylphosphate) (PBDPP) and bisphenol A bis(diphenylphosphate) (BPA-BDPP) have been proposed as potential replacements for brominated flame retardants

  13. RESEARCH OF HEAVY METALS, ORGANOCHLORINE AND ORGANOPHOSPHORUS PESTICIDES IN POWDERED INFANT FORMULA

    Directory of Open Access Journals (Sweden)

    M.C. Abete

    2009-02-01

    Full Text Available During the period between october 2007 and november 2008 were collected 60 samples of powdered infant formula. The analysis for the detection of heavy metals, organochlorine and organophosphorus pesticides show that the environmental situation is under control and powdered infant formula satisfies this health requisite.

  14. Dietary cumulative acute risk assessment of organophosphorus, carbamates and pyrethroids insecticides for the Brazilian population.

    NARCIS (Netherlands)

    Jardim, Andreia Nunes Oliveira; Brito, Alessandra Page; van Donkersgoed, Gerda; Boon, Polly E; Caldas, Eloisa Dutra

    Cumulative acute dietary risk assessments of organophosphorus (OPs), carbamates (CBs) and pyrethroids (PYs) were conducted for the Brazilian population. Residue data for 30786 samples of 30 foods were obtained from two national monitoring programs and one University laboratory, and consumption data

  15. Cumulative risk assessment of the exposure to organophosphorus and carbamate insecticides in the dutch diet

    NARCIS (Netherlands)

    Boon, P.E.; Voet, van der H.; Raaij, van M.T.M.; Klaveren, van J.D.

    2008-01-01

    We report the acute cumulative exposure to organophosphorus insecticides (OPs) and carbamates in the Dutch population and young children (1-6 years) via the diet. Residue data were derived from Dutch monitoring programmes performed during 2003-2005, and food consumption levels from the Dutch

  16. Small-scale purification of butyrylcholinesterase from human plasma and implementation of a μLC-UV/ESI MS/MS method to detect its organophosphorus adducts.

    Science.gov (United States)

    John, Harald; Breyer, Felicitas; Schmidt, Christian; Mizaikoff, Boris; Worek, Franz; Thiermann, Horst

    2015-10-01

    Human butyrylcholinesterase (hBChE) is a serine hydrolase (EC 3.1.1.8) present in all mammalian tissues and the bloodstream. Similar to acetylcholinesterase, the enzyme reacts with organophosphorus compounds (OP) like nerve agents or pesticides that cause enzyme inhibition (BChE adducts). These adducts represent valuable biomarkers for analytical verification of OP exposure. For establishment of these mass spectrometry based methods sufficient amounts of hBChE in high purity are required. Unfortunately, commercial lots are of inappropriate purity thus favouring in-house isolation. Therefore, we developed a small scale procedure to isolate hBChE from citrate plasma. After precipitation by polyethylene glycol (8% w/v and 20% w/v PEG 6000) hBChE was purified from plasma by four consecutive chromatographic steps including anion exchange, affinity extraction and size exclusion. Protein elution was monitored on-line by UV-absorbance (280 nm) followed by continuous fractionation for off-line analysis of (1) hBChE enzyme activity by Ellman assay, (2) protein purity by gel electrophoresis, and (3) protein identity by matrix-assisted laser desorption/ionization mass spectrometry (MALDI MS). Numerous major impurities separated from hBChE were identified. The purified material was used for in vitro incubation with diverse OP to establish a μ-liquid chromatography-ultra violet detection/electrospray ionization tandem-mass spectrometric method (μLC-UV/ESI MS/MS) for detection of hBChE adducts suitable for verification analysis. Analytical data for diverse OP pesticides including deuterated analogues as well as G- and V-type nerve agents and their precursor are summarized. This method was successfully applied to plasma samples provided by the Organisation for the Prohibition of Chemical Weapons (OPCW) for the 4th Biomedical Exercise. Copyright © 2015 John Wiley & Sons, Ltd.

  17. Conversion of cyclodextrin glycosyltransferase into a starch hydrolase by directed evolution : The role of alanine 230 in acceptor subsite+1

    NARCIS (Netherlands)

    Leemhuis, Hans; Rozeboom, Henriëtte J.; Wilbrink, Maarten; Euverink, Gert-Jan W.; Dijkstra, Bauke W.; Dijkhuizen, Lubbert

    2003-01-01

    Cycloclextrin glycosyltransferase (CGTase) preferably catalyzes transglycosylation reactions, whereas many other alpha-amylase family enzymes are hydrolases. Despite the availability of three-dimensional structures of several transglycosylases and hydrolases of this family, the factors that

  18. A new group of glycoside hydrolase family 13 α-amylases with an aberrant catalytic triad

    NARCIS (Netherlands)

    Sarian, Fean D; Janeček, Štefan; Pijning, Tjaard; Ihsanawati,; Nurachman, Zeily; Radjasa, Ocky K; Dijkhuizen, Lubbert; Natalia, Dessy; van der Maarel, Marc J E C

    2017-01-01

    α-Amylases are glycoside hydrolase enzymes that act on the α(1→4) glycosidic linkages in glycogen, starch, and related α-glucans, and are ubiquitously present in Nature. Most α-amylases have been classified in glycoside hydrolase family 13 with a typical (β/α)8-barrel containing two aspartic acid

  19. A polymorphism in the gene for microsomal epoxide hydrolase is associated with pre-eclampsia.

    NARCIS (Netherlands)

    Zusterzeel, P.L.M.; Peters, W.H.M.; Visser, W.A.; Hermsen, K.J.; Roelofs, H.M.J.; Steegers, E.A.P.

    2001-01-01

    OBJECTIVE: Microsomal epoxide hydrolase is an important enzyme involved in the metabolism of endogenous and exogenous toxicants. Polymorphic variants of the human epoxide hydrolase gene vary in enzyme activity. We determined whether genetic variability in the gene encoding for microsomal epoxide

  20. Salmonella regulates polyubiquitination and surface expression of MHC class II antigens.

    Science.gov (United States)

    Lapaque, Nicolas; Hutchinson, James L; Jones, Des C; Méresse, Stéphane; Holden, David W; Trowsdale, John; Kelly, Adrian P

    2009-08-18

    Salmonella typhimurium is a facultative pathogen capable of entering and replicating in both professional and non-professional antigen presenting cells. Control of infection requires MHC class II restricted CD4 T-helper cell responses. Here we show that Salmonella infection induced polyubiquitination of HLA-DR, a post-translational modification that led to removal of mature, peptide loaded, alphabeta dimers from the cell surface. Immature alphabetaIi complexes were unaffected. Surface expression of all class II isotypes, HLA-DP, -DQ, and -DR, was reduced in infected cells, but other cell-surface molecules that traffic through class II peptide loading compartments were unaffected. A Salmonella strain carrying a mutation in ssaV did not induce ubiquitination of class II, implicating Salmonella T3SS-2 effector proteins in the process. T3SS-2 effectors, with established or proposed roles in ubiquitination, were not required for class II down-regulation, suggesting that an additional T3SS-2 effector is involved in regulating MHC class II ubiquitination. Although recognized as a viral immune evasion strategy, here, we demonstrate that bacteria can control surface MHC expression through ubiquitination.

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

    Science.gov (United States)

    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.

  2. Degradation of Polyester Polyurethane by Bacterial Polyester Hydrolases

    Directory of Open Access Journals (Sweden)

    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.

  3. Marine extremophiles: a source of hydrolases for biotechnological applications.

    Science.gov (United States)

    Dalmaso, Gabriel Zamith Leal; Ferreira, Davis; Vermelho, Alane Beatriz

    2015-04-03

    The marine environment covers almost three quarters of the planet and is where evolution took its first steps. Extremophile microorganisms are found in several extreme marine environments, such as hydrothermal vents, hot springs, salty lakes and deep-sea floors. The ability of these microorganisms to support extremes of temperature, salinity and pressure demonstrates their great potential for biotechnological processes. Hydrolases including amylases, cellulases, peptidases and lipases from hyperthermophiles, psychrophiles, halophiles and piezophiles have been investigated for these reasons. Extremozymes are adapted to work in harsh physical-chemical conditions and their use in various industrial applications such as the biofuel, pharmaceutical, fine chemicals and food industries has increased. The understanding of the specific factors that confer the ability to withstand extreme habitats on such enzymes has become a priority for their biotechnological use. The most studied marine extremophiles are prokaryotes and in this review, we present the most studied archaea and bacteria extremophiles and their hydrolases, and discuss their use for industrial applications.

  4. Marine Extremophiles: A Source of Hydrolases for Biotechnological Applications

    Directory of Open Access Journals (Sweden)

    Gabriel Zamith Leal Dalmaso

    2015-04-01

    Full Text Available The marine environment covers almost three quarters of the planet and is where evolution took its first steps. Extremophile microorganisms are found in several extreme marine environments, such as hydrothermal vents, hot springs, salty lakes and deep-sea floors. The ability of these microorganisms to support extremes of temperature, salinity and pressure demonstrates their great potential for biotechnological processes. Hydrolases including amylases, cellulases, peptidases and lipases from hyperthermophiles, psychrophiles, halophiles and piezophiles have been investigated for these reasons. Extremozymes are adapted to work in harsh physical-chemical conditions and their use in various industrial applications such as the biofuel, pharmaceutical, fine chemicals and food industries has increased. The understanding of the specific factors that confer the ability to withstand extreme habitats on such enzymes has become a priority for their biotechnological use. The most studied marine extremophiles are prokaryotes and in this review, we present the most studied archaea and bacteria extremophiles and their hydrolases, and discuss their use for industrial applications.

  5. Acetylcarnitine hydrolase activity in bovine caudal epididymal spermatozoa

    Energy Technology Data Exchange (ETDEWEB)

    Bruns, K.; Foster, R.A.; Casillas, E.R.

    1986-05-01

    Recently, the authors identified mM concentrations of acetylcarnitine in epidiymal fluids and have investigated the metabolism of acetylcarnitine by bovine and hamster caudal epididymal spermatozoa. (1-/sup 14/C)acetyl-L-carnitine is oxidized to /sup 14/CO/sub 2/ by washed, intact hamster and bovine sperm at maximal rates of 8.4 and 15.2 nmol/hr/10/sup 7/ cells respectively. Conversely, the carnitine moiety of acetyl-L-(/sup 3/H-methyl)carnitine is not accumulated by sperm under similar conditions. Hydrolysis of (/sup 3/H)acetyl-L-carnitine and competition of uptake of (/sup 3/H)acetate by unlabeled acetate was demonstrated in incubations of intact cells of both species. The amount of (/sup 3/H)acetate accumulated in the incubation medium is time-dependent and also depends on the concentration of unlabeled acetate. A partial solubilization of acetylcarnitine hydrolase activity from washed, intact bovine caudal epididymal spermatozoa in buffer or 0.01% Triton X-100 is observed. There is an enrichment of acetylcarnitine hydrolase activity in purified plasma membranes from bovine caudal epididymal spermatozoa when compared to the activity present in broken cell preparations or other cellular fractions. The results suggest that acetylcarnitine is a substrate for spermatozoa as they traverse the epididymis.

  6. Cell-surface expression of Hsp70 on hematopoietic cancer cells after inhibition of HDAC activity

    DEFF Research Database (Denmark)

    Jensen, Helle

    frequently express Hsp70 on their cell surface, whereas the corresponding normal tissues do not. In addition, several clinically applied reagents, such as alkyl-lysophospholipides, chemotherapeutic agents, and anti-inflammatory reagents, have been found to enhance Hsp70 cell surface expression on cancer...

  7. Flumazenil decreases surface expression of α4β2δ GABAA receptors by increasing the rate of receptor internalization.

    Science.gov (United States)

    Kuver, Aarti; Smith, Sheryl S

    2016-01-01

    Increases in expression of α4βδ GABAA receptors (GABARs), triggered by fluctuations in the neurosteroid THP (3α-OH-5α[β]-pregnan-20-one), are associated with changes in mood and cognition. We tested whether α4βδ trafficking and surface expression would be altered by in vitro exposure to flumazenil, a benzodiazepine ligand which reduces α4βδ expression in vivo. We first determined that flumazenil (100 nM-100 μM, IC50=∼1 μM) acted as a negative modulator, reducing GABA (10 μM)-gated current in the presence of 100 nM THP (to increase receptor efficacy), assessed with whole cell patch clamp recordings of recombinant α4β2δ expressed in HEK-293 cells. Surface expression of recombinant α4β2δ receptors was detected using a 3XFLAG reporter at the C-terminus of α4 (α4F) using confocal immunocytochemical techniques following 48 h exposure of cells to GABA (10 μM)+THP (100 nM). Flumazenil (10 μM) decreased surface expression of α4F by ∼60%, while increasing its intracellular accumulation, after 48 h. Reduced surface expression of α4β2δ after flumazenil treatment was confirmed by decreases in the current responses to 100 nM of the GABA agonist gaboxadol. Flumazenil-induced decreases in surface expression of α4β2δ were prevented by the dynamin blocker, dynasore, and by leupeptin, which blocks lysosomal enzymes, suggesting that flumazenil is acting to increase endocytosis and lysosomal degradation of the receptor. Flumazenil increased the rate of receptor removal from the cell surface by 2-fold, assessed using botulinum toxin B to block insertion of new receptors. These findings may suggest new therapeutic strategies for regulation of α4β2δ expression using flumazenil. Copyright © 2015 Elsevier Inc. All rights reserved.

  8. Surface expression of intraplate postglacial faults in Sweden: from LiDAR data

    Science.gov (United States)

    Abduljabbar, Mawaheb; Ask, Maria; Bauer, Tobias; Lund, Björn; Smith, Colby; Mikko, Henrik; Munier, Raymond

    2016-04-01

    Large intraplate earthquakes, up to magnitude 8.0±0.3 (Lindblom et al. 2015) are inferred to have occurred in northern Fennoscandia at the end of, or just after the Weichselian deglaciation. More than a dozen large so-called postglacial faults (PGF) have been found in the region. The present-day microseismic activity is rather high in north Sweden, and there is a correlation between microseismicity and mapped PGF scarps: 71% of the observed earthquakes north of 66°N locate within 30 km to the southeast and 10 km to the northwest of PGFs (Lindblom et al., 2015). Surface expressions of PGFs in Sweden have mainly been mapped using aerial photogrammetry and trenching (e.g. Lagerbäck & Sundh 2008). Their detailed surface geometry may be investigated using the new high-resolution elevation model of Sweden (NNH) that has a vertical- and lateral resolution of 2 m and 0.25 m, respectively. With NNH data, known PGFs have been modified, and a number of new potential PGFs have been identified (Smith et al. 2014; Mikko et al. 2015). However, the detailed variation of their surface expression remains to be determined. Our main objective is to constrain the strike and surface offset (i.e., apparent vertical throw because of soil cover overlays the bedrock) across the PGF scarps. We anticipate using the results to constrain direction of fault motion and paleomagnitudes of PGFs, and in numerical analyzes to investigate the nature of PGFs. We have developed a methodology for analyzing PGF-geomorphology from LiDAR data using two main software platforms (Ask et al. 2015): (1) Move2015 by Midland Valley has been used for constructing 3D models of the surface traces of the PGFs to determine apparent vertical throw. The apparent hanging- and footwall cut off lines are digitized, and subsequent computation of coordinates is rather time efficient and provide continuous data of fault and soil geomorphology that can be statistically analyzed; and (2) ArcGIS 10.3 by Esri has mostly been

  9. Kettle holes formed by glacial outburst floods: identification when their surface expression has been removed?

    Science.gov (United States)

    Marren, Philip; Fay, Helen; Duller, Robert

    2014-05-01

    Kettle holes and obstacle marks formed by the transport, deposition and burial of ice-blocks during glacial outburst floods (jökulhlaups) are a common geomorphological feature on proglacial outwash plains. Indeed, they represent one of the few features which can unequivocally identify glacially-sourced flood deposits in the geomorphological and sedimentary record. Despite an abundance of work on the surface expression of jökulhlaup-generated ice-block structures, descriptions of the subsurface expression of these features in the sedimentary record are limited. There is currently no comprehensive model of the sedimentary characteristics of these features. This is a major gap in our knowledge, as the positive identification of ice-block features constitutes an unambiguous criterion for the identification of former jökulhlaup deposits in the Quaternary sedimentary record. We address this by describing several examples of ice-block impact in the sedimentary record from southern Iceland. Our work recognizes key criteria for the identification of ice-block impact in the sedimentary record, enabling them to be identified in sedimentary sections where their geomorphological expression has since been removed or buried. These key criterion combine: (1) structures formed by the interaction of water flow with the ice-block body during transportation and immobilization; (2) distinctive sedimentological features of surrounding deposits; and, (3) the post-burial mechanical disruption on the deposits. Formulating a suite of key criteria with which to positively identify the sedimentary impact of ice-blocks limits the possibility of misidentification in the sedimentary record, and provides a means of identifying previously unrecognized Quaternary catastrophic glacial floods.

  10. N-linked glycans are required on epithelial Na+ channel subunits for maturation and surface expression.

    Science.gov (United States)

    Kashlan, Ossama B; Kinlough, Carol L; Myerburg, Michael M; Shi, Shujie; Chen, Jingxin; Blobner, Brandon M; Buck, Teresa M; Brodsky, Jeffrey L; Hughey, Rebecca P; Kleyman, Thomas R

    2018-03-01

    Epithelial Na + channel (ENaC) subunits undergo N-linked glycosylation in the endoplasmic reticulum where they assemble into an αβγ complex. Six, 13, and 5 consensus sites (Asn-X-Ser/Thr) for N-glycosylation reside in the extracellular domains of the mouse α-, β-, and γ-subunits, respectively. Because the importance of ENaC N-linked glycans has not been fully addressed, we examined the effect of preventing N-glycosylation of specific subunits on channel function, expression, maturation, and folding. Heterologous expression in Xenopus oocytes or Fischer rat thyroid cells with αβγ-ENaC lacking N-linked glycans on a single subunit reduced ENaC activity as well as the inhibitory response to extracellular Na + . The lack of N-linked glycans on the β-subunit also precluded channel activation by trypsin. However, channel activation by shear stress was N-linked glycan independent, regardless of which subunit was modified. We also discovered that the lack of N-linked glycans on any one subunit reduced the total and surface levels of cognate subunits. The lack of N-linked glycans on the β-subunit had the largest effect on total levels, with the lack of N-linked glycans on the γ- and α-subunits having intermediate and modest effects, respectively. Finally, channels with wild-type β-subunits were more sensitive to limited trypsin proteolysis than channels lacking N-linked glycans on the β-subunit. Our results indicate that N-linked glycans on each subunit are required for proper folding, maturation, surface expression, and function of the channel.

  11. Calcium uptake by sarcoplasmic reticulum in the presence of organophosphorus insecticide methyl-parathion

    International Nuclear Information System (INIS)

    Blasiak, J.

    1995-01-01

    Using an isotope labelling technique it has been shown that an organophosphorus insecticide methyl parathion (0,0-diethyl 0-4-nitrophenyl phosphorothionate) depressed calcium uptake by sarcoplasmic reticulum isolated from rabbit hind leg muscle. The effect was significant for insecticide concentrations of 50 and 100 μM and was dose-dependent. The insecticide exerted a more pronounced effect on calcium uptake in the presence of ATP in the reticulum environment than in the absence of ATP. The inhibitory action of methyl parathion on Ca 2+ accumulation by sarcoplasmic reticulum can cause a rise in myoplasmic free Ca 2+ , the essential prerequisite for contracture activation. Because methyl parathion, as well as other organophosphorus insecticides, is primarily neurotoxic, evidence of non-specific effect could be important for assessing its environmental safety. (author). 20 refs, 2 figs

  12. Cumulative risk assessment of the intake of organophosphorus and carbamate pesticides in the Danish diet

    DEFF Research Database (Denmark)

    Jensen, A. F.; Petersen, Annette; Granby, Kit

    2003-01-01

    Reference Dose (ARfD) nor ADI was exceeded for any of the compounds studied. The results indicate that the Danish population is neither exposed to any cumulative chronic risk, nor at risk of acute exposure, from consumption of organophosphorus and carbamate pesticides from fruit, vegetables and cereals....... in the Danish nation-wide food consumption survey in 1995. The pesticide data are based on the Danish pesticide residue-monitoring programme from 1996-2001. The amount of 35 organophosphorus pesticides and carbamates were included in the cumulative risk assessment. Processing factors, such as reduction...... fruit, vegetables and cereals is for adults 0.8-2% of the Acceptable Daily Intake (ADI) in chlorpyrifos equivalents, and 0.03-11% of the ADI in methamidophos equivalents; and for children 2-5% of the ADI in the chlorpyrifos equivalents, and 0.07-27% of the ADI in methamidophos equivalents. Neither Acute...

  13. Sorafenib has soluble epoxide hydrolase inhibitory activity, which contributes to its effect profile in vivo.

    Science.gov (United States)

    Liu, Jun-Yan; Park, See-Hyoung; Morisseau, Christophe; Hwang, Sung Hee; Hammock, Bruce D; Weiss, Robert H

    2009-08-01

    The advent of multikinase inhibitors targeting the vascular endothelial growth factor (VEGF) receptor has revolutionized the treatment of highly angiogenic malignancies such as renal cell carcinoma. Interestingly, several such inhibitors are commercially available, and they each possess diverse specific beneficial and adverse effect profiles. In examining the structure of sorafenib, it was hypothesized that this compound would possess inhibitory effects on the soluble epoxide hydrolase, an enzyme with pleiotropic effects on inflammation and vascular disease. We now show that sorafenib but not another VEGF receptor targeted inhibitor sunitinib is a potent inhibitor of the human soluble epoxide hydrolase in vitro (K(I) = 17 +/- 4 nmol/L). Furthermore, sorafenib causes the expected in vivo shift in oxylipid profile resulting from soluble epoxide hydrolase inhibition, evidence of a reduction in the acute inflammatory response. Lipopolysaccharide-induced hypotension was reversed with sorafenib but not sunitinib treatment, suggesting that soluble epoxide hydrolase inhibition accounts for at least part of the anti-inflammatory effect of sorafenib. The pharmacokinetic studies presented here in light of the known potency of sorafenib as a soluble epoxide hydrolase inhibitor indicate that the soluble epoxide hydrolase will be largely inhibited at therapeutic doses of sorafenib. Thus, it is likely that soluble epoxide hydrolase inhibition contributes to the beneficial effects from the inhibition of the VEGF receptor and other kinases during treatment with sorafenib.

  14. Expanding the Cyanuric Acid Hydrolase Protein Family to the Fungal Kingdom

    Science.gov (United States)

    Dodge, Anthony G.; Preiner, Chelsea S.

    2013-01-01

    The known enzymes that open the s-triazine ring, the cyanuric acid hydrolases, have been confined almost exclusively to the kingdom Bacteria and are all homologous members of the rare cyanuric acid hydrolase/barbiturase protein family. In the present study, a filamentous fungus, Sarocladium sp. strain CA, was isolated from soil by enrichment culturing using cyanuric acid as the sole source of nitrogen. A reverse-genetic approach identified a fungal cyanuric acid hydrolase gene composed of two exons and one intron. The translated spliced sequence was 39 to 53% identical to previously characterized bacterial cyanuric acid hydrolases. The sequence was used to generate a gene optimized for expression in Escherichia coli and encoding an N-terminally histidine-tagged protein. The protein was purified by nickel affinity and anion-exchange chromatography. The purified protein was shown by 13C nuclear magnetic resonance (13C-NMR) to produce carboxybiuret as the product, which spontaneously decarboxylated to yield biuret and carbon dioxide. The protein was very narrow in substrate specificity, showing activity only with cyanuric acid and N-methyl cyanuric acid. Barbituric acid was an inhibitor of enzyme activity. Sequence analysis identified genes with introns in other fungi from the Ascomycota that, if spliced, are predicted to encode proteins with cyanuric acid hydrolase activity. The Ascomycota cyanuric acid hydrolase homologs are most closely related to cyanuric acid hydrolases from Actinobacteria. PMID:24039269

  15. Organophosphorus acid anhydrolase from Alteromonas macleodii: structural study and functional relationship to prolidases

    Czech Academy of Sciences Publication Activity Database

    Štěpánková, Andrea; Dušková, Jarmila; Skálová, Tereza; Hašek, Jindřich; Koval, Tomáš; Ostergaard, L. H.; Dohnálek, Jan

    2013-01-01

    Roč. 69, č. 4 (2013), s. 346-354 ISSN 1744-3091 R&D Projects: GA ČR GA310/09/1407; GA ČR GA305/07/1073; GA MŠk EE2.3.30.0029 Institutional research plan: CEZ:AV0Z40500505 Institutional support: RVO:61389013 Keywords : organophosphorus acid anhydrolase * prolidases * bifunctional Subject RIV: CE - Biochemistry Impact factor: 0.568, year: 2013

  16. Organophosphorus insecticides: Toxic effects and bioanalytical tests for evaluating toxicity during degradation processes

    Directory of Open Access Journals (Sweden)

    Čolović Mirjana B.

    2013-01-01

    Full Text Available Organophosphorus insecticides have been the most applied group of insecticides for the last two decades. Their main toxic effects are related to irreversible inactivation of acetylcholinesterase (AChE. Actually, they covalently bind to serine OH group in the enzyme active site forming phosphorylated enzyme that cannot hydrolyze acetylcholine. Organophosphorus insecticides in the environment undergo the natural degradation pathway including mainly homogeneous and heterogeneous hydrolysis (especially at high pH generating non-inhibiting products. Additionally, thio organophosphates are easily oxidized by naturally present oxidants and UV light, forming more toxic and stable oxons. Thus, oxidative degradation procedures, generally referred as advanced oxidation processes (AOP, have been applied for their efficient removal from contaminated waters. The most applied bioassays to monitor the organophosphate toxicity i.e. the detoxification degree during AOP are Vibrio fischeri and AChE bioassays. Vibrio fischeri toxicity test exploits bioluminescence as the measure of luciferase activity of this marine bacterium, whereas AChE bioassay is based on AChE activity inhibition. Both bioanalytical techniques are rapid (several minutes, simple, sensitive and reproducible. Vibrio fischeri test seems to be a versatile indicator of toxic compounds generated in AOP for organophosphorus insecticides degradation. However, detection of neurotoxic AChE inhibitors, which can be formed in AOP of some organophosphates, requires AChE bioassays. Therefore, AChE toxicity test is more appropriate for monitoring the degradation processes of thio organophosphates, because more toxic oxo organophosphates might be formed and overlooked by Vibrio fischeri bioluminescence inhibition. In addition, during organophosphates removal by AOP, compounds with strong genotoxic potential may be formed, which cannot be detected by standard toxicity tests. For this reason, determination of

  17. A Common Mechanism for Resistance to Oxime Reactivation of Acetylcholinesterase Inhibited by Organophosphorus Compounds

    Science.gov (United States)

    2013-01-01

    reactivators, we conducted a QSAR analysis for oxime reactivation of AChE inhibited by OP agents and their analogues. Our objective was to identify...reactivation as tabun-inhibited AChE. QSAR analysis of oxime reactivation of AChE inhibited by these OP compounds and others suggested that the presence of...organophosphorus; QSAR , quan- titative structure–activity relationship; VR, O-isobutyl methylphosphonofluoridate. ⇑ Corresponding author. Tel.: +1 410

  18. Estimated assessment of cumulative dietary exposure to organophosphorus residues from tea infusion in China

    OpenAIRE

    Cao, Pei; Yang, Dajin; Zhu, Jianghui; Liu, Zhaoping; Jiang, Dingguo; Xu, Haibin

    2018-01-01

    Background China has the world’s largest tea plantation area in the world. To sustain high yields of the tea, multiple pesticides are used on tea crops to control pests. Organophosphorus (OP) pesticides are among the most widely used types of agricultural pesticides in China. As tea is a significant potential source of exposure to pesticide residues, the public concern has increased in relation to pesticide residues found in tea in China. The aim of the study was to estimate cumulative dietar...

  19. Induction of plasma acetylcholinesterase activity in mice challenged with organophosphorus poisons

    International Nuclear Information System (INIS)

    Duysen, Ellen G.; Lockridge, Oksana

    2011-01-01

    The restoration of plasma acetylcholinesterase activity in mice following inhibition by organophosphorus pesticides and nerve agents has been attributed to synthesis of new enzyme. It is generally assumed that activity levels return to normal, are stable and do not exceed the normal level. We have observed over the past 10 years that recovery of acetylcholinesterase activity levels in mice treated with organophosphorus agents (OP) exceeds pretreatment levels and remains elevated for up to 2 months. The most dramatic case was in mice treated with tri-cresyl phosphate and tri-ortho-cresyl phosphate, where plasma acetylcholinesterase activity rebounded to a level 250% higher than the pretreatment activity. The present report summarizes our observations on plasma acetylcholinesterase activity in mice treated with chlorpyrifos, chlorpyrifos oxon, diazinon, tri-ortho-cresyl phosphate, tri-cresyl phosphate, tabun thiocholine, parathion, dichlorvos, and diisopropylfluorophosphate. We have developed a hypothesis to explain the excess acetylcholinesterase activity, based on published observations. We hypothesize that acetylcholinesterase activity is induced when cells undergo apoptosis and that consequently there is a rise in the level of plasma acetylcholinesterase. - Highlights: → Acetylcholinesterase activity is induced by organophosphorus agents. → AChE induction is related to apoptosis. → Induction of AChE activity by OP is independent of BChE.

  20. Degradation of organophosphorus pesticides and their transformation products in estuarine waters

    International Nuclear Information System (INIS)

    Lacorte, S.; Lartiges, S.B.; Garrigues, P.; Barcelo, D.

    1995-01-01

    The degradation of 10 organophosphorus pesticides in natural estuarine waters was studied. Estuarine water samples were spiked with organophosphorus pesticides at 50 μg/L level and were placed into 2-L Pyrexflasks being exposed outdoor to ambient sunlight and temperature. A sample of 10-75 mL of water was collected every week for analysis during a period of 5-6 weeks from January to March. The analytical determinations were performed by solid-phase extraction (SPE) with C 18 Emporedisks followed by GC-NPD and GC-MS with EI and by on-line SPE using PLRP-s exchangeable cartridges (Prospekt) followed by LC-DAD and LC-thermospray MS in PI mode. Five organophosphorus pesticides were stable for less than 1 week (disulfoton, fenamiphos, fenthion, malathion, and temephos), others had a half-life of ca. 1 week (chlorpyrifos-methyl, methidathion, and diazinon), and the rest showed a half-life of ca. 10 days (isofenphos and pyridafenthion). The half-life of three pesticidetransformation products: disulfoton sulfoxide, disulfoton sulfone, and fenthion sulfoxide varied from 7 to 12 days

  1. In situ activity-based protein profiling of serine hydrolases in E. coli

    Directory of Open Access Journals (Sweden)

    Dmitry Shamshurin

    2014-09-01

    Full Text Available A fluorophosphonate based alkyne activity probe was used for the selective labeling of active serine hydrolases in intact Escherichia coli cells. A biotin-azide tag was subsequently attached to the alkyne functionality of the probe with copper-catalyzed azide-alkyne cycloaddition (CuAAC reaction. Comparison of proteins from in-cell and lysate labeled preparations suggested qualitatively similar patterns of reactivity in both preparations. Approximately 68%, 30 of the total 44 serine hydrolases detectable in E. coli were labeled with the probe indicating significant coverage with a single probe. The methods described here offer a useful tool for profiling and monitoring serine hydrolase activity in situ.

  2. Adaptation of intestinal hydrolases to starvation in rats: effect of thyroid function

    DEFF Research Database (Denmark)

    Galluser, M; Belkhou, R; Freund, J N

    1991-01-01

    this process. These results indicate that intestinal hydrolases respond non-coordinately to long-term food deprivation. In addition, the thyroid status of the animals has a direct influence on the adaptation of several brush border hydrolases to starvation. This suggests that the drop in plasma thyroid...... hormones during fasting allows a better maintenance of protein content and of hydrolase activities in the brush border membranes of the small intestine. These adaptive processes seemed to be partly controlled at a post-transcriptional level....

  3. 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

  4. 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

    2015-05-01

    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.

  5. 2-deoxy D-glucose prevents cell surface expression of NKG2D ligands through inhibition of N-linked glycosylation

    DEFF Research Database (Denmark)

    Andresen, Lars; Skovbakke, Sarah Line; Persson, Gry

    2012-01-01

    NKG2D ligand surface expression is important for immune recognition of stressed and neotransformed cells. In this study, we show that surface expression of MICA/B and other NKG2D ligands is dependent on N-linked glycosylation. The inhibitor of glycolysis and N-linked glycosylation, 2-deoxy...

  6. Glycosylation of lactase-phlorizin hydrolase in rat small intestine during development

    NARCIS (Netherlands)

    Büller, H. A.; Rings, E. H.; Pajkrt, D.; Montgomery, R. K.; Grand, R. J.

    1990-01-01

    Age-specific changes in glycosylation of rat intestinal lactase-phlorizin hydrolase were analyzed using enzyme immunoprecipitated from microvillus membranes of suckling, weaning, and adult rats, and carbohydrate moieties were examined by lectin affinity binding, metabolic labeling, and neuraminidase

  7. Structural and Enzymatic Characterization of a Nucleoside Diphosphate Sugar Hydrolase from Bdellovibrio bacteriovorus.

    Directory of Open Access Journals (Sweden)

    Andres H de la Peña

    Full Text Available Given the broad range of substrates hydrolyzed by Nudix (nucleoside diphosphate linked to X enzymes, identification of sequence and structural elements that correctly predict a Nudix substrate or characterize a family is key to correctly annotate the myriad of Nudix enzymes. Here, we present the structure determination and characterization of Bd3179 -- a Nudix hydrolase from Bdellovibrio bacteriovorus-that we show localized in the periplasmic space of this obligate Gram-negative predator. We demonstrate that the enzyme is a nucleoside diphosphate sugar hydrolase (NDPSase and has a high degree of sequence and structural similarity to a canonical ADP-ribose hydrolase and to a nucleoside diphosphate sugar hydrolase (1.4 and 1.3 Å Cα RMSD respectively. Examination of the structural elements conserved in both types of enzymes confirms that an aspartate-X-lysine motif on the C-terminal helix of the α-β-α NDPSase fold differentiates NDPSases from ADPRases.

  8. Les lipases sont des hydrolases atypiques : principales caractéristiques et applications

    Directory of Open Access Journals (Sweden)

    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.

  9. A New Family of Biuret Hydrolases Involved in S-Triazine Ring Metabolism

    OpenAIRE

    Cameron, Stephan M.; Durchschein, Katharina; Richman, Jack E.; Sadowsky, Michael J.; Wackett, Lawrence P.

    2011-01-01

    Biuret is an intermediate in the bacterial metabolism of s-triazine ring compounds and is occasionally used as a ruminant feed supplement. We used bioinformatics to identify a biuret hydrolase, an enzyme that has previously resisted efforts to stabilize, purify and characterize. This newly discovered enzyme is a member of the cysteine hydrolase superfamily, a family of enzymes previously not found to be involved in s-triazine metabolism. The gene from Rhizobium leguminosarum bv. viciae strain...

  10. 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

  11. A New Family of Biuret Hydrolases Involved in S-Triazine Ring Metabolism.

    Science.gov (United States)

    Cameron, Stephan M; Durchschein, Katharina; Richman, Jack E; Sadowsky, Michael J; Wackett, Lawrence P

    2011-08-01

    Biuret is an intermediate in the bacterial metabolism of s-triazine ring compounds and is occasionally used as a ruminant feed supplement. We used bioinformatics to identify a biuret hydrolase, an enzyme that has previously resisted efforts to stabilize, purify and characterize. This newly discovered enzyme is a member of the cysteine hydrolase superfamily, a family of enzymes previously not found to be involved in s-triazine metabolism. The gene from Rhizobium leguminosarum bv. viciae strain 3841 encoding biuret hydrolase was synthesized, transformed into Escherichia coli, and expressed. The enzyme was purified and found to be stable. Biuret hydrolase catalyzed the hydrolysis of biuret to allophanate and ammonia. The k(cat)/K(M) of 1.7 × 10(5) M(-1)s(-1) and the relatively low K(M) of 23 ± 4 μM together suggested that this enzyme acts uniquely on biuret physiologically. This is supported by the fact that of the 34 substrate analogs of biuret tested, only two demonstrated reactivity, both at less than 5% of the rate determined for biuret. Biuret hydrolase does not react with carboxybiuret, the product of the enzyme immediately preceding biuret hydrolase in the metabolic pathway for cyanuric acid. This suggests an unusual metabolic strategy of an enzymatically-produced intermediate undergoing non-enzymatic decarboxylation to produce the substrate for the next enzyme in the pathway.

  12. Cloning and expression of a urate oxidase and creatinine hydrolase fusion gene in Escherichia coli.

    Science.gov (United States)

    Cheng, Xin; Liu, Fang; Zhang, Yanxin; Jiang, Yunsheng

    2013-01-01

    To construct a plasmid containing a urate oxidase and creatinine hydrolase fusion gene and transform the plasmid into Escherichia coli to decompose uric acid and creatinine. According to the GenBank data for the urate oxidase gene, specific primers were designed to amplify and remove the stop codon for the urate oxidase gene. The gene was then ligated into the plasmid pMG36e to construct pMG36e-U. Then, using the GenBank database for the creatinine hydrolase gene, primers were designed to amplify the creatinine hydrolase gene. This gene was ligated into pMG36e-U to form pMG36e-U/C. Next, this construct was transformed into E. coli, which was confirmed by screening the recombinant E. coli and sodium dodecylsulfonate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis. The engineered bacteria were cultured with a specific concentration of creatinine and uric acid for 24 h. Then, the concentrations of creatinine and uric acid in the culture fluid were measured. The recombinant gene fragment was approximately 1.68 kb, and it contained the urate oxidase and creatinine hydrolase genes. The transformed E. coli expressed creatinine hydrolase and uric acid oxidase. The creatinine decomposition rate increased by 43.5%, and the uric acid decomposition rate increased by 42.32%. The constructed recombinant plasmid containing a fusion gene of creatinine hydrolase and uric acid oxidase was transformed into E. coli, and the enzymatic activities were expressed.

  13. Expression of Nudix hydrolase genes in barley under UV irradiation

    Science.gov (United States)

    Tanaka, Sayuri; Sugimoto, Manabu; Kihara, Makoto

    Seed storage and cultivation should be necessary to self-supply foods when astronauts would stay and investigate during long-term space travel and habitation in the bases on the Moon and Mars. Thought the sunlight is the most importance to plants, both as the ultimate energy source and as an environmental signal regulating growth and development, UV presenting the sunlight can damage many aspects of plant processes at the physiological and DNA level. Especially UV-C, which is eliminated by the stratospheric ozone layer, is suspected to be extremely harmful and give a deadly injury to plants in space. However, the defense mechanism against UV-C irradiation damage in plant cells has not been clear. In this study, we investigated the expression of Nudix hydrolases, which defense plants from biotic / abiotic stress, in barley under UV irradiation. The genes encoding the amino acid sequences, which show homology to those of 28 kinds of Nudix hydrolases in Arabidopsis thaliana, were identified in the barley full-length cDNA library. BLAST analysis showed 14 kinds of barley genes (HvNUDX1-14), which encode the Nudix motif sequence. A phylogenetic tree showed that HvNUDX1, HvNUDX7, HvNUDX9 and HvNUDX11 belonged to the ADP-ribose pyrophosphohydrolase, ADP-sugar pyrophosphohydrolase, NAD(P)H pyrophosphohydrolase and FAD pyrophosphohydrolase subfamilies, respectively, HvNUDX3, HvNUDX6, and HvNUDX8 belonged to the Ap _{n}A pyrophosphohydrolase subfamilies, HvNUDX5 and HvNUDX14 belonged to the coenzyme A pyrophosphohydrolase subfamilies, HvNUDX12 and HvNUDX13 belonged to the Ap _{4}A pyrophosphohydrolase subfamilies. Induction of HvNUDX genes by UV-A (340nm), UV-B (312nm), and UV-C (260nm) were analyzed by quantitative RT-PCR. The results showed that HvNUDX4 was induced by UV-A and UV-B, HvNUDX6 was induced by UV-B and UV-C, and HvNUDX7 and HvNUDX14 were induced by UV-C, significantly. Our results suggest that the response of HvNUDXs to UV irradiation is different by UV

  14. Probabilistic assessment of the cumulative dietary acute exposure of the population of Denmark to organophosphorus and carbamate pesticides

    DEFF Research Database (Denmark)

    Jensen, Bodil Hamborg; Petersen, Annette; Christensen, Tue

    2009-01-01

    and cereals. The probabilistic approach was used in the assessments. Residue data obtained from the Danish monitoring programme carried out in the period 2004-2007, which included 6704 samples of fruit, vegetables and cereals, were used in the calculations. Food consumption data were obtained from......Organophosphorus and carbamate pesticides are acetylcholinesterase-inhibiting pesticides and as such have a common mode of action. We assessed the cumulative acute exposure of the population of Denmark to 25 organophosphorus and carbamate pesticide residues from the consumption of fruit, vegetables...

  15. Fractionation and Characterization of Tannin Acyl Hydrolase from Aspergillus niger

    Directory of Open Access Journals (Sweden)

    YUNITA ARIAN SANI ANWAR

    2009-09-01

    Full Text Available We previously produced tannin acyl hydrolase (tannase from Aspergillus niger isolated from cacao pod. In the present study the enzyme was subjected to fractionation by ammonium sulphate followed by dialysis process. The saturation level of ammonium sulphate used was 30–80% where the best enzyme activity was obtained at the saturation level of 60%. Compared to that of crude enzyme, specific activity of tannase after dialysis was four folds. Characterization results showed that optimum activity was at 35–50 °C and pH 6. Tannase was activated by K+ and Na+ at concentration of 0.01 and 0.05 M respectively. Mg2+ was found activate tannase only at 0.01 M. Addition of metal ions like Zn2+, Cu2+, Ca2+, Mn2+ and Fe2+ inhibited the enzyme activity. Kinetics analysis of various substrates tested showed that the Km value of tannic acid and gallotannin was 0.401 and 6.611 mM respectively. Vmax value of tannic acid was 10.804 U/ml and of gallotannin was 12.406 U/ml. Based on Michaelis-Menten constant (Km, the tannase obtained in the present study was more active in hydrolysing depside bonds rather than ester bonds.

  16. 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.

  17. Ubiquitin C-Terminal Hydrolase L1 in Tumorigenesis

    Directory of Open Access Journals (Sweden)

    Jennifer Hurst-Kennedy

    2012-01-01

    Full Text Available Ubiquitin carboxyl-terminal hydrolase L1 (UCH-L1, aka PGP9.5 is an abundant, neuronal deubiquitinating enzyme that has also been suggested to possess E3 ubiquitin-protein ligase activity and/or stabilize ubiquitin monomers in vivo. Recent evidence implicates dysregulation of UCH-L1 in the pathogenesis and progression of human cancers. Although typically only expressed in neurons, high levels of UCH-L1 have been found in many nonneuronal tumors, including breast, colorectal, and pancreatic carcinomas. UCH-L1 has also been implicated in the regulation of metastasis and cell growth during the progression of nonsmall cell lung carcinoma, colorectal cancer, and lymphoma. Together these studies suggest UCH-L1 has a potent oncogenic role and drives tumor development. Conversely, others have observed promoter methylation-mediated silencing of UCH-L1 in certain tumor subtypes, suggesting a potential tumor suppressor role for UCH-L1. In this paper, we provide an overview of the evidence supporting the involvement of UCH-L1 in tumor development and discuss the potential mechanisms of action of UCH-L1 in oncogenesis.

  18. 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.

  19. Structure and molecular characterization of barley nudix hydrolase genes.

    Science.gov (United States)

    Tanaka, Sayuri; Kihara, Makoto; Sugimoto, Manabu

    2015-01-01

    Putative nudix hydrolase (NUDX) genes, which encode amino acid sequences showing homology with those of Arabidopsis NUDXs and conserve nudix motif, were identified from barley. The 14 deduced barley NUDXs (HvNUDX1-14) were classified into established subfamilies, except for 8-oxo-deoxyguanosine 5'-triphosphate (8-oxo-dGTP) pyrophosphohydrolase and mRNA decapping enzyme subfamilies, and three substrate-unknown subfamilies. Drought and UV-C stresses, respectively, up-regulated 7 and 4 HvNUDX genes, but some homologs of Arabidopsis NUDXs showed different responses to abiotic stress. HvNUDX12 gene, belonging to diadenosine tetraphosphates (Ap₄A) pyrophosphohydrolase subfamily gene and up-regulated by UV-C, was expressed in Escherichia coli cells. The recombinant protein showed 8-oxo-dGTP, Ap₄A, and guanosine-3',5'-tetraphosphate (ppGpp) pyrophosphohydrolase activities, and the suppression of the lacZ amber mutation in a mutT-deficient E. coli cells caused by the incorporation of 8-oxo-GTP into mRNA was prevented to a significant degree. These results suggest that barley NUDXs have unique constitution and response of NUDX to abiotic stress.

  20. Determination of Twenty Organophosphorus Pesticides in Wheat Samples from Different Regions of Iran

    Directory of Open Access Journals (Sweden)

    Attaollah Shakoori *

    2017-09-01

    Full Text Available Background: Organophosphorus pesticides are widely used in agriculture, homes, gardens, and veterinary practices. Extensive application of pesticides in agriculture often results in residues of these compounds being absorbed into the foods, including wheat. The aim of this study was to evaluate the residue levels of 20 organophosphorus pesticides in wheat samples collected from different regions of Iran. Methods: This research reports a rapid, specific and sensitive multiresidue method based on the Quick Easy Cheap Effective Rugged and Safe (QuEChERS sample preparation method and gas chromatography with mass spectrometric detection in the selected ion monitoring mode (GC–SIM–MS to evaluate 20 organophosphorus pesticides in wheat samples. Results: In the concentration range of 20-200 ng/g, the calibration curves for each analyte was linear with a determination coefficient (R2 of 0.993 to 0.999. The limits of detection (LODs and quantitation (LOQs were between 2.5-6.7 and 7.5-20 ng/g, respectively. The mean recoveries obtained for three fortification levels (25, 50 and 100 ng/g, five replicates each were 80-114% with a satisfactory precision (RSD<20%. 31.1% samples contained residues of one or more target compounds. Chlorpyrifos was the most common residue (17.8%, followed by pirimiphos-methyl (6.7%, diazinon (4.4%, chlorpyrifos-methyl (1.1% and malathion (1.1%. Conclusion: Among the detected pesticides, only diazinon and malathion are permitted pesticides for wheat production in Iran. However, their concentrations were below the maximum residue levels (MRLs established by the Iranian National Standard Organization (INSO.

  1. Generation of murine tumor cell lines deficient in MHC molecule surface expression using the CRISPR/Cas9 system.

    Science.gov (United States)

    Das, Krishna; Eisel, David; Lenkl, Clarissa; Goyal, Ashish; Diederichs, Sven; Dickes, Elke; Osen, Wolfram; Eichmüller, Stefan B

    2017-01-01

    In this study, the CRISPR/Cas9 technology was used to establish murine tumor cell lines, devoid of MHC I or MHC II surface expression, respectively. The melanoma cell line B16F10 and the murine breast cancer cell line EO-771, the latter stably expressing the tumor antigen NY-BR-1 (EO-NY), were transfected with an expression plasmid encoding a β2m-specific single guide (sg)RNA and Cas9. The resulting MHC I negative cells were sorted by flow cytometry to obtain single cell clones, and loss of susceptibility of peptide pulsed MHC I negative clones to peptide-specific CTL recognition was determined by IFNγ ELISpot assay. The β2m knockout (KO) clones did not give rise to tumors in syngeneic mice (C57BL/6N), unless NK cells were depleted, suggesting that outgrowth of the β2m KO cell lines was controlled by NK cells. Using sgRNAs targeting the β-chain encoding locus of the IAb molecule we also generated several B16F10 MHC II KO clones. Peptide loaded B16F10 MHC II KO cells were insusceptible to recognition by OT-II cells and tumor growth was unaltered compared to parental B16F10 cells. Thus, in our hands the CRISPR/Cas9 system has proven to be an efficient straight forward strategy for the generation of MHC knockout cell lines. Such cell lines could serve as parental cells for co-transfection of compatible HLA alleles together with human tumor antigens of interest, thereby facilitating the generation of HLA matched transplantable tumor models, e.g. in HLAtg mouse strains of the newer generation, lacking cell surface expression of endogenous H2 molecules. In addition, our tumor cell lines established might offer a useful tool to investigate tumor reactive T cell responses that function independently from MHC molecule surface expression by the tumor.

  2. Generation of murine tumor cell lines deficient in MHC molecule surface expression using the CRISPR/Cas9 system.

    Directory of Open Access Journals (Sweden)

    Krishna Das

    Full Text Available In this study, the CRISPR/Cas9 technology was used to establish murine tumor cell lines, devoid of MHC I or MHC II surface expression, respectively. The melanoma cell line B16F10 and the murine breast cancer cell line EO-771, the latter stably expressing the tumor antigen NY-BR-1 (EO-NY, were transfected with an expression plasmid encoding a β2m-specific single guide (sgRNA and Cas9. The resulting MHC I negative cells were sorted by flow cytometry to obtain single cell clones, and loss of susceptibility of peptide pulsed MHC I negative clones to peptide-specific CTL recognition was determined by IFNγ ELISpot assay. The β2m knockout (KO clones did not give rise to tumors in syngeneic mice (C57BL/6N, unless NK cells were depleted, suggesting that outgrowth of the β2m KO cell lines was controlled by NK cells. Using sgRNAs targeting the β-chain encoding locus of the IAb molecule we also generated several B16F10 MHC II KO clones. Peptide loaded B16F10 MHC II KO cells were insusceptible to recognition by OT-II cells and tumor growth was unaltered compared to parental B16F10 cells. Thus, in our hands the CRISPR/Cas9 system has proven to be an efficient straight forward strategy for the generation of MHC knockout cell lines. Such cell lines could serve as parental cells for co-transfection of compatible HLA alleles together with human tumor antigens of interest, thereby facilitating the generation of HLA matched transplantable tumor models, e.g. in HLAtg mouse strains of the newer generation, lacking cell surface expression of endogenous H2 molecules. In addition, our tumor cell lines established might offer a useful tool to investigate tumor reactive T cell responses that function independently from MHC molecule surface expression by the tumor.

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

    NARCIS (Netherlands)

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

    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

  4. Effect of some organophosphorus pesticides on oxygen consumption of shrimp, Litopenaeus vannamei.

    Science.gov (United States)

    Galindo Reyes, José Guillermo; Dalla-Venezia, Luisella; Lazcano Alvarez, Maria Guadalupe

    2002-06-01

    Oxygen consumption was measured in adult specimens of Litopenaeus vannamei shrimps from a coastal lagoon of Sinaloa, Mexico, during exposure to sublethal concentrations of the organophosphorus pesticides, Diazinon, Folidol, and Gusathion. Each individual was used as a control of itself, to avoid differences between treatments being masked by individual variability. In all three treatments with pesticides, respiration rate, measured by a polarographic electrode, was significantly lower than in controls. This may, at least partly, explain the decrease in shrimp production observed in recent years in the coastal lagoons of Sinaloa. (c) 2002 Elsevier Science (USA).

  5. Paraoxonase 1 and cytochrome P450 polymorphisms in susceptibility to acute organophosphorus poisoning in Egyptians.

    Science.gov (United States)

    Zayed, Abeer Ahmed; Ahmed, Asmaa Ismail; Khattab, Amr Mohamed Tawfik; Mekdad, Alaa AbdeHamid; AbdelAal, Aly Gamaleldin

    2015-12-01

    Organophosphates are the basis of many insecticides, herbicides, and nerve agents. They were listed as highly acutely toxic agents. Findings in knockout mice suggest that paraoxonase 1 may modulate the toxicity resulting from exposure to organophosphorus compounds. In human, there is no enough data about genetic modulation of acute organophosphorus intoxication. CYP2D6 is involved in the metabolism of about 30% of xenobiotics. Prompt accurate management of OP acute intoxication can promote patient's survival. Forty acute organophosphorus intoxicated patients were divided according to presence of clinical toxicity manifestations and serum level of pseudo-cholinesterase into two groups of acute symptomatic and acute asymptomatic patients. A third group of 29 healthy volunteers served as control. Paraoxonase 1 Q192R and CYP2D6 G1934A polymorphisms, (QQ, QR, and RR for PON1) and (GG, GA, and AA for CYP2D6), were studied using polymerase chain reaction-restriction fragment length polymorphism technique. Serum paraoxonase 1 and pseudo-cholinesterase activities were measured spectrophotometrically. Serum pseudo-cholinesterase was significantly reduced in both acute intoxication groups compared to the controls (p=0.000). Paraoxonase 1 was significantly reduced in the symptomatic acute intoxication patients in comparison to the asymptomatic group (p=0.002). There was a significant increase in paraoxonase 1 192 RR genotype and R allele in the symptomatic patients in comparison to the controls and asymptomatic patients (p=0.006 and p=0.01, respectively). For CYP2D6 G1934A genotypes and alleles, no significant difference was found between groups (p=0.3 and p=0.18, respectively). However, one case of the two recorded fatalities was for a symptomatic female patient with the only traced AA genotype. The combination of both single nucleotide polymorphisms revealed a significant distribution difference between groups, with QQ+GG genotypes being more represented in the controls

  6. Comparative organophosphorus insecticide susceptibility in Caribbean populations of Aedes aegypti and Toxorhynchites moctezuma.

    Science.gov (United States)

    Rawlins, S C; Ragoonansingh, R

    1990-06-01

    Aedes aegypti larvae from Antigua, Jamaica, Puerto Rico, St. Lucia, Trinidad and Union Island and predatory larvae, Toxorhynchites moctezuma, from Trinidad were tested for susceptibility to temephos, malathion, fenthion, fenitrothion and chlorpyrifos. There was some organophosphorus resistance in all strains of Ae. aegypti, in the approximate order: Antigua greater than Jamaica greater than Puerto Rico greater than St. Lucia greater than Trinidad greater than Union Island. Toxorhynchites moctezuma was much less susceptible to temephos than the Ae. aegypti strain, indicating its possible usefulness in an integrated management program.

  7. Separation of Metal lons by Solvent Extraction with Organophosphorus Acid Compounds and Its Industrial Application

    OpenAIRE

    姚,秉華; 永長,幸雄; Yao,BINGHUA; NAGAOSA,Yukio

    1997-01-01

    Solvent extraction of iron(Ⅲ), zinc(Ⅱ), copper(Ⅱ),cadmium(Ⅱ),manganese(Ⅱ), cobalt(Ⅱ), magnesium(Ⅱ), and nickel(Ⅱ) from aqueous sufate media has been studied with organophosphorus acid compounds as extractants. The pH 1/2 va1ues for all metal ions described above have been discussed in tenns of the separatione : sfficiency between nickel(Ⅱ) and the other metal ions. As a practical application,the purification of unrefined nickel(Ⅱ) sulfate solution,containing the other metal ions as impurities...

  8. Simple, specific analysis of organophosphorus and carbamate pesticides in sediments using column extraction and gas chromatography

    Science.gov (United States)

    Belisle, A.A.; Swineford, D.M.

    1988-01-01

    A simple, specific procedure was developed for the analysis of organophosphorus and carbamate pesticides in sediment. The wet soil was mixed with anhydrous sodium sulfate to bind water and the residues were column extracted in acetone:methylene chloride (1:l,v/v). Coextracted water was removed by additional sodium sulfate packed below the sample mixture. The eluate was concentrated and analyzed directly by capillary gas chromatography using phosphorus and nitrogen specific detectors. Recoveries averaged 93 % for sediments extracted shortly after spiking, but decreased significantly as the samples aged.

  9. Pralidoxime in acute organophosphorus insecticide poisoning--a randomised controlled trial.

    Directory of Open Access Journals (Sweden)

    Michael Eddleston

    2009-06-01

    Full Text Available Poisoning with organophosphorus (OP insecticides is a major global public health problem, causing an estimated 200,000 deaths each year. Although the World Health Organization recommends use of pralidoxime, this antidote's effectiveness remains unclear. We aimed to determine whether the addition of pralidoxime chloride to atropine and supportive care offers benefit.We performed a double-blind randomised placebo-controlled trial of pralidoxime chloride (2 g loading dose over 20 min, followed by a constant infusion of 0.5 g/h for up to 7 d versus saline in patients with organophosphorus insecticide self-poisoning. Mortality was the primary outcome; secondary outcomes included intubation, duration of intubation, and time to death. We measured baseline markers of exposure and pharmacodynamic markers of response to aid interpretation of clinical outcomes. Two hundred thirty-five patients were randomised to receive pralidoxime (121 or saline placebo (114. Pralidoxime produced substantial and moderate red cell acetylcholinesterase reactivation in patients poisoned by diethyl and dimethyl compounds, respectively. Mortality was nonsignificantly higher in patients receiving pralidoxime: 30/121 (24.8% receiving pralidoxime died, compared with 18/114 (15.8% receiving placebo (adjusted hazard ratio [HR] 1.69, 95% confidence interval [CI] 0.88-3.26, p = 0.12. Incorporating the baseline amount of acetylcholinesterase already aged and plasma OP concentration into the analysis increased the HR for patients receiving pralidoxime compared to placebo, further decreasing the likelihood that pralidoxime is beneficial. The need for intubation was similar in both groups (pralidoxime 26/121 [21.5%], placebo 24/114 [21.1%], adjusted HR 1.27 [95% CI 0.71-2.29]. To reduce confounding due to ingestion of different insecticides, we further analysed patients with confirmed chlorpyrifos or dimethoate poisoning alone, finding no evidence of benefit.Despite clear reactivation of

  10. Platelet surface expression of SDF-1 is associated with clinical outcomes in the patients with cardiovascular disease.

    Science.gov (United States)

    Rath, Dominik; Chatterjee, Madhumita; Bongartz, Angela; Müller, Karin; Droppa, Michal; Stimpfle, Fabian; Borst, Oliver; Zuern, Christine; Vogel, Sebastian; Gawaz, Meinrad; Geisler, Tobias

    2017-01-01

    Platelet surface expression levels of stromal cell derived factor 1 (SDF-1) are elevated in acute coronary syndrome and associated with LVEF% improvement after myocardial infarction (MI). Platelet SDF-1 might facilitate thrombus formation and endomyocardial expression of SDF-1 is enhanced in inflammatory cardiomyopathy and positively correlates with myocardial fibrosis. The influence of platelet SDF-1 on outcome in the patients with symptomatic coronary artery disease (CAD) is to the best of our knowledge unknown. Blood samples of 608 consecutive CAD patients were collected during the percutaneous coronary intervention and analyzed for surface expression of SDF-1 by flow cytometry. The primary combined endpoint was defined as the composite of either MI, or ischemic stroke, or all-cause death. Secondary endpoints were defined as the aforementioned single events. The patients with baseline platelet SDF-1 levels above the third quartile showed a significantly worse cumulative event-free survival when compared to the patients with lower baseline SDF-1 levels (first to third quartile) (log rank 0.009 for primary combined endpoint and log rank 0.016 for secondary endpoint all-cause death). Multivariate Cox regression analysis showed that SDF-1 levels above the third quartile were independently associated with the primary combined endpoint and the secondary endpoint all-cause death. We provide first clinical evidence that high platelet expression levels of SDF-1 influence clinical outcomes in CAD patients in a negative way.

  11. Occupational levels of radiation exposure induce surface expression of interleukin-2 receptors in stimulated human peripheral blood lymphocytes

    International Nuclear Information System (INIS)

    Xu Yindong; Greenstock, C.L.; Trivedi, A.; Mitchel, R.E.J.

    1996-01-01

    Interleukin-2 (IL-2) is a cytokine responsible for a variety of immune and non-immune stimulatory and regulatory functions, including the activation and stimulation of cytotoxic cells able to recognize and kill human tumour cells and T-cell proliferation and differentiation. We show that low doses of radiation, in the range commonly received by atomic radiation workers or as a result of minor medical diagnostic procedures (0.25 to 10 mGy), stimulate the expression of IL-2 receptors (IL-2R) on the surface of peripheral blood lymphocytes (PBL) taken from normal human donors. This stimulated surface expression after in vitro irradiation is an indirect effect, resulting from the secretion into the medium of a soluble factor from the irradiated cells. This factor can also stimulate IL-2R surface expression in unirradiated cells. Consequently, radiation stimulation of IL-2R expression in a large population of PBL shows a triggered-type response rather than being proportional to dose. These results demonstrate that normal human cells can respond to doses of radiation in the range of common occupational or medical exposures. The data also demonstrate a possible defence mechanism against environmental stress by which a radiation-exposed cell can use an indirect signalling mechanism to communicate with and influence the biological processes in an unexposed cell. (orig.). With 1 fig., 4 tabs

  12. Distinct regions in the C-Terminus required for GLP-1R cell surface expression, activity and internalisation.

    Science.gov (United States)

    Thompson, Aiysha; Kanamarlapudi, Venkateswarlu

    2015-09-15

    The glucagon-like peptide-1 (GLP-1) receptor (GLP-1R), an important drug target in the treatment of type 2 diabetes, is a G-protein coupled receptor (GPCR) that mediates insulin secretion by GLP-1. The N-terminus controls GLP-1R biosynthetic trafficking to the cell surface but the C-terminus involvement in that trafficking is unknown. The aim of this study was to identify distinct regions within the C-terminal domain required for human GLP-1R (hGLP-1R) cell surface expression, activity and internalisation using a number of C-terminal deletions and site-directed mutations. The results of this study revealed that the residues 411-418 within the C-terminal domain of the hGLP-1R are critical in targeting the newly synthesised receptor to the plasma membrane. The residues 419-430 are important for cAMP producing activity of the receptor, most likely by coupling to Gαs. However, the residues 431-450 within the C-terminus are essential for agonist-induced hGLP-1R internalisation. In conclusion, these findings demonstrate the hGLP-1R has distinct regions within the C-terminal domain required for its cell surface expression, activity and agonist-induced internalisation. Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.

  13. Regulation of Pannexin 1 Surface Expression by Extracellular ATP: Potential Implications for Nervous System Function in Health and Disease

    Directory of Open Access Journals (Sweden)

    Leigh A. Swayne

    2017-08-01

    Full Text Available Pannexin 1 (Panx1 channels are widely recognized for their role in ATP release, and as follows, their function is closely tied to that of ATP-activated P2X7 purinergic receptors (P2X7Rs. Our recent work has shown that extracellular ATP induces clustering of Panx1 with P2X7Rs and their subsequent internalization through a non-canonical cholesterol-dependent mechanism. In other words, we have demonstrated that extracellular ATP levels can regulate the cell surface expression of Panx1. Here we discuss two situations in which we hypothesize that ATP modulation of Panx1 surface expression could be relevant for central nervous system function. The first scenario involves the development of new neurons in the ventricular zone. We propose that ATP-induced Panx1 endocytosis could play an important role in regulating the balance of cell proliferation, survival, and differentiation within this neurogenic niche in the healthy brain. The second scenario relates to the spinal cord, in which we posit that an impairment of ATP-induced Panx1 endocytosis could contribute to pathological neuroplasticity. Together, the discussion of these hypotheses serves to highlight important outstanding questions regarding the interplay between extracellular ATP, Panx1, and P2X7Rs in the nervous system in health and disease.

  14. 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

  15. Inactivation of chloramphenicol and florfenicol by a novel chloramphenicol hydrolase.

    Science.gov (United States)

    Tao, Weixin; Lee, Myung Hwan; Wu, Jing; Kim, Nam Hee; Kim, Jin-Cheol; Chung, Eunsook; Hwang, Eul Chul; Lee, Seon-Woo

    2012-09-01

    Chloramphenicol and florfenicol are broad-spectrum antibiotics. Although the bacterial resistance mechanisms to these antibiotics have been well documented, hydrolysis of these antibiotics has not been reported in detail. This study reports the hydrolysis of these two antibiotics by a specific hydrolase that is encoded by a gene identified from a soil metagenome. Hydrolysis of chloramphenicol has been recognized in cell extracts of Escherichia coli expressing a chloramphenicol acetate esterase gene, estDL136. A hydrolysate of chloramphenicol was identified as p-nitrophenylserinol by liquid chromatography-mass spectroscopy and proton nuclear magnetic resonance spectroscopy. The hydrolysis of these antibiotics suggested a promiscuous amidase activity of EstDL136. When estDL136 was expressed in E. coli, EstDL136 conferred resistance to both chloramphenicol and florfenicol on E. coli, due to their inactivation. In addition, E. coli carrying estDL136 deactivated florfenicol faster than it deactivated chloramphenicol, suggesting that EstDL136 hydrolyzes florfenicol more efficiently than it hydrolyzes chloramphenicol. The nucleotide sequences flanking estDL136 encode proteins such as amidohydrolase, dehydrogenase/reductase, major facilitator transporter, esterase, and oxidase. The most closely related genes are found in the bacterial family Sphingomonadaceae, which contains many bioremediation-related strains. Whether the gene cluster with estDL136 in E. coli is involved in further chloramphenicol degradation was not clear in this study. While acetyltransferases for chloramphenicol resistance and drug exporters for chloramphenicol or florfenicol resistance are often detected in numerous microbes, this is the first report of enzymatic hydrolysis of florfenicol resulting in inactivation of the antibiotic.

  16. Long-acting cocaine hydrolase for addiction therapy.

    Science.gov (United States)

    Chen, Xiabin; Xue, Liu; Hou, Shurong; Jin, Zhenyu; Zhang, Ting; Zheng, Fang; Zhan, Chang-Guo

    2016-01-12

    Cocaine abuse is a world-wide public health and social problem without a US Food and Drug Administration-approved medication. An ideal anticocaine medication would accelerate cocaine metabolism, producing biologically inactive metabolites by administration of an efficient cocaine-specific exogenous enzyme. Our recent studies have led to the discovery of the desirable, highly efficient cocaine hydrolases (CocHs) that can efficiently detoxify and inactivate cocaine without affecting normal functions of the CNS. Preclinical and clinical data have demonstrated that these CocHs are safe for use in humans and are effective for accelerating cocaine metabolism. However, the actual therapeutic use of a CocH in cocaine addiction treatment is limited by its short biological half-life (e.g., 8 h or shorter in rats). Here we demonstrate a novel CocH form, a catalytic antibody analog, which is a fragment crystallizable (Fc)-fused CocH dimer (CocH-Fc) constructed by using CocH to replace the Fab region of human IgG1. The CocH-Fc not only has a high catalytic efficiency against cocaine but also, like an antibody, has a considerably longer biological half-life (e.g., ∼107 h in rats). A single dose of CocH-Fc was able to accelerate cocaine metabolism in rats even after 20 d and thus block cocaine-induced hyperactivity and toxicity for a long period. Given the general observation that the biological half-life of a protein drug is significantly longer in humans than in rodents, the CocH-Fc reported in this study could allow dosing once every 2-4 wk, or longer, for treatment of cocaine addiction in humans.

  17. The role of epoxide hydrolases in health and disease.

    Science.gov (United States)

    El-Sherbeni, Ahmed A; El-Kadi, Ayman O S

    2014-11-01

    Epoxide hydrolases (EH) are ubiquitously expressed in all living organisms and in almost all organs and tissues. They are mainly subdivided into microsomal and soluble EH and catalyze the hydration of epoxides, three-membered-cyclic ethers, to their corresponding dihydrodiols. Owning to the high chemical reactivity of xenobiotic epoxides, microsomal EH is considered protective enzyme against mutagenic and carcinogenic initiation. Nevertheless, several endogenously produced epoxides of fatty acids function as important regulatory mediators. By mediating the formation of cytotoxic dihydrodiol fatty acids on the expense of cytoprotective epoxides of fatty acids, soluble EH is considered to have cytotoxic activity. Indeed, the attenuation of microsomal EH, achieved by chemical inhibitors or preexists due to specific genetic polymorphisms, is linked to the aggravation of the toxicity of xenobiotics, as well as the risk of cancer and inflammatory diseases, whereas soluble EH inhibition has been emerged as a promising intervention against several diseases, most importantly cardiovascular, lung and metabolic diseases. However, there is reportedly a significant overlap in substrate selectivity between microsomal and soluble EH. In addition, microsomal and soluble EH were found to have the same catalytic triad and identical molecular mechanism. Consequently, the physiological functions of microsomal and soluble EH are also overlapped. Thus, studying the biological effects of microsomal or soluble EH alterations needs to include the effects on both the metabolism of reactive metabolites, as well as epoxides of fatty acids. This review focuses on the multifaceted role of EH in the metabolism of xenobiotic and endogenous epoxides and the impact of EH modulations.

  18. 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

  19. Alterations of intestinal glycoprotein hydrolases in congenital diabetes

    Energy Technology Data Exchange (ETDEWEB)

    Najjar, S.M.

    1989-01-01

    The diabetic BioBreed (BB{sub d}) rat was used for the study of the molecular structure of intestinal brush border sucrase-{alpha}-dextrinase (SD) and aminooligopeptidase (AOP) in diabetes mellitus. The specific catalytic activity of S-D and AOP in the BB{sub d} rat is normal. However, solid-phase radioimmunoassay revealed loss of some antigenic determinants in the BB{sub d} rat. S-D and AOP migrated abnormally on 6% SDS-gel electrophoresis in the BB{sub d} rat. S was larger (+5 kDa), D was either smaller (-5 kDa) or unaltered, and AOP was smaller (-5 kDa) in the BB{sub d} than in the normal Wistar. The structural abnormalities were independent of hyperglycemia or ketoacidosis and restored to normal by daily insulin treatment (NPH, 3-4 units/rat) for two to three weeks. Newly-synthesized brush border hydrolases were examined after 6 hours of intraperitoneal injection of ({sup 35}S) methionine (2 mCi) and found to be altered, suggesting that structural abnormality appeared acutely during intracellular synthesis rather than being due to slow extracellular modifications such as non-enzymatic glycosylation. Deglycosylation of brush border proteins by trifluoromethanesulfonic acid resulted in an apoprotein with normal electrophoretic migration in BB{sub d}, indicating that the alteration was due to the carbohydrates component of the glycoprotein. Pulse-chase studies with ({sup 35}S) methionine were consistent with normal protein an co-translational and initial N-linked carbohydrate assembly in association with the endoplasmic reticulum in BB{sub d}. However, the post-translational maturation of N-linked and addition of 0-linked carbohydrate chains in Golgi were prolonged, and produced a larger single-chain precursor of S-D in BB{sub d} than normal.

  20. Alterations of intestinal glycoprotein hydrolases in congenital diabetes

    International Nuclear Information System (INIS)

    Najjar, S.M.

    1989-01-01

    The diabetic BioBreed (BB d ) rat was used for the study of the molecular structure of intestinal brush border sucrase-α-dextrinase (SD) and aminooligopeptidase (AOP) in diabetes mellitus. The specific catalytic activity of S-D and AOP in the BB d rat is normal. However, solid-phase radioimmunoassay revealed loss of some antigenic determinants in the BB d rat. S-D and AOP migrated abnormally on 6% SDS-gel electrophoresis in the BB d rat. S was larger (+5 kDa), D was either smaller (-5 kDa) or unaltered, and AOP was smaller (-5 kDa) in the BB d than in the normal Wistar. The structural abnormalities were independent of hyperglycemia or ketoacidosis and restored to normal by daily insulin treatment (NPH, 3-4 units/rat) for two to three weeks. Newly-synthesized brush border hydrolases were examined after 6 hours of intraperitoneal injection of [ 35 S] methionine (2 mCi) and found to be altered, suggesting that structural abnormality appeared acutely during intracellular synthesis rather than being due to slow extracellular modifications such as non-enzymatic glycosylation. Deglycosylation of brush border proteins by trifluoromethanesulfonic acid resulted in an apoprotein with normal electrophoretic migration in BB d , indicating that the alteration was due to the carbohydrates component of the glycoprotein. Pulse-chase studies with [ 35 S] methionine were consistent with normal protein an co-translational and initial N-linked carbohydrate assembly in association with the endoplasmic reticulum in BB d . However, the post-translational maturation of N-linked and addition of 0-linked carbohydrate chains in Golgi were prolonged, and produced a larger single-chain precursor of S-D in BB d than normal

  1. Epigenetic regulation of fatty acid amide hydrolase in Alzheimer disease.

    Directory of Open Access Journals (Sweden)

    Claudio D'Addario

    Full Text Available OBJECTIVE: Alzheimer disease (AD is a progressive, degenerative and irreversible neurological disorder with few therapies available. In search for new potential targets, increasing evidence suggests a role for the endocannabinoid system (ECS in the regulation of neurodegenerative processes. METHODS: We have studied the gene expression status and the epigenetic regulation of ECS components in peripheral blood mononuclear cells (PBMCs of subjects with late-onset AD (LOAD and age-matched controls (CT. RESULTS: We found an increase in fatty acid amide hydrolase (faah gene expression in LOAD subjects (2.30 ± 0.48 when compared to CT (1.00 ± 0.14; *p<0.05 and no changes in the mRNA levels of any other gene of ECS elements. Consistently, we also observed in LOAD subjects an increase in FAAH protein levels (CT: 0.75 ± 0.04; LOAD: 1.11 ± 0.15; *p<0.05 and activity (pmol/min per mg protein CT: 103.80 ± 8.73; LOAD: 125.10 ± 4.00; *p<0.05, as well as a reduction in DNA methylation at faah gene promoter (CT: 55.90 ± 4.60%; LOAD: 41.20 ± 4.90%; *p<0.05. CONCLUSIONS: Present findings suggest the involvement of FAAH in the pathogenesis of AD, highlighting the importance of epigenetic mechanisms in enzyme regulation; they also point to FAAH as a new potential biomarker for AD in easily accessible peripheral cells.

  2. Reward and Toxicity of Cocaine Metabolites Generated by Cocaine Hydrolase.

    Science.gov (United States)

    Murthy, Vishakantha; Geng, Liyi; Gao, Yang; Zhang, Bin; Miller, Jordan D; Reyes, Santiago; Brimijoin, Stephen

    2015-08-01

    Butyrylcholinesterase (BChE) gene therapy is emerging as a promising concept for treatment of cocaine addiction. BChE levels after gene transfer can rise 1000-fold above those in untreated mice, making this enzyme the second most abundant plasma protein. For months or years, gene transfer of a BChE mutated into a cocaine hydrolase (CocH) can maintain enzyme levels that destroy cocaine within seconds after appearance in the blood stream, allowing little to reach the brain. Rapid enzyme action causes a sharp rise in plasma levels of two cocaine metabolites, benzoic acid (BA) and ecgonine methyl ester (EME), a smooth muscle relaxant that is mildly hypotensive and, at best, only weakly rewarding. The present study, utilizing Balb/c mice, tested reward effects and cardiovascular effects of administering EME and BA together at molar levels equivalent to those generated by a given dose of cocaine. Reward was evaluated by conditioned place preference. In this paradigm, cocaine (20 mg/kg) induced a robust positive response but the equivalent combined dose of EME + BA failed to induce either place preference or aversion. Likewise, mice that had undergone gene transfer with mouse CocH (mCocH) showed no place preference or aversion after repeated treatments with a near-lethal 80 mg/kg cocaine dose. Furthermore, a single administration of that same high cocaine dose failed to affect blood pressure as measured using the noninvasive tail-cuff method. These observations confirm that the drug metabolites generated after CocH gene transfer therapy are safe even after a dose of cocaine that would ordinarily be lethal.

  3. Recognition of corn defense chitinases by fungal polyglycine hydrolases.

    Science.gov (United States)

    Naumann, Todd A; Bakota, Erica L; Price, Neil P J

    2017-06-01

    Polyglycine hydrolases (PGH)s are secreted fungal endoproteases that cleave peptide bonds in the polyglycine interdomain linker of ChitA chitinase, an antifungal protein from domesticated corn (Zea mays ssp. mays). These target-specific endoproteases are unusual because they do not cut a specific peptide bond but select one of many Gly-Gly bonds within the polyglycine region. Some Gly-Gly bonds are cleaved frequently while others are never cleaved. Moreover, we have previously shown that PGHs from different fungal pathogens prefer to cleave different Gly-Gly peptide bonds. It is not understood how PGHs selectively cleave the ChitA linker, especially because its polyglycine structure lacks peptide sidechains. To gain insights into this process we synthesized several peptide analogs of ChitA to evaluate them as potential substrates and inhibitors of Es-cmp, a PGH from the plant pathogenic fungus Epicoccum sorghi. Our results showed that part of the PGH recognition site for substrate chitinases is adjacent to the polyglycine linker on the carboxy side. More specifically, four amino acid residues were implicated, each spaced four residues apart on an alpha helix. Moreover, analogous peptides with selective Gly->sarcosine (N-methylglycine) mutations or a specific Ser->Thr mutation retained inhibitor activity but were no longer cleaved by PGH. Additonally, our findings suggest that peptide analogs of ChitA that inhibit PGH activity could be used to strengthen plant defenses. Published by Wiley-Blackwell. Published 2017. This article is a U.S. Government work and is in the public domain in the USA.

  4. Determination of polar organophosphorus pesticides in vegetables and fruits using liquid chromatography with tandem mass spectrometry: Selection of extraction solvent

    NARCIS (Netherlands)

    Mol, H.G.J.; Dam, R.C.J. van; Steijger, O.M.

    2003-01-01

    A method based on liquid chromatography (LC)-mass spectrometry (MS)/MS was developed for sensitive determination of a number of less gas chromatography (GC)-amenable organophosphorus pesticides (OPs; acephate, methamidophos, monocrotophos, omethoate, oxydemeton-methyl and vamidothion) in cabbage and

  5. DEVELOPMENT OF A RISK ASSESSMENT MODEL FOR THE EFFECTS OF ORGANOPHOSPHORUS PESTICIDES ON INFECTIOUS DISEASE SUSCEPTIBILITY AND THE IMMUNE SYSTEM

    Science.gov (United States)

    There is increased concern about the sublethal effects of organophosphorus (OP) pesticides on human and animal health. This class of chemicals has been shown to affect the immune function of macrophages and lymphocytes. Malathion, an OP compound, is one of the most widely used ...

  6. Draft Genome Sequence of the Organophosphorus Compound-Degrading Burkholderia zhejiangensis Strain CEIB S4-3

    OpenAIRE

    Hern?ndez-Mendoza, Armando; Mart?nez-Ocampo, Fernando; Lozano-Aguirre Beltr?n, Luis Fernando; Popoca-Ursino, Elida Carolina; Ortiz-Hern?ndez, Laura; S?nchez-Salinas, Enrique; Dant?n-Gonz?lez, Edgar

    2014-01-01

    Burkholderia species are widely distributed in the environment. A Burkholderia zhejiangensis strain was isolated from pesticide-contaminated soil from an agricultural field in Mexico and identified as an organophosphorus compound-degrading bacterium. In this study, we report the draft genome sequence of Burkholderia zhejiangensis strain CEIB S4-3.

  7. Metagenomic Functional Potential Predicts Degradation Rates of a Model Organophosphorus Xenobiotic in Pesticide Contaminated Soils

    Directory of Open Access Journals (Sweden)

    Thomas C. Jeffries

    2018-02-01

    Full Text Available Chemical contamination of natural and agricultural habitats is an increasing global problem and a major threat to sustainability and human health. Organophosphorus (OP compounds are one major class of contaminant and can undergo microbial degradation, however, no studies have applied system-wide ecogenomic tools to investigate OP degradation or use metagenomics to understand the underlying mechanisms of biodegradation in situ and predict degradation potential. Thus, there is a lack of knowledge regarding the functional genes and genomic potential underpinning degradation and community responses to contamination. Here we address this knowledge gap by performing shotgun sequencing of community DNA from agricultural soils with a history of pesticide usage and profiling shifts in functional genes and microbial taxa abundance. Our results showed two distinct groups of soils defined by differing functional and taxonomic profiles. Degradation assays suggested that these groups corresponded to the organophosphorus degradation potential of soils, with the fastest degrading community being defined by increases in transport and nutrient cycling pathways and enzymes potentially involved in phosphorus metabolism. This was against a backdrop of taxonomic community shifts potentially related to contamination adaptation and reflecting the legacy of exposure. Overall our results highlight the value of using holistic system-wide metagenomic approaches as a tool to predict microbial degradation in the context of the ecology of contaminated habitats.

  8. ACUTE EXOGENOUS INTOXICATIONS WITH ORGANOPHOSPHORUS PESTICIDES: DURATION OF THE HOSPITAL TREATMENT AND CLINICAL CRITERIA FOR PROGNOSIS

    Directory of Open Access Journals (Sweden)

    Petko Marinov

    2017-05-01

    Full Text Available Purpose: Several criteria have been suggested to estimate the intoxication severity, yet so far no system of clinical criteria has been developed to determine the duration of hospitalisation. The forecast is linked to the influence of the extended corrected QT interval and GCS (Glasgow Coma Scale on the frequency of the developing acute pulmonary insufficiency and lethality. The average duration of hospital treatment is also crucial. First of all, it is determined by the intoxication severity and lasts from 3 to 26 days. The aim of this study is to develop an individual forecast about the duration of hospitalisation for patients suffering from acute exogenous intoxication with organophosphorus pesticides (OPP. Materials/Methods: The subjects are 160 patients. We use statistical regression analysis to study the significance of 5 of the most typical clinical indicators of organophosphorus intoxication on the duration of hospitalisation: type of conscience, presence of spasms, pulmonary oedema, shock and multi-organ insufficiency syndrome (MOIS. To forecast the treatment length, we obtain simplified mathematical expressions in the form of score estimates. Results: The significance of the clinical indicators “MOIS”, “conscience” and “spasm” has been confirmed. A forecast matrix that gives the opportunity to forecast the personal duration of hospital treatment for each patient has been built.

  9. The analysis of common metabolites of organophosphorus pesticides in urine by gas chromatography/mass spectrometry

    International Nuclear Information System (INIS)

    Park, Seong Soo; Pyo, Hee Soo; Lee, Kang Jin; Park, Song Ja; Park, Taek Kyu

    1998-01-01

    Most organophosphorus pesticides may be metabolized to yield some common phosphates in human or in animals, and these metabolites may be used as the exposure biomarkers to pesticides. In this study, we developed the extraction method of four phosphate metabolites from the spiked human urine in high recovery by the solid phase extraction with a reverse-phase cartridge (cyclohexyl silica) followed by the elution with methanol. The extracted urinary metabolites were derivatized with hexamethyldisilazane/trimethyl-chlorosilane/pyridine (2:1:10, v/v/v) and identified by gas chromatography/mass spectrometry. Calibration curve obtained from each metabolite standard using by GC/MS/SIM has shown good linearity and detection limits of metabolites were the range of 0.05-0.1 μg/ml in urine. Phenthoate, one of the organophosphorus pesticides, was orally administrated to rats. Four metabolites were detected in the rat urine. The results of this study may be applied to development of exposure biomarkers for monitoring of environmental pollutants

  10. In vivo tracing of organophosphorus pesticides in cabbage (Brassica parachinensis) and aloe (Barbadensis)

    Energy Technology Data Exchange (ETDEWEB)

    Qiu, Junlang; Chen, Guosheng; Zhou, Hong; Xu, Jianqiao; Wang, Fuxin; Zhu, Fang, E-mail: ceszf@mail.sysu.edu.cn; Ouyang, Gangfeng, E-mail: cesoygf@mail.sysu.edu.cn

    2016-04-15

    In vivo solid-phase microextraction (SPME) sampling method coupled with gas chromatography–mass spectrometry (GC–MS) analysis was employed to trace the uptake and elimination of organophosphorus pesticides (OPPs) in two kinds of edible plants, cabbage (Brassica parachinensis) and aloe (Barbadensis). The metabolism of fenthion in aloe was also investigated by the liquid chromatography tandem mass spectrometry analysis (LC–MS/MS) to understand the fate of OPPs in living plants better. Transpiration stream concentration factor (TSCF) and depuration rate constants of the OPPs in living plants were obtained therein. The health risk of the OPPs treated aloe was estimated by the maximum residue limit (MRL) approach, and it revealed that the OPPs were rather safe for their fast degradable property. However, peak concentration of fenthion-sulfoxide was found to exceed the MRL and was higher than that of the parent fenthion, which indicated the potential risk of pesticide metabolites. This study highlighted the application of in vivo SPME for contaminant tracing in different living edible plants. The in vivo tracing method is very convenient and can provide more data to evaluate the risk of different pesticides, which are very important for the safety of agriculture production. - Highlights: • In vivo SPME was employed to sample organophosphorus pesticides in vegetables. • Uptake and elimination of OPPs were traced in cabbage and aloe. • In vivo tracing of fenthion demonstrated its metabolites could be rather dangerous. • The risks of OPPs were assessed based on the in vivo tracing data.

  11. High-Confidence Qualitative Identification of Organophosphorus Nerve Agent Adducts to Human Butyrylcholinesterase.

    Science.gov (United States)

    Mathews, Thomas P; Carter, Melissa D; Johnson, Darryl; Isenberg, Samantha L; Graham, Leigh Ann; Thomas, Jerry D; Johnson, Rudolph C

    2017-02-07

    In this study, a data-dependent, high-resolution tandem mass spectrometry (ddHRMS/MS) method capable of detecting all organophosphorus nerve agent (OPNA) adducts to human butyrylcholinesterase (BChE) was developed. After an exposure event, immunoprecipitation from blood with a BChE-specific antibody and digestion with pepsin produces a nine amino acid peptide containing the OPNA adduct. Signature product ions of this peptic BChE nonapeptide (FGES*AGAAS) offer a route to broadly screen for OPNA exposure. Taking this approach on an HRMS instrument identifies biomarkers, including unknowns, with high mass accuracy. Using a set of pooled human sera exposed to OPNAs as quality control (QC) materials, the developed method successfully identified precursor ions with 15 ng/mL. The developed method was applied to a convenience set of 96 unexposed serum samples and a blinded set of 80 samples treated with OPNAs. OPNA biomarkers were not observed in convenience set samples and no false positive or negative identifications were observed in blinded samples. All biomarkers in the blinded serum set >15 ng/mL were correctly identified. For the first time, this study reports a ddHRMS/MS method capable of complementing existing quantitative methodologies and suitable for identifying exposure to unknown organophosphorus agents.

  12. Confirmed organophosphorus and carbamate pesticide poisonings in South African wildlife (2009–2014

    Directory of Open Access Journals (Sweden)

    Christo J. Botha

    2015-12-01

    Full Text Available During a six-year period (from January 2009 to December 2014, specimens collected from 344 cases of suspected organophosphorus and carbamate pesticide poisonings in wildlife, including birds, were submitted to the Toxicology Laboratory (ARC-OVI for analysis. A positive diagnosis was made in 135 (39% of these cases. The majority of cases were from birds, which included Cape vultures (Gyps coprotheres and African white-backed vultures (Gyps africanus and bateleur eagles (Terathopius ecaudatus. In one incident 49 vultures were killed when a farmer intentionally laced carcasses with carbofuran in an attempt to control jackal predation. There were 22 incidents of poisoning in helmeted guineafowl (Numida meleagris. On nine different occasions blue cranes (Anthropoides paradiseus were poisoned, in one incident 14 birds were reported to have been killed. Over the period of investigation, there were 20 cases of poisoning involving mammalian species, the majority being vervet monkeys (Chlorocebus pygerythrus. The carbamate pesticides were responsible for 57 incidents of poisoning. Aldicarb, carbofuran and methomyl were detected in 26, 18 and 12 cases respectively. The majority of organophosphorus pesticide poisonings were caused by diazinon (n = 19, monocrotophos (n = 13 and methamidophos (n = 10.

  13. Separating esterase targets of organophosphorus compounds in the brain by preparative chromatography.

    Science.gov (United States)

    Mangas, I; Vilanova, E; Benabent, M; Estévez, J

    2014-02-10

    Low level exposure to organophosphorus esters (OPs) may cause long-term neurological effects and affect specific cognition domains in experimental animals and humans. Action on known targets cannot explain most of these effects by. Soluble carboxylesterases (EC 3.1.1.1) of chicken brain have been kinetically discriminated using paraoxon, mipafox and phenylmethyl sulfonylfluoride as inhibitors and phenyl valerate as a substrate. Three different enzymatic components were discriminated and called Eα, Eβ and Eγ. In this work, a fractionation procedure with various steps was developed using protein native separation methods by preparative HPLC. Gel permeation chromatography followed by ion exchange chromatography allowed enriched fractions with different kinetic behaviors. The soluble chicken brain fraction was fractionated, while total esterase activity, proteins and enzymatic components Eα, Eβ and Eγ were monitored in each subfraction. After the analysis, 13 fractions were pooled and conserved. Preincubation of the soluble chicken brain fraction of with the organophosphorus mipafox gave rise to a major change in the ion exchange chromatography profile, but not in the molecular exchanged chromatography profile, which suggest that mipafox permanently modifies the ionic properties of numerous proteins. Copyright © 2013 Elsevier Ireland Ltd. All rights reserved.

  14. In vivo tracing of organophosphorus pesticides in cabbage (Brassica parachinensis) and aloe (Barbadensis)

    International Nuclear Information System (INIS)

    Qiu, Junlang; Chen, Guosheng; Zhou, Hong; Xu, Jianqiao; Wang, Fuxin; Zhu, Fang; Ouyang, Gangfeng

    2016-01-01

    In vivo solid-phase microextraction (SPME) sampling method coupled with gas chromatography–mass spectrometry (GC–MS) analysis was employed to trace the uptake and elimination of organophosphorus pesticides (OPPs) in two kinds of edible plants, cabbage (Brassica parachinensis) and aloe (Barbadensis). The metabolism of fenthion in aloe was also investigated by the liquid chromatography tandem mass spectrometry analysis (LC–MS/MS) to understand the fate of OPPs in living plants better. Transpiration stream concentration factor (TSCF) and depuration rate constants of the OPPs in living plants were obtained therein. The health risk of the OPPs treated aloe was estimated by the maximum residue limit (MRL) approach, and it revealed that the OPPs were rather safe for their fast degradable property. However, peak concentration of fenthion-sulfoxide was found to exceed the MRL and was higher than that of the parent fenthion, which indicated the potential risk of pesticide metabolites. This study highlighted the application of in vivo SPME for contaminant tracing in different living edible plants. The in vivo tracing method is very convenient and can provide more data to evaluate the risk of different pesticides, which are very important for the safety of agriculture production. - Highlights: • In vivo SPME was employed to sample organophosphorus pesticides in vegetables. • Uptake and elimination of OPPs were traced in cabbage and aloe. • In vivo tracing of fenthion demonstrated its metabolites could be rather dangerous. • The risks of OPPs were assessed based on the in vivo tracing data.

  15. Evaluation of NHS carbamates as a potent and selective class of endocannabinoid hydrolase inhibitors.

    Science.gov (United States)

    Niphakis, Micah J; Cognetta, Armand B; Chang, Jae Won; Buczynski, Matthew W; Parsons, Loren H; Byrne, Frederika; Burston, James J; Chapman, Victoria; Cravatt, Benjamin F

    2013-09-18

    Monoacylglycerol lipase (MAGL) is a principal metabolic enzyme responsible for hydrolyzing the endogenous cannabinoid (endocannabinoid) 2-arachidonoylglycerol (2-AG). Selective inhibitors of MAGL offer valuable probes to further understand the enzyme's function in biological systems and may lead to drugs for treating a variety of diseases, including psychiatric disorders, neuroinflammation, and pain. N-Hydroxysuccinimidyl (NHS) carbamates have recently been identified as a promising class of serine hydrolase inhibitors that shows minimal cross-reactivity with other proteins in the proteome. Here, we explore NHS carbamates more broadly and demonstrate their potential as inhibitors of endocannabinoid hydrolases and additional enzymes from the serine hydrolase class. We extensively characterize an NHS carbamate 1a (MJN110) as a potent, selective, and in-vivo-active MAGL inhibitor. Finally, we demonstrate that MJN110 alleviates mechanical allodynia in a rat model of diabetic neuropathy, marking NHS carbamates as a promising class of MAGL inhibitors.

  16. The putative α/β-hydrolases of Dietzia cinnamea P4 strain as potential enzymes for biocatalytic applications

    NARCIS (Netherlands)

    Procopio da Silva, Luciano; Macrae, Andrew; van Elsas, Jan Dirk; Seldin, Lucy

    The draft genome of the soil actinomycete Dietzia cinnamea P4 reveals a versatile group of alpha/beta-hydrolase fold enzymes. Phylogenetic and comparative sequence analyses were used to classify the alpha/beta-hydrolases of strain P4 into six different groups: (i) lipases, (ii) esterases, (iii)

  17. Surface expression and limited proteolysis of ADAM10 are increased by a dominant negative inhibitor of dynamin

    Directory of Open Access Journals (Sweden)

    Slack Barbara E

    2011-05-01

    Full Text Available Abstract Background The amyloid precursor protein (APP is cleaved by β- and γ-secretases to generate toxic amyloid β (Aβ peptides. Alternatively, α-secretases cleave APP within the Aβ domain, precluding Aβ formation and releasing the soluble ectodomain, sAPPα. We previously showed that inhibition of the GTPase dynamin reduced APP internalization and increased release of sAPPα, apparently by prolonging the interaction between APP and α-secretases at the plasma membrane. This was accompanied by a reduction in Aβ generation. In the present study, we investigated whether surface expression of the α-secretase ADAM (a disintegrin and metalloprotease10 is also regulated by dynamin-dependent endocytosis. Results Transfection of human embryonic kidney (HEK cells stably expressing M3 muscarinic receptors with a dominant negative dynamin I mutant (dyn I K44A, increased surface expression of both immature, and mature, catalytically active forms of co-expressed ADAM10. Surface levels of ADAM10 were unaffected by activation of protein kinase C (PKC or M3 receptors, indicating that receptor-coupled shedding of the ADAM substrate APP is unlikely to be mediated by inhibition of ADAM10 endocytosis in this cell line. Dyn I K44A strongly increased the formation of a C-terminal fragment of ADAM10, consistent with earlier reports that the ADAM10 ectodomain is itself a target for sheddases. The abundance of this fragment was increased in the presence of a γ-secretase inhibitor, but was not affected by M3 receptor activation. The dynamin mutant did not affect the distribution of ADAM10 and its C-terminal fragment between raft and non-raft membrane compartments. Conclusions Surface expression and limited proteolysis of ADAM10 are regulated by dynamin-dependent endocytosis, but are unaffected by activation of signaling pathways that upregulate shedding of ADAM substrates such as APP. Modulation of ADAM10 internalization could affect cellular behavior in two

  18. Surface expression and limited proteolysis of ADAM10 are increased by a dominant negative inhibitor of dynamin.

    Science.gov (United States)

    Carey, Robyn M; Blusztajn, Jan K; Slack, Barbara E

    2011-05-17

    The amyloid precursor protein (APP) is cleaved by β- and γ-secretases to generate toxic amyloid β (Aβ) peptides. Alternatively, α-secretases cleave APP within the Aβ domain, precluding Aβ formation and releasing the soluble ectodomain, sAPPα. We previously showed that inhibition of the GTPase dynamin reduced APP internalization and increased release of sAPPα, apparently by prolonging the interaction between APP and α-secretases at the plasma membrane. This was accompanied by a reduction in Aβ generation. In the present study, we investigated whether surface expression of the α-secretase ADAM (a disintegrin and metalloprotease)10 is also regulated by dynamin-dependent endocytosis. Transfection of human embryonic kidney (HEK) cells stably expressing M3 muscarinic receptors with a dominant negative dynamin I mutant (dyn I K44A), increased surface expression of both immature, and mature, catalytically active forms of co-expressed ADAM10. Surface levels of ADAM10 were unaffected by activation of protein kinase C (PKC) or M3 receptors, indicating that receptor-coupled shedding of the ADAM substrate APP is unlikely to be mediated by inhibition of ADAM10 endocytosis in this cell line. Dyn I K44A strongly increased the formation of a C-terminal fragment of ADAM10, consistent with earlier reports that the ADAM10 ectodomain is itself a target for sheddases. The abundance of this fragment was increased in the presence of a γ-secretase inhibitor, but was not affected by M3 receptor activation. The dynamin mutant did not affect the distribution of ADAM10 and its C-terminal fragment between raft and non-raft membrane compartments. Surface expression and limited proteolysis of ADAM10 are regulated by dynamin-dependent endocytosis, but are unaffected by activation of signaling pathways that upregulate shedding of ADAM substrates such as APP. Modulation of ADAM10 internalization could affect cellular behavior in two ways: by altering the putative signaling activity of

  19. Peptidoglycan Hydrolases of Local Lactic Acid Bacteria from Kazakh Traditional Food

    Directory of Open Access Journals (Sweden)

    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.

  20. Sequential changes of lamellar body hydrolases during ozone-induced alveolar injury and repair

    Energy Technology Data Exchange (ETDEWEB)

    Glew, R.H.; Basu, A.; Shelley, S.A.; Paterson, J.F.; Diven, W.F.; Montgomery, M.R.; Balis, J.U.

    1989-05-01

    Lamellar body hydrolases in acutely damaged and regenerating type II cells were determined using an established rat model with well-defined stages of bronchiolo-alveolar injury and repair. Lamellar bodies were isolated from control and ozone-exposed (3.0 ppm for 8 hours) adult male rats by sucrose density gradient centrifugation and analyzed for their content of six different lysosomal hydrolases. Immediately after 3 ppm ozone exposure (zero-time) there was a significant decrease in specific enzyme activity (units/mg protein) of five lamellar body hydrolases and these activities remained depressed for at least 24 hours after exposure. In addition, total enzyme activity (units/lung) was reduced at zero-time for beta-hexosaminidase and at 24 hours postexposure for alpha-mannosidase and alpha-L-fucosidase. During the reparative and recovery stages (48 to 96 hours) the hydrolases demonstrated variable elevations in both specific activity and total activity (units/lung). Characteristically, beta-hexosaminidase and beta-galactosidase reached supranormal values at 96 hours, whereas alpha-mannosidase remained below normal levels through the recovery stage. Moreover, at 24 to 48 hours the lamellar body fraction demonstrated prominent enzyme depletion relative to the expanding pool of stored surfactant. It is concluded that acute ozone stress initiates the development of hydrolase deficiency within the lamellar bodies of injured and regenerating type II cells. This deficiency state is followed by asynchronous lamellar body hydrolase elevations that reflect distinct patterns of response rather than uniform return to normal condition. The lysosomal enzyme changes of lamellar bodies may be pathogenetically linked to the development of associated alterations in the storage and secretion of surfactant.

  1. Peptidase family U34 belongs to the superfamily of N-terminal nucleophile hydrolases

    Science.gov (United States)

    Pei, Jimin; Grishin, Nick V.

    2003-01-01

    Peptidase family U34 consists of enzymes with unclear catalytic mechanism, for instance, dipeptidase A from Lactobacillus helveticus. Using extensive sequence similarity searches, we infer that U34 family members are homologous to penicillin V acylases (PVA) and thus potentially adopt the N-terminal nucleophile (Ntn) hydrolase fold. Comparative sequence and structural analysis reveals a cysteine as the catalytic nucleophile as well as other conserved residues important for catalysis. The PVA/U34 family is variable in sequence and exhibits great diversity in substrate specificity, to include enzymes such as choloyglycine hydrolases, acid ceramidases, isopenicillin N acyltransferases, and a subgroup of eukaryotic proteins with unclear function. PMID:12717035

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

    Science.gov (United States)

    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.

  3. Impaired cell surface expression of HLA-B antigens on mesenchymal stem cells and muscle cell progenitors

    DEFF Research Database (Denmark)

    Isa, Adiba; Nehlin, Jan; Sabir, Hardee Jawad

    2010-01-01

    HLA class-I expression is weak in embryonic stem cells but increases rapidly during lineage progression. It is unknown whether all three classical HLA class-I antigens follow the same developmental program. In the present study, we investigated allele-specific expression of HLA-A, -B, and -C...... at the mRNA and protein levels on human mesenchymal stem cells from bone marrow and adipose tissue as well as striated muscle satellite cells and lymphocytes. Using multicolour flow cytometry, we found high cell surface expression of HLA-A on all stem cells and PBMC examined. Surprisingly, HLA-B was either...... undetectable or very weakly expressed on all stem cells protecting them from complement-dependent cytotoxicity (CDC) using relevant human anti-B and anti-Cw sera. IFNgamma stimulation for 48-72 h was required to induce full HLA-B protein expression. Quantitative real-time RT-PCR showed that IFNgamma induced...

  4. Recent Developments in Organophosphorus Flame Retardants Containing P-C Bond and Their Applications

    Directory of Open Access Journals (Sweden)

    Sophie Wendels

    2017-07-01

    Full Text Available Organophosphorus compounds containing P-C bonds are increasingly developed as flame retardant additives due to their excellent thermal and hydrolytic stability and ease of synthesis. The latest development (since 2010 in organophosphorus flame retardants containing P-C bonds summarized in this review. In this review, we have broadly classified such phosphorus compounds based on the carbon unit linked to the phosphorus atom i.e., could be a part of either an aliphatic or an aromatic unit. We have only considered those published literature where a P-C bond was created as a part of synthetic strategy to make either an intermediate or a final organophosphorus compound with an aim to use it as a flame retardant. General synthetic strategies to create P-C bonds are briefly discussed. Most popular synthetic strategies used for developing P-C containing phosphorus based flame retardants include Michael addition, Michaelis–Arbuzov, Friedels–Crafts and Grignard reactions. In general, most flame retardant derivatives discussed in this review have been prepared via a one- to two-step synthetic strategy with relatively high yields greater than 80%. Specific examples of P-C containing flame retardants synthesized via suitable synthetic strategy and their applications on various polymer systems are described in detail. Aliphatic phosphorus compounds being liquids or low melting solids are generally applied in polymers via coatings (cellulose or are incorporated in the bulk of the polymers (epoxy, polyurethanes during their polymerization as reactive or non-reactive additives. Substituents on the P atoms and the chemistry of the polymer matrix greatly influence the flame retardant behavior of these compounds (condensed phase vs. the gas phase. Recently, aromatic DOPO based phosphinate flame retardants have been developed with relatively higher thermal stabilities (>250 °C. Such compounds have potential as flame retardants for high temperature processable

  5. Trace Detection of Organophosphorus Chemical Warfare Agents in Wastewater and Plants by Luminescent UIO-67(Hf) and Evaluating the Bioaccumulation of Organophosphorus Chemical Warfare Agents.

    Science.gov (United States)

    Lian, Xiao; Yan, Bing

    2018-04-17

    Organophosphorus chemical warfare agents (OPCWAs) are a group of organic pollutants characterized by high toxicity and chemical stability, and they are very difficult to be degraded. The trace quality of OPCWAs in water and food will cause great harm to the human body. Therefore, the detection of OPCWAs is a difficult challenge, which has become the research hotspot over the world. In this work, a Hf-based luminescent metal-organic framework (Eu@1) is prepared, and the reactivity of Hf 12 results in a methanephosphonic acid (MPA)-induced luminescence quenching and the charge transfer from MPA to Hf(IV) and generated exciplexes which are responsible for this quenching effect. The excellent performance of Eu@1 in the detection of MPA, with its finer selectivity, high sensitivity (LOD = 0.4 ppm), and large linear range (10 -7 to 10 -3 M), is encouraging for application in wastewater detection. Importantly, MPA is a pollutant that can be absorbed by plants and causes the bioaccumulation effect, and thus, the detection of MPA in real plant samples is a purposeful topic. Eu@1 also achieved satisfactory results in actual plant sample testing, and the bioaccumulation of MPA in onions, turnips, and cabbages is determined via our sensor. This fabricated detector provides a feasible path for the detection of ppm-level OPCWAs in a complex environment, which will help humans to avoid OPCWA-contaminated foods.

  6. 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

  7. Organophosphorus pentavalent compounds: history, synthetic methods of preparation and application as insecticides and antitumor agents; Compostos organofosforados pentavalentes: historico, metodos sinteticos de preparacao e aplicacoes como inseticidas e agentes antitumorais

    Energy Technology Data Exchange (ETDEWEB)

    Santos, Viviane Martins Rebello dos; Donnici, Claudio Luis [Universidade Federal de Minas Gerais, Belo Horizonte, MG (Brazil). Dept. de Quimica; DaCosta, Joao Batista Neves; Caixeiro, Janaina Marques Rodrigues [Universidade Federal Rural do Rio de Janeiro, Seropedica, RJ (Brazil). Dept. de Quimica]. E-mail: dacosta@ufrrj.br

    2007-01-15

    This paper is a review of the history, synthesis and application of organophosphorus compounds, especially of those of pentavalent phosphorus, such as phosphoramidates, phosphorothioates, phosphonates and phosphonic acids with insecticide and anticancer activities. The organophosphorus compounds with agrochemical applications show great structural variety, They include not only insecticides, but also fungicides, herbicides, and others. The large variety of commercially available organophosphorus pesticides is remarkable. Even more interesting is the high efficiency of some organophosphorus compounds as anticancer agents such as cyclophosphamide and its derivatives. (author)

  8. Influence of modifiers on the separation of dysprosium from neodymium using organophosphorus acid derivates

    Energy Technology Data Exchange (ETDEWEB)

    Elwert, Tobias; Schwarz, Sabrina; Goldmann, Daniel [TU Clausthal, Clausthal-Zellerfeld (Germany). Lehrstuhl fuer Rohstoffaufbereitung und Recycling

    2016-01-15

    The aim of this study was to investigate the applicability of three organophosphorus acid derivates (D2EHPA, EHEHPA and Cyanex 572) for the separation of terbium and dysprosium from praseodymium and neodymium from NdFeB magnets in chloride solution. A special focus was put on the effect of phase modifiers. The investigations revealed that all extractants show in general a similar extraction behavior but the extraction is shifted to higher pH values in the order D2EHPA < EHEHPA < Cyanex 572. Therefore, and due to higher realizable loadings, EHEHPA and Cyanex 572 are more suitable for the investigated separation problem than D2EHPA. Whereas EHEHPA requires 1-decanol as phase modifier, Cyanex 572 can be employed without modifier addition.

  9. Nicotine And The Risk Of Exposure To The Organophosphorus Insecticide Cyolane

    International Nuclear Information System (INIS)

    Hassanin M, M.; Bahig Mervat, E.

    1999-01-01

    The present investigation was undertaken to show the effect of repeated oral doses of nicotine (0.5 mg/kg) on rats pretreated or during treatment with cyolane (0.5 mg/kg 1/20 LD 5 0). The results revealed that exposure of smokers to organophosphorus pesticides exerted slight changes in GOT and GPT. Alkaline phosphatase was significantly increased to reach maximal (228.54 and 148.64%) after nicotine + cyolane and/or nicotine were given for a week to rats pretreated with cyolane for 2 weeks. Total protein, albumin and Albumin/Globulin (A/G) ratio were also affected. Administration of nicotine along with cyolane to rats showed a significant increase in cholesterol content compared to that of cyolane treated rats. Values of thyroxine (T 4 ) and triiodothyronine (T 3 ) also fluctuated

  10. Assessment of some biochemical and hematological parameters in treated with the organophosphorus insecticide cyanox

    International Nuclear Information System (INIS)

    ElMahdy, A.A.; Fayz, V.; Hassanin, M.M.

    2002-01-01

    The effect of single acute oral doses of 29 and 58 mg/kg of cyanox (an organophosphorus pesticide) on selected biochemical and hematological parameters in male rats were investigated. This compound inhibited blood acetylcholine esterase (AChE) significantly, over 72 hours post-treatment with the higher dose level. Serum alkaline phosphatase (ALP) was significantly depressed, 48 hr after treatment with 58 mg/kg. Serum bilirubin was elevated significantly after 48 and 24 hr of treatment with 29 and 58 mg/kg, respectively. Serum cholesterol increased significantly over 48 hr post-treatment with both dose levels.Hypertriglyceridemia was noticed only in the higher dose group. Serum total lipids were increased significantly at both dose levels. A slight treatment-related effect was observed in serum creatinine level. The hematological investigations revealed the occurrence of macrocytic normochromic anemia that was more evident in the lower dose group

  11. Multipathway organophosphorus pesticide exposures of preschool children living in agricultural and nonagricultural communities

    International Nuclear Information System (INIS)

    Lu Chensheng; Kedan, Golan; Fisker-Andersen, Jennifer; Kissel, J.C.; Fenske, R.A.

    2004-01-01

    Environmental measurements of organophosphorus (OP) pesticides were conducted in the homes of 13 children, who lived either in the Seattle metropolitan area or in the agricultural region of Washington State, to ascertain exposure through multiple pathways. Each home was sampled for two 24-h periods during two seasons, summer and fall. Samples included 24-h indoor air, drinking water, soil, house dust, and hand and toy wipes and 24-h duplicate diets. At least one OP pesticide was found in each of the matrices sampled except for drinking water. Half of the indoor air samples contained detectable levels of chlorpyrifos or diazinon. Detectable levels of diazinon and azinphosmethyl in house dust were found in most of the agricultural homes, whereas only diazinon was found in the Seattle homes in the summer. Quantifiable chlorpyrifos and azinphosmethyl were found on either agricultural children's hands or their toys. These findings suggest different exposure pathways for children living in agricultural and nonagricultural regions

  12. Application of laccase-mediator system (LMS) for the degradation of organophosphorus compounds.

    Science.gov (United States)

    Trovaslet-Leroy, Marie; Jolivalt, Claude; Froment, Marie-Thérèse; Brasme, Bernardin; Lefebvre, Bertrand; Daveloose, Denis; Nachon, Florian; Masson, Patrick

    2010-09-06

    Degradation of organophosphorus compounds was achieved in the presence of purified fungal laccase from Trametes versicolor and a small molecular weight redox mediator (ABTS). This laccase-mediator system (LMS) catalyzed degradation of VX, PhX and VR while had no apparent effect on CVX, ecothiophate or demeton. Inhibition of ABTS oxidation was shown with VX, PhX, VR and CVX. Results with CVX suggest either no degradation subsequent to interaction with the laccase active site or the formation of a new toxic compound. PhX degradation was also monitored by mass spectroscopy, a method that allowed us to identify certain intermediates formed during OP degradation. Altogether, results underline the importance of the OP nitrogen atom at beta-position and of its substituents, even though the intimate mechanism of laccase-catalyzed degradation is not yet known. Copyright (c) 2010 Elsevier Ireland Ltd. All rights reserved.

  13. Estimation of solubility of organo-phosphorus extractants by P determination using molybdovanadophosphoric acid method

    International Nuclear Information System (INIS)

    Gill, J.S.; Kotekar, M.K.; Singh, H.

    2005-01-01

    Solvent extraction processes have been found to be suitable for uranium recovery from phosphoric acid. Various extractants like di-2-ethyl hexylphosphoric acid (D2EHPA), di-nonylphenyl phosphoric acid (DNPPA) and synergistic agents like tri-butyl phosphate (TBP), tri-octyl phosphine oxide (TOPO) have been used in liquid-liquid extraction of uranium from phosphoric acid. Contents of these organo-phosphorus compounds in aqueous raffinates need estimation for process requirements. Solubility of Tri-butyl phosphate (TBP) and Di-2-ethylhexyl phosphoric acid (D2EHPA) extractants have been determined in different media of water, oxalic acid (0.6M) and sulphuric acid (3.75M) solutions. These compounds were estimated by determining their phosphorus (P) contents employing molybdovanadophosphoric acid method, after digesting and solubalizing them in nitric and perchloric acid. (author)

  14. Concentration of Organochlorine and Organophosphorus Pesticides in Different Molluscs from Tighra Reservoir, Gwalior, India.

    Science.gov (United States)

    Mamta; Rao, R J; Wani, Khursheed Ahmad

    2015-09-01

    Concentration of organochlorine and organophosphorus pesticides and their metabolites were determined in whole body homogenate of L. acuminata, I. exustus, V. dissimilis, V. bengalensis, from Tighra Reservoir during summer and post monsoon season using GC-MS technique. The different types of pesticides viz., HCB, heptachlor, aldrin, endosulfan, dieldrin, p,p-DDT, choloropyrifos, methyl parathion, dicholorovos, ethion, malathion, parathion were found in resident molluscs studied. Concentration of these pesticides varied independently during the summer and the post monsoon season at monitored sites. The study concluded that presence of pesticides in Tighra Reservoir is a major concern on public and ecosystem health and use of biopesticides in the adjacent area of Reservoir is highly recommended.

  15. Is the stoichiometry of the europium nitrate complexes with neutral organophosphorus extractants be anticipated?

    International Nuclear Information System (INIS)

    Beudaert, Ph.; Lamare, V.; Wipff, G.

    2001-01-01

    Molecular dynamics simulations have been performed in water on europium nitrate complexes with three neutral organophosphorus extractants (TBP, TPPO and CMPO) in order to determine on what criteria it is possible to obtain by simulations the experimental 1:3 stoichiometry in organic solution. This stoichiometry was investigated by progressive saturation of the cation coordination sphere. When the nitrate counter-ions are bidentate, the 1:3 stoichiometry corresponds to the degree of saturation where the interaction energy between europium and water becomes repulsive. Beyond this stoichiometry, complexes with TPPO and CMPO are unstable, although a 1:4 complex with TBP may exist but its formation appears to be energetically unfavored. (author)

  16. Determination of carbamates and organophosphorus pesticides by SDME-GC in natural water.

    Science.gov (United States)

    López-Blanco, Carmen; Gómez-Alvarez, Sonia; Rey-Garrote, María; Cancho-Grande, Beatriz; Simal-Gándara, Jesús

    2005-10-01

    Water contamination due to the wide variety of pesticides used in agriculture practices is a global environmental pollution problem. Analytical methods with low quantification limits are necessary. The application of a new extraction technique, solvent drop microextraction (SDME), followed by gas chromatography with a nitrogen-phosphorus detector, was assessed for determining carbamates and organophosphorus pesticides in natural water. Experimental parameters which control the performance of SDME such as selection of microextraction solvent, optimization of organic drop volume, effects of sample stirring, salt addition, and, finally, sorption time profiles were studied. Once SDME was optimized, analytical parameters such as linearity (r 2>0.99), precision (pesticides from water in 14 min; the use of organic solvents and water samples for SDME is negligible compared to other extraction techniques.

  17. Acetylcholinesterase-reduced graphene oxide hybrid films for organophosphorus neurotoxin sensing via quartz crystal microbalance

    Science.gov (United States)

    Tang, Shi; Ma, Wenying; Xie, Guangzhong; Su, Yuanjie; Jiang, Yadong

    2016-09-01

    An acetylcholinesterase (AChE)-reduced graphene oxide (RGO) hybrid films based biosensor enabled by quartz crystal microbalance (QCM) has been developed for the detection of organophosphorus neurotoxin in gas phase at room temperature. To improve the sensing performance, RGO was used to immobilize large quantities of enzyme and provide a favorable microenvironment to maintain the enzyme activity. The experimental results reveal that the response of AChE-RGO/glutaraldehyde based sensors is about 8 times larger than that of the AChE with the sensitivity of 1.583 Hz/mg/m3. 1.0 mg amount of RGO, 5% concentration of glutaraldehyde and pH 6.8 is the optimal condition of this biosensor.

  18. Application of graphene for the SPE clean-up of organophosphorus pesticides residues from apple juices.

    Science.gov (United States)

    Han, Qiang; Wang, Zonghua; Xia, Jianfei; Zhang, Xiaoqiong; Wang, Hongwu; Ding, Mingyu

    2014-01-01

    In this paper, an effective graphene-based SPE clean-up procedure coupled with GC-MS was developed for the determination of organophosphorus pesticide residues in apple juices. The apple juice samples were diluted with water and could be loaded onto the cartridge directly. Several parameters affecting the extraction efficiency were investigated, including the type of elution, washing solution, and sample pH. Under the optimized conditions, excellent limits of quantitation for the target analytes were found to be 0.15-1.18 ng/mL, and the average recoveries of the analytes at two spiked levels for real-sample analysis ranged from 69.8 to 106.2% with RSDs less than 7.3%. Furthermore, the graphene-based cartridges exhibited superior reusability for juice sample analysis. The proposed method is sensitive, simple, and cost saving, and provides a detection platform for the monitoring of pesticide residues.

  19. Removal of acetic acid from simulated hemicellulosic hydrolysates by emulsion liquid membrane with organophosphorus extractants.

    Science.gov (United States)

    Lee, Sang Cheol

    2015-09-01

    Selective removal of acetic acid from simulated hemicellulosic hydrolysates containing xylose and sulfuric acid was attempted in a batch emulsion liquid membrane (ELM) system with organophosphorus extractants. Various experimental variables were used to develop a more energy-efficient ELM process. Total operation time of an ELM run with a very small quantity of trioctylphosphine oxide as the extractant was reduced to about a third of those required to attain almost the same extraction efficiency as obtained in previous ELM works without any extractant. Under specific conditions, acetic acid was selectively separated with a high degree of extraction and insignificant loss of xylose, and its purity and enrichment ratio in the stripping phase were higher than 92% and 6, respectively. Also, reused organic membrane solutions exhibited the extraction efficiency as high as fresh organic solutions did. These results showed that the current ELM process would be quite practical. Copyright © 2015 Elsevier Ltd. All rights reserved.

  20. Fungal lytic polysaccharide monooxygenases bind starch and β-cyclodextrin similarly to amylolytic hydrolases

    DEFF Research Database (Denmark)

    Nekiunaite, Laura; Isaksen, Trine; Vaaje-Kolstad, Gustav

    2016-01-01

    , the clustering of CBM20s from starch-targeting LPMOs and hydrolases was in accord with taxonomy and did not correlate to appended catalytic activity. Altogether, these results demonstrate that the CBM20-binding scaffold is retained in the evolution of hydrolytic and oxidative starch-degrading activities....

  1. Improvement of enantioselectivity by immobilized imprinting of epoxide hydrolase from Rhodotorula glutinis

    NARCIS (Netherlands)

    Kronenburg, N.A.E.; Bont, de J.A.M.; Fischer, L.

    2001-01-01

    The yeast Rhodotorula glutinis contains an enantioselective, membrane-associated epoxide hydrolase (EH). Partially purified EH was immobilized in a two-step procedure. In the first step, the proteins were derivatized with itaconic anhydride. In the second step, the derivatized proteins were

  2. Primary structure and catalytic mechanism of the epoxide hydrolase from Agrobacterium radiobacter AD1

    NARCIS (Netherlands)

    Rink, R; Fennema, M; Smids, M; Dehmel, U; Janssen, DB

    1997-01-01

    The epoxide hydrolase gene from Agrobacterium radiobacter AD1, a bacterium that is able to grow on epichlorohydrin as the sole carbon source, was cloned by means of the polymerase chain reaction with two degenerate primers based on the N-terminal and C-terminal sequences of the enzyme, The epoxide

  3. Genetically lowered microsomal epoxide hydrolase activity and tobacco-related cancer in 47,000 individuals

    DEFF Research Database (Denmark)

    Lee, Julie; Dahl, Morten; Nordestgaard, Børge G

    2011-01-01

    Two functional polymorphisms of the microsomal epoxide hydrolase (mEH) gene (EPHX1), Tyr113His (rs1051740) and His139Arg (rs2234922), have variably been found to influence susceptibility to various cancer forms. We tested whether genetically lowered mEH activity affects risk of developing cancer ...

  4. In Silico Investigation of Flavonoids as Potential Trypanosomal Nucleoside Hydrolase Inhibitors

    Directory of Open Access Journals (Sweden)

    Christina Hung Hung Ha

    2015-01-01

    Full Text Available Human African Trypanosomiasis is endemic to 37 countries of sub-Saharan Africa. It is caused by two related species of Trypanosoma brucei. Current therapies suffer from resistance and public accessibility of expensive medicines. Finding safer and effective therapies of natural origin is being extensively explored worldwide. Pentamidine is the only available therapy for inhibiting the P2 adenosine transporter involved in the purine salvage pathway of the trypanosomatids. The objective of the present study is to use computational studies for the investigation of the probable trypanocidal mechanism of flavonoids. Docking experiments were carried out on eight flavonoids of varying level of hydroxylation, namely, flavone, 5-hydroxyflavone, 7-hydroxyflavone, chrysin, apigenin, kaempferol, fisetin, and quercetin. Using AutoDock 4.2, these compounds were tested for their affinity towards inosine-adenosine-guanosine nucleoside hydrolase and the inosine-guanosine nucleoside hydrolase, the major enzymes of the purine salvage pathway. Our results showed that all of the eight tested flavonoids showed high affinities for both hydrolases (lowest free binding energy ranging from −10.23 to −7.14 kcal/mol. These compounds, especially the hydroxylated derivatives, could be further studied as potential inhibitors of the nucleoside hydrolases.

  5. High-throughput screening for gene libraries expressing carbohydrate hydrolase activity

    NARCIS (Netherlands)

    Leemhuis, Hans; Euverink, Gert-Jan W.; Dijkhuizen, Lubbert

    2003-01-01

    A simple and fast method is described allowing screening of large number of Escherichia coli clones (4000 per day) for the presence of functional or improved carbohydrate hydrolase enzymes. The procedure is relatively cheap and has the advantage that carbohydrate degrading activity can be directly

  6. O-hydroxyacetamide carbamates as a highly potent and selective class of endocannabinoid hydrolase inhibitors.

    Science.gov (United States)

    Niphakis, Micah J; Johnson, Douglas S; Ballard, T Eric; Stiff, Cory; Cravatt, Benjamin F

    2012-05-16

    The two major endocannabinoid transmitters, anandamide (AEA) and 2-arachidonoylglycerol (2-AG), are degraded by distinct enzymes in the nervous system, fatty acid amide hydrolase (FAAH) and monoacylglycerol lipase (MAGL), respectively. FAAH and MAGL inhibitors cause elevations in brain AEA and 2-AG levels, respectively, and reduce pain, anxiety, and depression in rodents without causing the full spectrum of psychotropic behavioral effects observed with direct cannabinoid receptor-1 (CB1) agonists. These findings have inspired the development of several classes of endocannabinoid hydrolase inhibitors, most of which have been optimized to show specificity for either FAAH or MAGL or, in certain cases, equipotent activity for both enzymes. Here, we investigate an unusual class of O-hydroxyacetamide carbamate inhibitors and find that individual compounds from this class can serve as selective FAAH or dual FAAH/MAGL inhibitors in vivo across a dose range (0.125-12.5 mg kg(-1)) suitable for behavioral studies. Competitive and click chemistry activity-based protein profiling confirmed that the O-hydroxyacetamide carbamate SA-57 is remarkably selective for FAAH and MAGL in vivo, targeting only one other enzyme in brain, the additional 2-AG hydrolase ABHD6. These data designate O-hydroxyacetamide carbamates as a versatile chemotype for creating endocannabinoid hydrolase inhibitors that display excellent in vivo activity and tunable selectivity for FAAH-anandamide versus MAGL (and ABHD6)-2-AG pathways.

  7. Proteomic Analysis of a Novel Bacillus Jumbo Phage Revealing Glycoside Hydrolase As Structural Component.

    Science.gov (United States)

    Yuan, Yihui; Gao, Meiying

    2016-01-01

    Tailed phages with genomes of larger than 200 kbp are classified as Jumbo phages and exhibited extremely high uncharted diversity. The genomic annotation of Jumbo phage is often disappointing because most of the predicted proteins, including structural proteins, failed to make good hits to the sequences in the databases. In this study, 23 proteins of a novel Bacillus Jumbo phage, vB_BpuM_BpSp, were identified as phage structural proteins by the structural proteome analysis, including 14 proteins of unknown function, 5 proteins with predicted function as structural proteins, a glycoside hydrolase, a Holliday junction resolvase, a RNA-polymerase β-subunit, and a host-coding portal protein, which might be hijacked from the host strain during phage virion assembly. The glycoside hydrolase (Gp255) was identified as phage virion component and was found to interact with the phage baseplate protein. Gp255 shows specific lytic activity against the phage host strain GR8 and has high temperature tolerance. In situ peptidoglycan-hydrolyzing activities analysis revealed that the expressed Gp255 and phage structural proteome exhibited glycoside hydrolysis activity against the tested GR8 cell extracts. This study identified the first functional individual structural glycoside hydrolase in phage virion. The presence of activated glycoside hydrolase in phage virions might facilitate the injection of the phage genome during infection by forming pores on the bacterial cell wall.

  8. Purification and characterization of TrzF: biuret hydrolysis by allophanate hydrolase supports growth.

    Science.gov (United States)

    Shapir, Nir; Cheng, Gang; Sadowsky, Michael J; Wackett, Lawrence P

    2006-04-01

    TrzF, the allophanate hydrolase from Enterobacter cloacae strain 99, was cloned, overexpressed in the presence of a chaperone protein, and purified to homogeneity. Native TrzF had a subunit molecular weight of 65,401 and a subunit stoichiometry of alpha(2) and did not contain significant levels of metals. TrzF showed time-dependent inhibition by phenyl phosphorodiamidate and is a member of the amidase signature protein family. TrzF was highly active in the hydrolysis of allophanate but was not active with urea, despite having been previously considered a urea amidolyase. TrzF showed lower activity with malonamate, malonamide, and biuret. The allophanate hydrolase from Pseudomonas sp. strain ADP, AtzF, was also shown to hydrolyze biuret slowly. Since biuret and allophanate are consecutive metabolites in cyanuric acid metabolism, the low level of biuret hydrolase activity can have physiological significance. A recombinant Escherichia coli strain containing atzD, encoding cyanuric acid hydrolase that produces biuret, and atzF grew slowly on cyanuric acid as a source of nitrogen. The amount of growth produced was consistent with the liberation of 3 mol of ammonia from cyanuric acid. In vitro, TrzF was shown to hydrolyze biuret to liberate 3 mol of ammonia. The biuret hydrolyzing activity of TrzF might also be physiologically relevant in native strains. E. cloacae strain 99 grows on cyanuric acid with a significant accumulation of biuret.

  9. 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

  10. Improved annotation of conjugated bile acid hydrolase superfamily members in Gram-positive bacteria.

    NARCIS (Netherlands)

    Lambert, J.M.; Siezen, R.J.; Vos, W.M. de; Kleerebezem, M.

    2008-01-01

    Most Gram-positive bacteria inhabiting the gastrointestinal tract are capable of hydrolysing bile salts. Bile salt hydrolysis is thought to play an important role in various biological processes in the host. Therefore, correct annotation of bacterial bile salt hydrolases (Bsh) in public databases

  11. The alpha/beta-Hydrolase Fold 3DM Database (ABHDB) as a Tool for Protein Engineering

    NARCIS (Netherlands)

    Kourist, R.; Jochens, H.; Bartsch, S.; Kuipers, R.K.P.; Padhi, S.K.; Gall, M.; Bottcher, D.; Joosten, H.J.; Bornscheuer, U.T.

    2010-01-01

    Aligning the haystack to expose the needle: The 3DM method was used to generate a comprehensive database of the a/ß-hydrolase fold enzyme superfamily. This database facilitates the analysis of structure–function relationships and enables novel insights into this superfamily to be made. In addition

  12. Soluble epoxide hydrolase in the generation and maintenance of high blood pressure in spontaneously hypertensive rats

    NARCIS (Netherlands)

    Koeners, Maarten P.; Wesseling, Sebastiaan; Ulu, Arzu; Lopez Sepulveda, Rocio; Morisseau, Christophe; Braam, Branko; Hammock, Bruce D.; Joles, Jaap A.

    Koeners MP, Wesseling S, Ulu A, Sepulveda RL, Morisseau C, Braam B, Hammock BD, Joles JA. Soluble epoxide hydrolase in the generation and maintenance of high blood pressure in spontaneously hypertensive rats. Am J Physiol Endocrinol Metab 300: E691-E698, 2011. First published January 25, 2011; doi:

  13. Esterase SeE of Streptococcus equi ssp. equi is a novel nonspecific carboxylic ester hydrolase.

    Science.gov (United States)

    Xie, Gang; Liu, Mengyao; Zhu, Hui; Lei, Benfang

    2008-12-01

    Extracellular carboxylic ester hydrolases are produced by many bacterial pathogens and have been shown recently to be important for virulence of some pathogens. However, these hydrolases are poorly characterized in enzymatic activity. This study prepared and characterized the secreted ester hydrolase of Streptococcus equi ssp. equi (designated SeE for S. equi esterase). SeE hydrolyzes ethyl acetate, acetylsalicylic acid, and tributyrin but not ethyl butyrate. This substrate specificity pattern does not match those of the three conventional types of nonspecific carboxylic ester hydrolases (carboxylesterases, arylesterases, and acetylesterases). To determine whether SeE has lipase activity, a number of triglycerides and vinyl esters were tested in SeE-catalyzed hydrolysis. SeE does not hydrolyze triglycerides and vinyl esters of long-chain carboxylic acids nor display interfacial activation, indicating that SeE is not a lipase. Like the conventional carboxylesterases, SeE is inhibited by di-isopropylfluorophosphate. These findings indicate that SeE is a novel carboxylesterase with optimal activity for acetyl esters.

  14. Characterization of the epoxide hydrolase from an epichlorohydrin-degrading Pseudomonas sp.

    NARCIS (Netherlands)

    Jacobs, Mariken H.J.; van den Wijngaard, Abraham; Pentenga, Marjan; Janssen, Dick B.

    1991-01-01

    An epoxide hydrolase was purified to homogeneity from the epichlorohydrin-utilizing bacterium Pseudomonas sp. strain AD1. The enzyme was found to be a monomeric protein with a molecular mass of 35 kDa. With epichlorohydrin as the substrate, the enzyme followed Michaelis-Menten kinetics with a Km

  15. Nanophotocatalytic UV degradation system for organophosphorus pesticides in water samples and analysis by Kubista model

    Directory of Open Access Journals (Sweden)

    Amit Kumar Sharma

    2016-11-01

    Full Text Available The photocatalytic degradation of organophosphorus pesticides like methyl parathion (MP and parathion (PA, under UV irradiation is studied using synthesized Zinc oxide (ZnO nanocatalyst. Two cases were considered for removal of organophosphorus (OP pesticides such as only UV (i.e. direct photolysis and UV–ZnO nanocrystal. The Langmuir–Hinshelwood (L–H relationship between the initial rate and the initial concentration indicates that the reaction occurs with the adsorbed substrate. Factors affecting the kinetics of the process such as quantum yield, electrical energy per order (EEO, dosage of nanophotocatalyst and L–H model have been discussed. The optimal conditions for degradation were obtained using 85 mg l−1 ZnO, 40% O2 (v/v with rate constants L–H adsorption equilibrium constant (Kp = 0.127 for MP and 0.122 for PA and rate constant of surface reaction (Kc = 0.212 for MP and 0.204 for PA. The kinetics of disappearance of OP pesticides (i.e. MP and PA in water suspension using ZnO nanophotocatalyst illuminated at λ = 253.7 nm and examined. The quantum yield values for photodegradation of pesticides by photolysis and UV–ZnO process under the illumination of UV at 253.7 nm is calculated as 0.00072, 0.012 for MP; 0.00068 and 0.01 for PA. The EEO values for degradation of pesticide by photolysis for UV–ZnO was found to be 20,006, 1392.4 kWh m−3 for MP; 19,089 and 1387.2 kWh m−3 for PA.

  16. 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.

  17. 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.

  18. Increased fetal endocardial echogenicity mimicking endocardial fibroelastosis following maternal organophosphorus poisoning and its complete regression in utero

    International Nuclear Information System (INIS)

    Balakumar, Karippaliyil; Misha, Kannan; Milind, Karippaliyil

    2013-01-01

    Fetal endocardial fibroelastosis (EFE) has been diagnosed by antenatal ultrasonography in the past few years. A typical case of isolated endocardial fibroelastosis is illustrated here, in a fetus of 22 weeks of gestational age exposed to maternal organophosphorus poisoning at 20 weeks. No other structural cardiac or other systemic anomalies were detected in this fetus. The abnormal fetal echocardiographic features mimicking endocardial fibroelastosis completely regressed after 14 weeks and a normal full-term baby was delivered. Postnatal echocardiogram showed normal cardiac parameters. The diagnostic features mimicking EFE following maternal organophosphorus poisoning at 20 weeks of gestational age and the subsequent complete reversal of these changes after 14 weeks of diagnosis are reported for the first time in the literature

  19. Synergistic Extraction of Copper from Nitrate Solutions Using β-Hydroxy-Naphthaldoxime and Organophosphorus Compounds into Carbon-Tetrachloride

    Science.gov (United States)

    Dey, Pulak; Basu, Sukalyan

    2011-12-01

    The extraction behavior of Cu(II) from an aqueous nitrate medium employing β-hydroxy-naphthaldoxime in carbon tetrachloride has been investigated in the presence of several organophosphorus donors like tri-octyl phosphine oxide, tri-butyl phosphine oxide, and tri-butyl phosphate at pH 1.5. The concentration of the metal was measured by atomic absorption spectrophotometry. Synergism was observed when neutral donor was added because of the formation of the adduct [Cu(L)2.(S)] in CCl4 (S denotes neutral donor). The equilibrium constants of the binary system using β-hydroxy-naphthaldoxime and the ternary system involving another addition of an organophosphorus compound were calculated from the extraction date obtained. Furthermore, the temperature dependence of the equilibrium constants was also investigated to evaluate standard enthalpy (Δ H°), entropy (Δ S°), and free energy (Δ G°) of the reactions proposed.

  20. Degradation of Organophosphorus Pesticides in Water during UV/H2O2 Treatment: Role of Sulphate and Bicarbonate Ions

    OpenAIRE

    Fadaei, Am; Dehghani, Mh; Mahvi, Ah; Nasseri, S.; Rastkari, N.; Shayeghi, M.

    2012-01-01

    The photodegradation of two organophosphorus pesticides, malathian and diazinon, by sulfate radicals and bicarbonate radicals in aqueous solution were studied. The effect of the operational parameters such as pH, salt concentration, water type, H2O2 concentration and initial concentration of pesticides was studied. Gas chromatography mass spectroscopy (GC–MS) was used for analyses of pesticides. When salt effect was studied, it was found that sodium bicarbonate was the most powerful inhibitor...

  1. Expression of organophosphorus-degradation gene ( opd) in aggregating and non-aggregating filamentous nitrogen-fixing cyanobacteria

    Science.gov (United States)

    Li, Qiong; Tang, Qing; Xu, Xudong; Gao, Hong

    2010-11-01

    Genetic engineering in filamentous N2-fixing cyanobacteria usually involves Anabaena sp. PCC 7120 and several other non-aggregating species. Mass culture and harvest of such species are more energy consuming relative to aggregating species. To establish a gene transfer system for aggregating species, we tested many species of Anabaena and Nostoc, and identified Nostoc muscorum FACHB244 as a species that can be genetically manipulated using the conjugative gene transfer system. To promote biodegradation of organophosphorus pollutants in aquatic environments, we introduced a plasmid containing the organophosphorus-degradation gene ( opd) into Anabaena sp. PCC 7120 and Nostoc muscorum FACHB244 by conjugation. The opd gene was driven by a strong promoter, P psbA . From both species, we obtained transgenic strains having organophosphorus-degradation activities. At 25°C, the whole-cell activities of the transgenic Anabaena and Nostoc strains were 0.163±0.001 and 0.289±0.042 unit/μg Chl a, respectively. However, most colonies resulting from the gene transfer showed no activity. PCR and DNA sequencing revealed deletions or rearrangements in the plasmid in some of the colonies. Expression of the green fluorescent protein gene from the same promoter in Anabaena sp. PCC 7120 showed similar results. These results suggest that there is the potential to promote the degradation of organophosphorus pollutants with transgenic cyanobacteria and that selection of high-expression transgenic colonies is important for genetic engineering of Anabaena and Nostoc species. For the first time, we established a gene transfer and expression system in an aggregating filamentous N2-fixing cyanobacterium. The genetic manipulation system of Nostoc muscorum FACHB244 could be utilized in the elimination of pollutants and large-scale production of valuable proteins or metabolites.

  2. Quantum dot-DNA aptamer conjugates coupled with capillary electrophoresis: A universal strategy for ratiometric detection of organophosphorus pesticides.

    Science.gov (United States)

    Tang, Tingting; Deng, Jingjing; Zhang, Min; Shi, Guoyue; Zhou, Tianshu

    2016-01-01

    Based on the highly sensitivity and stable-fluorescence of water-soluble CdTe/CdS core-shell quantum dots (QDs) with broad-specificity DNA aptamers, a novel ratiometric detection strategy was proposed for the sensitive detection of organophosphorus pesticides by capillary electrophoresis with laser-induced fluorescence (CE-LIF). The as-prepared QDs were first conjugated with the amino-modified oligonucleotide (AMO) by amidation reaction, which is partial complementary to the DNA aptamer of organophosphorus pesticides. Then QD-labeled AMO (QD-AMO) was incubated with the DNA aptamer to form QD-AMO-aptamer duplex. When the target organophosphorus pesticides were added, they could specifically bind the DNA aptamer, leading to the cleavage of QD-AMO-aptamer duplex, accompany with the release of QD-AMO. As a result, the ratio of peak height between QD-AMO and QD-AMO-aptamer duplex changed in the detection process of CE-LIF. This strategy was subsequently applied for the detection of phorate, profenofos, isocarbophos, and omethoate with the detection limits of 0.20, 0.10, 0.17, and 0.23μM, respectively. This is the first report about using QDs as the signal indicators for organophosphorus pesticides detection based on broad-specificity DNA aptamers by CE-LIF, thus contributing to extend the scope of application of QDs in different fields. The proposed method has great potential to be a universal strategy for rapid detection of aptamer-specific small molecule targets by simply changing the types of aptamer sequences. Copyright © 2015 Elsevier B.V. All rights reserved.

  3. A study on oxidative stress and antioxidant status of agricultural workers exposed to organophosphorus insecticides during spraying

    OpenAIRE

    Rastogi S; Satyanarayan P.V.V; Ravishankar D; Tripathi Sachin

    2009-01-01

    Oxidative stress status and Acetylcholinesterase (AChE) activity were studied in blood samples obtained from 61 agricultural workers engaged in spraying organophosphorus (OP) insecticides in the mango plantation, with a minimum work history of one year, in the age range of 12-55 years. Controls were age-matched, unexposed workers, who never had any exposure to OP pesticides. They were evaluated for oxidative stress markers MDA (end product of lipid peroxidation), reduced glutathione (GSH), an...

  4. The Brugada syndrome mutation A39V does not affect surface expression of neuronal rat Cav1.2 channels

    Directory of Open Access Journals (Sweden)

    Simms Brett A

    2012-03-01

    Full Text Available Abstract Background A loss of function of the L-type calcium channel, Cav1.2, results in a cardiac specific disease known as Brugada syndrome. Although many Brugada syndrome channelopathies reduce channel function, one point mutation in the N-terminus of Cav1.2 (A39V has been shown to elicit disease a phenotype because of a loss of surface trafficking of the channel. This lack of cell membrane expression could not be rescued by the trafficking chaperone Cavβ. Findings We report that despite the striking loss of trafficking described previously in the cardiac Cav1.2 channel, the A39V mutation while in the background of the brain isoform traffics and functions normally. We detected no differences in biophysical properties between wild type Cav1.2 and A39V-Cav1.2 in the presence of either a cardiac (Cavβ2b, or a neuronal beta subunit (Cavβ1b. In addition, the A39V-Cav1.2 mutant showed a normal Cavβ2b mediated increase in surface expression in tsA-201 cells. Conclusions The Brugada syndrome mutation A39V when introduced into rat brain Cav1.2 does not trigger the loss-of-trafficking phenotype seen in a previous study on the human heart isoform of the channel.

  5. Ionizing radiation modulates the surface expression of human leukocyte antigen-G in a human melanoma cell line

    Energy Technology Data Exchange (ETDEWEB)

    Michelin, S.; Gallegos, C.E.; Dubner, D. [Radiopathology Laboratory, Nuclear Regulatory Authority, Buenos Aires (Argentina); Favier, B.; Carosella, E.D. [CEA, I2BM, Hopital Saint-Louis, IUH, Service de Recherches en Hemato-Immunologie, Paris (France)

    2009-07-01

    Human leukocyte antigen G (HLA-G) is a nonclassical HLA class I molecule involved in fetus protection from the maternal immune system, transplant tolerance, and viral and tumoral immune escape. Tumor-specific HLA-G expression has been described for a wide variety of malignancies, including melanomas. The aim of this study was to evaluate whether ionizing radiation (IR) could modulate the surface expression of HLA-G1 in a human melanoma cell line that expresses endogenously membrane-bound HLA-G1. For this purpose, cells were exposed to increasing doses of {gamma}-irradiation (0-20 Gy) and HLA-G1 levels at the plasma membrane were analyzed at different times postirradiation by flow cytometry. HLA-G total expression and the presence of the soluble form of HLA-G1 (sHLA-G1) in the culture medium of irradiated cells were also evaluated. IR was capable of down regulating cell surface and total HLA-G levels, with a concomitant increase of sHLA-G1 in the medium. These results could indicate that {gamma}-irradiation decreases HLA-G1 surface levels by enhancing the proteolytic cleavage of this molecule. (authors)

  6. Cell cycle phase-specific surface expression of nerve growth factor receptors TrkA and p75(NTR).

    Science.gov (United States)

    Urdiales, J L; Becker, E; Andrieu, M; Thomas, A; Jullien, J; van Grunsven, L A; Menut, S; Evan, G I; Martín-Zanca, D; Rudkin, B B

    1998-09-01

    Expression of the nerve growth factor (NGF) receptors TrkA and p75(NTR) was found to vary at the surface of PC12 cells in a cell cycle phase-specific manner. This was evidenced by using flow cytometric and microscopic analysis of cell populations labeled with antibodies to the extracellular domains of both receptors. Differential expression of these receptors also was evidenced by biotinylation of surface proteins and Western analysis, using antibodies specific for the extracellular domains of TrkA and p75(NTR). TrkA is expressed most strongly at the cell surface in M and early G1 phases, whereas p75(NTR) is expressed mainly in late G1, S, and G2 phases. This expression reflects the molecular and cellular responses to NGF in specific phases of the cell cycle; in the G1 phase NGF elicits both the anti-mitogenic effect, i.e., inhibition of the G1 to S transition, and the differentiation response whereas a survival effect is provoked elsewhere in the cell cycle. A model is proposed relating these responses to the surface expression of the two receptors. These observations open the way for novel approaches to the investigation of the mechanism of NGF signal transduction.

  7. Cell surface expression and function of the macromolecular C1 complex on the surface of human monocytes

    Directory of Open Access Journals (Sweden)

    Kinga K Hosszu

    2012-03-01

    Full Text Available The synthesis of the subunits of the C1 complex (C1q, C1s, C1r, and its regulator C1 inhibitor (C1-Inh by human monocytes has been previously established. However, surface expression of these molecules by monocytes has not been shown. Using flow cytometry and antigen-capture ELISA, we show here for the first time that, in addition to C1q, PB monocytes and the monocyte-derived U937 cells express C1s and C1r, as well as Factor B and C1-Inh on their surface. C1s and C1r immunoprecipitated with C1q, suggesting that at least some of the C1q on these cells is part of the C1 complex. Furthermore, the C1 complex on U937 cells was able to trigger complement activation via the classical pathway. The presence of C1-Inh may ensure that an unwarranted autoactivation of the C1 complex does not take place. Since C1-Inh closely monitors the activation of the C1 complex in a sterile or infectious inflammatory environment, further elucidation of the role of C1 complex is crucial to dissect its function in monocyte, DC and T cell activities, and its implications in host defense and tolerance.

  8. Kinetoplastid membrane protein-11 is present in promastigotes and amastigotes of Leishmania amazonensis and its surface expression increases during metacyclogenesis

    Directory of Open Access Journals (Sweden)

    Denise CS Matos

    2010-05-01

    Full Text Available Kinetoplastid membrane protein-11 (KMP-11, a protein present in all kinetoplastid protozoa, is considered a potential candidate for a leishmaniasis vaccine. A suitable leishmaniasis vaccine candidate molecule must be expressed in amastigotes, the infective stage for mammals. However, the expression of KMP-11 in Leishmania amastigotes has been a subject of controversy. We evaluated the expression of this molecule in logarithmic and stationary growth phase promastigotes, as well as in amastigotes, of Leishmania amazonensis by immunoblotting, flow cytometry and immunocytochemistry, using a monoclonal antibody against KMP-11. We found that KMP-11 is present in promastigotes and amastigotes. In both stages, the protein was found in association with membrane structures (at the cell surface, flagellar pocket and intracellular vesicles. More importantly, its surface expression is higher in amastigotes than in promastigotes and increases during metacyclogenesis. The increased expression of KMP-11 in metacyclic promastigotes, and especially in amastigotes, indicates a role for this molecule in the parasite relationship with the mammalian host. The presence of this molecule in amastigotes is consistent with the previously demonstrated immunoprotective capacity of vaccine prototypes based on the KMP-11-coding gene and the presence of humoral and cellular immune responses to KMP-11 in Leishmania-infected humans and animals.

  9. Cigarette Smoke Suppresses the Surface Expression of c-kit and FcεRI on Mast Cells

    Directory of Open Access Journals (Sweden)

    M. E. Givi

    2013-01-01

    Full Text Available Chronic obstructive pulmonary disease (COPD is a multicomponent disease characterized by emphysema and/or chronic bronchitis. COPD is mostly associated with cigarette smoking. Cigarette smoke contains over 4,700 chemical compounds, including free radicals and LPS (a Toll-Like Receptor 4 agonist at concentrations which may contribute to the pathogenesis of diseases like COPD. We have previously shown that short-term exposure to cigarette smoke medium (CSM can stimulate several inflammatory cells via TLR4 and that CSM reduces the degranulation of bone-marrow-derived mast cells (BMMCs. In the current study, the effect of CSM on mast cells maturation and function was investigated. Coculturing of BMMC with CSM during the development of bone marrow progenitor cells suppressed the granularity and the surface expression of c-kit and FcεRI receptors. Stimulation with IgE/antigen resulted in decreased degranulation and release of Th1 and Th2 cytokines. The effects of CSM exposure could not be mimicked by the addition of LPS to the culture medium. In conclusion, this study shows that CSM may affect mast cell development and subsequent response to allergic activation in a TLR4-independent manner.

  10. Identification and isolation of bacteria containing OPH enzyme for biodegradation of organophosphorus pesticide diazinon from contaminated agricultural soil

    Directory of Open Access Journals (Sweden)

    Sara Mobarakpoor

    2015-04-01

    Full Text Available Background: Organophosphorus insecticide diazinon has been widely used in agriculture and has the ability to transfer and accumulate in soil, water and animal tissues, and to induce toxicity in plants, animals and humans. In humans, diazinon inhibits nerve transmission by inactivating acetylcholinesterase enzyme. The present study was carried out to identify bacteria containing OPH enzyme for biodegradation of diazinon from contaminated agricultural soil. Methods: In this study, 8 contaminated agricultural soil samples that were exposed to pesticides, especially diazinon in the last two decades, were collected from the farms of Hamedan province. After preparing the media, for isolation of several bacterial strains containing OPH enzyme that are capable of biodegrading organophosphorus pesticides by diazinon enzymatic hydrolysis, bacterial genomic DNA extraction, plasmid product sequencing, phylogenetic sequence processing and phylogenetic tree drawing were carried out. Results: Eight bacterial strains, capable of secreting OPH enzyme, were isolated from soil samples, one of which named BS-1 with 86% similarity to Bacillus safensis displayed the highest organophosphate-hydrolyzing capability and can be used as a source of carbon and phosphorus. Conclusion: It can be concluded that the isolated bacterial strain identified in this study with OPH enzyme secretion has the potential for biodegradation of organophosphorus pesticides, especially diazinon in invitro conditions. Also, further studies such as the environmental stability and interaction, production strategies, safety, cost-benefit, environmental destructive parameters, and, toxicological, genetic and biochemical aspects are recommended prior to the application of bacterial strains in the field-scale bioremediation.

  11. The mode of inhibitor binding to peptidyl-tRNA hydrolase: binding studies and structure determination of unbound and bound peptidyl-tRNA hydrolase from Acinetobacter baumannii.

    Science.gov (United States)

    Kaushik, Sanket; Singh, Nagendra; Yamini, Shavait; Singh, Avinash; Sinha, Mau; Arora, Ashish; Kaur, Punit; Sharma, Sujata; Singh, Tej P

    2013-01-01

    The incidences of infections caused by an aerobic Gram-negative bacterium, Acinetobacter baumannii are very common in hospital environments. It usually causes soft tissue infections including urinary tract infections and pneumonia. It is difficult to treat due to acquired resistance to available antibiotics is well known. In order to design specific inhibitors against one of the important enzymes, peptidyl-tRNA hydrolase from Acinetobacter baumannii, we have determined its three-dimensional structure. Peptidyl-tRNA hydrolase (AbPth) is involved in recycling of peptidyl-tRNAs which are produced in the cell as a result of premature termination of translation process. We have also determined the structures of two complexes of AbPth with cytidine and uridine. AbPth was cloned, expressed and crystallized in unbound and in two bound states with cytidine and uridine. The binding studies carried out using fluorescence spectroscopic and surface plasmon resonance techniques revealed that both cytidine and uridine bound to AbPth at nanomolar concentrations. The structure determinations of the complexes revealed that both ligands were located in the active site cleft of AbPth. The introduction of ligands to AbPth caused a significant widening of the entrance gate to the active site region and in the process of binding, it expelled several water molecules from the active site. As a result of interactions with protein atoms, the ligands caused conformational changes in several residues to attain the induced tight fittings. Such a binding capability of this protein makes it a versatile molecule for hydrolysis of peptidyl-tRNAs having variable peptide sequences. These are the first studies that revealed the mode of inhibitor binding in Peptidyl-tRNA hydrolases which will facilitate the structure based ligand design.

  12. The mode of inhibitor binding to peptidyl-tRNA hydrolase: binding studies and structure determination of unbound and bound peptidyl-tRNA hydrolase from Acinetobacter baumannii.

    Directory of Open Access Journals (Sweden)

    Sanket Kaushik

    Full Text Available The incidences of infections caused by an aerobic Gram-negative bacterium, Acinetobacter baumannii are very common in hospital environments. It usually causes soft tissue infections including urinary tract infections and pneumonia. It is difficult to treat due to acquired resistance to available antibiotics is well known. In order to design specific inhibitors against one of the important enzymes, peptidyl-tRNA hydrolase from Acinetobacter baumannii, we have determined its three-dimensional structure. Peptidyl-tRNA hydrolase (AbPth is involved in recycling of peptidyl-tRNAs which are produced in the cell as a result of premature termination of translation process. We have also determined the structures of two complexes of AbPth with cytidine and uridine. AbPth was cloned, expressed and crystallized in unbound and in two bound states with cytidine and uridine. The binding studies carried out using fluorescence spectroscopic and surface plasmon resonance techniques revealed that both cytidine and uridine bound to AbPth at nanomolar concentrations. The structure determinations of the complexes revealed that both ligands were located in the active site cleft of AbPth. The introduction of ligands to AbPth caused a significant widening of the entrance gate to the active site region and in the process of binding, it expelled several water molecules from the active site. As a result of interactions with protein atoms, the ligands caused conformational changes in several residues to attain the induced tight fittings. Such a binding capability of this protein makes it a versatile molecule for hydrolysis of peptidyl-tRNAs having variable peptide sequences. These are the first studies that revealed the mode of inhibitor binding in Peptidyl-tRNA hydrolases which will facilitate the structure based ligand design.

  13. 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

  14. CC chemokine receptor 5 cell-surface expression in relation to CC chemokine receptor 5 genotype and the clinical course of HIV-1 infection

    NARCIS (Netherlands)

    de Roda Husman, A. M.; Blaak, H.; Brouwer, M.; Schuitemaker, H.

    1999-01-01

    CCR5 cell-surface expression was studied in relation to CCR5 genotype and clinical course of HIV-1 infection. HIV-1 infected CCR5+/+ individuals had higher percentages of CCR5-expressing CD4+ T cells as compared with HIV-1-infected CCR532/+ individuals. For both genotypic groups, the percentages of

  15. Crystallization and preliminary X-ray diffraction analysis of cyclic imide hydrolase (CIH) from Pseudomonas putida YZ-26

    OpenAIRE

    Fan, Zheng; Qi, Jianxun; Shi, Yawei; Liu, Yiwei

    2011-01-01

    A recombinant cyclic imide hydrolase from P. putida YZ-26 has been crystallized by the hanging-drop method. This will be helpful in understanding the role of CIH in pyrimidine metabolism and organic acid bioconversion.

  16. Defining Sequence Space and Reaction Products within the Cyanuric Acid Hydrolase (AtzD)/Barbiturase Protein Family

    OpenAIRE

    Seffernick, Jennifer L.; Erickson, Jasmine S.; Cameron, Stephan M.; Cho, Seunghee; Dodge, Anthony G.; Richman, Jack E.; Sadowsky, Michael J.; Wackett, Lawrence P.

    2012-01-01

    Cyanuric acid hydrolases (AtzD) and barbiturases are homologous, found almost exclusively in bacteria, and comprise a rare protein family with no discernible linkage to other protein families or an X-ray structural class. There has been confusion in the literature and in genome projects regarding the reaction products, the assignment of individual sequences as either cyanuric acid hydrolases or barbiturases, and spurious connection of this family to another protein family. The present study h...

  17. Inhibiting Inosine Hydrolase and Alanine Racemase to Enhance the Germination of Bacillus anthracis Sterne Spores: Potential Spore Decontamination Strategies

    Science.gov (United States)

    2015-06-19

    2015): << Inhibiting inosine hydrolase and alanine racemase to enhance the germination of Bacillus anthracis Sterne spores: potential spore...display a currently valid OMB control number. 1. REPORT DATE 02 OCT 2015 2. REPORT TYPE N/A 3. DATES COVERED 4. TITLE AND SUBTITLE Inhibiting...inosine hydrolase and alanine racemase to enhance the germination of Bacillus anthracis Sterne spores potential spore decontamination strategies 5a

  18. β-Secretase BACE1 Promotes Surface Expression and Function of Kv3.4 at Hippocampal Mossy Fiber Synapses.

    Science.gov (United States)

    Hartmann, Stephanie; Zheng, Fang; Kyncl, Michele C; Karch, Sandra; Voelkl, Kerstin; Zott, Benedikt; D'Avanzo, Carla; Lomoio, Selene; Tesco, Giuseppina; Kim, Doo Y; Alzheimer, Christian; Huth, Tobias

    2018-04-04

    The β-secretase β-site APP-cleaving enzyme 1 (BACE1) is deemed a major culprit in Alzheimer's disease, but accumulating evidence indicates that there is more to the enzyme than driving the amyloidogenic processing of the amyloid precursor protein. For example, BACE1 has emerged as an important regulator of neuronal activity through proteolytic and, most unexpectedly, also through nonproteolytic interactions with several ion channels. Here, we identify and characterize the voltage-gated K + channel 3.4 (Kv3.4) as a new and functionally relevant interaction partner of BACE1. Kv3.4 gives rise to A-type current with fast activating and inactivating kinetics and serves to repolarize the presynaptic action potential. We found that BACE1 and Kv3.4 are highly enriched and remarkably colocalized in hippocampal mossy fibers (MFs). In BACE1 -/- mice of either sex, Kv3.4 surface expression was significantly reduced in the hippocampus and, in synaptic fractions thereof, Kv3.4 was specifically diminished, whereas protein levels of other presynaptic K + channels such as K Ca 1.1 and K Ca 2.3 remained unchanged. The apparent loss of presynaptic Kv3.4 affected the strength of excitatory transmission at the MF-CA3 synapse in hippocampal slices of BACE1 -/- mice when probed with the Kv3 channel blocker BDS-I. The effect of BACE1 on Kv3.4 expression and function should be bidirectional, as predicted from a heterologous expression system, in which BACE1 cotransfection produced a concomitant upregulation of Kv3.4 surface level and current based on a physical interaction between the two proteins. Our data show that, by targeting Kv3.4 to presynaptic sites, BACE1 endows the terminal with a powerful means to regulate the strength of transmitter release. SIGNIFICANCE STATEMENT The β-secretase β-site APP-cleaving enzyme 1 (BACE1) is infamous for its crucial role in the pathogenesis of Alzheimer's disease, but its physiological functions in the intact nervous system are only gradually

  19. Uroguanylin inhibits H-ATPase activity and surface expression in renal distal tubules by a PKG-dependent pathway.

    Science.gov (United States)

    da Silva Lima, Vanessa; Crajoinas, Renato O; Carraro-Lacroix, Luciene R; Godinho, Alana N; Dias, João L G; Dariolli, Rafael; Girardi, Adriana C C; Fonteles, Manassés C; Malnic, Gerhard; Lessa, Lucília M A

    2014-09-15

    Cumulative evidence suggests that guanylin peptides play an important role on electrolyte homeostasis. We have previously reported that uroguanylin (UGN) inhibits bicarbonate reabsorption in a renal distal tubule. In the present study, we tested the hypothesis that the bicarbonaturic effect of UGN is at least in part attributable to inhibition of H(+)-ATPase-mediated hydrogen secretion in the distal nephron. By in vivo stationary microperfusion experiments, we were able to show that UGN inhibits H(+)-ATPase activity by a PKG-dependent pathway because KT5823 (PKG inhibitor) abolished the UGN effect on distal bicarbonate reabsorption and H89 (PKA inhibitor) was unable to prevent it. The in vivo results were confirmed by the in vitro experiments, where we used fluorescence microscopy to measure intracellular pH (pHi) recovery after an acid pulse with NH4Cl. By this technique, we observed that UGN and 8 bromoguanosine-cGMP (8Br-cGMP) inhibited H(+)-ATPase-dependent pHi recovery and that the UGN inhibitory effect was abolished in the presence of the PKG inhibitor. In addition, by using RT-PCR technique, we verified that Madin-Darby canine kidney (MDCK)-C11 cells express guanylate cyclase-C. Besides, UGN stimulated an increase of both cGMP content and PKG activity but was unable to increase the production of cellular cAMP content and PKA activity. Furthermore, we found that UGN reduced cell surface abundance of H+-ATPase B1 subunit in MDCK-C11 and that this effect was abolished by the PKG inhibitor. Taken together, our data suggest that UGN inhibits H(+)-ATPase activity and surface expression in renal distal cells by a cGMP/PKG-dependent pathway. Copyright © 2014 the American Physiological Society.

  20. Lovastatin enhances ecto-5'-nucleotidase activity and cell surface expression in endothelial cells: implication of rho-family GTPases.

    Science.gov (United States)

    Ledoux, S; Laouari, D; Essig, M; Runembert, I; Trugnan, G; Michel, J B; Friedlander, G

    2002-03-08

    Extracellular adenosine production by the GPI-anchored Ecto-5'-Nucleotidase (Ecto-5'-Nu) plays an important role in the cardiovascular system, notably in defense against hypoxia. It has been previously suggested that HMG-CoA reductase inhibitors (HRIs) could potentiate the hypoxic stimulation of Ecto-5'Nu in myocardial ischemia. In order to elucidate the mechanism of Ecto-5'-Nu stimulation by HRIs, Ecto-5'-Nu activity and expression were determined in an aortic endothelial cell line (SVAREC) incubated with lovastatin. Lovastatin enhanced Ecto-5'-Nu activity in a dose-dependent manner. This increase was not supported by de novo synthesis of the enzyme because neither the mRNA content nor the total amount of the protein were modified by lovastatin. By contrast, lovastatin enhanced cell surface expression of Ecto-5'-Nu and decreased endocytosis of Ecto-5'-Nu, as evidenced by immunostaining. This effect appeared unrelated to modifications of cholesterol content or Ecto-5'-Nu association with detergent-resistant membranes. The effect of lovastatin was reversed by mevalonate, the substrate of HMG-CoA reductase, by its isoprenoid derivative, geranyl-geranyl pyrophosphate, and by cytotoxic necrotizing factor, an activator of Rho-GTPases. Stimulation of Ecto-5'-Nu by lovastatin enhanced the inhibition of platelet aggregation induced by endothelial cells. In conclusion, lovastatin enhances Ecto-5'-Nu activity and membrane expression in endothelial cells. This effect seems independent of lowering cholesterol content but could be supported by an inhibition of Ecto-5'-Nu endocytosis through a decrease of Rho-GTPases isoprenylation.

  1. Looped host defense peptide CLP-19 binds to microtubules and inhibits surface expression of TLR4 on mouse macrophages.

    Science.gov (United States)

    Li, Di; Liu, Yao; Yang, Ya; Chen, Jian-hong; Yang, Jie; Zou, Lin-yun; Tian, Zhi-qiang; Lv, Jun; Xia, Pei-yuan

    2013-06-15

    The looped host defense peptide CLP-19 is derived from a highly functional core region of the Limulus anti-LPS factor and exerts robust anti-LPS activity by directly interacting with LPS in the extracellular space. We previously showed that prophylactic administration of CLP-19 even 20 h prior to LPS challenge might significantly increase the survival rate in a lethal endotoxin shock mouse model. Such an effect may be associated with immune regulation of CLP-19. To investigate the underlying mechanisms, peptide affinity chromatography, immunofluorescence, and Western blotting procedures were used to identify α- and β-tubulin as direct and specific binding partners of CLP-19 in the mouse macrophage cell line RAW 264.7. Bioinformatic analysis using the AutoDock Vina molecular docking and PyMOL molecular graphics system predicted that CLP-19 would bind to the functional residues of both α- and β-tubulin and would be located within the groove of microtubules. Tubulin polymerization assay revealed that CLP-19 might induce polymerization of microtubules and prevent depolymerization. The immunoregulatory effect of CLP-19 involving microtubules was investigated by flow cytometry, immunofluorescence, and Western blotting, which showed that CLP-19 prophylactic treatment of RAW 264.7 cells significantly inhibited LPS-induced surface expression of TLR4. Taken together, these results suggest that CLP-19 binding to microtubules disrupts the dynamic equilibrium of microtubules, reducing the efficacy of microtubule-dependent vesicular transport that would otherwise translocate TLR4 from the endoplasmic reticulum to the cell surface.

  2. CXCR3 surface expression in human airway epithelial cells: cell cycle dependence and effect on cell proliferation.

    Science.gov (United States)

    Aksoy, Mark O; Yang, Yi; Ji, Rong; Reddy, P J; Shahabuddin, Syed; Litvin, Judith; Rogers, Thomas J; Kelsen, Steven G

    2006-05-01

    We recently demonstrated that human bronchial epithelial cells (HBEC) constitutively express the CXC chemokine receptor CXCR3, which when activated, induces directed cell migration. The present study in HBEC examined the relative expression of the CXCR3 splice variants CXCR3-A and -B, cell cycle dependence of CXCR3 expression, and the effects of the CXCR3 ligand, the interferon-gamma-inducible CXC chemokine I-TAC/CXCL11, on DNA synthesis and cell proliferation. Both CXCR3-A and -B mRNA, assessed by real-time RT-PCR, were expressed in normal HBEC (NHBEC) and the HBEC line 16-HBE. However, CXCR3-B mRNA was 39- and 6-fold greater than CXCR3-A mRNA in NHBEC and 16-HBE, respectively. Although most HBEC (>80%) assessed by flow cytometry and immunofluorescence microscopy contained intracellular CXCR3, only a minority (75%) were in the S + G(2)/M phases of the cell cycle. Stimulation of CXCR3 with I-TAC enhanced thymidine incorporation and cell proliferation and increased p38 and ERK1/2 phosphorylation. These data indicate that 1) human airway epithelial cells primarily express CXCR3-B mRNA, 2) surface expression of CXCR3 is largely confined to the S + G(2)/M phases of the cell cycle, and 3) activation of CXCR3 induces DNA synthesis, cell proliferation, and activation of MAPK pathways. We speculate that activation of CXCR3 exerts a mitogenic effect in HBEC, which may be important during airway mucosal injury in obstructive airway diseases such as asthma and chronic obstructive pulmonary disease.

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

    International Nuclear Information System (INIS)

    Hu, Wenxin; Wang, Qihai; Bi, Ruchang

    2005-01-01

    The 31.3 kDa Ap 4 A hydrolase from Shigella flexneri 2a has been cloned, expressed and purified using an Escherichia coli expression system. Crystals of Ap 4 A hydrolase have been obtained by the hanging-drop technique at 291 K using PEG 550 MME as precipitant. Diadenosine tetraphosphate (Ap 4 A) hydrolase (EC 3.6.1.41) hydrolyzes Ap 4 A symmetrically in prokaryotes. It plays a potential role in organisms by regulating the concentration of Ap 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 4 A hydrolase from Shigella flexneri 2a has been cloned, expressed and purified using an Escherichia coli expression system. Crystals of Ap 4 A hydrolase have been obtained by the hanging-drop technique at 291 K using PEG 550 MME as precipitant. Ap 4 A hydrolase crystals diffract X-rays to 3.26 Å and belong to space group P2 1 , with unit-cell parameters a = 118.9, b = 54.6, c = 128.5 Å, β = 95.7°

  4. [Research on rapid and quantitative detection method for organophosphorus pesticide residue].

    Science.gov (United States)

    Sun, Yuan-Xin; Chen, Bing-Tai; Yi, Sen; Sun, Ming

    2014-05-01

    The methods of physical-chemical inspection is adopted in the traditional pesticide residue detection, which require a lot of pretreatment processes, are time-consuming and complicated. In the present study, the authors take chlorpyrifos applied widely in the present agricultural field as the research object and propose a rapid and quantitative detection method for organophosphorus pesticide residues. At first, according to the chemical characteristics of chlorpyrifos and comprehensive chromogenic effect of several colorimetric reagents and secondary pollution, the pretreatment of the scheme of chromogenic reaction of chlorpyrifos with resorcin in a weak alkaline environment was determined. Secondly, by analyzing Uv-Vis spectrum data of chlorpyrifos samples whose content were between 0. 5 and 400 mg kg-1, it was confirmed that the characteristic information after the color reaction mainly was concentrated among 360 approximately 400 nm. Thirdly, the full spectrum forecasting model was established based on the partial least squares, whose correlation coefficient of calibration was 0. 999 6, correlation coefficient of prediction reached 0. 995 6, standard deviation of calibration (RMSEC) was 2. 814 7 mg kg-1, and standard deviation of verification (RMSEP) was 8. 012 4 mg kg-1. Fourthly, the wavelengths whose center wavelength is 400 nm was extracted as characteristic region to build a forecasting model, whose correlation coefficient of calibration was 0. 999 6, correlation coefficient of prediction reached 0. 999 3, standard deviation of calibration (RMSEC) was 2. 566 7 mg kg-1 , standard deviation of verification (RMSEP) was 4. 886 6 mg kg-1, respectively. At last, by analyzing the near infrared spectrum data of chlorpyrifos samples with contents between 0. 5 and 16 mg kg-1, the authors found that although the characteristics of the chromogenic functional group are not obvious, the change of absorption peaks of resorcin itself in the neighborhood of 5 200 cm

  5. Crystal structure of human leukotriene A(4) hydrolase, a bifunctional enzyme in inflammation.

    Science.gov (United States)

    Thunnissen, M M; Nordlund, P; Haeggström, J Z

    2001-02-01

    Leukotriene (LT) A(4) hydrolase/aminopeptidase (LTA4H) is a bifunctional zinc enzyme that catalyzes the biosynthesis of LTB4, a potent lipid chemoattractant involved in inflammation, immune responses, host defense against infection, and PAF-induced shock. The high resolution crystal structure of LTA4H in complex with the competitive inhibitor bestatin reveals a protein folded into three domains that together create a deep cleft harboring the catalytic Zn(2+) site. A bent and narrow pocket, shaped to accommodate the substrate LTA(4), constitutes a highly confined binding region that can be targeted in the design of specific anti-inflammatory agents. Moreover, the structure of the catalytic domain is very similar to that of thermolysin and provides detailed insight into mechanisms of catalysis, in particular the chemical strategy for the unique epoxide hydrolase reaction that generates LTB(4).

  6. Genetically reduced soluble epoxide hydrolase activity and risk of stroke and other cardiovascular disease

    DEFF Research Database (Denmark)

    Lee, Julie; Dahl, Morten; Grande, Peer

    2010-01-01

    epoxide hydrolase activity is associated with risk of ischemic stroke, myocardial infarction, and ischemic heart disease. METHODS: We genotyped participants from the Copenhagen City Heart Study (n=10 352), the Copenhagen General Population Study (n=26 042), the Copenhagen Carotid Stroke Study (n=398 cases......+796 control subjects), and the Copenhagen Ischemic Heart Disease Study (n=4901 cases+9798 control subjects) for the R103C, R287Q, and Arg(402-403ins) variants in the EPHX2 gene and recorded hospital admissions due to ischemic stroke, myocardial infarction, and ischemic heart disease. RESULTS......=0.08 to 1.00). Similar results were obtained for myocardial infarction and ischemic heart disease in the 3 studies. CONCLUSIONS: Our results show with significant power that genetically reduced soluble epoxide hydrolase activity is not a major risk factor for ischemic stroke, myocardial infarction...

  7. 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...

  8. Biosynthesis of intestinal microvillar proteins. Intracellular processing of lactase-phlorizin hydrolase

    DEFF Research Database (Denmark)

    Danielsen, E M; Skovbjerg, H; Norén, Ove

    1984-01-01

    The biosynthesis of pig small intestinal lactase-phlorizin hydrolase (EC 3.2.1.23-62) was studied by labelling of organ cultured mucosal explants with [35S]methionine. The earliest detactable form of the enzyme was an intracellular, membrane-bound polypeptide of Mr 225 000, sensitive to endo H...... 000 polypeptide is of the same size as the mature lactase-phlorizin hydrolase and was the only form expressed in the microvillar membrane. Together, these data are indicative of an intracellular proteolytic cleavage during transport. The presence of leupeptin during labelling prevented the appearance...... of the Mr 160 000 form but not that of the Mr 245 000 polypeptide, suggesting that the proteolytic cleavage takes place after trimming and complex glycosylation. The proteolytic cleavage was not essential for the transport since the precursor was expressed in the microvillar membrane in the presence...

  9. Screening brazilian macrophomina phaseolina isolates for alkaline lipases and other extracellular hydrolases

    OpenAIRE

    Schinke, Cláudia; Germani, Jose Carlos

    2012-01-01

    Macrophomina phaseolina, phylum Ascomycota, is a phytopathogenic fungus distributed worldwide in hot dry areas. There are few studies on its secreted lipases and none on its colony radial growth rate, an indicator of fungal ability to use nutrients for growth, on media other than potato-dextrose agar. In this study, 13 M. phaseolina isolates collected in different Brazilian regions were screened for fast-growth and the production of hydrolases of industrial interest, especially alkaline lipas...

  10. Organophosphate Hydrolase in Conductometric Biosensor for the Detection of Organophosphate Pesticides

    OpenAIRE

    Ani Mulyasuryani; Sasangka Prasetyawan

    2015-01-01

    The research has developed an enzyme biosensor for the detection organophosphate pesticide residues. The biosensor consists of a pair of screen-printed carbon electrode (SPCEs). One of electrodes contains immobilized organophosphate hydrolase (OPH) on a chitosan membrane by cross-linking it with glutaraldehyde. The area of the electrodes was optimized to 3, 5, and 7 mm2. The OPH was isolated from Pseudomonas putida, and was purified by the ammonium sulfate precipitation method, with 6444 ppm ...

  11. Functional analysis of the Escherichia coli genome for members of the alpha/beta hydrolase family.

    Science.gov (United States)

    Zhang, L; Godzik, A; Skolnick, J; Fetrow, J S

    1998-01-01

    Database-searching methods based on sequence similarity have become the most commonly used tools for characterizing newly sequenced proteins. Due to the often underestimated functional diversity in protein families and superfamilies, however, it is difficult to make the characterization specific and accurate. In this work, we have extended a method for active-site identification from predicted protein structures. The structural conservation and variation of the active sites of the alpha/beta hydrolases with known structures were studied. The similarities were incorporated into a three-dimensional motif that specifies essential requirements for the enzymatic functions. A threading algorithm was used to align 651 Escherichia coli open reading frames (ORFs) to one of the members of the alpha/beta hydrolase fold family. These ORFs were then screened according to our three-dimensional motif and with an extra requirement that demands conservation of the key active-site residues among the proteins that bear significant sequence similarity to the ORFs. 17 ORFs from E. coli were predicted to have hydrolase activity and their putative active-site residues were identified. Most were in agreement with the experiments and results of other database-searching methods. The study further suggests that YHET_ECOLI, a hypothetical protein classified as a member of the UPF0017 family (an uncharacterized protein family), bears all the hallmarks of the alpha/beta hydrolase family. The novel feature of our method is that it uses three-dimensional structural information for function prediction. The results demonstrate the importance and necessity of such a method to fill the gap between sequence alignment and function prediction; furthermore, the method provides a way to verify the structure predictions, which enables an expansion of the applicable scope of the threading algorithms.

  12. Gastric Lavage in Acute Organophosphorus Pesticide poisoning (GLAOP – a randomised controlled trial of multiple vs. single gastric lavage in unselected acute organophosphorus pesticide poisoning

    Directory of Open Access Journals (Sweden)

    Cao YuPing

    2006-10-01

    Full Text Available Abstract Background Organophosphorus (OP pesticide poisoning is the most common form of pesticide poisoning in many Asian countries. Guidelines in western countries for management of poisoning indicate that gastric lavage should be performed only if two criteria are met: within one hour of poison ingestion and substantial ingested amount. But the evidence on which these guidelines are based is from medicine overdoses in developed countries and may be irrelevant to OP poisoning in Asia. Chinese clinical experience suggests that OP remains in the stomach for several hours or even days after ingestion. Thus, there may be reasons for doing single or multiple gastric lavages for OP poisoning. There have been no randomised controlled trials (RCTs to assess this practice of multiple lavages. Since it is currently standard therapy in China, we cannot perform a RCT of no lavage vs. a single lavage vs. multiple lavages. We will compare a single gastric lavage with three gastric lavages as the first stage to assess the role of gastric lavage in OP poisoning. Methods/Design We have designed an RCT assessing the effectiveness of multiple gastric lavages in adult OP self-poisoning patients admitted to three Chinese hospitals within 12 hrs of ingestion. Patients will be randomised to standard treatment plus either a single gastric lavage on admission or three gastric lavages at four hour intervals. The primary outcome is in-hospital mortality. Analysis will be on an intention-to-treat basis. On the basis of the historical incidence of OP at the study sites, we expect to enroll 908 patients over three years. This projected sample size provides sufficient power to evaluate the death rate; and a variety of other exposure and outcome variables, including particular OPs and ingestion time. Changes of OP level will be analyzed in order to provide some toxic kinetic data. Discussion the GLAOP study is a novel, prospective cohort study that will explore to the toxic

  13. 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

  14. Characterization of tunable piperidine and piperazine carbamates as inhibitors of endocannabinoid hydrolases

    Science.gov (United States)

    Long, Jonathan Z.; Jin, Xin; Adibekian, Alexander; Li, Weiwei; Cravatt, Benjamin F.

    2010-01-01

    Monoacylglycerol lipase (MAGL) and fatty acid amide hydrolase (FAAH) are two enzymes from the serine hydrolase superfamily that degrade the endocannabinoids 2-arachidonoylglycerol and anandamide, respectively. We have recently discovered that MAGL and FAAH are both inhibited by carbamates bearing an N-piperidine/piperazine group. Piperidine/piperazine carbamates show excellent in vivo activity, raising brain endocannabinoid levels and producing CB1-dependent behavioral effects in mice, suggesting that they represent a promising class of inhibitors for studying the endogenous functions of MAGL and FAAH. Herein, we disclose a full account of the syntheses, structure-activity relationships, and inhibitory activities of piperidine/piperazine carbamates against members of the serine hydrolase family. These scaffolds can be tuned for MAGL-selective or dual MAGL-FAAH inhibition by the attachment of an appropriately substituted bisarylcarbinol or aryloxybenzyl moiety, respectively, on the piperidine/piperazine ring. Modifications to the piperidine/piperazine ring ablated inhibitory activity, suggesting a strict requirement for a six-member ring to maintain potency. PMID:20099888

  15. 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.

  16. Isolation and characterization of arginine ester hydrolase from Heloderma horridum (beaded lizard) venom.

    Science.gov (United States)

    Nikai, T; Imai, K; Komori, Y; Sugihara, H

    1992-03-01

    1. An arginine ester hydrolase was isolated from Heloderma horridum (beaded lizard) venom by Sephadex G-75, DEAE-Sephacel and Q-Sepharose column chromatography, resulting in 5.4 mg of purified enzyme from 320.0 mg of crude venom. 2. The enzyme was shown to be homogeneous by both SDS and non-SDS disc electrophoresis on polyacrylamide gel at pH 8.3. 3. The enzyme possesses arginine ester hydrolase and transglutaminase-like activities, but did not exhibit clotting activity. 4. Molecular weight was determined to be ca 29 kDa, with an isoelectric point of 4.4. 5. The enzyme was stable to heat treatment (95 degrees C, 10 min) and to pH changes over the range 2-11. 6. The arginine ester hydrolase was inactivated by diisopropylfluorophosphate (DFP), beta-mercaptoethanol and N-bromosuccinimide, suggesting that serine, disulfide bonds and tryptophan are involved in enzymatic activity. 7. Amino terminal sequences were determined and appear to be similar to porcine pancreatic kallikrein.

  17. Regulation of catalytic behaviour of hydrolases through interactions with functionalized carbon-based nanomaterials

    Energy Technology Data Exchange (ETDEWEB)

    Pavlidis, Ioannis V. [University of Ioannina, Laboratory of Biotechnology, Department of Biological Applications and Technologies (Greece); Vorhaben, Torge [Institute of Biochemistry, Greifswald University, Department of Biotechnology and Enzyme Catalysis (Germany); Gournis, Dimitrios [University of Ioannina, Department of Materials Science and Engineering (Greece); Papadopoulos, George K. [Epirus Institute of Technology, Laboratory of Biochemistry and Biophysics, Faculty of Agricultural Technology (Greece); Bornscheuer, Uwe T. [Institute of Biochemistry, Greifswald University, Department of Biotechnology and Enzyme Catalysis (Germany); Stamatis, Haralambos, E-mail: hstamati@cc.uoi.gr [University of Ioannina, Laboratory of Biotechnology, Department of Biological Applications and Technologies (Greece)

    2012-05-15

    The interaction of enzymes with carbon-based nanomaterials (CBNs) is crucial for the function of biomolecules and therefore for the design and development of effective nanobiocatalytic systems. In this study, the effect of functionalized CBNs, such as graphene oxide (GO) and multi-wall carbon nanotubes (CNTs), on the catalytic behaviour of various hydrolases of biotechnological interest was monitored and the interactions between CBNs and proteins were investigated. The enzyme-nanomaterial interactions significantly affect the catalytic behaviour of enzymes, resulting in an increase up to 60 % of the catalytic efficiency of lipases and a decrease up to 30 % of the esterase. Moreover, the use of CNTs and GO derivatives, especially those that are amine-functionalized, led to increased thermal stability of most the hydrolases tested. Fluorescence and circular dichroism studies indicated that the altered catalytic behaviour of enzymes in the presence of CBNs arises from specific enzyme-nanomaterial interactions, which can lead to significant conformational changes. In the case of lipases, the conformational changes led to a more active and rigid structure, while in the case of esterases this led to destabilization and unfolding. Kinetic and spectroscopic studies indicated that the extent of the interactions between CBNs and hydrolases can be mainly controlled by the functionalization of nanomaterials than by their geometry.

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

    Energy Technology Data Exchange (ETDEWEB)

    Bera, Asim K; Aukema, Kelly G.; Elias, Mikael; Wackett, Lawrence P. (UMM)

    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.

  19. Brucella abortus choloylglycine hydrolase affects cell envelope composition and host cell internalization.

    Directory of Open Access Journals (Sweden)

    María Inés Marchesini

    Full Text Available Choloylglycine hydrolase (CGH, E.C. 3.5.1.24 is a conjugated bile salt hydrolase that catalyses the hydrolysis of the amide bond in conjugated bile acids. Bile salt hydrolases are expressed by gastrointestinal bacteria, and they presumably decrease the toxicity of host's conjugated bile salts. Brucella species are the causative agents of brucellosis, a disease affecting livestock and humans. CGH confers Brucella the ability to deconjugate and resist the antimicrobial action of bile salts, contributing to the establishment of a successful infection through the oral route in mice. Additionally, cgh-deletion mutant was also attenuated in intraperitoneally inoculated mice, which suggests that CGH may play a role during systemic infection other than hydrolyzing conjugated bile acids. To understand the role CGH plays in B. abortus virulence, we infected phagocytic and epithelial cells with a cgh-deletion mutant (Δcgh and found that it is defective in the internalization process. This defect along with the increased resistance of Δcgh to the antimicrobial action of polymyxin B, prompted an analysis of the cell envelope of this mutant. Two-dimensional electrophoretic profiles of Δcgh cell envelope-associated proteins showed an altered expression of Omp2b and different members of the Omp25/31 family. These results were confirmed by Western blot analysis with monoclonal antibodies. Altogether, the results indicate that Brucella CGH not only participates in deconjugation of bile salts but also affects overall membrane composition and host cell internalization.

  20. Brucella abortus choloylglycine hydrolase affects cell envelope composition and host cell internalization.

    Science.gov (United States)

    Marchesini, María Inés; Connolly, Joseph; Delpino, María Victoria; Baldi, Pablo C; Mujer, Cesar V; DelVecchio, Vito G; Comerci, Diego J

    2011-01-01

    Choloylglycine hydrolase (CGH, E.C. 3.5.1.24) is a conjugated bile salt hydrolase that catalyses the hydrolysis of the amide bond in conjugated bile acids. Bile salt hydrolases are expressed by gastrointestinal bacteria, and they presumably decrease the toxicity of host's conjugated bile salts. Brucella species are the causative agents of brucellosis, a disease affecting livestock and humans. CGH confers Brucella the ability to deconjugate and resist the antimicrobial action of bile salts, contributing to the establishment of a successful infection through the oral route in mice. Additionally, cgh-deletion mutant was also attenuated in intraperitoneally inoculated mice, which suggests that CGH may play a role during systemic infection other than hydrolyzing conjugated bile acids. To understand the role CGH plays in B. abortus virulence, we infected phagocytic and epithelial cells with a cgh-deletion mutant (Δcgh) and found that it is defective in the internalization process. This defect along with the increased resistance of Δcgh to the antimicrobial action of polymyxin B, prompted an analysis of the cell envelope of this mutant. Two-dimensional electrophoretic profiles of Δcgh cell envelope-associated proteins showed an altered expression of Omp2b and different members of the Omp25/31 family. These results were confirmed by Western blot analysis with monoclonal antibodies. Altogether, the results indicate that Brucella CGH not only participates in deconjugation of bile salts but also affects overall membrane composition and host cell internalization.

  1. 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.

  2. Exported Epoxide Hydrolases Modulate Erythrocyte Vasoactive Lipids during Plasmodium falciparum Infection

    Directory of Open Access Journals (Sweden)

    Natalie J. Spillman

    2016-10-01

    Full Text Available Erythrocytes are reservoirs of important epoxide-containing lipid signaling molecules, including epoxyeicosatrienoic acids (EETs. EETs function as vasodilators and anti-inflammatory modulators in the bloodstream. Bioactive EETs are hydrolyzed to less active diols (dihydroxyeicosatrienoic acids by epoxide hydrolases (EHs. The malaria parasite Plasmodium falciparum infects host red blood cells (RBCs and exports hundreds of proteins into the RBC compartment. In this study, we show that two parasite epoxide hydrolases, P. falciparum epoxide hydrolases 1 (PfEH1 and 2 (PfEH2, both with noncanonical serine nucleophiles, are exported to the periphery of infected RBCs. PfEH1 and PfEH2 were successfully expressed in Escherichia coli, and they hydrolyzed physiologically relevant erythrocyte EETs. Mutations in active site residues of PfEH1 ablated the ability of the enzyme to hydrolyze an epoxide substrate. Overexpression of PfEH1 or PfEH2 in parasite-infected RBCs resulted in a significant alteration in the epoxide fatty acids stored in RBC phospholipids. We hypothesize that the parasite disruption of epoxide-containing signaling lipids leads to perturbed vascular function, creating favorable conditions for binding and sequestration of infected RBCs to the microvascular endothelium.

  3. Organophosphorus esters in the oceans and possible relation with ocean gyres

    International Nuclear Information System (INIS)

    Cheng, Wenhan; Xie, Zhouqing; Blais, Jules M.; Zhang, Pengfei; Li, Ming; Yang, Chengyun; Huang, Wen; Ding, Rui; Sun, Liguang

    2013-01-01

    Four organophosphorus esters (OPEs) were detected in aerosol samples collected in the West Pacific, the Indian Ocean and the Southern Ocean from 2009 to 2010, suggesting their circumpolar and global distribution. In general, the highest concentrations were detected near populated regions in China, Australia and New Zealand. OPE concentrations in the Southern Ocean were about two orders of magnitude lower than those near major continents. Additionally, relatively high OPE concentrations were detected at the Antarctic Peninsula, where several scientific survey stations are located. The four OPEs investigated here are significantly correlated with each other, suggesting they may derive from the same source. In the circumpolar transect, OPE concentrations were associated with ocean gyres in the open ocean. Their concentrations were positively related with average vorticity in the sampling area suggesting that a major source of OPEs may be found in ocean gyres where plastic debris is known to accumulate. -- Highlights: •We provide OPE concentrations in aerosols in a circumpolar expedition. •We find strong anthropogenic source of OPE pollution. •We suggest potential relationship between ocean gyres and OPE pollution. -- Our work provides a circumpolar investigation on OPEs in the Southern Ocean and we suggest a possibility that ocean currents and gyres may act as important roles in global transport of OPEs

  4. Simulated solar light phototransformation of organophosphorus azinphos methyl at the surface of clays and goethite.

    Science.gov (United States)

    Menager, Matthieu; Sarakha, Mohamed

    2013-01-15

    The photochemical behavior of the pesticide azinphos methyl at the surface of clays (kaolinite, bentonite) and goethite was studied using Suntest setup (λ > 300 nm). The quantum yield on the clays was found to be roughly three times lower than that in aqueous solution. However, the photochemical efficiency was much higher at the surface of goethite owing to its photocatalytic activity through the hydroxyl radical production. The added humic substances on kaolonite show an inhibition of azinphos methyl degradation while the incorporation of iron(III) aquacomplexes leads to an important increase of the disappearance together with the formation of iron(II). Hydroxyl radical species were found to be formed either by excitation of goethite or clays. The goethite support acts as a more efficient catalyst for the formation of these reactive oxygen species. The photodecomposition reactions observed were (i) hydrolysis process leading to the formation of benzotriazone and the oxidation of the P = S bond giving rise to the formation of the oxon derivative, and (ii) homolytic cleavage of the N-C and C-S bonds of the organophosphorus bridge leading to the formation of dimers that appear to be specific to the irradiation at the surface of solid supports since they were not observed when the irradiation was performed in aqueous media: a statement that is related to the presence of aggregates at the surface of solid supports.

  5. Occurrences of organophosphorus esters and phthalates in the microplastics from the coastal beaches in north China.

    Science.gov (United States)

    Zhang, Haibo; Zhou, Qian; Xie, Zhiyong; Zhou, Yang; Tu, Chen; Fu, Chuancheng; Mi, Wenying; Ebinghaus, Ralf; Christie, Peter; Luo, Yongming

    2018-03-01

    Chemical pollution in the microplastics has been concerned worldwide as pollutants might potentially transfer from the environment to living organisms via plastics. Here, we investigate organophosphorus esters (OPEs) and phthalic acid esters (PAEs) in the beached microplastics collected from 28 coastal beaches of the Bohai and Yellow Sea in north China. The analyzed microplastics included polyethylene (PE) pellets and fragments, polypropylene (PP) flakes and fragments and polystyrene (PS) foams. The tris-(2-chloroethyl)-phosphate (TCEP), tris (1-chloro-2-propyl) phosphate (TCPP) and di-(2-ethylhexyl) phthalate (DEHP) were the three predominant compounds found overall. The maximum Σ4 OPEs concentration was 84,595.9ngg -1 , almost three orders of magnitude higher than the maximum Σ9 PAEs concentration. The PP flakes and PS foams contained the highest concentrations of the additives in contrast to the PE pellets which contained the lowest. The high concentration level of carcinogenic chlorinated OPEs and DEHP with endocrine disrupting effects implied the suggested potential hazards to coastal organisms. Spatial differences and compositional variation of the additives among the different microplastics suggests different origins and residence times in the coastal environment. This indicates that the characteristics of chemical additives might be a useful approach when tracing sources of microplastics in the environment. Copyright © 2017 Elsevier B.V. All rights reserved.

  6. New Cinchona Oximes Evaluated as Reactivators of Acetylcholinesterase and Butyrylcholinesterase Inhibited by Organophosphorus Compounds

    Directory of Open Access Journals (Sweden)

    Maja Katalinić

    2017-07-01

    Full Text Available For the last six decades, researchers have been focused on finding efficient reactivators of organophosphorus compound (OP-inhibited acetylcholinesterase (AChE and butyrylcholinesterase (BChE. In this study, we have focused our research on a new oxime scaffold based on the Cinchona structure since it was proven to fit the cholinesterases active site and reversibly inhibit their activity. Three Cinchona oximes (C1, C2, and C3, derivatives of the 9-oxocinchonidine, were synthesized and investigated in reactivation of various OP-inhibited AChE and BChE. As the results showed, the tested oximes were more efficient in the reactivation of BChE and they reactivated enzyme activity to up to 70% with reactivation rates similar to known pyridinium oximes used as antidotes in medical practice today. Furthermore, the oximes showed selectivity towards binding to the BChE active site and the determined enzyme-oxime dissociation constants supported work on the future development of inhibitors in other targeted studies (e.g., in treatment of neurodegenerative disease. Also, we monitored the cytotoxic effect of Cinchona oximes on two cell lines Hep G2 and SH-SY5Y to determine the possible limits for in vivo application. The cytotoxicity results support future studies of these compounds as long as their biological activity is targeted in the lower micromolar range.

  7. Susceptibility of nine organophosphorus pesticides in skimmed milk towards inoculated lactic acid bacteria and yogurt starters.

    Science.gov (United States)

    Zhou, Xin-Wei; Zhao, Xin-Huai

    2015-01-01

    Previous research has shown that fresh milk might be polluted by some organophosphorus pesticides (OPPs). In this study the dissipation of nine OPPs, namely chlorpyrifos, chlorpyrifos-methyl, diazinon, dichlorvos, fenthion, malathion, phorate, pirimiphos-methyl and trichlorphon, in skimmed milk was investigated to clarify their susceptibility towards lactic acid bacteria (LAB) and yogurt starters. Skimmed milk was spiked with nine OPPs, inoculated with five strains of LAB and two commercial yogurt starters at 42 °C for 24 and 5 h respectively and subjected to quantitative OPP analysis by gas chromatography. Degradation kinetic constants of these OPPs were calculated based on a first-order reaction model. OPP dissipation in the milk was enhanced by the inoculated strains and starters, resulting in OPP concentrations decreasing by 7.0-64.6 and 7.4-19.2% respectively. Totally, the nine OPPs were more susceptible to Lactobacillus bulgaricus, as it enhanced their degradation rate constants by 18.3-133.3%. Higher phosphatase production of the assayed stains was observed to bring about greater OPP degradation in the milk. Both LAB and yogurt starters could enhance OPP dissipation in skimmed milk, with the nine OPPs studied having different susceptibilities towards them. Phosphatase was a key factor governing OPP dissipation. The LAB of higher phosphatase production have more potential to decrease OPPs in fermented foods. © 2014 Society of Chemical Industry.

  8. Organophosphorus pesticide exposure in agriculture: effects of temperature, ultraviolet light and abrasion on PVC gloves

    Science.gov (United States)

    ISMAIL, Ismaniza; GASKIN, Sharyn; PISANIELLO, Dino; EDWARDS, John W.

    2017-01-01

    Elbow length PVC gloves are often recommended for protection against organophosphorus pesticide (OP) exposure in agriculture. However, performance may be reduced due to high temperature, UV exposure and abrasion. We sought to assess these impacts for two OPs under normal use and reasonable worst-case scenarios. Glove permeation tests were conducted using ASTM cells with two PVC glove brands at 23°C and 45°C for up to 8 h. Technical grade dichlorvos and formulated diazinon were used undiluted and at application strength. Breakthough of undiluted dichlorvos occurred at both 23°C and 45°C, but only at 45°C for application strength. Breakthrough of diazinon was not achieved, except when undiluted at 45°C. UV-exposed and abraded gloves showed reduced performance, with the effect being approximately two-fold for dichlorvos. Only small differences were noted between glove brands. Extra precautions should be taken when handling concentrated OPs at high temperature, or when using abraded or sunlight-exposed gloves. PMID:29199264

  9. Revealing chemophoric sites in organophosphorus insecticides through the MIA-QSPR modeling of soil sorption data.

    Science.gov (United States)

    Daré, Joyce K; Silva, Cristina F; Freitas, Matheus P

    2017-10-01

    Soil sorption of insecticides employed in agriculture is an important parameter to probe the environmental fate of organic chemicals. Therefore, methods for the prediction of soil sorption of new agrochemical candidates, as well as for the rationalization of the molecular characteristics responsible for a given sorption profile, are extremely beneficial for the environment. A quantitative structure-property relationship method based on chemical structure images as molecular descriptors provided a reliable model for the soil sorption prediction of 24 widely used organophosphorus insecticides. By means of contour maps obtained from the partial least squares regression coefficients and the variable importance in projection scores, key molecular moieties were targeted for possible structural modification, in order to obtain novel and more environmentally friendly insecticide candidates. The image-based descriptors applied encode molecular arrangement, atoms connectivity, groups size, and polarity; consequently, the findings in this work cannot be achieved by a simple relationship with hydrophobicity, usually described by the octanol-water partition coefficient. Copyright © 2017 Elsevier Inc. All rights reserved.

  10. Electrochemical Biosensor Based on Optimized Biocomposite for Organophosphorus and Carbamates Pesticides Detection

    Directory of Open Access Journals (Sweden)

    R. Montes

    2018-01-01

    Full Text Available This paper presents the characterization and optimization of biosensors based on graphite-epoxy which incorporates the enzyme acetylcholinesterase (AChE. By means of advanced electrochemical techniques, such as electrochemical impedance spectroscopy (EIS and cyclic voltammetry (CV, the characterization and optimization of graphite-epoxy-AChE biosensors have been performed. In order to obtain sensitive electrodes, the optimal composition of the transducer material (graphite-epoxy-enzyme ratio was studied. The optimization of the conductive particles distribution inside the biomaterial has allowed an improvement of the electrochemical properties. Optimal composition guarantees improving electrochemical properties required, such as high electron-transfer rate, high signal-to-noise ratio, and suitable sensitivity. The optimal biocomposite composition range was obtained between 16% and 17% of graphite and 0.12% of AChE. The biosensors were applied to the analysis of different pesticides, organophosphorus and carbamates, using indirect measurements based on enzymatic inhibition process. These optimized biosensors present detection limit one order of magnitude lower compared to the standard composition (nonoptimized and allow achieving concentrations lower than the established ones by the pesticides regulation. Finally, spiked tap water samples with pesticides were analyzed with the optimized biosensors.

  11. Organophosphorus flame retardants in mangrove sediments from the Pearl River Estuary, South China.

    Science.gov (United States)

    Hu, Yong-Xia; Sun, Yu-Xin; Li, Xiao; Xu, Wei-Hai; Zhang, Ying; Luo, Xiao-Jun; Dai, Shou-Hui; Xu, Xiang-Rong; Mai, Bi-Xian

    2017-08-01

    Forty-eight surface sediments were collected from three mangrove wetlands in the Pearl River Estuary (PRE) of South China to investigate the distribution of organophosphorus flame retardants (OPFRs) and the relationship between OPFRs and microbial community structure determined by phospholipid fatty acid. Concentrations of ΣOPFRs in mangrove sediments of the PRE ranged from 13.2 to 377.1 ng g -1 dry weight. Levels of ΣOPFRs in mangrove sediments from Shenzhen and Guangzhou were significantly higher than those from Zhuhai, indicating that OPFRs were linked to industrialization and urbanization. Tris(chloropropyl)phosphate was the predominant profile of OPFRs in mangrove sediments from Shenzhen (38.9%) and Guangzhou (35.0%), while the composition profile of OPFRs in mangrove sediments from Zhuhai was dominated by tris(2-chloroethyl) phosphate (25.5%). The mass inventories of OPFRs in the mangrove sediments of Guangzhou, Zhuhai and Shenzhen were 439.5, 133.5 and 662.3 ng cm -2 , respectively. Redundancy analysis revealed that OPFRs induced a shift in the structure of mangrove sediment microbial community and the variations were significantly correlated with tris(1,3-dichloro-2-propyl)phosphate and tris(2-butoxyethyl) phosphate. Copyright © 2017 Elsevier Ltd. All rights reserved.

  12. Separation and concentration of lanthanoids using microcapsules containing acidic organophosphorus compounds as an extractant

    Energy Technology Data Exchange (ETDEWEB)

    Kamio, Eiji; Kondo, Kazuo [Doshisha Univ., Department of Chemical Engineering and Materials Science, Kyoto (Japan)

    2002-06-01

    In this study, we measured the extraction equilibria of lanthanoids with microcapsules containing acidic organophosphorus compound as an extractant and discuss their mutual separation by using a column packed with the microcapsules. The extraction equilibria of lanthanoids into the microcapsules containing 2-ethylhexylphosphonic acid mono-2-ethylhexyl ester (EHPNA) were elucidated and the extraction equilibrium constants were calculated by slope-analysis method. It was suggested that the lanthanoid ions are extracted in the microcapsules in a high loading state. Furthermore, the adsorption behavior of lanthanoids into the column packed with the microcapsules containing EHPNA was observed. It was found that adsorption and elution of lanthanoids are briefly achieved by selecting pH of the feed aqueous solution. However, it was impossible to separate them only in adsorption or elution operation. So, the mutual separation of lanthanoids was investigated using the adsorption column connected to the development column containing microcapsules. By selecting pH of the eluent, each metal was separated mutually in more than 95% of purity. The metal ions in the eluent from the development column could be concentrated by treating it with a column packed with the microcapsules containing di(2-ethylhexyl) phosphoric acid (D2EHPA). Considering these information, it will be possible to design a continuous extracting, separating and concentrating reactor of lanthanoids using a column packed with the microcapsules. (author)

  13. Determination of organophosphorus pesticides using molecularly imprinted polymer solid phase extraction

    International Nuclear Information System (INIS)

    Mohd Marsin Sanagi; Syairah Salleh; Wan Aini Wan Ibrahim

    2011-01-01

    Molecularly imprinted polymer solid phase extraction (MIP-SPE) method has been developed for the determination of organophosphorus pesticides (OPPs) in water samples. The MIP was prepared by thermo-polymerization method using methacrylic acid (MAA) as functional monomer, ethylene glycol dimethacrylate (EGDMA) as crosslinker, acetonitrile as porogenic solvent and quinalphos as the template molecule. The three OPPs (diazinon, quinalphos and chloropyrifos) were selected as target analytes as they are widely used in agriculture sector. Various parameters affecting the extraction efficiency of the imprinted polymers have been evaluated to optimize the selective preconcentration of OPPs from aqueous samples. The characteristics of the MIP-SPE method were validated by high performance liquid chromatography (HPLC). The accuracy and selectivity of the MIP-SPE process developed were verified using non-imprinted polymer solid phase extraction (NIP-SPE) and a commercial C 18 -SPE was used for comparison. The recoveries of the target analytes obtained using the MIPs as the solid phase sorbent ranged from 83% to 98% (RSDs 1.05 - 1.98 %; n=3) for water sample. The developed MIP-SPE method demonstrates that it could be applied for the determination of OPPs in water samples. (author)

  14. Cholinesterase reactivators and bioscavengers for pre- and post-exposure treatments of organophosphorus poisoning.

    Science.gov (United States)

    Masson, Patrick; Nachon, Florian

    2017-08-01

    Organophosphorus agents (OPs) irreversibly inhibit acetylcholinesterase (AChE) causing a major cholinergic syndrome. The medical counter-measures of OP poisoning have not evolved for the last 30 years with carbamates for pretreatment, pyridinium oximes-based AChE reactivators, antimuscarinic drugs and neuroprotective benzodiazepines for post-exposure treatment. These drugs ensure protection of peripheral nervous system and mitigate acute effects of OP lethal doses. However, they have significant limitations. Pyridostigmine and oximes do not protect/reactivate central AChE. Oximes poorly reactivate AChE inhibited by phosphoramidates. In addition, current neuroprotectants do not protect the central nervous system shortly after the onset of seizures when brain damage becomes irreversible. New therapeutic approaches for pre- and post-exposure treatments involve detoxification of OP molecules before they reach their molecular targets by administrating catalytic bioscavengers, among them phosphotriesterases are the most promising. Novel generation of broad spectrum reactivators are designed for crossing the blood-brain barrier and reactivate central AChE. This is an article for the special issue XVth International Symposium on Cholinergic Mechanisms. © 2017 International Society for Neurochemistry.

  15. Computational Enzymology and Organophosphorus Degrading Enzymes: Promising Approaches Toward Remediation Technologies of Warfare Agents and Pesticides.

    Science.gov (United States)

    Ramalho, Teodorico C; de Castro, Alexandre A; Silva, Daniela R; Silva, Maria Cristina; Franca, Tanos C C; Bennion, Brian J; Kuca, Kamil

    2016-01-01

    The re-emergence of chemical weapons as a global threat in hands of terrorist groups, together with an increasing number of pesticides intoxications and environmental contaminations worldwide, has called the attention of the scientific community for the need of improvement in the technologies for detoxification of organophosphorus (OP) compounds. A compelling strategy is the use of bioremediation by enzymes that are able to hydrolyze these molecules to harmless chemical species. Several enzymes have been studied and engineered for this purpose. However, their mechanisms of action are not well understood. Theoretical investigations may help elucidate important aspects of these mechanisms and help in the development of more efficient bio-remediators. In this review, we point out the major contributions of computational methodologies applied to enzyme based detoxification of OPs. Furthermore, we highlight the use of PTE, PON, DFP, and BuChE as enzymes used in OP detoxification process and how computational tools such as molecular docking, molecular dynamics simulations and combined quantum mechanical/molecular mechanics have and will continue to contribute to this very important area of research.

  16. Theoretical and experimental study of actinide complexes with monoamides and organophosphorus ligands in solution

    International Nuclear Information System (INIS)

    Ribokaite, Kristina

    2013-01-01

    Monoamides and organophosphate are of great interest for the nuclear fuel cycle. Such ligands can selectively extract actinides in liquid-liquid extraction processes. The structure of the extractant (its functional group and its alkyl substituents) has a predominant role in the selective separation of actinides. This thesis concerns the theoretical and experimental studies of model systems in the aim of better understanding of the effect on molecular structures of the complexes. Structures of actinides complexes formed with model ligands in simple media (water or methanol in the presence of nitrate ions) have been characterized. At first, the complexation of uranyl by monoamide and phosphine oxide was studied in water and methanol. Molecular Dynamics simulations and DFT calculations were used to quantify the stability of uranyl complexes with those ligands, and to determine their structural properties. The theoretical results were then compared with experimental results obtained by UV-visible, infrared, Raman and EXAFS on the same chemical systems. The results were used to highlight the greater stability of uranyl complexes with phosphine oxide and monoamides. Further spectroscopic measurements combined with molecular modeling were used to gain a better understanding of the coordination mode of nitrate ion around the uranyl in both water and methanol. Finally, DFT calculations were used to study the influence of the structure of the monoamide or organophosphorus ligand and their interaction with the actinides (IV, VI) including steric effects in the first coordination sphere. (author) [fr

  17. Partitioning of organophosphorus pesticides into phosphatidylcholine small unilamellar vesicles studied by second-derivative spectrophotometry.

    Science.gov (United States)

    Takegami, Shigehiko; Kitamura, Keisuke; Ohsugi, Mayuko; Ito, Aya; Kitade, Tatsuya

    2015-06-15

    In order to quantitatively examine the lipophilicity of the widely used organophosphorus pesticides (OPs) chlorfenvinphos (CFVP), chlorpyrifos-methyl (CPFM), diazinon (DZN), fenitrothion (FNT), fenthion (FT), isofenphos (IFP), profenofos (PFF) and pyraclofos (PCF), their partition coefficient (Kp) values between phosphatidylcholine (PC) small unilamellar vesicles (SUVs) and water (liposome-water system) were determined by second-derivative spectrophotometry. The second-derivative spectra of these OPs in the presence of PC SUV showed a bathochromic shift according to the increase in PC concentration and distinct derivative isosbestic points, demonstrating the complete elimination of the residual background signal effects that were observed in the absorption spectra. The Kp values were calculated from the second-derivative intensity change induced by addition of PC SUV and obtained with a good precision of R.S.D. below 10%. The Kp values were in the order of CPFM>FT>PFF>PCF>IFP>CFVP>FNT⩾DZN and did not show a linear correlation relationship with the reported partition coefficients obtained using an n-octanol-water system (R(2)=0.530). Also, the results quantitatively clarified the effect of chemical-group substitution in OPs on their lipophilicity. Since the partition coefficient for the liposome-water system is more effective for modeling the quantitative structure-activity relationship than that for the n-octanol-water system, the obtained results are toxicologically important for estimating the accumulation of these OPs in human cell membranes. Copyright © 2015 Elsevier B.V. All rights reserved.

  18. A portable liquid crystal-based polarized light system for the detection of organophosphorus nerve gas

    Science.gov (United States)

    He, Feng Jie; Liu, Hui Long; Chen, Long Cong; Xiong, Xing Liang

    2018-03-01

    Liquid crystal (LC)-based sensors have the advantageous properties of being fast, sensitive, and label-free, the results of which can be accessed directly only through the naked eye. However, the inherent disadvantages possessed by LC sensors, such as relying heavily on polarizing microscopes and the difficulty to quantify, have limited the possibility of field applications. Herein, we have addressed these issues by constructing a portable polarized detection system with constant temperature control. This system is mainly composed of four parts: the LC cell, the optics unit, the automatic temperature control unit, and the image processing unit. The LC cell was based on the ordering transitions of LCs in the presence of analytes. The optics unit based on the imaging principle of LCs was designed to substitute the polarizing microscope for the real-time observation. The image processing unit is expected to quantify the concentration of analytes. The results have shown that the presented system can detect dimethyl methyl phosphonate (a stimulant for organophosphorus nerve gas) within 25 s, and the limit of detection is about 10 ppb. In all, our portable system has potential in field applications.

  19. [Contamination of Organophosphorus Pesticides Residue in Fresh Vegetables and Related Health Risk Assessment in Changchun, China].

    Science.gov (United States)

    Yu, Rui; Liu, Jing-shuang; Wang, Qi-cun; Liu, Qiang; Wang, Yang

    2015-09-01

    This study aims to investigate the concentrations of organophosphorus pesticides (OPs) in fresh vegetables. A total of Z14 samples from seven types of vegetables were collected from the suburb in Changchun City. The OPs were analyzed by gas chromatography coupled with flame photometric detector (GC-FPD). Target hazard quotients (THQ) were applied to estimate the potential health risk to inhabitants. Results showed that OPs concentrations exceeded the Maximum Residue Limit (MRL) in more than 23. 4% samples, and were not detected in only 7. 9% samples. Detection rates of OPs was as follow in the decreased order: diazinon (82. 2%) > phorate (45. 8%) > dimethoate (29. 4%) > parathion-methyl (27. 6%) > omethoate (23. 8%) > dichlorvos (22. 9%) > fenitrothion (21%) > fenthion (18. 7%) > parathion (18. 2%) > methamidophos (17. 3%) > malathion (12. 1%). The percentages above MRL for leaves were higher than for non-leafy vegetables. The order of percentages of OPs above MRL was as follows: green onion (82. 5%) > radish (37. 5%) > red pepper (17. 2%) > Chinese vegetable (14. 3%) > cucumber (3. 2%) > eggplant (2. 9%) > tomato (0%). 49. 5% vegetables samples showed more than one OP. The average target hazard quotients (ave THQ) were all less than one and the average Hazard Index (ave HI) was 0. 462, so that inhabitants who expose average OP levels may not experience adverse health effects.

  20. Organophosphorus pesticide residues in milled rice (Oryza sativa) on the Chinese market and dietary risk assessment.

    Science.gov (United States)

    Chen, Chen; Li, Yun; Chen, Mingxue; Chen, Zhijun; Qian, Yongzhong

    2009-03-01

    The present study investigates the occurrence of acetylcholinesterase (AChE)-inhibiting organophosphorus (OP) pesticide residues in milled rice samples obtained form local markets in China during the period 2004-2006 and estimates their cumulative exposure. Concentrations of OP pesticides were determined by gas chromatography with flame photometric detection (GC-FPD). The results showed that 9.3% of the samples contained detectable residues of at least one of the seven target OP pesticides (chlorpyrifos, dichlorvos, omethoate, methamidophos, parathion-methyl, parathion and triazophos) mainly used for agriculture in China, with concentrations ranging 0.011-1.756 mg kg(-1). Rice consumption data was obtained from an individual food consumption survey. Relative potency factors (RPFs) for each pesticide were calculated with methamidophos as the index compound (IC), using 1- or 2-year chronic non-observed adverse effect levels (NOAEL) for AChE inhibition, mostly in rat brain, obtained from international evaluations of pesticides. Exposure to AChE-inhibiting pesticides for the population above 7 years old at P99.9 represented 52-94.5% of the acceptable daily intake (ADI) expressed as methamidophos. Estimated exposure for children aged 2-4 and 4-7 years at P99.9 were 119 and 104.3% of the ADI level, respectively. This study suggests that a yearly monitoring program for OP pesticide residues and strict implementation of the national safety standard for milled rice is necessary.

  1. Removal of actinides from nuclear fuel reprocessing waste solutions with bidentate organophosphorus extractants

    International Nuclear Information System (INIS)

    Schulz, W.W.; McIsaac, L.D.

    1975-08-01

    The neutral bidentate organophosphorus reagents DBDECMP (dibutyl-N,N-diethylcarbamylmethylenephosphonate) and its dihexyl analogue DHDECMP are candidate extractants for removal of actinides from certain acidic waste streams produced at the U. S. ERDA Hanford and Idaho Falls sites. Various chemical and physical properties including availability, cost, purification, alpha radiolysis, and aqueous phase solubility of DBDECMP and DHDECMP are reviewed. A conceptual flowsheet employing a 15 percent DBDECMP (or DHDECMP)--CCl 4 extractant for removal (and recovery) of Am and Pu from Hanford's Plutonium Reclamation Facility acid waste stream (CAW solution) was successfully demonstrated in laboratory-scale mixer-settler tests; this extraction scheme can be used to produce an actinide-free waste. A 30 percent DBDECMP-xylene flowsheet is being tested at the Idaho Falls site for removal of U, Np, Pu, and Am from Idaho Chemical Processing Plant first-cycle high-level raffinate to produce an actinide-free (less than 10 nCi alpha activity/gram) waste. (auth)

  2. Development of apple certified reference material for quantification of organophosphorus and pyrethroid pesticides.

    Science.gov (United States)

    Otake, Takamitsu; Yarita, Takashi; Aoyagi, Yoshie; Kuroda, Youko; Numata, Masahiko; Iwata, Hitoshi; Watai, Masatoshi; Mitsuda, Hitoshi; Fujikawa, Takashi; Ota, Hidekazu

    2013-06-01

    An apple certified reference material for the analysis of pesticide residues was issued by the National Metrology Institute of Japan. Organophosphorus and pyrethroid pesticides were sprayed on apples, and these were used as raw materials of certified reference material. The harvested apples were cut into small pieces, freeze-dried, pulverized, sieved, placed into 200 brown glass bottles (3g each), and sterilized by γ-irradiation. Stability and homogeneity assessment was performed, and the relative uncertainties due to instability (for an expiry date of 32 months) and inhomogeneity were 10.3-25.0% and 4.0-6.8%, respectively. The characterization was carried out using multiple analytical methods to ensure the reliability of analytical results; the values of target pesticides were obtained by isotope dilution mass spectrometry. Certified values were 2.28 ± 0.82 mg/kg for diazinon, 3.14 ± 0.79 mg/kg for fenitrothion, 1.55 ± 0.81 mg/kg for cypermethrin, and 2.81 ± 0.70 mg/kg for permethrin. Copyright © 2012 Elsevier Ltd. All rights reserved.

  3. Removal of atrazine and four organophosphorus pesticides from environmental waters by diatomaceous earth-remediation method.

    Science.gov (United States)

    Agdi, K; Bouaid, A; Esteban, A M; Hernando, P F; Azmani, A; Camara, C

    2000-10-01

    A new viable remediation technique based on the use of diatomaceous earth is proposed to improve the ecological system. Its ability to remove atrazine and the four organophosphorus pesticides parathion-methyl, chlorpyriphos, fenamiphos and methidathion from river and waste waters has been proven. A series of experiments including variable conditions, such as temperature, pH, contact time, pesticide concentration and adsorbent quantity, were performed to demonstrate the efficiency of pesticide removal from three different water samples. The batch experiments showed that diatomaceous earth was able to remove 95% of chlorpyriphos, 75% of methidathion and parathion-methyl and 55% of atrazine and fenamiphos from all types of waters tested. The individual adsorption of each pesticide on diatomaceous earth could be described by the Freundlich isotherm and a tentative adsorption mechanism was proposed. The Freundlich coefficient (Kf) and Freundlich constant (1/n) appeared to be closely related to the physicochemical properties (Kow, solubility) of the compounds. The actual results support the conclusion that diatomaceous earth has the potential to serve as an extractant in remediation techniques.

  4. Biosensors and their applications in detection of organophosphorus pesticides in the environment.

    Science.gov (United States)

    Hassani, Shokoufeh; Momtaz, Saeideh; Vakhshiteh, Faezeh; Maghsoudi, Armin Salek; Ganjali, Mohammad Reza; Norouzi, Parviz; Abdollahi, Mohammad

    2017-01-01

    This review discusses the past and recent advancements of biosensors focusing on detection of organophosphorus pesticides (OPs) due to their exceptional use during the last decades. Apart from agricultural benefits, OPs also impose adverse toxicological effects on animal and human population. Conventional approaches such as chromatographic techniques used for pesticide detection are associated with several limitations. A biosensor technology is unique due to the detection sensitivity, selectivity, remarkable performance capabilities, simplicity and on-site operation, fabrication and incorporation with nanomaterials. This study also provided specifications of the most OPs biosensors reported until today based on their transducer system. In addition, we highlighted the application of advanced complementary materials and analysis techniques in OPs detection systems. The availability of these new materials associated with new sensing techniques has led to introduction of easy-to-use analytical tools of high sensitivity and specificity in the design and construction of OPs biosensors. In this review, we elaborated the achievements in sensing systems concerning innovative nanomaterials and analytical techniques with emphasis on OPs.

  5. Differential protein adduction by seven organophosphorus pesticides in both brain and thymus.

    Science.gov (United States)

    Carter, Wayne G; Tarhoni, Mabruka; Rathbone, Alexandra J; Ray, David E

    2007-04-01

    There is a need for mechanistic understanding of the lasting ill health reported in several studies of workers exposed to organophosphorus (OP) pesticide. Although the acute toxicity is largely explicable by acetylcholinesterase inhibition and the lasting effects of frank poisoning by direct excitotoxicity or indirect consequences of the cholinergic syndrome, effects at lower levels of exposure would not be predicted from these mechanisms. Similarly, reversible interactions with nicotinic and muscarinic receptors in adults would not predict continuing ill health. Many OP pesticides produce protein adduction, and the lasting nature of this makes it a candidate mechanism for the production of continuing ill health. We found significant adduction of partially characterized protein targets in both rat brain and thymus by azamethiphos, chlorfenvinphos, chlorpyrifos-oxon, diazinon-oxon, dichlorvos and malaoxon, in vitro and pirimiphos-methyl in vivo. The diversity in the adduction pattern seen across these agents at low dose levels means that any longer term effects of adduction would be specific to specific organophosphates, rather than generic. This presents a challenge to epidemiology, as most exposures are to different agents over time. However, some adducted proteins are also expressed in blood, notably albumin, and so may provide exposure measures to increase the power of future epidemiological studies.

  6. In vitro human skin penetration model for organophosphorus compounds with different physicochemical properties.

    Science.gov (United States)

    Thors, L; Koch, B; Koch, M; Hägglund, L; Bucht, A

    2016-04-01

    A flow-through diffusion cell was validated for in vitro human epidermal penetration studies of organophosphorus compounds (OPCs) applied by infinite dosing. By testing OPCs with similar molecular weight but different physicochemical properties, it was shown that hydrophilic and lipophilic properties are major determinants for the penetration rate. Lipophilic OPCs displayed maximum cumulative penetration in the 20-75% agent concentration range whereas the hydrophilic OPCs displayed maximum cumulative penetration at 10 or 20% agent concentration. Low penetration was observed for all agents at 1% agent concentration or when applied as neat agents. The impact of the receptor solution composition was evaluated by comparing the penetration using receptor solutions of different ratios of ethanol and water. For diluted OPCs, a high concentration of ethanol in the receptor solution significantly increased the penetration compared to lower concentrations. When OPCs were applied as neat agents, the composition of the receptor solution only affected the penetration for one of four tested compounds. In conclusion, the flow-through diffusion cell was useful for examining the penetration of OPCs through the epidermal membrane. It was also demonstrated that the penetration rates of OPCs are strongly influenced by dilution in water and the receptor fluid composition. Copyright © 2016 Elsevier B.V. All rights reserved.

  7. Degradation of Organophosphorus and Pyrethroid Insecticides in Beverages: Implications for Risk Assessment

    Directory of Open Access Journals (Sweden)

    Samantha A. Radford

    2018-02-01

    Full Text Available Since urinary insecticide metabolites are commonly used as biomarkers of exposure, it is important that we quantify whether insecticides degrade in food and beverages in order to better perform risk assessment. This study was designed to quantify degradation of organophosphorus and pyrethroid insecticides in beverages. Purified water, white grape juice, orange juice, and red wine were fortified with 500 ng/mL diazinon, malathion, chlorpyrifos, permethrin, cyfluthrin, cypermethrin, and deltamethrin, and aliquots were extracted several times over a 15-day storage period at 2.5 °C. Overall, statistically significant loss of at least one insecticide was observed in each matrix, and at least five out of seven insecticides demonstrated a statistically significant loss in all matrices except orange juice. An investigation of an alternative mechanism of insecticide loss—adsorption onto the glass surface of the storage jars—was carried out, which indicated that this mechanism of loss is insignificant. Results of this work suggest that insecticides degrade in these beverages, and this degradation may lead to pre-existing insecticide degradates in the beverages, suggesting that caution should be exercised when using urinary insecticide metabolites to assess exposure and risk.

  8. Sensitive spectrophotometric methods for determination of some organophosphorus pesticides in vegetable samples

    Directory of Open Access Journals (Sweden)

    MAGDA A. AKL

    2010-03-01

    Full Text Available Three rapid, simple, reproducible and sensitive spectrophotometric methods (A, B and C are described for the determination of two organophosphorus pesticides, (malathion and dimethoate in formulations and vegetable samples. The methods A and B involve the addition of an excess of Ce4+ into sulphuric acid medium and the determination of the unreacted oxidant by decreasing the red color of chromotrope 2R (C2R at a suitable lmax = 528 nm for method A, or a decrease in the orange pink color of rhodamine 6G (Rh6G at a suitable lmax = = 525 nm. The method C is based on the oxidation of malathion or dimethoate with the slight excess of N-bromosuccinimide (NBS and the determination of unreacted oxidant by reacting it with amaranth dye (AM in hydrochloric acid medium at a suitable lmax = 520 nm. A regression analysis of Beer-Lambert plots showed a good correlation in the concentration range of 0.1-4.2 μg mL−1. The apparent molar absorptivity, Sandell sensitivity, the detection and quantification limits were calculated. For more accurate analysis, Ringbom optimum concentration ranges are 0.25-4.0 μg mL−1. The developed methods were successfully applied to the determination of malathion, and dimethoate in their formulations and environmental vegetable samples.

  9. Evaluation of working air quality by using semipermeable membrane devices. Analysis of organophosphorus pesticides.

    Science.gov (United States)

    Esteve-Turrillas, Francesc A; Pastor, Agustín; de la Guardia, Miguel

    2008-09-19

    It has been evaluated the use of semipermeable membrane devices (SPMDs) as passive samplers of organophosphorus pesticides from air, in order to determine the contamination of working environments. Additionally, the use of SPMDs as portable samplers has been also considered. The analytical methodology for the determination of diazinon, chlorpyrifos-methyl, pirimiphos-methyl, chlorpyrifos and fenthion in SPMDs exposed to contaminated air was based on microwave-assisted extraction and gas chromatography with mass spectrometry determination. Limit of detection (LOD) values from 2 to 4ngSPMD(-1) and repeatability from 2 to 7% were obtained by using the aforementioned methodology. Theoretical calculated sampling rates were employed for the estimation of the pesticide concentration in air, by using the pesticide mass retained in the deployed SPMD. The obtained LOD values, for a sampling time of 7 days, were from 1 to 2ngm(-3). The evaluation of the air quality of a pesticide laboratory with an intensive use of diazinon and chlorpyrifos has been made in order to check the operation safety conditions.

  10. Effects of organophosphorus flame retardant TDCPP on normal human corneal epithelial cells: Implications for human health.

    Science.gov (United States)

    Xiang, Ping; Liu, Rong-Yan; Li, Chao; Gao, Peng; Cui, Xin-Yi; Ma, Lena Q

    2017-11-01

    Tris(1,3-dichloro-2-propyl) phosphate (TDCPP) is one of the most detected organophosphorus flame retardants (OPFRs) in the environment, especially in indoor dust. Continuous daily exposure to TDCPP-containing dust may adversely impact human cornea. However, its detrimental effects on human corneal epithelium are largely unknown. In this study, we investigated the cell apoptosis in normal human corneal epithelial cells (HCECs) after TDCPP exposure and elucidated the underlying molecular mechanisms. Our data indicated a dose-dependent decrease of cell viability after TDCPP exposure with LC 50 at 202 μg/mL. A concentration-dependent apoptotic sign was observed in HCECs after exposing to ≥2 μg/mL TDCPP. Endoplasmic reticulum stress induction was evidenced by up-regulation of its biomarker genes (ATF-4, CHOP, BiP, and XBP1). Furthermore, alternation of Bcl-2/Bax expression, mitochondrial membrane potential loss, cellular ATP content decrease, and caspase-3 and -9 activity increase were observed after exposing to 2 or 20 μg/mL TDCPP. Taken together, the data implicated the involvement of endoplasmic reticulum stress in TDCPP-induced HCEC apoptosis, probably mediated by mitochondrial apoptotic pathway. Our findings showed TDCPP exposure induced toxicity to human cornea. Due to TDCPP's presence at high levels in indoor dust, further study is warranted to evaluate its health risk on human corneas. Published by Elsevier Ltd.

  11. Detection of sarin hydrolysis products from sarin-like organophosphorus agent-exposed human erythrocytes.

    Science.gov (United States)

    Nagao, M; Takatori, T; Matsuda, Y; Nakajima, M; Niijima, H; Iwase, H; Iwadate, K; Amano, T

    1997-11-07

    A sarin-like organophosphorus agent, [bis(isopropyl methyl)phosphonate; BIMP], was synthesized. This agent has the same phosphonate group as sarin and also has the same anti-acetylcholinesterase activity potency as sarin. The ID50 and LD50 values of BIMP in mice after intravenous injection were 3.9 nM and 0.8 mg/kg, respectively. The AChE activities of their red blood cells and brains were dose-dependently reduced by intravenous BIMP. After preparation of experimental BIMP-exposed human red blood cells, BIMP-bound acetylcholinesterase (AChE) was solubilized from erythrocyte membranes, purified by immunoaffinity chromatography, digested with trypsin, and the sarin hydrolysis products bound to AChE were released by alkaline phosphatase digestion. The digested sarin hydrolysis products were subjected to trimethylsilyl (TMS) derivatization and detected by gas chromatography-mass spectrometry. Isopropyl methylphosphonic- and methylphosphonic acids, which are the sarin hydrolysis products, were detected in experimental BIMP-exposed human red blood cells. This new method, which enables sarin's hydrolysis products to be detected in BIMP-exposed erythrocytes, is a useful tool for studying sarin-poisoning victims.

  12. Cavitand-functionalized porous silicon as an active surface for organophosphorus vapor detection.

    Science.gov (United States)

    Tudisco, Cristina; Betti, Paolo; Motta, Alessandro; Pinalli, Roberta; Bombaci, Luigi; Dalcanale, Enrico; Condorelli, Guglielmo G

    2012-01-24

    This paper reports on the preparation of a porous silicon-based material covalently functionalized with cavitand receptors suited for the detection of organophosphorus vapors. Two different isomeric cavitands, both containing one acid group at the upper rim, specifically designed for covalent anchoring on silicon, were grafted on H-terminated porous silicon (PSi) by thermal hydrosilylation. The covalently functionalized surfaces and their complexation properties were characterized by combining different analytical techniques, namely X-ray photoelectron spectroscopy (XPS), Fourier transform infrared spectroscopy (FTIR), and mass spectroscopy analysis coupled with thermal desorption experiments. Complexation experiments were performed by exposing both active surfaces and a control surface consisting of PSi functionalized with a structurally similar but inactive methylene-bridged cavitand (MeCav) to dimethyl methylphosphonate (DMMP) vapors. Comparison between active and inactive surfaces demonstrated the recognition properties of the new surfaces. Finally, the nature of the involved interactions, the energetic differences between active and inactive surfaces toward DMMP complexation, and the comparison with a true nerve gas agent (sarin) were studied by DFT modeling. The results revealed the successful grafting reaction, the specific host-guest interactions of the PSi-bonded receptors, and the reversibility of the guest complexation.

  13. Improvement of Some Biochemical Activities of Rats Treated with The Organophosphorus Insecticide Cytrolane Using Soyabean

    International Nuclear Information System (INIS)

    Abdel-Fattah, A.M.; Elmahdy, A.A.

    2005-01-01

    This Study aims to evaluate the role of soyabean diet in improvement of some biochemical activities of female rats received daily oral dose 0.89 mg/kg (1/10 LD 50 )of cytrolane as organophosphorus insecticide on some biochemical activities in female rats for ten consecutive days. Prior pesticide administration, female rats received soyabean at dose 58 g/kg body weight for 2 and 4 weeks. Under the effect of cytrolane administration, the results showed significant increase in serum triglycerides, total cholesterol, HDL-cholesterol and CPK mean levels. Total serum protein and progesterone levels were significantly decreased compared to the control. On the other hand, by the administration of soyabean prior cytrolane, it reduced considerably the changes produced by cytrolane on levels of serum cholesterol, triglyceride, HDL-cholesterol which decreased more or less compared to control, total protein level was within normal value. Also, serum progesterone concentration increased. Therefore, soyabean administration accelerated the normalization and improvement processes of the altered lipid profiles, protein and progesterone hormone

  14. Role of Site-Specific N-Glycans Expressed on GluA2 in the Regulation of Cell Surface Expression of AMPA-Type Glutamate Receptors.

    Directory of Open Access Journals (Sweden)

    Yusuke Takeuchi

    Full Text Available The AMPA-type glutamate receptor (AMPAR, which is a tetrameric complex composed of four subunits (GluA1-4 with several combinations, mediates the majority of rapid excitatory synaptic transmissions in the nervous system. Cell surface expression levels of AMPAR modulate synaptic plasticity, which is considered one of the molecular bases for learning and memory formation. To date, a unique trisaccharide (HSO3-3GlcAβ1-3Galβ1-4GlcNAc, human natural killer-1 (HNK-1 carbohydrate, was found expressed specifically on N-linked glycans of GluA2 and regulated the cell surface expression of AMPAR and the spine maturation process. However, evidence that the HNK-1 epitope on N-glycans of GluA2 directly affects these phenomena is lacking. Moreover, it is thought that other N-glycans on GluA2 also have potential roles in the regulation of AMPAR functions. In the present study, using a series of mutants lacking potential N-glycosylation sites (N256, N370, N406, and N413 within GluA2, we demonstrated that the mutant lacking the N-glycan at N370 strongly suppressed the intracellular trafficking of GluA2 from the endoplasmic reticulum (ER in HEK293 cells. Cell surface expression of GluA1, which is a major subunit of AMPAR in neurons, was also suppressed by co-expression of the GluA2 N370S mutant. The N370S mutant and wild-type GluA2 were co-immunoprecipitated with GluA1, suggesting that N370S was properly associated with GluA1. Moreover, we found that N413 was the main potential site of the HNK-1 epitope that promoted the interaction of GluA2 with N-cadherin, resulting in enhanced cell surface expression of GluA2. The HNK-1 epitope on N-glycan at the N413 of GluA2 was also involved in the cell surface expression of GluA1. Thus, our data suggested that site-specific N-glycans on GluA2 regulate the intracellular trafficking and cell surface expression of AMPAR.

  15. 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

  16. Inhibitory effects of two G protein-coupled receptor kinases on the cell surface expression and signaling of the human adrenomedullin receptor

    International Nuclear Information System (INIS)

    Kuwasako, Kenji; Sekiguchi, Toshio; Nagata, Sayaka; Jiang, Danfeng; Hayashi, Hidetaka; Murakami, Manabu; Hattori, Yuichi; Kitamura, Kazuo; Kato, Johji

    2016-01-01

    Receptor activity-modifying protein 2 (RAMP2) enables the calcitonin receptor-like receptor (CLR, a family B GPCR) to form the type 1 adrenomedullin receptor (AM 1 receptor). Here, we investigated the effects of the five non-visual GPCR kinases (GRKs 2 through 6) on the cell surface expression of the human (h)AM 1 receptor by cotransfecting each of these GRKs into HEK-293 cells that stably expressed hRAMP2. Flow cytometric analysis revealed that when coexpressed with GRK4 or GRK5, the cell surface expression of the AM 1 receptor was markedly decreased prior to stimulation with AM, thereby attenuating both the specific [ 125 I]AM binding and AM-induced cAMP production. These inhibitory effects of both GRKs were abolished by the replacement of the cytoplasmic C-terminal tail (C-tail) of CLR with that of the calcitonin receptor (a family B GPCR) or β 2 -adrenergic receptor (a family A GPCR). Among the sequentially truncated CLR C-tail mutants, those lacking the five residues 449–453 (Ser-Phe-Ser-Asn-Ser) abolished the inhibition of the cell surface expression of CLR via the overexpression of GRK4 or GRK5. Thus, we provided new insight into the function of GRKs in agonist-unstimulated GPCR trafficking using a recombinant AM 1 receptor and further determined the region of the CLR C-tail responsible for this GRK function. - Highlights: • We discovered a novel function of GRKs in GPCR trafficking using human CLR/RAMP2. • GRKs 4 and 5 markedly inhibited the cell surface expression of human CLR/RAMP2. • Both GRKs exhibited highly significant receptor signaling inhibition. • Five residues of the C-terminal tail of CLR govern this function of GRKs.

  17. Inhibitory effects of two G protein-coupled receptor kinases on the cell surface expression and signaling of the human adrenomedullin receptor

    Energy Technology Data Exchange (ETDEWEB)

    Kuwasako, Kenji, E-mail: kuwasako@med.miyazaki-u.ac.jp [Frontier Science Research Center, University of Miyazaki, Miyazaki, 889-1692 (Japan); Sekiguchi, Toshio [Noto Marine Laboratory, Division of Marine Environmental Studies, Institute of Nature and Environmental Technology, Kanazawa University, Ishikawa, 927-0553 (Japan); Nagata, Sayaka [Division of Circulatory and Body Fluid Regulation, Faculty of Medicine, University of Miyazaki, Miyazaki, 889-1692 (Japan); Jiang, Danfeng; Hayashi, Hidetaka [Frontier Science Research Center, University of Miyazaki, Miyazaki, 889-1692 (Japan); Murakami, Manabu [Department of Pharmacology, Hirosaki University, Graduate School of Medicine, Hirosaki, 036-8562 (Japan); Hattori, Yuichi [Department of Molecular and Medical Pharmacology, Graduate School of Medicine and Pharmaceutical Sciences, University of Toyama, Toyama, 930-0194 (Japan); Kitamura, Kazuo [Division of Circulatory and Body Fluid Regulation, Faculty of Medicine, University of Miyazaki, Miyazaki, 889-1692 (Japan); Kato, Johji [Frontier Science Research Center, University of Miyazaki, Miyazaki, 889-1692 (Japan)

    2016-02-19

    Receptor activity-modifying protein 2 (RAMP2) enables the calcitonin receptor-like receptor (CLR, a family B GPCR) to form the type 1 adrenomedullin receptor (AM{sub 1} receptor). Here, we investigated the effects of the five non-visual GPCR kinases (GRKs 2 through 6) on the cell surface expression of the human (h)AM{sub 1} receptor by cotransfecting each of these GRKs into HEK-293 cells that stably expressed hRAMP2. Flow cytometric analysis revealed that when coexpressed with GRK4 or GRK5, the cell surface expression of the AM{sub 1} receptor was markedly decreased prior to stimulation with AM, thereby attenuating both the specific [{sup 125}I]AM binding and AM-induced cAMP production. These inhibitory effects of both GRKs were abolished by the replacement of the cytoplasmic C-terminal tail (C-tail) of CLR with that of the calcitonin receptor (a family B GPCR) or β{sub 2}-adrenergic receptor (a family A GPCR). Among the sequentially truncated CLR C-tail mutants, those lacking the five residues 449–453 (Ser-Phe-Ser-Asn-Ser) abolished the inhibition of the cell surface expression of CLR via the overexpression of GRK4 or GRK5. Thus, we provided new insight into the function of GRKs in agonist-unstimulated GPCR trafficking using a recombinant AM{sub 1} receptor and further determined the region of the CLR C-tail responsible for this GRK function. - Highlights: • We discovered a novel function of GRKs in GPCR trafficking using human CLR/RAMP2. • GRKs 4 and 5 markedly inhibited the cell surface expression of human CLR/RAMP2. • Both GRKs exhibited highly significant receptor signaling inhibition. • Five residues of the C-terminal tail of CLR govern this function of GRKs.

  18. An investigation of interactions between hypocretin/orexin signaling and glutamate receptor surface expression in the rat nucleus accumbens under basal conditions and after cocaine exposure.

    Science.gov (United States)

    Plaza-Zabala, Ainhoa; Li, Xuan; Milovanovic, Mike; Loweth, Jessica A; Maldonado, Rafael; Berrendero, Fernando; Wolf, Marina E

    2013-12-17

    Hypocretin peptides are critical for the effects of cocaine on excitatory synaptic strength in the ventral tegmental area (VTA). However, little is known about their role in cocaine-induced synaptic plasticity in the nucleus accumbens (NAc). First, we tested whether hypocretin-1 by itself could acutely modulate glutamate receptor surface expression in the NAc, given that hypocretin-1 in the VTA reproduces cocaine's effects on glutamate transmission. We found no effect of hypocretin-1 infusion on AMPA or NMDA receptor surface expression in the NAc, measured by biotinylation, either 30 min or 3h after the infusion. Second, we were interested in whether changes in hypocretin receptor-2 (Hcrtr-2) expression contribute to cocaine-induced plasticity in the NAc. As a first step towards addressing this question, Hcrtr-2 surface expression was compared in the NAc after withdrawal from extended-access self-administration of saline (control) versus cocaine. We found that surface Hcrtr-2 levels remain unchanged following 14, 25 or 48 days of withdrawal from cocaine, a time period in which high conductance GluA2-lacking AMPA receptors progressively emerge in the NAc. Overall, our results fail to support a role for hypocretins in acute modulation of glutamate receptor levels in the NAc or a role for altered Hcrtr-2 expression in withdrawal-dependent synaptic adaptations in the NAc following cocaine self-administration. Copyright © 2013 Elsevier Ireland Ltd. All rights reserved.

  19. Differential down-modulation of HLA-G and HLA-A2 or -A3 cell surface expression following human cytomegalovirus infection.

    Science.gov (United States)

    Pizzato, Nathalie; Garmy-Susini, Barbara; Le Bouteiller, Philippe; Lenfant, Françoise

    2004-06-01

    During pregnancy, the non-classical major histocompatibility complex (MHC) class I HLA-G molecule is specifically expressed in trophoblast cells at the materno-fetal interface and may exert a local control of the immune response against viral infections. Human cytomegalovirus (HCMV) infection, which is the major cause of congenital defects, encodes multiple glycoproteins (US2, US3, US6, US10 and US11) that interrupt the MHC class I pathway of antigen presentation. The effect of some of these unique short (US) proteins on HLA-G expression has been previously studied, but little is known about the modulation of HLA-G cell surface expression during the course of HCMV infection which ensures expression of all of these US proteins. Using flow cytometry analysis, HLA-G cell surface expression was evaluated in HCMV-infected U373-HLA-G transfectant cells and compared with the modulation of the endogenous classical HLA-A2 molecules. The results indicated that HCMV infection down-modulated HLA-G cell surface expression, but later after infection and to a lesser extent than HLA-A2. Using various HLA-G/HLA-A2 chimeras, we showed that the unique structure of HLA-G cytoplasmic tail was partly involved in the resistance of HLA-G to viral down-modulation. Such limited down-modulation of HLA-G may have functional consequences in term of innate immunity against congenital HCMV infection.

  20. Effects of the organophosphorus pesticide Folisuper 600 (methyl parathion on the heart function of bullfrog tadpoles, Lithobates catesbeianus (Shaw, 1802

    Directory of Open Access Journals (Sweden)

    M. J. Costa

    Full Text Available Abstract The aim of this work was to evaluate whether the heart function of bullfrog tadpoles (25 Gosner stage is affected by their acute exposure (48 h to a sub-lethal concentration (10 µg.L–1 of the active principle of the organophosphorus pesticide Folisuper 600R (methyl parathion - MP. Our results demonstrated that MP causes not only a reduction in tadpoles’ cardiac ventricular mass, resulting in a marked reduction in their cardiac twitch force, but also impairs their swimming performance, irrespective of increasing their heart rate. Together, these findings indicate that low and realistic concentration of MP have a negative impact on tadpoles’ performance, jeopardizing their survival.

  1. Defining sequence space and reaction products within the cyanuric acid hydrolase (AtzD)/barbiturase protein family.

    Science.gov (United States)

    Seffernick, Jennifer L; Erickson, Jasmine S; Cameron, Stephan M; Cho, Seunghee; Dodge, Anthony G; Richman, Jack E; Sadowsky, Michael J; Wackett, Lawrence P

    2012-09-01

    Cyanuric acid hydrolases (AtzD) and barbiturases are homologous, found almost exclusively in bacteria, and comprise a rare protein family with no discernible linkage to other protein families or an X-ray structural class. There has been confusion in the literature and in genome projects regarding the reaction products, the assignment of individual sequences as either cyanuric acid hydrolases or barbiturases, and spurious connection of this family to another protein family. The present study has addressed those issues. First, the published enzyme reaction products of cyanuric acid hydrolase are incorrectly identified as biuret and carbon dioxide. The current study employed (13)C nuclear magnetic resonance (NMR) spectroscopy and mass spectrometry to show that cyanuric acid hydrolase releases carboxybiuret, which spontaneously decarboxylates to biuret. This is significant because it revealed that homologous cyanuric acid hydrolases and barbiturases catalyze completely analogous reactions. Second, enzymes that had been annotated incorrectly in genome projects have been reassigned here by bioinformatics, gene cloning, and protein characterization studies. Third, the AtzD/barbiturase family has previously been suggested to consist of members of the amidohydrolase superfamily, a large class of metallohydrolases. Bioinformatics and the lack of bound metals both argue against a connection to the amidohydrolase superfamily. Lastly, steady-state kinetic measurements and observations of protein stability suggested that the AtzD/barbiturase family might be an undistinguished protein family that has undergone some resurgence with the recent introduction of industrial s-triazine compounds such as atrazine and melamine into the environment.

  2. Decreased surface expression of the δ subunit of the GABAA receptor contributes to reduced tonic inhibition in dentate granule cells in a mouse model of fragile X syndrome.

    Science.gov (United States)

    Zhang, Nianhui; Peng, Zechun; Tong, Xiaoping; Lindemeyer, A Kerstin; Cetina, Yliana; Huang, Christine S; Olsen, Richard W; Otis, Thomas S; Houser, Carolyn R

    2017-11-01

    While numerous changes in the GABA system have been identified in models of Fragile X Syndrome (FXS), alterations in subunits of the GABA A receptors (GABA A Rs) that mediate tonic inhibition are particularly intriguing. Considering the key role of tonic inhibition in controlling neuronal excitability, reduced tonic inhibition could contribute to FXS-associated disorders such as hyperactivity, hypersensitivity, and increased seizure susceptibility. The current study has focused on the expression and function of the δ subunit of the GABA A R, a major subunit involved in tonic inhibition, in granule cells of the dentate gyrus in the Fmr1 knockout (KO) mouse model of FXS. Electrophysiological studies of dentate granule cells revealed a marked, nearly four-fold, decrease in tonic inhibition in the Fmr1 KO mice, as well as reduced effects of two δ subunit-preferring pharmacological agents, THIP and DS2, supporting the suggestion that δ subunit-containing GABA A Rs are compromised in the Fmr1 KO mice. Immunohistochemistry demonstrated a small but statistically significant decrease in δ subunit labeling in the molecular layer of the dentate gyrus in Fmr1 KO mice compared to wildtype (WT) littermates. The discrepancy between the large deficits in GABA-mediated tonic inhibition in granule cells in the Fmr1 KO mice and only modest reductions in immunolabeling of the δ subunit led to studies of surface expression of the δ subunit. Cross-linking experiments followed by Western blot analysis demonstrated a small, non-significant decrease in total δ subunit protein in the hippocampus of Fmr1 KO mice, but a four-fold decrease in surface expression of the δ subunit in these mice. No significant changes were observed in total or surface expression of the α4 subunit protein, a major partner of the δ subunit in the forebrain. Postembedding immunogold labeling for the δ subunit demonstrated a large, three-fold, decrease in the number of symmetric synapses with

  3. Insect nicotinic receptor interactions in vivo with neonicotinoid, organophosphorus, and methylcarbamate insecticides and a synergist

    Science.gov (United States)

    Shao, Xusheng; Xia, Shanshan; Durkin, Kathleen A.; Casida, John E.

    2013-01-01

    The nicotinic acetylcholine (ACh) receptor (nAChR) is the principal insecticide target. Nearly half of the insecticides by number and world market value are neonicotinoids acting as nAChR agonists or organophosphorus (OP) and methylcarbamate (MC) acetylcholinesterase (AChE) inhibitors. There was no previous evidence for in vivo interactions of the nAChR agonists and AChE inhibitors. The nitromethyleneimidazole (NMI) analog of imidacloprid, a highly potent neonicotinoid, was used here as a radioligand, uniquely allowing for direct measurements of house fly (Musca domestica) head nAChR in vivo interactions with various nicotinic agents. Nine neonicotinoids inhibited house fly brain nAChR [3H]NMI binding in vivo, corresponding to their in vitro potency and the poisoning signs or toxicity they produced in intrathoracically treated house flies. Interestingly, nine topically applied OP or MC insecticides or analogs also gave similar results relative to in vivo nAChR binding inhibition and toxicity, but now also correlating with in vivo brain AChE inhibition, indicating that ACh is the ultimate OP- or MC-induced nAChR active agent. These findings on [3H]NMI binding in house fly brain membranes validate the nAChR in vivo target for the neonicotinoids, OPs and MCs. As an exception, the remarkably potent OP neonicotinoid synergist, O-propyl O-(2-propynyl) phenylphosphonate, inhibited nAChR in vivo without the corresponding AChE inhibition, possibly via a reactive ketene metabolite reacting with a critical nucleophile in the cytochrome P450 active site and the nAChR NMI binding site. PMID:24108354

  4. Catalytic efficiencies of directly evolved phosphotriesterase variants with structurally different organophosphorus compounds in vitro.

    Science.gov (United States)

    Goldsmith, Moshe; Eckstein, Simone; Ashani, Yacov; Greisen, Per; Leader, Haim; Sussman, Joel L; Aggarwal, Nidhi; Ovchinnikov, Sergey; Tawfik, Dan S; Baker, David; Thiermann, Horst; Worek, Franz

    2016-11-01

    The nearly 200,000 fatalities following exposure to organophosphorus (OP) pesticides each year and the omnipresent danger of a terroristic attack with OP nerve agents emphasize the demand for the development of effective OP antidotes. Standard treatments for intoxicated patients with a combination of atropine and an oxime are limited in their efficacy. Thus, research focuses on developing catalytic bioscavengers as an alternative approach using OP-hydrolyzing enzymes such as Brevundimonas diminuta phosphotriesterase (PTE). Recently, a PTE mutant dubbed C23 was engineered, exhibiting reversed stereoselectivity and high catalytic efficiency (k cat /K M ) for the hydrolysis of the toxic enantiomers of VX, CVX, and VR. Additionally, C23's ability to prevent systemic toxicity of VX using a low protein dose has been shown in vivo. In this study, the catalytic efficiencies of V-agent hydrolysis by two newly selected PTE variants were determined. Moreover, in order to establish trends in sequence-activity relationships along the pathway of PTE's laboratory evolution, we examined k cat /K M values of several variants with a number of V-type and G-type nerve agents as well as with different OP pesticides. Although none of the new PTE variants exhibited k cat /K M values >10 7  M -1  min -1 with V-type nerve agents, which is required for effective prophylaxis, they were improved with VR relative to previously evolved variants. The new variants detoxify a broad spectrum of OPs and provide insight into OP hydrolysis and sequence-activity relationships.

  5. Dose-response effects of atropine and HI-6 treatment of organophosphorus poisoning in guinea pigs

    Energy Technology Data Exchange (ETDEWEB)

    Koplovitz, I.; Menton, R.; Matthews, C.; Shutz, M.; Nalls, C.

    1995-12-31

    H1-6 (1-2-hydrnxyiminomethyl-1 pyridino-3-(4-carbameyl- 1--pyddino)-2- oxaprnpane dichioride) has been evaluated as an oxime alternative to pralidoxime, and toxogonin in the treatment of organophosphorus (OP) poisoning. The dose response effects of atropine (ATR) and HI-6 were investigated to more fully explore the interaction of these compounds in the treatment of OP poisoning. ATR, HI-6 and various combinations of the two drugs were evaluated against lethal poisoning by soman (GD) and tabun (GA) in guinea pigs. The effect of adjunctive diazepam treatment on the efficacy of atropine and HI-6 against soman was also investigated. Animals of either sex were challenged s.c. with OP and treated i.m. 1 min later with ATR and/or HI-6. When used, diazepam was injected immediately after ATR+HI6. LD50s of each treatment were calculated from probit models based on 24-hour survival against 5 levels of nerve agent and 6 animals per challenge level. A protective index (PI) was calculated by dividing the nerve agent LD50 in the presence of treatment by the LD50 in the absence of treatment. Treatment with HI-6 alone had little effect on the toxicity of either OP. Treatment with ATR alone was more effective than HI-6 alone and was significantly more effective against soman than against tabun. When used in combination atropine and HI-6 had a strong synergistic effect against both agents. The dose of atropine used with HI-6 was critical in determining the efficacy of HI-6 against either agent. The slopes of the dose-lethality curves were minimally affected by the dose of ATR or HI-6. Adjunctive treatment with diazepam enhanced the efficacy of HI-6 and atropine against soman.

  6. Dialkyl phosphate metabolites of organophosphorus in applicators of agricultural pesticides in Majes – Arequipa (Peru

    Directory of Open Access Journals (Sweden)

    Chung Arturo

    2006-12-01

    Full Text Available Abstract Background Organophosphorus (OPs pesticides are the most commonly used pesticides in Peruvian agriculture. The population at risk for OPs exposure includes formulators, applicators and farmers. Majes Valley is the most important agricultural center of the Southern region of Peru. The present study was aimed to determine the knowledge about using OPs, safety practice and urinary dialkylphosphate metabolites on OP applicators in the Majes Valley, Peru. Methods This study was based on a questionnaire which included socio-demographic characteristics, knowledge of safety practices to handling OPs, characteristics of pesticide application and use of protective measures to avoid pesticide contamination. Exposure was assessed by measuring six urinary OP metabolites (DMP, DMTP, DMDTP, DEP, DETP, and DEDTP by gas chromatography using a single flame photometric detector. The sample consisted of 31 men and 2 women aged 20 – 65 years old. Results 76% of applicators had at least one urinary dialkylphosphate metabolite above the limit of detection. The geometric mean (GM and the geometric standard deviation (GSD of DMP and DEP were 5.73 ug/g cr. (GSD 2.51, and 6.08 ug/g cr. (GSD 3.63, respectively. The percentage of applicators with detectable DMP, DMDTP, and DMTP in urine was 72.72%, 3.03%, and 15.15%, respectively, while the corresponding figures for DEP, DETP, and DEDTP were 48.48%, 36.36% and 15.15%, respectively. There was no significant association between the use of protection practices and the absence of urine OPs metabolites suggesting inadequate protection practices. Conclusion The pesticide applicators in Majes Valley have significant exposure to OP pesticides, probably due to inappropriate protective practices. Future work should evaluate possible health effects.

  7. Oximes in acute organophosphorus pesticide poisoning: a systematic review of clinical trials.

    Science.gov (United States)

    Eddleston, M; Szinicz, L; Eyer, P; Buckley, N

    2002-05-01

    Acute organophosphorus (OP) pesticide poisoning is widespread in the developing world. Standard treatment involves the administration of intravenous atropine and an oxime to counter acetylcholinesterase inhibition at the synapse, but the usefulness of oximes is uncertain. To assess the evidence on the use of oximes in OP poisoning. Systematic review. We searched Medline, Embase, and Cochrane databases (last check 01/02/02) for 'organophosphate' or 'oxime' together with 'poisoning' or 'overdose'. We cross-referenced from other articles, and contacted experts to identify unpublished studies. A Web search engine [www.google.com] was also used, with the keywords 'organophosphate', 'oxime', and 'trial' (last check 01/02/02). We found two randomized controlled trials (RCTs) involving 182 patients treated with pralidoxime. The RCTs found no benefit with pralidoxime, and have been used to argue that pralidoxime should not be used in OP poisoning. The RCT authors must be congratulated for attempting important studies in a difficult environment. However, their studies did not take into account recently clarified issues regarding outcome, and their methodology is unclear. A generalized statement that pralidoxime should not be used in OP poisoning is not supported by the published results. Oximes may well be irrelevant in the overwhelming self-poisoning typical of the tropics, but a large RCT comparing the current WHO-recommended pralidoxime regimen (>30 mg/kg bolus followed by >8 mg/kg/h infusion) with placebo is needed for a definitive answer. Such a study should be designed to identify any patient subgroups that might benefit from oximes.

  8. Spatial learning impairment in prepubertal guinea pigs prenatally exposed to the organophosphorus pesticide chlorpyrifos: Toxicological implications

    Science.gov (United States)

    Mamczarz, Jacek; Pescrille, Joseph D.; Gavrushenko, Lisa; Burke, Richard D.; Fawcett, William P.; DeTolla, Louis J.; Chen, Hegang; Pereira, Edna F.R.; Albuquerque, Edson X.

    2017-01-01

    Exposure of the developing brain to chlorpyrifos (CPF), an organophosphorus (OP) pesticide used extensively in agriculture worldwide, has been associated with increased prevalence of cognitive deficits in children, particularly boys. The present study was designed to test the hypothesis that cognitive deficits induced by prenatal exposure to sub-acute doses of CPF can be reproduced in precocial small species. To address this hypothesis, pregnant guinea pigs were injected daily with CPF (25 mg/kg, s.c.) or vehicle (peanut oil) for 10 days starting on presumed gestation day (GD) 53–55. Offspring were born around GD 65, weaned on postnatal day (PND) 20, and subjected to behavioral tests starting around PND 30. On the day of birth, butyrylcholinesterase (BuChE), an OP bioscavenger used as a biomarker of OP exposures, and acetylcholinesterase (AChE), a major molecular target of OP compounds, were significantly inhibited in the blood of CPF-exposed offspring. In their brains, BuChE, but not AChE, was significantly inhibited. Prenatal CPF exposure had no significant effect on locomotor activity or on locomotor habituation, a form of non-associative memory assessed in open fields. Spatial navigation in the Morris water maze (MWM) was found to be sexually dimorphic among guinea pigs, with males outperforming females. Prenatal CPF exposure impaired spatial learning more significantly among male than female guinea pigs and, consequently, reduced the sexual dimorphism of the task. The results presented here, which strongly support the test hypothesis, reveal that the guinea pig is a valuable animal model for preclinical assessment of the developmental neurotoxicity of OP pesticides. These findings are far reaching as they lay the groundwork for future studies aimed at identifying therapeutic interventions to treat and/or prevent the neurotoxic effects of CPF in the developing brain. PMID:27296654

  9. A phase II clinical trial to assess the safety of clonidine in acute organophosphorus pesticide poisoning

    Directory of Open Access Journals (Sweden)

    Karunatilake Harindra

    2009-08-01

    Full Text Available Abstract Background An estimated 2–3 million people are acutely poisoned by organophosphorus pesticides each year, mostly in the developing world. There is a pressing need for new affordable antidotes and clonidine has been shown to be effective in animal studies. Our aim was to determine the safety of clonidine given as an antidote in adult patients presenting with signs or symptoms of acute organophosphate ingestion. Methods This study was a dose finding, open-label, multicentre, phase II trial. Forty eight patients with acute organophosphate poisoning were randomized to receive either clonidine or placebo: Four to receive placebo and twelve to receive clonidine at each dose level. The first dose level was an initial loading dose of 0.15 mg followed by an infusion of 0.5 mg of clonidine over 24 hours. The initial loading dose was increased to 0.3 mg, 0.45 and 0.6 mg. at all dosing levels however the subsequent infusion remained at 0.5 mg of clonidine over 24 hours. Results The baseline characteristics of both groups were similar. The trial was stopped after completion of the 3rd dosing level. At the 1st and 2nd dosing level there were no reported adverse drug reactions. At the 3rd dosing level 5 patients (42% developed significant hypotension during clonidine treatment that responded to intravenous fluids. There were no statistical differences in ventilation rate, pre and post GCS, and mortality rates over all levels. Conclusion Our findings suggest use of moderate doses of clonidine in acute organophosphate poisoning can be used without causing frequent clinical problems but that higher doses are associated with a high incidence of hypotension requiring intervention. Further studies are needed to study the efficacy of clonidine as an antidote in organophosphate poisoning. Trial registration Current Controlled Trial ISRCTN89917816.

  10. Predictors of Detected Organophosphorus Pesticides Among Orang Asli Children Living in Malaysia.

    Science.gov (United States)

    Mat Sutris, Juliana; Md Isa, Zaleha; Sumeri, Siti Arfah; Ghazi, Hasanain Faisal

    Increasing use of pesticides in agriculture to control pest may result in permanent damage to the environment and consequently cause harmful health problems especially among infant and children. Due to pesticide's natural toxicity and its widespread use, it causes a serious threat to public health especially to this vulnerable group. The purpose of this study was to determine the organophosphorus pesticide urinary metabolite levels and its predictors among Orang Asli children of the Mah Meri tribe living in an agricultural island in Kuala Langat, Selangor. Data collection was carried out at an island in Kuala Langat, Selangor, where a total of 180 Orang Asli children of the Mah Meri tribe voluntarily participated in the study. Data were collected via a validated, modified questionnaire. Urinary organophosphate metabolites, namely dimethylphosphate, diethylphosphate, dimethylthiophosphate, dimethyldithiophosphate, diethylthiophosphate, and diethyldithiophosphate were measured to assess organophosphate pesticide exposure in children. Eighty-four (46.7%) of the respondents were positive for urine dialkyl phosphate metabolites. In multivariable analysis, children who frequently consumed apples had 4 times higher risk of pesticide detection than those who consumed apple less frequently. In addition, those who frequently ate cucumbers had 4 times higher risk for pesticide detection than those who ate cucumbers less frequently. Children with a father whose occupation involved high exposure to pesticides (agriculture) had 3 times higher risk of pesticide detection than those with a father in a low-risk occupation (nonagriculture). Almost half of the children (46.7%) in the study area tested positive for urinary dialkyl phosphate metabolite levels. Most of the metabolite levels were equal to or higher than that reported in other previous studies. Major factors associated with pesticide detection in children in this study were frequent intake of apple and cucumber and

  11. Organophosphorus pollutants (OPP) in aquatic environment at Damietta Governorate, Egypt: implications for monitoring and biomarker responses.

    Science.gov (United States)

    Abdel-Halim, K Y; Salama, A K; El-Khateeb, E N; Bakry, N M

    2006-06-01

    The study was carried out from spring 1999 to spring 2001 to monitor the residue levels of organophosphorus pollutants (OPP) in aquatic environment of the drainage canal surrounding a pesticide factory at Damietta Governorate. Water, sediment, and fish samples were collected at six different seasonal periods. OPPs were analyzed by GLC and confirmed using GC-MS. Chlorpyrifos, chlorpyrifos-methyl, malathion, diazinon, pirimiphos-methyl and profenofos were detected in most samples. Chlorpyrifos was dominant in all water and sediment samples. It was ranged from 24.5 to 303.8 and 0.9 to 303.8 ppb in water and sediment samples, respectively. Diazinon level was slightly similar to chlorpyrifos in fish samples. Data based on the grand total concentration of OPP showed that the most polluted samples were collected either at spring 1999 or autumn 2000. They were 675.5 and 303.8 ppb in water samples and 43.0 and 52.2 ppb in fish collected at spring 1999 and autumn 2000, respectively. The obtained results are in parallel to that found in case of cholinesterase activity where the activity of both acetylcholinesterase (AChE) and butyrylcholinesterase (BuChE) was declined at these seasonal period. The activity levels of AChE and BuChE were found to be 77.18% and 59.67% of control at spring 1999 and 78.62% and 85.80% of control, at autumn 2000, respectively. Thus, AChE and BuChE could be used as biomarkers for tracing and biomonitoring OPP pollution.

  12. Phytase production by Aspergillus niger NCIM 563 for a novel application to degrade organophosphorus pesticides.

    Science.gov (United States)

    Shah, Parin C; Kumar, V Ravi; Dastager, Syed G; Khire, Jayant M

    2017-12-01

    The production of phytase using Aspergillus niger NCIM 563 under submerged fermentation conditions was studied using protein rich chickpea flour as substrate. Employing a hybrid statistical media optimization strategy of Plackett-Burman and Box-Behnken experimental designs in shake-flasks gave an increased phytase activity from an initial 66 IU/mL in 216 h to 160 IU/mL in a reduced time of 132 h. Productivity, thus increased by 3.97 times from 7.3 to 29 IU/mL/day. Using the optimized media, the production was successfully scaled-up further and improved up to 164 IU/mL in 96 h by studies carried out employing 2 and 10-L fermenters. The enzyme supernatant was recovered using centrifugal separation of biomass and the stability of the produced phytase was tested for animal feed applications under gastric conditions. In vitro degradation studies of water soluble monocrotophos, methyl parathion and water insoluble chlorpyrifos, pesticides used extensively in agriculture was carried out. It was observed by HPLC analysis that phytase could degrade 72% of chlorpyrifos at pH 7.0, 35 °C. Comparable results were obtained with monocrotophos and methyl parathion. With chlorpyrifos at higher temperature 50 °C as much as 91% degradation could be obtained. The degradation of chlorpyrifos was further validated by spraying phytase on harvested green chilli (Capsicum annuum L) under normal conditions of pH 7.0, 35 °C and the degradation products obtained analyzed by LCMS. Thus, the present study brings out a potentially novel application of phytase for biodegradation of organophosphorus pesticides.

  13. Evaluation of medical countermeasures against organophosphorus compounds: the value of experimental data and computer simulations.

    Science.gov (United States)

    Worek, Franz; Aurbek, Nadine; Herkert, Nadja M; John, Harald; Eddleston, Michael; Eyer, Peter; Thiermann, Horst

    2010-09-06

    Despite extensive research for more than six decades on medical countermeasures against poisoning by organophosphorus compounds (OP) the treatment options are meagre. The presently established acetylcholinesterase (AChE) reactivators (oximes), e.g. obidoxime and pralidoxime, are insufficient against a number of nerve agents and there is ongoing debate on the benefit of oxime treatment in human OP pesticide poisoning. Up to now, the therapeutic efficacy of oximes was mostly evaluated in animal models but substantial species differences prevent direct extrapolation of animal data to humans. Hence, it was considered essential to establish relevant experimental in vitro models for the investigation of oximes as antidotes and to develop computer models for the simulation of oxime efficacy in different scenarios of OP poisoning. Kinetic studies on the various interactions between erythrocyte AChE from various species, structurally different OP and different oximes provided a basis for the initial assessment of the ability of oximes to reactivate inhibited AChE. In the present study, in vitro enzyme-kinetic and pharmacokinetic data from a minipig model of dimethoate poisoning and oxime treatment were used to calculate dynamic changes of AChE activities. It could be shown that there is a close agreement between calculated and in vivo AChE activities. Moreover, computer simulations provided insight into the potential and limitations of oxime treatment. In the end, such data may be a versatile tool for the ongoing discussion of the pros and cons of oxime treatment in human OP pesticide poisoning. Copyright (c) 2009 Elsevier Ireland Ltd. All rights reserved.

  14. A proton wire and water channel revealed in the crystal structure of isatin hydrolase

    DEFF Research Database (Denmark)

    Bjerregaard-Andersen, Kaare; Sommer, Theis; Jensen, Jan Kristian

    2014-01-01

    to a novel family of metalloenzymes that include the bacterial kynurenine formamidase. The product state, mimicked by bound thioisatinate, reveals a water molecule that bridges the thioisatinate to a proton wire in an adjacent water channel and thus allows the proton released by the reaction to escape only...... when the product is formed. The functional proton wire present in IH-b represents a unique catalytic feature common to all hydrolases is here trapped and visualized for the first time. The local molecular environment required to coordinate thioisatinate allows stronger and more confident identification...

  15. Studies on whole cell fluorescence-based screening for epoxide hydrolases and Baeyer-Villiger monooxygenases

    Energy Technology Data Exchange (ETDEWEB)

    Bicalho, Beatriz; Chen, Lu S.; Marsaioli, Anita J. [Universidade Estadual de Campinas, SP (Brazil). Inst. de Quimica]. E-mail: anita@iqm.unicamp.br; Grognux, Johann; Reymond, Jean-Louis [University of Berne (Switzerland). Dept. of Chemistry and Biochemistry

    2004-12-01

    Biocatalysis reactions were performed on microtiter plates (200 {mu}L) aiming at the utilization of fluorogenic substrates (100 {mu}mol L{sup -1}) for rapid whole cell screening for epoxide hydrolases (EHs) and Baeyer-Villiger monooxygenases (BVMOs). A final protocol was achieved for EHs, with 3 new enzymatic sources being detected (Agrobacterium tumefaciens, Pichia stipitis, Trichosporom cutaneum). The fluorogenic assay for BVMO did not work as expected. However, an approach to possible variables involved (aeration; pH) provided the first detection of a BVMO activity in T. cutaneum. (author)

  16. Novel Cell Wall Hydrolase CwlC from Bacillus thuringiensis Is Essential for Mother Cell Lysis.

    Science.gov (United States)

    Chen, Xiaomin; Gao, Tantan; Peng, Qi; Zhang, Jie; Chai, Yunrong; Song, Fuping

    2018-04-01

    In this study, a sporulation-specific gene (tentatively named cwlC ) involved in mother cell lysis in Bacillus thuringiensis was characterized. The encoded CwlC protein consists of an N-terminal N -acetylmuramoyl-l-alanine amidase (Mur N Ac-LAA) domain and a C-terminal amidase02 domain. The recombinant histidine-tagged CwlC proteins purified from Escherichia coli were able to directly bind to and digest the B. thuringiensis cell wall. The CwlC point mutations at the two conserved glutamic acid residues (Glu-24 and Glu-140) shown to be critical for the catalytic activity in homologous amidases resulted in a complete loss of cell wall lytic activity, suggesting that CwlC is an N -acetylmuramoyl-l-alanine amidase. Results of transcriptional analyses indicated that cwlC is transcribed as a monocistronic unit and that its expression is dependent on sporulation sigma factor K (σ K ). Deletion of cwlC completely blocked mother cell lysis during sporulation without impacting the sporulation frequency, Cry1Ac protein production, and insecticidal activity. Taken together, our data suggest that CwlC is an essential cell wall hydrolase for B. thuringiensis mother cell lysis during sporulation. Engineered B. thuringiensis strains targeting cwlC , which allows the crystal inclusion to remain encapsulated in the mother cell at the end of sporulation, may have the potential to become more effective biological control agents in agricultural applications since the crystal inclusion remains encapsulated in the mother cell at the end of sporulation. IMPORTANCE Mother cell lysis has been well studied in Bacillus subtilis , which involves three distinct yet functionally complementary cell wall hydrolases. In this study, a novel cell wall hydrolase, CwlC, was investigated and found to be essential for mother cell lysis in Bacillus thuringiensis CwlC of B. thuringiensis only shows 9 and 21% sequence identity with known B. subtilis mother cell hydrolases CwlB and CwlC, respectively

  17. Studies on whole cell fluorescence-based screening for epoxide hydrolases and Baeyer-Villiger monooxygenases

    International Nuclear Information System (INIS)

    Bicalho, Beatriz; Chen, Lu S.; Marsaioli, Anita J.; Grognux, Johann; Reymond, Jean-Louis

    2004-01-01

    Biocatalysis reactions were performed on microtiter plates (200 μL) aiming at the utilization of fluorogenic substrates (100 μmol L -1 ) for rapid whole cell screening for epoxide hydrolases (EHs) and Baeyer-Villiger monooxygenases (BVMOs). A final protocol was achieved for EHs, with 3 new enzymatic sources being detected (Agrobacterium tumefaciens, Pichia stipitis, Trichosporom cutaneum). The fluorogenic assay for BVMO did not work as expected. However, an approach to possible variables involved (aeration; pH) provided the first detection of a BVMO activity in T. cutaneum. (author)

  18. Acyl hydrolases from trans-AT polyketide synthases target acetyl units on acyl carrier proteins.

    Science.gov (United States)

    Jenner, Matthew; Afonso, Jose P; Kohlhaas, Christoph; Karbaum, Petra; Frank, Sarah; Piel, Jörn; Oldham, Neil J

    2016-04-18

    Acyl hydrolase (AH) domains are a common feature of trans-AT PKSs. They have been hypothesised to perform a proofreading function by removing acyl chains from stalled sites. This study determines the substrate tolerance of the AH PedC for a range of acyl-ACPs. Clear preference towards short, linear acyl-ACPs is shown, with acetyl-ACP the best substrate. These results imply a more targeted housekeeping role for PedC: namely the removal of unwanted acetyl groups from ACP domains caused by erroneous transfer of acetyl-CoA, or possibly by decarboxylation of malonyl-ACP.

  19. Purification and Characterization of TrzF: Biuret Hydrolysis by Allophanate Hydrolase Supports Growth

    OpenAIRE

    Shapir, Nir; Cheng, Gang; Sadowsky, Michael J.; Wackett, Lawrence P.

    2006-01-01

    TrzF, the allophanate hydrolase from Enterobacter cloacae strain 99, was cloned, overexpressed in the presence of a chaperone protein, and purified to homogeneity. Native TrzF had a subunit molecular weight of 65,401 and a subunit stoichiometry of α2 and did not contain significant levels of metals. TrzF showed time-dependent inhibition by phenyl phosphorodiamidate and is a member of the amidase signature protein family. TrzF was highly active in the hydrolysis of allophanate but was not acti...

  20. Neutron diffraction analysis of Pseudomonas aeruginosa peptidyl-tRNA hydrolase 1.

    Science.gov (United States)

    McFeeters, Hana; Vandavasi, Venu Gopal; Weiss, Kevin L; Coates, Leighton; McFeeters, Robert L

    2016-03-01

    Perdeuterated peptidyl-tRNA hydrolase 1 from Pseudomonas aeruginosa was crystallized for structural analysis using neutron diffraction. Crystals of perdeuterated protein were grown to 0.15 mm(3) in size using batch crystallization in 22.5% polyethylene glycol 4000, 100 mM Tris pH 7.5, 10%(v/v) isopropyl alcohol with a 20-molar excess of trilysine as an additive. Neutron diffraction data were collected from a crystal at room temperature using the MaNDi single-crystal diffractometer at Oak Ridge National Laboratory.

  1. Chemical constituents from the root of Polygonum multiflorum and their soluble epoxide hydrolase inhibitory activity.

    Science.gov (United States)

    Sun, Ya Nan; Li, Wei; Kim, Jang Hoon; Yan, Xi Tao; Kim, Ji Eun; Yang, Seo Young; Kim, Young Ho

    2015-06-01

    Fourteen compounds were isolated from a methanol extract of Polygonum multiflorum roots, and their structures were elucidated by comparing spectroscopic data to published spectra. The inhibitory effects of the isolated compounds on soluble epoxide hydrolase (sEH) were then evaluated. Compounds 1-7 inhibited sEH activity potently, with IC50 values ranging from 6.2 ± 0.5 to 48.6 ± 3.1 μM. Moreover, a kinetic analysis of compounds 1-7 revealed that the inhibitory actions of compounds 1, 3 and 4 were non-competitive, whereas those of compounds 2 and 5-7 were mixed-type.

  2. Cocaine Hydrolase Gene Transfer Demonstrates Cardiac Safety and Efficacy against Cocaine-Induced QT Prolongation in Mice.

    Science.gov (United States)

    Murthy, Vishakantha; Reyes, Santiago; Geng, Liyi; Gao, Yang; Brimijoin, Stephen

    2016-03-01

    Cocaine addiction is associated with devastating medical consequences, including cardiotoxicity and risk-conferring prolongation of the QT interval. Viral gene transfer of cocaine hydrolase engineered from butyrylcholinesterase offers therapeutic promise for treatment-seeking drug users. Although previous preclinical studies have demonstrated benefits of this strategy without signs of toxicity, the specific cardiac safety and efficacy of engineered butyrylcholinesterase viral delivery remains unknown. Here, telemetric recording of electrocardiograms from awake, unrestrained mice receiving a course of moderately large cocaine doses (30 mg/kg, twice daily for 3 weeks) revealed protection against a 2-fold prolongation of the QT interval conferred by pretreatment with cocaine hydrolase vector. By itself, this prophylactic treatment did not affect QT interval duration or cardiac structure, demonstrating that viral delivery of cocaine hydrolase has no intrinsic cardiac toxicity and, on the contrary, actively protects against cocaine-induced QT prolongation. Copyright © 2016 by The American Society for Pharmacology and Experimental Therapeutics.

  3. Dust Measurement of Two Organophosphorus Flame Retardants, Resorcinol Bis(diphenylphosphate) (RBDPP) and Bisphenol A Bis(diphenylphosphate) (BPA-BDPP), Used as Alternatives for BDE-209

    NARCIS (Netherlands)

    Brandsma, S.H.; Sellström, U.; de Wit, C.A.; de Boer, J.; Leonards, P.E.G.

    2013-01-01

    Resorcinol bis(diphenylphosphate) (RBDPP) and bisphenol A bis(diphenylphosphate) (BPA-BDPP) are two halogen-free organophosphorus flame retardant (PFRs) that are used as an alternative for the decabromodiphenyl ether (Deca-BDE) technical mixture in TV/flatscreen housing and other electronic consumer

  4. Analysis of two alternative organophosphorus flame retardants in electronic and plastic consumer products: resorcinol bis-(diphenylphosphate) (PBDPP) and bisphenol A bis (diphenylphosphate) (BPA-BDPP)

    NARCIS (Netherlands)

    Ballesteros Gomez, A.M.; Brandsma, S.H.; de Boer, J.; Leonards, P.E.G.

    2014-01-01

    Following the phase-out of polybrominated diphenyl ethers (PBDEs), organophosphorus flame retardants (PFRs) are increasingly used as alternative flame retardants in many products. Data on the presence of two alternative PFRs in consumer products, resorcinol bis (diphenylphosphate) (PBDPP or RDP) and

  5. Synthesis of organophosphorus modified nanoparticles and their reinforcements on the fire safety and mechanical properties of polyurea

    International Nuclear Information System (INIS)

    Qian, Xiaodong; Song, Lei; Wang, Bibo; Hu, Yuan; Yuen, Richard K.K.

    2013-01-01

    Novel organophosphorus modified nanoparticles (FRs-nanoparticles) were synthesized by the hydrolysis and condensation of both 9,10-dihydro-9-oxa-10-phosphaphenanthrene-10-oxide modified vinyl trimethoxy silane (DOPO-VTS) and 3-triethoxysilylpropylamine. FRs-nanoparticles were incorporated into the polyurea matrix in different ratios via in situ polymerization, resulting in the formation of organic/inorganic nanocomposites. The nanocomposites were characterized by thermogravimetric analysis (TGA), real-time fourier transform infrared spectra (RTIR), microscale combustion calorimeter (MCC) and tensile testing machine. The TGA results revealed that FRs-nanoparticles could slightly catalyze the thermal degradation of the nanocomposites in nitrogen atmosphere, but significantly improve the thermal stability of the nanocomposites in air atmosphere. The T 0.5 (50wt.% weight loss) of the nanocomposites was delayed by 32 °C in air atmosphere. Moreover, the char yield increased from 2.3wt.% to 8.9 wt.% at 550 °C when the loadings of FRs-nanoparticles was 10wt.%, indicating the catalyzing charring effect of FRs-nanoparticles. The MCC results revealed that all the nanocomposites exhibited much lower flammability compared with virgin polyurea. Furthermore, the tensile test indicated that the FRs-nanoparticles could also improve the mechanical properties of polyurea. - Highlights: ► Novel organophosphorus modified nanoparticles (FRs-nanoparticles) were synthesized. ► FRs-nanoparticles were incorporated into the polyurea. ► Both the thermal stability and mechanical properties of polyurea were improved

  6. Monitoring of organochlorine and organophosphorus pesticide residues in water during different seasons of Tighra reservoir Gwalior, Madhya Pradesh, India.

    Science.gov (United States)

    Mamta; Rao, R J; Wani, Khursheed Ahmad

    2015-11-01

    Analysis of pesticides during different seasons for pesticidal contamination in water samples of Tighra reservoir was carried out on gas chromatograph-electron capture detector with capillary columns following multiresidual analytical technique. Organochlorine pesticides, viz., hexachlorobenzene (HCB), alpha-benzene hexachloride (BHC), beta-BHC, γ-BHC, heptachlor, aldrin, alpha-endosulfan, beta-endosulfan, p,p-DDE, dieldrin, o,p-dichlorodiphenyldichloroethane (DDD), p,p-DDD, p,p-dichlorodiphenyltrichloroethane (DDT), and endrin, and organophosphorus pesticides, viz., choloropyrifos, methyl parathion, diazion, dicholorovos, ethion, malathion, and parathion, were detected in water samples during different seasons. However, the pesticide concentration varied during different seasons at different sites. The prominent reason of contamination is the use of pesticides (both organochlorine and organophosphorus) in the agricultural fields near Tighra reservoir. On the basis of our observations, more extensive monitoring studies need to be carried out, covering all wetlands of Madhya Pradesh to enforce the policies for the restricted application of pesticides in agricultural fields adjacent to wetlands.

  7. Metabolic activation of three arylamines and two organophosphorus insecticides by coriander (Coriandrum sativum) a common edible vegetable.

    Science.gov (United States)

    Cortés-Eslava, J; Gómez-Arroyo, S; Villalobos-Pietrini, R; Espinosa-Aguirre, J J

    2001-12-15

    Organophosphorus insecticides and arylamines, widely distributed in the environment, can be activated into mutagens by plants. Plant activation of three aromatic amines, 4-nitro-o-phenylenediamine (NOP), m-phenylenediamine (m-PDA) and 2-aminofluorene (2AF), and two organophosphorus insecticides, dimethoate and methyl parathion has been the focus of this study. The plant cell/microbe coincubation assay was used employing coriander (Coriandrum sativum) suspended cell cultures as the activating system. Interestingly, this vegetable is included in the Mexican diet and ingested generally uncooked and could have epidemiological consequences. As a genetic end point, the Salmonella typhimurium tester strain TA98 was used. Protein contents, as well as peroxidase activity and peroxidase activity inhibited by diethyldithiocarbamate (DEDTC) of coriander cultures were determined after the coculture. Coriander cells highly activated three aromatic amines, NOP, m-PDA and 2-AF to mutagenic products detected in Salmonella. On the other hand, insecticides were only lightly activated, probably because peroxidase activity of coriander cells was inhibited, corroborated by DEDTC peroxidase inhibition. In all the assays, NOP was the more potent mutagenic compound. The results demonstrated that coriander cells were metabolically competent and suitable for a plant cell microbe coincubation assay, developed to analyze the promutagen activation by plant systems and can be used as a indicator of potential genetic effects.

  8. Novel organophosphorus scaffolds of urease inhibitors obtained by substitution of Morita-Baylis-Hillman adducts with phosphorus nucleophiles.

    Science.gov (United States)

    Ntatsopoulos, Vassilis; Vassiliou, Stamatia; Macegoniuk, Katarzyna; Berlicki, Łukasz; Mucha, Artur

    2017-06-16

    The reactivity of Morita-Baylis-Hillman allyl acetates was employed to introduce phosphorus-containing functionalities to the side chain of the cinnamic acid conjugated system by nucleophilic displacement. The proximity of two acidic groups, the carboxylate and phosphonate/phosphinate groups, was necessary to form interactions in the active site of urease by recently described inhibitor frameworks. Several organophosphorus scaffolds were obtained and screened for inhibition of the bacterial urease, an enzyme that is essential for survival of urinary and gastrointestinal tract pathogens. α-Substituted phosphonomethyl- and 2-phosphonoethyl-cinnamate appeared to be the most potent and were further optimized. As a result, one of the most potent organophosphorus inhibitors of urease, α-phosphonomethyl-p-methylcinnamic acid, was identified, with K i  = 0.6 μM for Sporosarcina pasteurii urease. High complementarity to the enzyme active site was achieved with this structure, as any further modifications significantly decreased its affinity. Finally, this work describes the challenges faced in developing ligands for urease. Copyright © 2017 Elsevier Masson SAS. All rights reserved.

  9. [Multiresidue analysis of organophosphorus pesticides in vegetables and fruits using dual-column GC-FPD, -NPD].

    Science.gov (United States)

    Ueno, E; Oshima, H; Saito, I; Matsumoto, H

    2001-12-01

    We investigated simultaneous and consecutive analytical methods for pesticide residues in large numbers of vegetable and fruit samples. Extraction of the sample with acetonitrile was followed by a salting-out step using a graduated cylinder. Co-extractives were removed by gel permeation chromatography (GPC) and the pesticide eluate was separated to 2 fractions. Firstly, the second fraction (85-125 mL) was passed through a silica-gel mini-column. After putting a Florisil mini-column before the silica-gel mini-column, the first fraction (55-85 mL) was passed through the tandem mini-columns, which were then eluted with 15 mL of 50% acetone/petroleum ether. The eluate was subjected to dual-column GC with dual FPD (P mode, column Rtx-OPPesticides) and NPD (column Rtx-200) detectors. Recoveries of 63 organophosphorus pesticides from fortified spinach, tomato, apple and strawberry, ranged from 71 to 126% with RSD values of 1-18%, except for the RSDs of omethoate, isoxathion, and pyraclofos (20% <). Detection limits of pesticides were 0.5-2 ng/g. Surveillance of pesticides in vegetables and fruits was carried out by using the present method. From 20 out of 30 samples, 15 pesticides (39 in total) were detected. The results indicated that the present method can be applied as an efficient and reliable tool for monitoring organophosphorus pesticide residues in vegetables and fruits.

  10. 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...

  11. Partial Purification and Characterization of an Inducible Indole-3-Acetyl-L-Aspartic Acid Hydrolase from Enterobacter agglomerans.

    Science.gov (United States)

    Chou, J. C.; Kuleck, G. A.; Cohen, J. D.; Mulbry, W. W.

    1996-11-01

    Indole-3-acetyl-amino acid conjugate hydrolases are believed to be important in the regulation of indole-3-acetic acid (IAA) metabolism in plants and therefore have potential uses for the alteration of plant IAA metabolism. To isolate bacterial strains exhibiting significant indole-3-acetyl-aspartate (IAA-Asp) hydrolase activity, a sewage sludge inoculation was cultured under conditions in which IAA-Asp served as the sole source of carbon and nitrogen. One isolate, Enterobacter agglomerans, showed hydrolase activity inducible by IAA-L-Asp or N-acetyl-L-Asp but not by IAA, (NH4)2SO4, urea, or indoleacetamide. Among a total of 17 IAA conjugates tested as potential substrates, the enzyme had an exclusively high substrate specificity for IAA-L-Asp. Substrate concentration curves and Lineweaver-Burk plots of the kinetic data showed a Michaelis constant value for IAA-L-Asp of 13.5 mM. The optimal pH for this enzyme was between 8.0 and 8.5. In extraction buffer containing 0.8 mM Mg2+ the hydrolase activity was inhibited to 80% by 1 mM dithiothreitol and to 60% by 1 mm CuSO4; the activity was increased by 40% with 1 mM MnSO4. However, in extraction buffer with no trace elements, the hydrolase activity was inhibited to 50% by either 1 mM dithiothreitol or 1% Triton X-100 (Sigma). These results suggest that disulfide bonding might be essential for enzyme activity. Purification of the hydrolase by hydroxyapatite and TSK-phenyl (HP-Genenchem, South San Francisco, CA) preparative high-performance liquid chromatography yielded a major 45-kD polypeptide as shown by sodium dodecyl sulfate-polyacrylamide gel electrophoresis.

  12. Partial purification and characterization of an inducible indole-3-acetyl-L-aspartic acid hydrolase from Enterobacter agglomerans

    Energy Technology Data Exchange (ETDEWEB)

    Chou, Jyh-Ching [Department of Agriculture, Beltsville, MD (United States)]|[Univ. of Maryland, College Park, MD (United States); Cohen, J.D.; Mulbry, W.W. [Department of Agriculture, Beltsville, MD (United States)] [and others

    1996-11-01

    Indole-3-acetyl-amino acid conjugate hydrolases are believed to be important in the regulation of indole-3-acetic acid (IAA) metabolism in plants and therefore have potential uses for the alteration of plant IAA metabolism. To isolate bacterial strains exhibiting significant indole-3-acetyl-aspartate (IAA-Asp) hydrolase activity, a sewage sludge inoculation was cultured under conditions in which IAA-Asp served as the sole source of carbon and nitrogen. One isolate, Enterobacter agglomerans, showed hydrolase activity inducible by IAA-L-Asp or N-acetyl-L-Asp but not by IAA, (NH{sub 4}){sub 2}SO{sub 4}, urea, or indoleacetamide. Among a total of 17 IAA conjugates tested as potential substrates, the enzyme had an exclusively high substrate specificity for IAA-L-Asp of 13.5 mM. The optimal pH for this enzyme was between 8.0 and 8.5. In extraction buffer containing 0.8 mM Mg{sup 2+} the hydrolase activity was inhibited to 80% by 1 mM dithiothreitol and to 60% by 1 mm CuSO{sub 4}; the activity was increased by 40% with 1mM MnSO{sub 4}. However, in extraction buffer with no trace elements, the hydrolase activity was inhibited to 50% by either 1 mM dithiothreitol or 1% Triton X-100 (Sigma). These results suggest that disulfide bonding might be essential for enzyme activity. Purification of the hydrolase by hydroxyapatite and TSK-phenyl (HP-Genenchem, South San Francisco, CA) preparative high-performance liquid chromatography yielded a major 45-kD polypeptide as shown by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. 45 refs., 5 figs., 3 tabs.

  13. Evidence for biosynthesis of lactase-phlorizin hydrolase as a single-chain high-molecular weight precursor

    DEFF Research Database (Denmark)

    Skovbjerg, H; Danielsen, E M; Noren, Ove

    1984-01-01

    Precursor forms of lactase-phlorizin hydrolase, sucrase-isomaltase and aminopeptidase N were studied by pulse-labelling of organ-cultured human intestinal biopsies. After labelling the biopsies were fractionated by the Ca2+-precipitation method and the enzymes isolated by immunoprecipitation....... The results indicate that the lactase-phlorizin hydrolase is synthesized as a Mr 245 000 polypeptide, which is intracellularly cleaved into its mature Mr 160 000 form. Sucrase-isomaltase is shown to be synthesized as a single chain precursor (Mr 245 000 and 265 000) while the precursor of aminopeptidase N...

  14. Signal peptide cleavage is essential for surface expression of a regulatory T cell surface protein, leucine rich repeat containing 32 (LRRC32

    Directory of Open Access Journals (Sweden)

    Sugiyama Hideaki

    2011-05-01

    Full Text Available Abstract Background Elevated numbers of regulatory T cells (Tregs have been implicated in certain cancers. Depletion of Tregs has been shown to increase anti-tumor immunity. Tregs also play a critical role in the suppression of autoimmune responses. The study of Tregs has been hampered by a lack of adequate surface markers. Leucine Rich Repeat Containing 32 (LRRC32, also known as Glycoprotein A Repetitions Predominant (GARP, has been postulated as a novel surface marker of activated Tregs. However, there is limited information regarding the processing of LRRC32 or the regulatory phenotype and functional activity of Tregs expressing LRRC32. Results Using naturally-occurring freshly isolated Tregs, we demonstrate that low levels of LRRC32 are present intracellularly prior to activation and that freshly isolated LRRC32+ Tregs are distinct from LRRC32- Tregs with respect to the expression of surface CD62L. Using LRRC32 transfectants of HEK cells, we demonstrate that the N-terminus of LRRC32 is cleaved prior to expression of the protein at the cell surface. Furthermore, we demonstrate using a construct containing a deleted putative signal peptide region that the presence of a signal peptide region is critical to cell surface expression of LRRC32. Finally, mixed lymphocyte assays demonstrate that LRRC32+ Tregs are more potent suppressors than LRRC32- Tregs. Conclusions A cleaved signal peptide site in LRRC32 is necessary for surface localization of native LRRC32 following activation of naturally-occurring freshly-isolated regulatory T cells. LRRC32 expression appears to alter the surface expression of activation markers of T cells such as CD62L. LRRC32 surface expression may be useful as a marker that selects for more potent Treg populations. In summary, understanding the processing and expression of LRRC32 may provide insight into the mechanism of action of Tregs and the refinement of immunotherapeutic strategies aimed at targeting these cells.

  15. Differential recognition and hydrolysis of host carbohydrate antigens by Streptococcus pneumoniae family 98 glycoside hydrolases.

    Science.gov (United States)

    Higgins, Melanie A; Whitworth, Garrett E; El Warry, Nahida; Randriantsoa, Mialy; Samain, Eric; Burke, Robert D; Vocadlo, David J; Boraston, Alisdair B

    2009-09-18

    The presence of a fucose utilization operon in the Streptococcus pneumoniae genome and its established importance in virulence indicates a reliance of this bacterium on the harvesting of host fucose-containing glycans. The identities of these glycans, however, and how they are harvested is presently unknown. The biochemical and high resolution x-ray crystallographic analysis of two family 98 glycoside hydrolases (GH98s) from distinctive forms of the fucose utilization operon that originate from different S. pneumoniae strains reveal that one enzyme, the predominant type among pneumococcal isolates, has a unique endo-beta-galactosidase activity on the LewisY antigen. Altered active site topography in the other species of GH98 enzyme tune its endo-beta-galactosidase activity to the blood group A and B antigens. Despite their different specificities, these enzymes, and by extension all family 98 glycoside hydrolases, use an inverting catalytic mechanism. Many bacterial and viral pathogens exploit host carbohydrate antigens for adherence as a precursor to colonization or infection. However, this is the first evidence of bacterial endoglycosidase enzymes that are known to play a role in virulence and are specific for distinct host carbohydrate antigens. The strain-specific distribution of two distinct types of GH98 enzymes further suggests that S. pneumoniae strains may specialize to exploit host-specific antigens that vary from host to host, a factor that may feature in whether a strain is capable of colonizing a host or establishing an invasive infection.

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

    Energy Technology Data Exchange (ETDEWEB)

    Danielsen, E.M. (Univ. of Cophenhagen (Denmark))

    1990-01-09

    The pig intestinal brush border enzymes aminopeptidase and lactase-phlorizin hydrolase are present in the microvilla membrane as homodimers. Dimethyl adipimidate was used to cross-link the two ({sup 35}S)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 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 cleavage of its high molecular weight precursor. Since only the mature form and not the precursor of this enzyme could be cross-linked, formation of tightly associated dimers only takes place after transport out of the endoplasmic reticulum. Dimerization of the two brush border enzymes therefore seems to occur in different organelles of the enterocyte.

  17. Acd, a peptidoglycan hydrolase of Clostridium difficile with N-acetylglucosaminidase activity.

    Science.gov (United States)

    Dhalluin, Anne; Bourgeois, Ingrid; Pestel-Caron, Martine; Camiade, Emilie; Raux, Gregory; Courtin, Pascal; Chapot-Chartier, Marie-Pierre; Pons, Jean-Louis

    2005-07-01

    A gene encoding a putative peptidoglycan hydrolase was identified by sequence similarity searching in the Clostridium difficile 630 genome sequence, and the corresponding protein, named Acd (autolysin of C. difficile) was expressed in Escherichia coli. The deduced amino acid sequence of Acd shows a modular structure with two main domains: an N-terminal domain exhibiting repeated sequences and a C-terminal catalytic domain. The C-terminal domain exhibits sequence similarity with the glucosaminidase domains of Staphylococcus aureus Atl and Bacillus subtilis LytD autolysins. Purified recombinant Acd produced in E. coli was confirmed to be a cell-wall hydrolase with lytic activity on the peptidoglycan of several Gram-positive bacteria, including C. difficile. The hydrolytic specificity of Acd was studied by RP-HPLC analysis and MALDI-TOF MS using B. subtilis cell-wall extracts. Muropeptides generated by Acd hydrolysis demonstrated that Acd hydrolyses peptidoglycan bonds between N-acetylglucosamine and N-acetylmuramic acid, confirming that Acd is an N-acetylglucosaminidase. The transcription of the acd gene increased during vegetative cellular growth of C. difficile 630. The sequence of the acd gene appears highly conserved in C. difficile strains. Regarding deduced amino acid sequences, the C-terminal domain with enzymic function appears to be the most conserved of the two main domains. Acd is the first known autolysin involved in peptidoglycan hydrolysis of C. difficile.

  18. 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.

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

    Science.gov (United States)

    Matsuzawa, Tomohiko; Mitsuishi, Yasushi; Kameyama, Akihiko; Yaoi, Katsuro

    2016-03-04

    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. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

  20. COMPARATIVE MODELLING AND LIGAND BINDING SITE PREDICTION OF A FAMILY 43 GLYCOSIDE HYDROLASE FROM Clostridium thermocellum

    Directory of Open Access Journals (Sweden)

    Shadab Ahmed

    2012-06-01

    Full Text Available The phylogenetic analysis of Clostridium thermocellum family 43 glycoside hydrolase (CtGH43 showed close evolutionary relation with carbohydrate binding family 6 proteins from C. cellulolyticum, C. papyrosolvens, C. cellulyticum, and A. cellulyticum. Comparative modeling of CtGH43 was performed based on crystal structures with PDB IDs 3C7F, 1YIF, 1YRZ, 2EXH and 1WL7. The structure having lowest MODELLER objective function was selected. The three-dimensional structure revealed typical 5-fold beta–propeller architecture. Energy minimization and validation of predicted model with VERIFY 3D indicated acceptability of the proposed atomic structure. The Ramachandran plot analysis by RAMPAGE confirmed that family 43 glycoside hydrolase (CtGH43 contains little or negligible segments of helices. It also showed that out of 301 residues, 267 (89.3% were in most favoured region, 23 (7.7% were in allowed region and 9 (3.0% were in outlier region. IUPred analysis of CtGH43 showed no disordered region. Active site analysis showed presence of two Asp and one Glu, assumed to form a catalytic triad. This study gives us information about three-dimensional structure and reaffirms the fact that it has the similar core 5-fold beta–propeller architecture and so probably has the same inverting mechanism of action with the formation of above mentioned catalytic triad for catalysis of polysaccharides.

  1. Expanded insecticide catabolic activity gained by a single nucleotide substitution in a bacterial carbamate hydrolase gene.

    Science.gov (United States)

    Öztürk, Başak; Ghequire, Maarten; Nguyen, Thi Phi Oanh; De Mot, René; Wattiez, Ruddy; Springael, Dirk

    2016-12-01

    Carbofuran-mineralizing strain Novosphingobium sp. KN65.2 produces the CfdJ enzyme that converts the N-methylcarbamate insecticide to carbofuran phenol. Purified CfdJ shows a remarkably low K M towards carbofuran. Together with the carbaryl hydrolase CehA of Rhizobium sp. strain AC100, CfdJ represents a new protein family with several uncharacterized bacterial members outside the proteobacteria. Although both enzymes differ by only four amino acids, CehA does not recognize carbofuran as a substrate whereas CfdJ also hydrolyzes carbaryl. None of the CfdJ amino acids that differ from CehA were shown to be silent regarding carbofuran hydrolytic activity but one particular amino acid substitution, i.e., L152 to F152, proved crucial. CfdJ is more efficient in degrading methylcarbamate pesticides with an aromatic side chain whereas CehA is more efficient in degrading the oxime carbamate nematicide oxamyl. The presence of common flanking sequences suggest that the cfdJ gene is located on a remnant of the mobile genetic element Tnceh carrying cehA. Our results suggest that these enzymes can be acquired through horizontal gene transfer and can evolve to degrade new carbamate substrates by limited amino acid substitutions. We demonstrate that a carbaryl hydrolase can gain the additional capacity to degrade carbofuran by a single nucleotide transversion. © 2016 Society for Applied Microbiology and John Wiley & Sons Ltd.

  2. Novel Strategies for Upstream and Downstream Processing of Tannin Acyl Hydrolase

    Directory of Open Access Journals (Sweden)

    Luis V. Rodríguez-Durán

    2011-01-01

    Full Text Available Tannin acyl hydrolase also referred as tannase is an enzyme with important applications in several science and technology fields. Due to its hydrolytic and synthetic properties, tannase could be used to reduce the negative effects of tannins in beverages, food, feed, and tannery effluents, for the production of gallic acid from tannin-rich materials, the elucidation of tannin structure, and the synthesis of gallic acid esters in nonaqueous media. However, industrial applications of tannase are still very limited due to its high production cost. Thus, there is a growing interest in the production, recovery, and purification of this enzyme. Recently, there have been published a number of papers on the improvement of upstream and downstream processing of the enzyme. These papers dealt with the search for new tannase producing microorganisms, the application of novel fermentation systems, optimization of culture conditions, the production of the enzyme by recombinant microorganism, and the design of efficient protocols for tannase recovery and purification. The present work reviews the state of the art of basic and biotechnological aspects of tannin acyl hydrolase, focusing on the recent advances in the upstream and downstream processing of the enzyme.

  3. 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.

  4. Identification of the chain-dispersing peptidoglycan hydrolase LytB of Streptococcus gordonii.

    Directory of Open Access Journals (Sweden)

    Riccardo Arrigucci

    Full Text Available Bacterial cell division ends with the separation of the daughter cells, a process that requires peptidoglycan hydrolases (PGHs. Bacteria lacking cell separating PGHs are impaired in cell separation with the formation of long chains or clusters. We identified a gene in Streptococcus gordonii encoding for a putative glucosaminidase (lytB. The lytB isogenic mutant grew in long bacterial chains and resulted in impaired biofilm formation. Purified recombinant LytB showed a murolytic activity on Micrococcus lysodeikticus cell suspension and was able to disperse the long chains of the mutant, restoring the wild type diplococci/short chain phenotype. LytB protein was localized only in culture supernatant cell fraction of S. gordonii, and co-cultures of wild type and lytB mutant showed a significant reduction of bacterial chain length, indicating that LytB is a secreted enzyme. Our results demonstrate that LytB is a secreted peptidoglycan hydrolase required for S. gordonii cell separation.

  5. α/β-Hydrolase Domain 6 in the Ventromedial Hypothalamus Controls Energy Metabolism Flexibility

    Directory of Open Access Journals (Sweden)

    Alexandre Fisette

    2016-10-01

    Full Text Available α/β-Hydrolase domain 6 (ABHD6 is a monoacylglycerol hydrolase that degrades the endocannabinoid 2-arachidonoylglycerol (2-AG. Although complete or peripheral ABHD6 loss of function is protective against diet-induced obesity and insulin resistance, the role of ABHD6 in the central control of energy balance is unknown. Using a viral-mediated knockout approach, targeted endocannabinoid measures, and pharmacology, we discovered that mice lacking ABHD6 from neurons of the ventromedial hypothalamus (VMHKO have higher VMH 2-AG levels in conditions of endocannabinoid recruitment and fail to physiologically adapt to key metabolic challenges. VMHKO mice exhibited blunted fasting-induced feeding and reduced food intake, energy expenditure, and adaptive thermogenesis in response to cold exposure, high-fat feeding, and dieting (transition to a low-fat diet. Our findings identify ABHD6 as a regulator of the counter-regulatory responses to major metabolic shifts, including fasting, nutrient excess, cold, and dieting, thereby highlighting the importance of ABHD6 in the VMH in mediating energy metabolism flexibility.

  6. Screening Brazilian Macrophomina phaseolina isolates for alkaline lipases and other extracellular hydrolases.

    Science.gov (United States)

    Schinke, Claudia; Germani, José C

    2012-03-01

    Macrophomina phaseolina, phylum Ascomycota, is a phytopathogenic fungus distributed worldwide in hot dry areas. There are few studies on its secreted lipases and none on its colony radial growth rate, an indicator of fungal ability to use nutrients for growth, on media other than potato-dextrose agar. In this study, 13 M. phaseolina isolates collected in different Brazilian regions were screened for fast-growth and the production of hydrolases of industrial interest, especially alkaline lipases. Hydrolase detection and growth rate determination were done on citric pectin, gelatin, casein, soluble starch, and olive oil as substrates. Ten isolates were found to be active on all substrates tested. The most commonly detected enzymes were pectinases, amylases, and lipases. The growth rate on pectin was significantly higher (P oil was followed for 4 days by measuring the activity in the cultivation broth. The specific lipolytic activity of isolate PEL was significantly higher at 96 h (130 mU mg protein(-1)). The broth was active at 37 °C, pH 8, indicating the potential utility of the lipases of this isolate in mild alkaline detergents. There was a strong and positive correlation (0.86) between radial growth rate and specific lipolytic activity.

  7. Novel Strategies for Upstream and Downstream Processing of Tannin Acyl Hydrolase

    Science.gov (United States)

    Rodríguez-Durán, Luis V.; Valdivia-Urdiales, Blanca; Contreras-Esquivel, Juan C.; Rodríguez-Herrera, Raúl; Aguilar, Cristóbal N.

    2011-01-01

    Tannin acyl hydrolase also referred as tannase is an enzyme with important applications in several science and technology fields. Due to its hydrolytic and synthetic properties, tannase could be used to reduce the negative effects of tannins in beverages, food, feed, and tannery effluents, for the production of gallic acid from tannin-rich materials, the elucidation of tannin structure, and the synthesis of gallic acid esters in nonaqueous media. However, industrial applications of tannase are still very limited due to its high production cost. Thus, there is a growing interest in the production, recovery, and purification of this enzyme. Recently, there have been published a number of papers on the improvement of upstream and downstream processing of the enzyme. These papers dealt with the search for new tannase producing microorganisms, the application of novel fermentation systems, optimization of culture conditions, the production of the enzyme by recombinant microorganism, and the design of efficient protocols for tannase recovery and purification. The present work reviews the state of the art of basic and biotechnological aspects of tannin acyl hydrolase, focusing on the recent advances in the upstream and downstream processing of the enzyme. PMID:21941633

  8. Organophosphate and pyrethroid hydrolase activities of mutant Esterases from the cotton bollworm Helicoverpa armigera.

    Science.gov (United States)

    Li, Yongqiang; Farnsworth, Claire A; Coppin, Chris W; Teese, Mark G; Liu, Jian-Wei; Scott, Colin; Zhang, Xing; Russell, Robyn J; Oakeshott, John G

    2013-01-01

    Two mutations have been found in five closely related insect esterases (from four higher Diptera and a hymenopteran) which each confer organophosphate (OP) hydrolase activity on the enzyme and OP resistance on the insect. One mutation converts a Glycine to an Aspartate, and the other converts a Tryptophan to a Leucine in the enzymes' active site. One of the dipteran enzymes with the Leucine mutation also shows enhanced activity against pyrethroids. Introduction of the two mutations in vitro into eight esterases from six other widely separated insect groups has also been reported to increase substantially the OP hydrolase activity of most of them. These data suggest that the two mutations could contribute to OP, and possibly pyrethroid, resistance in a variety of insects. We therefore introduced them in vitro into eight Helicoverpa armigera esterases from a clade that has already been implicated in OP and pyrethroid resistance. We found that they do not generally enhance either OP or pyrethroid hydrolysis in these esterases but the Aspartate mutation did increase OP hydrolysis in one enzyme by about 14 fold and the Leucine mutation caused a 4-6 fold increase in activity (more in one case) of another three against some of the most insecticidal isomers of fenvalerate and cypermethrin. The Aspartate enzyme and one of the Leucine enzymes occur in regions of the H. armigera esterase isozyme profile that have been previously implicated in OP and pyrethroid resistance, respectively.

  9. Organophosphate and pyrethroid hydrolase activities of mutant Esterases from the cotton bollworm Helicoverpa armigera.

    Directory of Open Access Journals (Sweden)

    Yongqiang Li

    Full Text Available Two mutations have been found in five closely related insect esterases (from four higher Diptera and a hymenopteran which each confer organophosphate (OP hydrolase activity on the enzyme and OP resistance on the insect. One mutation converts a Glycine to an Aspartate, and the other converts a Tryptophan to a Leucine in the enzymes' active site. One of the dipteran enzymes with the Leucine mutation also shows enhanced activity against pyrethroids. Introduction of the two mutations in vitro into eight esterases from six other widely separated insect groups has also been reported to increase substantially the OP hydrolase activity of most of them. These data suggest that the two mutations could contribute to OP, and possibly pyrethroid, resistance in a variety of insects. We therefore introduced them in vitro into eight Helicoverpa armigera esterases from a clade that has already been implicated in OP and pyrethroid resistance. We found that they do not generally enhance either OP or pyrethroid hydrolysis in these esterases but the Aspartate mutation did increase OP hydrolysis in one enzyme by about 14 fold and the Leucine mutation caused a 4-6 fold increase in activity (more in one case of another three against some of the most insecticidal isomers of fenvalerate and cypermethrin. The Aspartate enzyme and one of the Leucine enzymes occur in regions of the H. armigera esterase isozyme profile that have been previously implicated in OP and pyrethroid resistance, respectively.

  10. Microbial biodegradation of biuret: defining biuret hydrolases within the isochorismatase superfamily.

    Science.gov (United States)

    Robinson, Serina L; Badalamenti, Jonathan P; Dodge, Anthony G; Tassoulas, Lambros J; Wackett, Lawrence P

    2018-03-12

    Biuret is a minor component of urea fertilizer and an intermediate in s-triazine herbicide biodegradation. The microbial metabolism of biuret has never been comprehensively studied. Here, we enriched and isolated bacteria from a potato field that grew on biuret as a sole nitrogen source. We sequenced the genome of the fastest-growing isolate, Herbaspirillum sp. BH-1 and identified genes encoding putative biuret hydrolases (BHs). We purified and characterized a functional BH enzyme from Herbaspirillum sp. BH-1 and two other bacteria from divergent phyla. The BH enzymes reacted exclusively with biuret in the range of 2-11 µmol min -1 mg -1 protein. We then constructed a global protein superfamily network to map structure-function relationships in the BH subfamily and used this to mine > 7000 genomes. High-confidence BH sequences were detected in Actinobacteria, Alpha- and Beta-proteobacteria, and some fungi, archaea and green algae, but not animals or land plants. Unexpectedly, no cyanuric acid hydrolase homologs were detected in > 90% of genomes with BH homologs, suggesting BHs may have arisen independently of s-triazine ring metabolism. This work links genotype to phenotype by enabling accurate genome-mining to predict microbial utilization of biuret. Importantly, it advances understanding of the microbial capacity for biuret biodegradation in agricultural systems. © 2018 Society for Applied Microbiology and John Wiley & Sons Ltd.

  11. Structural and kinetic insights into the mechanism of 5-hydroxyisourate hydrolase from Klebsiella pneumoniae

    Energy Technology Data Exchange (ETDEWEB)

    French, Jarrod B.; Ealick, Steven E., E-mail: see3@cornell.edu [Cornell University, Ithaca, NY 14853-1301 (United States)

    2011-08-01

    The crystal structure of 5-hydroxyisourate hydrolase from K. pneumoniae and the steady-state kinetic parameters of the native enzyme as well as several mutants provide insights into the catalytic mechanism of this enzyme and the possible roles of the active-site residues. The stereospecific oxidative degradation of uric acid to (S)-allantoin has recently been demonstrated to proceed via two unstable intermediates and requires three separate enzymatic reactions. The second step of this reaction, the conversion of 5-hydroxyisourate (HIU) to 2-oxo-4-hydroxy-4-carboxy-5-ureidoimidazoline, is catalyzed by HIU hydrolase (HIUH). The high-resolution crystal structure of HIUH from the opportunistic pathogen Klebsiella pneumoniae (KpHIUH) has been determined. KpHIUH is a homotetrameric protein that, based on sequence and structural similarity, belongs to the transthyretin-related protein family. In addition, the steady-state kinetic parameters for this enzyme and four active-site mutants have been measured. These data provide valuable insight into the functional roles of the active-site residues. Based upon the structural and kinetic data, a mechanism is proposed for the KpHIUH-catalyzed reaction.

  12. Molecular Basis of Prodrug Activation by Human Valacyclovirase, an [alpha]-Amino Acid Ester Hydrolase

    Energy Technology Data Exchange (ETDEWEB)

    Lai, Longsheng; Xu, Zhaohui; Zhou, Jiahai; Lee, Kyung-Dall; Amidon, Gordon L. (Michigan)

    2008-07-08

    Chemical modification to improve biopharmaceutical properties, especially oral absorption and bioavailability, is a common strategy employed by pharmaceutical chemists. The approach often employs a simple structural modification and utilizes ubiquitous endogenous esterases as activation enzymes, although such enzymes are often unidentified. This report describes the crystal structure and specificity of a novel activating enzyme for valacyclovir and valganciclovir. Our structural insights show that human valacyclovirase has a unique binding mode and specificity for amino acid esters. Biochemical data demonstrate that the enzyme hydrolyzes esters of {alpha}-amino acids exclusively and displays a broad specificity spectrum for the aminoacyl moiety similar to tricorn-interacting aminopeptidase F1. Crystal structures of the enzyme, two mechanistic mutants, and a complex with a product analogue, when combined with biochemical analysis, reveal the key determinants for substrate recognition; that is, a flexible and mostly hydrophobic acyl pocket, a localized negative electrostatic potential, a large open leaving group-accommodating groove, and a pivotal acidic residue, Asp-123, after the nucleophile Ser-122. This is the first time that a residue immediately after the nucleophile has been found to have its side chain directed into the substrate binding pocket and play an essential role in substrate discrimination in serine hydrolases. These results as well as a phylogenetic analysis establish that the enzyme functions as a specific {alpha}-amino acid ester hydrolase. Valacyclovirase is a valuable target for amino acid ester prodrug-based oral drug delivery enhancement strategies.

  13. Probing the mechanisms for the selectivity and promiscuity of methyl parathion hydrolase.

    Science.gov (United States)

    Purg, Miha; Pabis, Anna; Baier, Florian; Tokuriki, Nobuhiko; Jackson, Colin; Kamerlin, Shina Caroline Lynn

    2016-11-13

    Diverse organophosphate hydrolases have convergently evolved the ability to hydrolyse man-made organophosphates. Thus, these enzymes are attractive model systems for studying the factors shaping enzyme functional evolution. Methyl parathion hydrolase (MPH) is an enzyme from the metallo-β-lactamase superfamily, which hydrolyses a wide range of organophosphate, aryl ester and lactone substrates. In addition, MPH demonstrates metal-ion-dependent selectivity patterns. The origins of this remain unclear, but are linked to open questions about the more general role of metal ions in functional evolution and divergence within enzyme superfamilies. Here, we present detailed mechanistic studies of the paraoxonase and arylesterase activities of MPH complexed with five different transition metal ions, and demonstrate that the hydrolysis reactions proceed via similar pathways and transition states. However, while it is possible to discern a clear structural origin for the selectivity between different substrates, the selectivity between different metal ions appears to lie instead in the distinct electrostatic properties of the metal ions themselves, which causes subtle changes in transition state geometries and metal-metal distances at the transition state rather than significant structural changes in the active site. While subtle, these differences can be significant for shaping the metal-ion-dependent activity patterns observed for this enzyme.This article is part of the themed issue 'Multiscale modelling at the physics-chemistry-biology interface'. © 2016 The Authors.

  14. Molecular cloning, characterization and comparison of bile salt hydrolases from Lactobacillus johnsonii PF01.

    Science.gov (United States)

    Chae, J P; Valeriano, V D; Kim, G-B; Kang, D-K

    2013-01-01

    To clone, characterize and compare the bile salt hydrolase (BSH) genes of Lactobacillus johnsonii PF01. The BSH genes were amplified by polymerase chain reaction (PCR) using specific oligonucleotide primers, and the products were inserted into the pET21b expression vector. Escherichia coli BLR (DE3) cells were transformed with pET21b vectors containing the BSH genes and induced using 0·1 mmol l(-1) isopropylthiolgalactopyranoside. The overexpressed BSH enzymes were purified using a nickel-nitrilotriacetic acid (Ni(2+) -NTA) agarose column and their activities characterized. BSH A hydrolysed tauro-conjugated bile salts optimally at pH 5·0 and 55°C, whereas BSH C hydrolysed glyco-conjugated bile salts optimally at pH 5·0 and 70°C. The enzymes had no preferential activities towards a specific cholyl moiety. BSH enzymes vary in their substrate specificities and characteristics to broaden its activity. Despite the lack of conservation in their putative substrate-binding sites, these remain functional through motif conservation. This is to our knowledge the first report of isolation of BSH enzymes from a single strain, showing hydrolase activity towards either glyco-conjugated or tauro-conjugated bile salts. Future structural homology studies and site-directed mutagenesis of sites associated with substrate specificity may elucidate specificities of BSH enzymes. © 2012 The Society for Applied Microbiology.

  15. Immobilization of organophosphate hydrolase on an amyloid fibril nanoscaffold: towards bioremediation and chemical detoxification.

    Science.gov (United States)

    Raynes, Jared K; Pearce, F Grant; Meade, Susie J; Gerrard, Juliet A

    2011-01-01

    Organophosphate hydrolase has potential as a bioremediation and chemical detoxification enzyme, but the problems of reusability and stability need to be addressed to use this enzyme on an industrial scale. Immobilizing the enzyme to a nanoscaffold may help to solve these problems. Amyloid fibrils generated from insulin and crystallin provided a novel nanoscaffold for the immobilization of organophosphate hydrolase, using glutaraldehyde as the crosslinking reagent. Electrophoretic, centrifugation, and temperature stability experiments, together with transmission electron microscopy were undertaken to verify that crosslinking had successfully occurred. The resulting fibrils remained active towards the substrate paraoxon and when immobilized to the insulin amyloid fibrils, the enzyme exhibited a significant (∼ 300%) increase in the relative temperature stability at 40, 45, and 50°C (as measured by comparing the initial enzyme activity to the activity remaining after heating), compared to free enzyme. This confirms that amyloid fibrils could provide a new type of nanoscaffold for enzyme immobilization. Copyright © 2010 American Institute of Chemical Engineers (AIChE).

  16. Differential Recognition and Hydrolysis of Host Carbohydrate Antigens by Streptococcus pneumoniae Family 98 Glycoside Hydrolases

    Energy Technology Data Exchange (ETDEWEB)

    Higgins, M.; Whitworth, G; El Warry, N; Randriantsoa, M; Samain, E; Burke, R; Vocadlo, D; Boraston, A

    2009-01-01

    The presence of a fucose utilization operon in the Streptococcus pneumoniae genome and its established importance in virulence indicates a reliance of this bacterium on the harvesting of host fucose-containing glycans. The identities of these glycans, however, and how they are harvested is presently unknown. The biochemical and high resolution x-ray crystallographic analysis of two family 98 glycoside hydrolases (GH98s) from distinctive forms of the fucose utilization operon that originate from different S. pneumoniae strains reveal that one enzyme, the predominant type among pneumococcal isolates, has a unique endo-{beta}-galactosidase activity on the LewisY antigen. Altered active site topography in the other species of GH98 enzyme tune its endo-{beta}-galactosidase activity to the blood group A and B antigens. Despite their different specificities, these enzymes, and by extension all family 98 glycoside hydrolases, use an inverting catalytic mechanism. Many bacterial and viral pathogens exploit host carbohydrate antigens for adherence as a precursor to colonization or infection. However, this is the first evidence of bacterial endoglycosidase enzymes that are known to play a role in virulence and are specific for distinct host carbohydrate antigens. The strain-specific distribution of two distinct types of GH98 enzymes further suggests that S. pneumoniae strains may specialize to exploit host-specific antigens that vary from host to host, a factor that may feature in whether a strain is capable of colonizing a host or establishing an invasive infection.

  17. Systematic Survey of Serine Hydrolase Activity in Mycobacterium tuberculosis Defines Changes Associated with Persistence

    Energy Technology Data Exchange (ETDEWEB)

    Ortega, Corrie; Anderson, Lindsey N.; Frando, Andrew; Sadler, Natalie C.; Brown, Robert W.; Smith, Richard D.; Wright, Aaron T.; Grundner, Christoph

    2016-02-01

    The transition between replication and non-replication underlies much of Mycobacterium tuberculosis (Mtb) pathogenicity, as non- or slowly replicating Mtb are responsible for persistence and poor treatment outcomes. Therapeutic targeting of non-replicating, persistent populations is a priority for tuberculosis treatment, but only few drug targets in non-replicating Mtb are currently known. Here, we directly measure the activity of the highly diverse and druggable serine hydrolases (SHs) during active replication and non-replication by activity-based proteomics. We predict serine hydrolase activity for 78 proteins, including 27 proteins with previously unknown function, and identify 37 SHs that remain active even in the absence of replication, providing a set of candidate persistence targets. Non-replication was associated with large shifts in the activity of the majority of SHs. These activity changes were largely independent of SH abundance, indicating extensive post-translational regulation. By probing a large cross-section of druggable Mtb enzyme space during replication and non-replication, we identify new SHs and suggest new persistence targets.

  18. 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.

  19. Edmonston Measles Virus Prevents Increased Cell Surface Expression of Peptide-Loaded Major Histocompatibility Complex Class II Proteins in Human Peripheral Monocytes

    OpenAIRE

    Yilla, Mamadi; Hickman, Carole; McGrew, Marcia; Meade, Elizabeth; Bellini, William J.

    2003-01-01

    Gamma interferon (IFN-γ) induces expression of the gene products of the major histocompatibility complex (MHC), whereas IFN-α/β can interfere with or suppress class II protein expression. In separate studies, measles virus (MV) was reported to induce IFN-α/β and to up-regulate MHC class II proteins. In an attempt to resolve this paradox, we examined the surface expression of MHC class I and class II proteins in MV-infected peripheral monocytes in the presence and absence of IFN-α/β. Infection...

  20. Mutations within the Pathogenic Region of Herpes Simplex Virus 1 gK Signal Sequences Alter Cell Surface Expression and Neurovirulence

    Science.gov (United States)

    Matundan, Harry H.; Mott, Kevin R.; Akhtar, Aslam Abbasi; Breunig, Joshua J.

    2014-01-01

    ABSTRACT To investigate the role of the signal sequences of herpes simplex virus 1 (HSV-1) gK on virus replication and viral pathogenesis, we constructed recombinant viruses with or without mutations within the signal sequences of gK. These recombinant viruses expressed two additional copies of the mutated (MgK) or native (NgK) form of the gK gene in place of the latency-associated transcript with a myc epitope tag to facilitate detection at their 3′ ends. The replication of MgK virus was similar to that of NgK both in vitro and in vivo, as well as in the trigeminal ganglia (TG) of latently infected mice. The levels of gB and gK transcripts in the corneas, TG, and brains of infected mice on days 3 and 5 postinfection were markedly virus and time dependent, as well as tissue specific. Mutation in the signal sequence of gK in MgK virus blocked cell surface expression of gK-myc in rabbit skin cells, increased 50% lethal dose, and decreased corneal scarring in ocularly infected mice compared to the NgK or revertant (RgK) virus. MgK and NgK viruses, and not the RgK virus, showed a reduced extent of explant reactivation at the lower dose of ocular infection but not at the higher dose. However, the time of reactivation was not affected by overexpression of the different forms of gK. Taken together, these results strongly suggest that the 8mer peptide (ITAYGLVL) within the signal sequence of gK promotes cell surface expression of gK in infected cells and ocular pathogenesis in infected mice. IMPORTANCE In this study, we show for the first time that mutations within the signal sequence of gK blocked cell surface expression of inserted recombinant gK in vitro. Furthermore, this blockage in cell surface expression was correlated with higher 50% lethal dose and less corneal scarring in vivo. Thus, these studies point to a key role for the 8mer within the signal sequence of gK in HSV-1-induced pathogenicity. PMID:25505072

  1. Microbial Disease Spectrum Linked to a Novel IL-12Rβ1 N-Terminal Signal Peptide Stop-Gain Homozygous Mutation with Paradoxical Receptor Cell-Surface Expression

    Science.gov (United States)

    Louvain de Souza, Thais; de Souza Campos Fernandes, Regina C.; Azevedo da Silva, Juliana; Gomes Alves Júnior, Vladimir; Gomes Coelho, Adelia; Souza Faria, Afonso C.; Moreira Salomão Simão, Nabia M.; Souto Filho, João T.; Deswarte, Caroline; Boisson-Dupuis, Stéphanie; Torgerson, Dara; Casanova, Jean-Laurent; Bustamante, Jacinta; Medina-Acosta, Enrique

    2017-01-01

    Patients with Mendelian Susceptibility to Mycobacterial Diseases (MSMD) exhibit variable vulnerability to infections by mycobacteria and other intramacrophagic bacteria (e.g., Salmonella and Klebsiella) and fungi (e.g., Histoplasma, Candida, Paracoccidioides, Coccidioides, and Cryptococcus). The hallmark of MSMD is the inherited impaired production of interferon gamma (IFN-γ) or the lack of response to it. Mutations in the interleukin (IL)-12 receptor subunit beta 1 (IL12RB1) gene accounts for 38% of cases of MSMD. Most IL12RB1 pathogenic allele mutations, including ten known stop-gain variants, cause IL-12Rβ1 complete deficiency (immunodeficiency-30, IMD30) by knocking out receptor cell-surface expression. IL12RB1 loss-of-function genotypes impair both IL-12 and IL-23 responses. Here, we assess the health effects of a rare, novel IL12RB1 stop-gain homozygous genotype with paradoxical IL-12Rβ1 cell-surface expression. We appraise four MSMD children from three unrelated Brazilian kindreds by clinical consultation, medical records, and genetic and immunologic studies. The clinical spectrum narrowed down to Bacillus Calmette-Guerin (BCG) vaccine-related suppurative adenitis in all patients with one death, and recrudescence in two, histoplasmosis, and recurrence in one patient, extraintestinal salmonellosis in one child, and cutaneous vasculitis in another. In three patients, we established the homozygous Trp7Ter predicted loss-of-function inherited genotype and inferred it from the heterozygote parents of the fourth case. The Trp7Ter mutation maps to the predicted IL-12Rβ1 N-terminal signal peptide sequence. BCG- or phytohemagglutinin-blasts from the three patients have reduced cell-surface expression of IL-12Rβ1 with impaired production of IFN-γ and IL-17A. Screening of 227 unrelated healthy subjects from the same geographic region revealed one heterozygous genotype (allele frequency 0.0022) vs. one in over 841,883 public genome/exomes. We also show that the

  2. Genetic Variations in the Human G Protein-coupled Receptor Class C, Group 6, Member A (GPRC6A) Control Cell Surface Expression and Function

    DEFF Research Database (Denmark)

    Jorgensen, Stine; Have, Christian Theil; Underwood, Christina Rye

    2017-01-01

    GPRC6A is a G protein-coupled receptor activated by l-amino acids, which, based on analyses of knock-out mice, has been suggested to have physiological functions in metabolism and testicular function. The human ortholog is, however, mostly retained intracellularly in contrast to the cell surface...... of the human ortholog. Genetic analyses of the 1000 genome database and the Inter99 cohort of 6,000 Danes establish the distribution of genotypes among ethnic groups, showing that the cell surface-expressed and functional variant is much more prevalent in the African population than in European and Asian...

  3. Organophosphorus pesticides enhance the genotoxicity of benzo(a)pyrene by modulating its metabolism.

    Science.gov (United States)

    Hreljac, Irena; Filipic, Metka

    2009-12-01

    Organophosphorus compounds (OPs) are widely used as pesticides. They act primarily as neurotoxins, but there is increasing evidence for secondary mechanisms of their toxicity. We have shown that the model OPs, methyl parathion (PT) and methyl paraoxon (PO), are genotoxic. Benzo(a)pyrene (BaP) is a widespread environmental genotoxin found in cigarette smoke, polluted air and grilled food. As people are constantly exposed to low concentrations of BaP and also to OPs, the aim of this study was to determine possible synergistic effects of PT and PO on BaP-induced genotoxicity. In the bacterial reverse mutation assay, PT and PO increased the number of BaP-induced mutations. The comet assay with human hepatoma HepG2 cells showed that BaP-induced DNA strand breaks were increased by PT but slightly decreased by PO. Using the acellular comet assay with UVC-induced DNA strand breaks, we observed a decrease in DNA migration, indicating that OPs cause cross-linking, thus interfering with comet assay results. In HepG2 cells the two OPs induced micronuclei formation at very low doses (0.01 microg/ml) and together with BaP, a more than additive increase of micronuclei was observed, confirming their co-genotoxic effect. We demonstrated for the first time that PT and PO modulate the metabolic activation of BaP. Addition of PT or PO increased aldo-keto reductase (AKR1C1/2) levels in the presence of BaP, while cytochrome 1A (CYP1A) mRNA expression and activity were decreased. Further, specific inhibition of CYP1A had no effect on BaP or OP+BaP-induced micronuclei, whereas inhibition of AKR1C dramatically decreased the number of micronuclei induced by BaP or OP+BaP. Based on these results we propose that co-genotoxicity results from OPs mediated modulation of BaP metabolism, favouring the induction of AKR1C enzymes known to catalyse the formation of DNA reactive BaP o-quinones and the production of reactive oxygen species.

  4. Swine models in the design of more effective medical countermeasures against organophosphorus poisoning

    International Nuclear Information System (INIS)

    Dorandeu, F.; Mikler, J.R.; Thiermann, H.; Tenn, C.; Davidson, C.; Sawyer, T.W.; Lallement, G.; Worek, F.

    2007-01-01

    Although the three most commonly used large mammal species in the safety assessment of drugs remain the dog, the macaque and the marmoset, swine, especially minipigs, have also been widely used over the years in many toxicological studies. Swine present a number of interesting biological and physiological characteristics. Similarities in skin properties with humans have led to extensive in vitro and in vivo studies. There is a specific interest in cardiovascular research, as well as in anaesthesiology and critical care medicine due to common features of swine and human physiology. Although knowledge of swine brain structure and functions remains incomplete, data does exist. The multiple blood sampling that is necessary in pharmacokinetic and toxicokinetic studies are possible, as well as multiparametric monitoring and interventions with equipment used in human clinical settings. Practicality (handling), scientific (stress reduction) and ethical (invasive monitoring) reasons have led research teams to incorporate anaesthesia into their paradigms which makes the analysis of data increasingly difficult. Although not substantiated by scientific data, the swine appears to have an intermediate position in the scale of public perception between non-human primates and animals commonly referred to as pets (i.e. dogs and cats) and rodents. The benefits of the swine model justify the use of these animals in the design of more effective medical countermeasures against known chemical warfare agents (nerve agents, vesicants and lung damaging agents). Exposure to organophosphorus (OP) pesticides represents a severe health issue in developing countries, while OP intoxication with the more lethal military nerve agents is not only of military concern but also a terrorist threat. Tailoring therapeutic regimens to the reality of OP poisoning is of the utmost importance when little experimental data and sparse human clinical data are available in the decision making process. We will

  5. Organophosphorus flame retardants and heavy metals in municipal landfill leachate treatment system in Guangzhou, China.

    Science.gov (United States)

    Deng, Mingjun; Kuo, Dave T F; Wu, Qihang; Zhang, Ying; Liu, Xinyu; Liu, Shengyu; Hu, Xiaodong; Mai, Bixian; Liu, Zhineng; Zhang, Haozhi

    2018-05-01

    The occurrence, distribution and removal efficiencies of organophosphorus flame retardants (OPFRs) and metals were examined in a municipal landfill leachate treatment system in Guangzhou, China. Five OPFRs and thirty-five metals were detected in wastewater samples collected at different treatment stages. ∑OPFRs was reduced from 4807.02 ng L -1 to 103.91 ng L -1 through the treatment system, with close to 98% removed from the dissolved phase. Tris(clorisopropyl) phosphates (TCPPs) dominated through the treatment process and accounted for over 80% and 50% of ∑OPFRs at the influent and the effluent, respectively. TCPPs were most efficiently removed (98.6%) followed by tris(2-chloroethyl) phosphate (TCEP) (96.6%) and triphenyl phosphate (TPP) (88.5%). For metals, Fe, Cr, and Rb were dominant in the raw leachate, detected at 7.55, 2.82, and 4.50 mg L -1 , respectively. Thirteen regulated heavy metals - including eight major pollutants (i.e., As. Cd, Cr, Cu, Hg, Ni, Pb, and Zn) - have been detected in all wastewater samples at sub-mg L -1 levels. Over 99.5% removal was achieved for Cr, Ni, and Fe, and close to 95% removal efficiency was observed for Rb. For the eight major heavy metals, over 99% removal was observed; the only exception was Cu, which was removed at 89%. It was found that microfiltration/reverse osmosis was critical for the removal of OPFRs and heavy metals while the core biological treatment played a minor role towards their removal. Remobilization of Co, Cu, Fe, Hg, Mn, Ni, Sb, and Sr from the returned sludge occurred during the second denitrification, indicating the need for additional post-biological process for effective removal of both contaminants. This study highlights the critical need to develop cheap, effective treatment technologies for contaminants-laden leachate generated from open dumps and under-designed landfills. Copyright © 2018 Elsevier Ltd. All rights reserved.

  6. Estimated assessment of cumulative dietary exposure to organophosphorus residues from tea infusion in China.

    Science.gov (United States)

    Cao, Pei; Yang, Dajin; Zhu, Jianghui; Liu, Zhaoping; Jiang, Dingguo; Xu, Haibin

    2018-02-14

    China has the world's largest tea plantation area in the world. To sustain high yields of the tea, multiple pesticides are used on tea crops to control pests. Organophosphorus (OP) pesticides are among the most widely used types of agricultural pesticides in China. As tea is a significant potential source of exposure to pesticide residues, the public concern has increased in relation to pesticide residues found in tea in China. The aim of the study was to estimate cumulative dietary exposure to OP residues from tea infusion for Chinese tea consumers to determine whether exposure to OP residues from tea infusion is a cause of health concern for tea consumers in China. OP residue data were obtained from the China National Monitoring Program on Food Safety (2013-2014), encompassing 1687 tea samples from 12 provinces. Tea consumption data were obtained from the China National Nutrient and Health Survey (2002), comprising 506 tea consumers aged 15-82 years. The transfer rates of residues from tea leaves into tea infusions were obtained from the literature. The relative potency factor (RPF) approach was used to estimate acute cumulative exposure to 20 OP residues from tea infusion using methamidophos as the index compound. Dietary exposure was calculated in a probabilistic way. For tea consumers, the mean and the 99.9th percentile (P99.9) of cumulative dietary exposure to OP residues from tea infusion equalled 0.08 and 1.08 μg/kg bw/d. When compared with the acute reference dose (ARfD), 10 μg/kg bw/d for methamidophos, this accounts for 0.8 and 10.8% of the ARfD. Even when considering OP residues from vegetables, fruits and other foods, there are no health concerns based on acute dietary exposure to OP residues from tea infusion. However, it is necessary to strengthen the management of the OP pesticides used on tea in China to reduce the risk of chronic dietary exposure to OPs from tea infusion.

  7. Molecular and immunological responses of the giant freshwater prawn, Macrobrachium rosenbergii, to the organophosphorus insecticide, trichlorfon.

    Science.gov (United States)

    Chang, Chin-Chyuan; Rahmawaty, Atiek; Chang, Zhong-Wen

    2013-04-15

    Trichlorfon is an organophosphorus (OP) insecticide that is used as an agriculture pesticide to destroy insects, a human medicine to combat internal parasites, and an ectoparasiticide in the livestock and aquaculture industries, but which has caused aquatic toxicity in the prawn industry. The aim of this study was to investigate the effects of trichlorfon on molecular and enzymatic processes of the immunological response of the giant freshwater prawn, Macrobrachium rosenbergii, at 0, 0.2, and 0.4mgL(-1) with 0, 3, 6, 12, and 24h of exposure. The total hemocyte count (THC), respiratory bursts (RBs), phenoloxidase (PO) activity, and superoxide dismutase (SOD) activity were examined to evaluate immunological responses and oxidative stress. Results showed that THCs of the prawn exposed to trichlorfon at both concentrations (0.2 and 0.4mgL(-1)) had increased after 12 and 24h; SOD and PO activities had significantly increased at 3h, whereas production of RBs had dramatically increased as oxidative stress at each sampling time after exposure to trichlorfon compared to the control. A potential biomarker of OPs, acetylcholinesterase (AChE) revealed a significant decrease after exposure for 6h, and showed a time-dependent tendency. Immune gene expressions, including prophenoloxidase (proPO), the lipopolysaccharide- and β-1,3-glucan-binding protein (LGBP), peroxinectin (PE), α2-macroglubulin (α2M), transglutaminase (TG), and copper, zinc (Cu,Zn)-SOD, of prawns exposed to trichlorfon at 0, 0.2, and 0.4mgL(-1) for 0, 6, and 24h were further evaluated. Expressions of all of the immune genes significantly decreased when prawns were exposed to 0.4mgL(-1) trichlorfon for 24h, and among them, an increase in SOD expression was seen after exposure to 0.4mgL(-1) for 6h. Prawns exposed to trichlorfon within 24h exhibited the decrease of circulating hemocytes, and also the induction of oxidative stress, which caused subsequent damage to DNA formation of immune genes. From these

  8. Enzymatic Analysis of G- and V-Agents and Their Degradation Products

    National Research Council Canada - National Science Library

    Elashvili, Ilya

    2003-01-01

    .... The nerve agents can be hydrolyzed to their respective methylphosphonate alkyl ester (h-agent) products by alkali treatment or by specific hydrolytic enzymes, such as organophosphorus hydrolase...

  9. Analysis of monomeric and oligomeric organophosphorus flame retardants in fish muscle tissues using liquid chromatography–electrospray ionization tandem mass spectrometry: Application to Nile tilapia (Oreochromis niloticus) from an e-waste processing area in northern Vietnam

    OpenAIRE

    Matsukami, Hidenori; Suzuki, Go; Tue, Nguyen Minh; Tuyen, Le Huu; Viet, Pham Hung; Takahashi, Shin; Tanabe, Shinsuke; Takigami, Hidetaka

    2016-01-01

    Using electrospray ionization tandem mass spectrometry combined with liquid chromatography (LC), a novel analytical method was developed to quantify eight monomeric organophosphorus flame retardants (m-PFRs) and three oligomeric organophosphorus flame retardants (o-PFRs) in fish muscle samples. The optimization and validation experiments indicate that the developed method can determine accurately the concentrations of analytes in fish muscle samples. The recoveries of analytes in fish muscle ...

  10. Low Constitutive Cell Surface Expression of HLA-B Is Caused by a Posttranslational Mechanism Involving Glu180 and Arg239

    DEFF Research Database (Denmark)

    Dellgren, Christoffer; Ekwelum, Vanessa A. C.; Ormhøj, Maria

    2016-01-01

    most of the variability of expression between common HLA-A and -B allomorphs. In this study, we present a fine mapping of the structural determinants that allow such variability by exchanging key amino acids located within the C-terminal part of the α2 domain and the α3 domain of HLA-A2 and -B8......, including Glu/Asp at position 177, Gln/Glu at position 180, Gly/Arg at position 239, and Pro/Ser at position 280. We found that the HLA-A2 and -B8 expression profiles could be interconverted to a large extent by mutual exchange of Gln/Glu at position 180 or by Gly/Arg at position 239. The presence of Gln......(180) and Gly(239), as in HLA-A2, led to higher cell surface expression levels when compared with the presence of Glu(180) and Arg(239), as in HLA-B8. This indicates that the amino acids at positions 180 and 239 determine the level of cell surface expression of common HLA-A and -B allomorphs, probably...

  11. The extracellular membrane-proximal domain of membrane-bound IgE restricts B cell activation by limiting B cell antigen receptor surface expression.

    Science.gov (United States)

    Vanshylla, Kanika; Opazo, Felipe; Gronke, Konrad; Wienands, Jürgen; Engels, Niklas

    2018-03-01

    Immunoglobulin E (IgE) antibodies are key mediators of allergic reactions. Due to their potentially harmful anaphylactic properties, their production is tightly regulated. The membrane-bound isoform of IgE (mIgE), which is an integral component of the B cell antigen receptor, has been shown to be critical for the regulation of IgE responses in mice. In primate species including humans, mIgE can be expressed in two isoforms that are produced by alternative splicing of the primary ε Ig heavy chain transcript, and differ in the absence or presence of an extracellular membrane-proximal domain (EMPD) consisting of 52 amino acids. However, the function of the EMPD remains unclear. Here, we demonstrate that the EMPD restricts surface expression of mIgE-containing BCRs in human and murine B cells. The EMPD does not interfere with BCR assembly but acts as an autonomous endoplasmic reticulum retention domain. Limited surface expression of EMPD-containing mIgE-BCRs caused impaired activation of intracellular signaling cascades and hence represents a regulatory mechanism that may control the production of potentially anaphylactic IgE antibodies in primate species. © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  12. Potential of the virion-associated peptidoglycan hydrolase HydH5 and its derivative fusion proteins in milk biopreservation

    Science.gov (United States)

    Bacteriophage lytic enzymes have recently attracted considerable interest as novel antimicrobials against Gram-positive bacteria. In this work, antimicrobial activity in milk of HydH5 [(a virion-associated peptidoglycan hydrolase (VAPGH) encoded by the Staphylococcus aureus bacteriophage vB_SauS-ph...

  13. Crystal Structure of α-1,4-Glucan Lyase, a Unique Glycoside Hydrolase Family Member with a Novel Catalytic Mechanism

    NARCIS (Netherlands)

    Rozeboom, Henriëtte J.; Yu, Shukun; Madrid, Susan; Kalk, Kor H.; Zhang, Ran; Dijkstra, Bauke W.

    2013-01-01

    α-1,4-Glucan lyase (EC 4.2.2.13) from the red seaweed Gracilariopsis lemaneiformis cleaves α-1,4-glucosidic linkages in glycogen, starch, and malto-oligosaccharides, yielding the keto-monosaccharide 1,5-anhydro-D-fructose. The enzyme belongs to glycoside hydrolase family 31 (GH31) but degrades

  14. Comparative expression of the mRNA for three intestinal hydrolases during postnatal development in the rat

    DEFF Research Database (Denmark)

    Freund, J N; Torp, N; Duluc, I

    1990-01-01

    The distribution of the mRNA for intestinal aminopeptidase-N, lactase-phlorizin hydrolase and sucrase-isomaltase was compared during rat postnatal development as well as along the longitudinal axis of the intestinal tract including small-intestine and colon. We found out that each mRNA exhibited...

  15. Genetic and biochemical characterization of a novel monoterpene epsilon-lactone hydrolase from Rhodococcus erythropolis DCL14

    NARCIS (Netherlands)

    Vlugt-Bergmans, van der C.J.B.; Werf, van der M.J.

    2001-01-01

    A monoterpene ε-lactone hydrolase (MLH) from Rhodococcus erythropolis DCL14, catalyzing the ring opening of lactones which are formed during degradation of several monocyclic monoterpenes, including carvone and menthol, was purified to apparent homogeneity. It is a monomeric enzyme of 31 kDa that is

  16. Genetic and biochemical characterization of a novel monoterpene e-lactone hydrolase from Rhodococcus erythropolis DCL14

    NARCIS (Netherlands)

    Vlugt-Bergmans, C.J.B. van der; Werf, M.J. van der

    2001-01-01

    A monoterpene ε-lactone hydrolase (MLH) from Rhodococcus erythropolis DCL14, catalyzing the ring opening of lactones which are formed during degradation of several monocyclic monoterpenes, including carvone and menthol, was purified to apparent homogeneity. It is a monomeric enzyme of 31 kDa that is

  17. Genetic basis for the resistance of Staphylococcus aureus to peptidoglycan hydrolase by comparative transcriptome and whole genome sequence analysis

    Science.gov (United States)

    Background: Lysostaphin is a glycyl-glycine bacteriocin peptidoglycan hydrolase secreted by Staphylococcus simulans for degrading the peptidoglycan moieties in Staphylococcus aureus cell walls which result in cell lysis. There are known mechanisms of resistance to lysostaphin, e.g. serine in place...

  18. 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.…

  19. Acetobacter turbidans α-Amino Acid Ester Hydrolase. How a Single Mutation Improves an Antibiotic-Producing Enzyme

    NARCIS (Netherlands)

    Barends, Thomas R.M.; Polderman-Tijmes, Jolanda J.; Jekel, Peter A.; Williams, Christopher; Wybenga, Gjalt; Janssen, Dick B.; Dijkstra, Bauke W.

    2006-01-01

    The α-amino acid ester hydrolase (AEH) from Acetobacter turbidans is a bacterial enzyme catalyzing the hydrolysis and synthesis of β-lactam antibiotics. The crystal structures of the native enzyme, both unliganded and in complex with the hydrolysis product D-phenylglycine are reported, as well as

  20. The influence of ferric (III citrate on ATP-hydrolases of Desulfuromonas acetoxidans ІМV В-7384

    Directory of Open Access Journals (Sweden)

    O. Maslovska

    2013-02-01

    Full Text Available Desulfuromonas acetoxidans obtains energy for growth by the anaerobic oxidation of organic compounds with the carbon dioxide formation. It was found that ferrum and manganese are used as terminal electron acceptors in the processes of anaerobic respiration, such as dissimilative Fe3+- and Mn4+-reduction, carried out by these bacteria (Lovely, 1991. D. acetoxidans ІМV B-7384 can be used as anode biocatalyst in microbial fuel cell with high electron recovery through acetate oxidation to the electric current as a result of electron transfer to the anode or 3d-type transition metals, such as ferrum and manganese, in the process of their reduction. Investigation of biochemical changes of D. acetoxidans ІМV B-7384 under the influence of Fe (III compounds is important for optimization of the process of bacterial electricity generation. ATP-hydrolase is located in cytoplasmic membrane, and its subunits are exposed to both the cytoplasm and the external environment. Therefore, the changes of that enzyme activity can be used as an indicator of various stress exposure. Presence of ferric iron ions in the bacterial growth medium could catalyze generation of organic reactive oxygen species, such as peroxyl (ROO- and alkoxyl (RO- radicals. Lipid peroxidation is one of the main reasons of cell damage and it’s following death under the influence of reactive oxygen metabolites. It is known that lipid peroxidation and membrane transport processes are somehow interrelated, but mechanisms of such interaction are still unidentified. In our previous researche we have shown the influence of ferric (III citrate on the intensity of lipid peroxidation of D. аcetoxidans ІМV В-7384. Significant increase of the content of lipid peroxidation products (lipid hydroperoxides, conjugated dienes and malondialdehyde in bacterial cells has been observed under the addition of ferric (III citrate into the cultural medium. The increase of the concentration of lipid

  1. Compostos organofosforados pentavalentes: histórico, métodos sintéticos de preparação e aplicações como inseticidas e agentes antitumorais Organophosphorus pentavalent compounds: history, synthetic methods of preparation and application as insecticides and antitumor agents

    Directory of Open Access Journals (Sweden)

    Viviane Martins Rebello dos Santos

    2007-02-01

    Full Text Available This paper is a review of the history, synthesis and application of organophosphorus compounds, especially of those of pentavalent phosphorus, such as phosphoramidates, phosphorothioates, phosphonates and phosphonic acids with insecticide and anticancer activities. The organophosphorus compounds with agrochemical applications show great structural variety, They include not only insecticides, but also fungicides, herbicides, and others. The large variety of commercially available organophosphorus pesticides is remarkable. Even more interesting is the high efficiency of some organophosphorus compounds as anticancer agents such as cyclophosphamide and its derivatives.

  2. Applied clinical pharmacology and public health in rural Asia – preventing deaths from organophosphorus pesticide and yellow oleander poisoning

    Science.gov (United States)

    Eddleston, Michael

    2013-01-01

    Self-poisoning with pesticides or plants is a major clinical problem in rural Asia, killing several hundred thousand people every year. Over the last 17 years, our clinical toxicology and pharmacology group has carried out clinical studies in the North Central Province of Sri Lanka to improve treatment and reduce deaths. Studies have looked at the effectiveness of anti-digoxin Fab in cardiac glycoside plant poisoning, multiple dose activated charcoal in all poisoning, and pralidoxime in moderate toxicity organophosphorus insecticide poisoning. More recently, using a Haddon matrix as a guide, we have started conducting public health and animal studies to find strategies that may work outside of the hospital. Based on the 2009 GSK Research in Clinical Pharmacology prize lecture, this review shows the evolution of the group's research from a clinical pharmacology approach to one that studies possible interventions at multiple levels, including the patient, the community and government legislation. PMID:22943579

  3. Effect of two organophosphorus insecticides on the growth, respiration and (14C)-glucose metabolism of Azobacter chroococcum Beij

    International Nuclear Information System (INIS)

    Balasubramanian, A.; Narayanan, R.

    1980-01-01

    The two organophosphorus insecticides, commonly applied to soil, viz., disulfoton (0,0-diethyl S-2-ethyl thio ethyl phosphorodithioate) and fensulfothion (0,0-diethyl 0-4-methyl sulphinyl phenyl phosphorothioate) did not affect the in vitro growth of Azotobacter chroococcum Beij., the free-living, nitrogen fixing soil bacterium, at 2 ppm (lower level), while the normal dose (5 ppm) and the higher level (10 ppm) suppressed the growth. Respiration of the organism (glucose oxidation) was adversely affected by the insecticides in the growth medium and the inhibition increased with the concentration of the chemical. Both the insecticides suppressed the assimilation of ( 14 C)-glucose in the cold-TCA soluble, hot-TCA soluble fractions and insoluble residue of the cells whereas the 14 C-incorporation in the alcohol soluble and alcohol-ether soluble fractions was enhanced indicating that the insecticides considerably altered the glucose metabolism of the bacterium. (author)

  4. Degradation of Organophosphorus Pesticides in Water during UV/H2O2 Treatment: Role of Sulphate and Bicarbonate Ions

    Directory of Open Access Journals (Sweden)

    Am Fadaei

    2012-01-01

    Full Text Available The photodegradation of two organophosphorus pesticides, malathian and diazinon, by sulfate radicals and bicarbonate radicals in aqueous solution were studied. The effect of the operational parameters such as pH, salt concentration, water type, H2O2 concentration and initial concentration of pesticides was studied. Gas chromatography mass spectroscopy (GC–MS was used for analyses of pesticides. When salt effect was studied, it was found that sodium bicarbonate was the most powerful inhibitor used, while sodium sulfate was the weakest one. The highest degradation in UV/H2O2 process for malathion was found in alkaline condition and for diazinon in acidic condition. The photodegradation in all waters used in this work exhibited first order kinetics. Photodegradation rate in distilled water was higher than real water. The degradation of pesticides increased with increasing of H2O2 concentration.

  5. Determination of some organophosphorus pesticides in water and watermelon samples by microextraction prior to high-performance liquid chromatography.

    Science.gov (United States)

    Wang, Chun; Wu, Qiuhua; Wu, Chunxia; Wang, Zhi

    2011-11-01

    A novel method based on simultaneous liquid-liquid microextraction and carbon nanotube reinforced hollow fiber microporous membrane solid-liquid phase microextraction has been developed for the determination of six organophosphorus pesticides, i.e. isocarbophos, phosmet, parathion-methyl, triazophos, fonofos and phoxim, in water and watermelon samples prior to high-performance liquid chromatography (HPLC). Under the optimum conditions, the method shows a good linearity within a range of 1-200 ng/mL for water samples and 5-200 ng/g for watermelon samples, with the correlation coefficients (r) varying from 0.9990 to 0.9997 and 0.9986 to 0.9995, respectively. The limits of detection (LODs) were in the range between 0.1 and 0.3 ng/mL for water samples and between 1.0 and 1.5 ng/g for watermelon samples. The recoveries of the method at spiking levels of 5.0 and 50.0 ng/mL for water samples were between 85.4 and 100.8%, and at spiking levels of 5.0 and 50.0 ng/g for watermelon samples, they were between 82.6 and 92.4%, with the relative standard deviations (RSDs) varying from 4.5-6.9% and 5.2-7.4%, respectively. The results suggested that the developed method represents a simple, low-cost, high analytes preconcentration and excellent sample cleanup procedure for the determination of organophosphorus pesticides in water and watermelon samples. Copyright © 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  6. Luminescence recognition of different organophosphorus pesticides by the luminescent Eu(III)-pyridine-2,6-dicarboxylic acid probe.

    Science.gov (United States)

    Azab, Hassan A; Duerkop, Axel; Anwar, Z M; Hussein, Belal H M; Rizk, Moustafa A; Amin, Tarek

    2013-01-08

    Luminescence quenching of a novel long lived Eu(III)-pyridine-2,6-dicarboxylic acid probe of 1:2 stoichiometric ratio has been studied in 0.10 volume fraction ethanol-water mixture at pH 7.5 (HEPES buffer) in the presence of the organophosphorus pesticides chlorfenvinphos (P1), malathion (P2), azinphos (P3), and paraxon ethyl (P4). The luminescence intensity of Eu(III)-(PDCA)(2) probe decreases as the concentration of the pesticide increases. It was observed that the quenching due to P3 and P4 proceeds via both diffusional and static quenching processes. Direct methods for the determination of the pesticides under investigation have been developed using the luminescence quenching of Eu(III)-pyridine-2,6-dicarboxylic acid probe in solution. The linear range for determination of the selected pesticides is 1.0-35.0 μM. The detection limits were 0.24-0.55 μM for P3, P4, and P1 and 2.5 μM for P2, respectively. The binding constants (K), and thermodynamic parameters of the OPs with Eu(III)-(PDCA)(2) were evaluated. Positive and negative values of entropy (ΔS) and enthalpy (ΔH) changes for Eu(III)-(PDCA)(2)-P1 ternary complex were calculated. As the waters in this study do not contain the above mentioned OPs over the limit detectable by the method, a recovery study was carried out after the addition of the adequate amounts of the organophosphorus pesticides under investigation. Copyright © 2012 Elsevier B.V. All rights reserved.

  7. Determination of no-observed effect level (NOEL-biomarker equivalents to interpret biomonitoring data for organophosphorus pesticides in children

    Directory of Open Access Journals (Sweden)

    Bouchard Michèle

    2009-02-01

    Full Text Available Abstract Background Environmental exposure to organophosphorus pesticides has been characterized in various populations, but interpretation of these data from a health risk perspective remains an issue. The current paper proposes biological reference values to help interpret biomonitoring data related to an exposure to organophosphorus pesticides in children for which measurements of alkylphosphate metabolites are available. Methods Published models describing the kinetics of malathion and chlorpyrifos in humans were used to determine no-observed effect level – biomarker equivalents for methylphosphates and ethylphosphates, respectively. These were expressed in the form of cumulative urinary amounts of alkylphosphates over specified time periods corresponding to an absorbed no-observed effect level dose (derived from a published human exposure dose and assuming various plausible exposure scenarios. Cumulative amounts of methylphosphate and ethylphosphate metabolites measured in the urine of a group of Quebec children were then compared to the proposed biological reference values. Results From a published no-observed effect level dose for malathion and chlorpyrifos, the model predicts corresponding oral biological reference values for methylphosphate and ethylphosphate derivatives of 106 and 52 nmol/kg of body weight, respectively, in 12-h nighttime urine collections, and dermal biological reference values of 40 and 32 nmol/kg of body weight. Out of the 442 available urine samples, only one presented a methylphosphate excretion exceeding the biological reference value established on the basis of a dermal exposure scenario and none of the methylphosphate and ethylphosphate excretion values were above the obtained oral biological reference values, which reflect the main exposure route in children. Conclusion This study is a first step towards the development of biological guidelines for organophophorus pesticides using a toxicokinetic modeling

  8. Rapid simultaneous determination of organophosphorus pesticides in human serum and urine by liquid chromatography-mass spectrometry

    Directory of Open Access Journals (Sweden)

    Zlatković Milica

    2010-01-01

    Full Text Available Background/Aim. Analysis of organophosphosphorus compounds and their metabolites in a biological material includes the use of numerous methods, covering both preparation of samples for analysis and their identification that is considered to be very complex. Low concentrations monitoring requires implementation of highly sensitive analytical techniques. The aim of this study was to develop and validate an original and sensitive method for the detection and quantitation of organophosphorus pesticides (dimethoate, diazinon, malathion and malaoxon in human biological matrices (serum, urine. Methods. This method was based on a solid-phase extraction procedure, a chromatographic separation using an ACQUITY UPLC ® HSST3 column and mass spectrometric detection in the positve ion mode. Mobile phase: was consited of Solvent A (5 mM ammonium formate pH 3.0 and Solvent B (0.1% acetic formate in methanol, in a linear gradient (constant flow-rate 0.3 mL/min. Results. The standard curve was linear in the range of 0.05-5.00 mg/L for malathion and malaoxon, 0.10-5.00 mg/L for dimethoate and 0.05-2.50 mg/L for diazinon. The correlation coefficient was r ≥ 0.99. Extraction recoveries were satisfactory and ranged between 90-99%. The limits of detection (LOD was between 0.007- 0.07 mg/L and the limits of quantitation (LOQ ranged between 0.022-0.085 mg/L. Intra- and interassay precision and accuracy were satisfactory for all of the pesticides analyzed. Conclusion. The method of liquid chromatography - mass spectrometry is simple, accurate, and useful for the determination of organophosphorus pesticides in both clinical and forensic toxicology.

  9. In vitro study of the neuropathic potential of the organophosphorus compounds fenamiphos and profenofos: Comparison with mipafox and paraoxon.

    Science.gov (United States)

    Emerick, Guilherme L; Fernandes, Laís S; de Paula, Eloísa Silva; Barbosa, Fernando; dos Santos, Neife Aparecida Guinaim; dos Santos, Antonio Cardozo

    2015-08-01

    Organophosphorus-induced delayed neuropathy (OPIDN) is a central-peripheral distal axonopathy that develops 8-14 days after poisoning by a neuropathic organophosphorus compound (OP). Several OPs that caused OPIDN were withdrawn from the agricultural market due to induction of serious delayed effects. Therefore, the development of in vitro screenings able to differentiate neuropathic from non-neuropathic OPs is of crucial importance. Thus, the aim of this study was to evaluate the differences in the neurotoxic effects of mipafox (neuropathic OP) and paraoxon (non-neuropathic OP) in SH-SY5Y human neuroblastoma cells, using the inhibition and aging of neuropathy target esterase (NTE), inhibition of acetylcholinesterase (AChE), activation of calpain, neurite outgrowth, cytotoxicity and intracellular calcium as indicators. Additionally, the potential of fenamiphos and profenofos to cause acute and/or delayed effects was also evaluated. Mipafox had the lowest IC50 and induced the highest percentage of aging of NTE among the OPs evaluated. Only mipafox was able to cause calpain activation after 24 h of incubation. Concentrations of mipafox and fenamiphos which inhibited at least 70% of NTE were also able to reduce neurite outgrowth. Cytotoxicity was higher in non-neuropathic than in neuropathic OPs while the intracellular calcium levels were higher in neuropathic than in non-neuropathic OPs. In conclusion, the SH-SY5Y cellular model was selective to differentiate neuropathic from non-neuropathic OPs; fenamiphos, but not profenofos presented results compatible with the induction of OPIDN. Copyright © 2015 Elsevier Ltd. All rights reserved.

  10. The Antioxidant and Starch Hydrolase Inhibitory Activity of Ten Spices in an In Vitro Model of Digestion: Bioaccessibility of Anthocyanins and Carotenoids

    Science.gov (United States)

    Jayawardena, Nilakshi; Watawana, Mindani I.; Jayathilaka, Ruchini T.; Waisundara, Viduranga Y.

    2015-01-01

    The antioxidant and starch hydrolase inhibitory activities of cardamom, cloves, coriander, cumin seeds, curry leaves, fenugreek, mustard seeds, nutmeg, sweet cumin, and star anise extracts were investigated in an in vitro model of digestion mimicking the gastric and duodenal conditions. The total phenolic contents in all spice extracts had statistically significantly (P spice extracts showed statistically significant changes in the initial starch hydrolase enzyme inhibitory values prior to gastric and duodenal digestion. In conclusion, this study was able to prove that the 10 spices were a significant source of total phenolics, antioxidant, and starch hydrolase inhibitory activities. PMID:26693245

  11. 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.

  12. Antibiotic growth promoters enhance animal production by targeting intestinal bile salt hydrolase and its producers

    Directory of Open Access Journals (Sweden)

    Jun eLin

    2014-02-01

    Full Text Available The growth-promoting effect of antibiotic growth promoters (AGPs was correlated with the decreased activity of bile salt hydrolase (BSH, an intestinal bacteria-produced enzyme that exerts negative impact on host fat digestion and utilization. Consistent with this finding, independent chicken studies have demonstrated that AGP usage significantly reduced population of Lactobacillus species, the major BSH-producers in the intestine. Recent finding also demonstrated that some AGPs, such as tetracycline and roxarsone, display direct inhibitory effect on BSH activity. Therefore, BSH is a promising microbiome target for developing novel alternatives to AGPs. Specifically, dietary supplementation of BSH inhibitor may promote host lipid metabolism and energy harvest, consequently enhancing feed efficiency and body weight gain in food animals.

  13. Synthesis of novel bioactive lactose-derived oligosaccharides by microbial glycoside hydrolases

    Science.gov (United States)

    Díez-Municio, Marina; Herrero, Miguel; Olano, Agustín; Moreno, F Javier

    2014-01-01

    Prebiotic oligosaccharides are increasingly demanded within the Food Science domain because of the interesting healthy properties that these compounds may induce to the organism, thanks to their beneficial intestinal microbiota growth promotion ability. In this regard, the development of new efficient, convenient and affordable methods to obtain this class of compounds might expand even further their use as functional ingredients. This review presents an overview on the most recent interesting approaches to synthesize lactose-derived oligosaccharides with potential prebiotic activity paying special focus on the microbial glycoside hydrolases that can be effectively employed to obtain these prebiotic compounds. The most notable advantages of using lactose-derived carbohydrates such as lactosucrose, galactooligosaccharides from lactulose, lactulosucrose and 2-α-glucosyl-lactose are also described and commented. PMID:24690139

  14. Hydrolysis of Polysorbate 20 and 80 by a Range of Carboxylester Hydrolases.

    Science.gov (United States)

    McShan, Andrew C; Kei, Pervina; Ji, Junyan A; Kim, Daniel C; Wang, Y John

    2016-01-01

    Degradation of the surfactant polysorbate (PS) by enzyme impurities has been previously suggested as a mechanism for the formation of visible and subvisible particles that affect product quality. Although chemical degradation pathways of PS, such as oxidation and acid/base hydrolysis, have been previously characterized, enzymatic degradation of PS remains poorly understood. In this report, enzyme-mediated hydrolysis of the major components of PS was monitored using an evaporative light scattering detection-high-performance liquid chromatography method. PS20 and PS80 tested contained 99% of laurate and 98% oleate esters, respectively, were heterogeneous with respect to head group, and contained a distribution of ester types. Carboxylester hydrolases tested included those from Pseudomonas cepacia, Thermomyces lanuginosus, Candida antarctica, rabbit liver, and pig pancreas. PS hydrolysis was monitored by observing the change in the peak area of major PS components over time and quantified using a parameter called t50, which was defined as the time required for each peak to reach 50% of its initial value. Time course experiments suggested that PS hydrolysis was dependent on the order of esters (mono-, di-, or triester), the identity of the hydrophilic head group (sorbitan or isosorbide), and the identity of the fatty acid ester tail (C12 vs C18:1). In addition, the pattern of PS hydrolysis was unique to the type of enzyme used. Importantly, we observed that no PS component was completely resistant to the carboxylester hydrolases tested here. Our results illustrate a potential fingerprint approach that could be useful in verifying enzyme-mediated PS degradation in drug substance and provide an improved understanding of the complexity of PS degradation in the presence of enzymes. Degradation of the non-ionic surfactant polysorbate (PS) has been reported to lead to the formation of visible and subvisible particles that affect product quality. Chemical degradation pathways

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

    Science.gov (United States)

    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.

  16. Protein features as determinants of wild-type glycoside hydrolase thermostability

    DEFF Research Database (Denmark)

    Geertz-Hansen, Henrik Marcus; Kiemer, Lars; Nielsen, Morten

    2017-01-01

    Thermostable enzymes for conversion of lignocellulosic biomass into biofuels have significant advantages over enzymes with more moderate themostability due to the challenging application conditions. Experimental discovery of thermostable enzymes is highly cost intensive, and the development of in......-silico methods guiding the discovery process would be of high value. To develop such an in-silico method and provide the data foundation of it, we determined the melting temperatures of 602 fungal glycoside hydrolases from the families GH5, 6, 7, 10, 11, 43 and AA9 (formerly GH61). We, then used sequence...... their biological significance. The presented prediction method is made publicly available at http://www.cbs.dtu.dk/services/ThermoP. This article is protected by copyright. All rights reserved....

  17. The use of neutron scattering to determine the functional structure of glycoside hydrolase.

    Science.gov (United States)

    Nakamura, Akihiko; Ishida, Takuya; Samejima, Masahiro; Igarashi, Kiyohiko

    2016-10-01

    Neutron diffraction provides different information from X-ray diffraction, because neutrons are scattered by atomic nuclei, whereas X-rays are scattered by electrons. One of the key advantages of neutron crystallography is the ability to visualize hydrogen and deuterium atoms, making it possible to observe the protonation state of amino acid residues, hydrogen bonds, networks of water molecules and proton relay pathways in enzymes. But, because of technical difficulties, less than 100 enzyme structures have been evaluated by neutron crystallography to date. In this review, we discuss the advantages and disadvantages of neutron crystallography as a tool to investigate the functional structure of glycoside hydrolases, with some examples. Copyright © 2016 Elsevier Ltd. All rights reserved.

  18. 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

    -to-handle, multiplexed and highly reproducible method using CPH assays where different fruits have been screened for enzyme activity. Additionally, the importance and impact of the extraction method and buffer conditions on the assay are investigated. We will show that one experimental setup can be used for testing all...... an important role in fruit development and ripening processes by modulating the plant cell wall. Knowledge about these enzymes is important for research in fruit development and also important for industry regarding postharvest properties. Although advances in genetic control and cell wall biochemistry have...... 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...

  19. Use of nanostructure initiator mass spectrometry (NIMS to deduce selectivity of reaction in glycoside hydrolases

    Directory of Open Access Journals (Sweden)

    Kai eDeng

    2015-10-01

    Full Text Available Chemically synthesized nanostructure-initiator mass spectrometry (NIMS probes derivatized with tetrasaccharides were used to study the reactivity of representative Clostridium thermocellum β-glucosidase, endoglucanases and cellobiohydrolase. Diagnostic patterns for reactions of these different classes of enzymes were observed. Results show sequential removal of glucose by the β-glucosidase and a progressive increase in specificity of reaction from endoglucanases to cellobiohydrolase. Time-dependent reactions of these polysaccharide-selective enzymes were modeled by numerical integration, which provides a quantitative basis to make functional distinctions among a continuum of naturally evolved catalytic properties. Consequently, our method, which combines automated protein translation with high-sensitivity and time-dependent detection of multiple products, provides a new approach to annotate glycoside hydrolase phylogenetic trees with functional measurements.

  20. Structural studies of the nudix hydrolase DR1025 from deinococcus radiodurans and its ligand complexes

    Energy Technology Data Exchange (ETDEWEB)

    Ranatunga, Wasantha; Hill, Emma E.; Mooster, Jana L.; Holbrook, Elizabeth L.; Schulze-Gahmen, Ursula; Xu, WenLian; Bessman, Maurice J.; Brenner, Steve n E.; Holbrook, Stephen R.

    2004-01-29

    We have determined the crystal structure, at 1.4, of the Nudix hydrolase DR1025 from the extremely radiation resistant bacterium Deinococcus radiodurans. The protein forms an intertwined homodimer by exchanging N-terminal segments between chains. We have identified additional conserved elements of the Nudix fold, including the metal-binding motif, a kinked b-strand characterized by a proline two positions upstream of the Nudix consensus sequence, and participation of the N-terminal extension in the formation of the substrate-binding pocket. Crystal structures were also solved of DR1025 crystallized in the presence of magnesium and either a GTP analog or Ap4A (both at 1.6 resolution). In the Ap4Aco-crystal, the electron density indicated that the product of asymmetric hydrolysis, ATP, was bound to the enzyme. The GTP analog bound structure showed that GTP was bound almost identically as ATP. Neither nucleoside triphosphate was further cleaved.

  1. Purification and Properties of a Glycerol Ester Hydrolase (Lipase) from Propionibacterium shermanii

    Science.gov (United States)

    Oterholm, Anders; Ordal, Z. John; Witter, Lloyd D.

    1970-01-01

    An intracellular glycerol ester hydrolase (lipase) from Propionibacterium shermanii was recovered from cell-free extracts and purified by ammonium sulfate precipitation, gel filtration, and ion-exchange chromatography on diethylaminoethylcellulose. Maximum enzyme activity was observed at pH 7.2 and 47 C when an emulsion of tributyrin was used as substrate. The enzyme was stable between pH 5.5 and 8. Heating the enzyme solution at 45 C for 10 min resulted in a 75% decrease in activity. Maximum rate of hydrolysis of triglycerides was observed on tripropionin, followed in order by tributyrin, tricaproin, and tricaprylin. The lipase was strongly inhibited by mercury and arsenicals, but specific sulfhydryl reagents had little or no inhibiting effect on the enzyme activity. The enzyme also showed some esterase activity, but the hydrolysis of substrates in solution was small as compared to the hydrolysis of substrates in emulsion. PMID:5456938

  2. Localization of acid hydrolases in protoplasts. Examination of the proposed lysosomal function of the mature vacuole

    Energy Technology Data Exchange (ETDEWEB)

    Butcher, H.C.; Wagner, G.J.; Siegelman, H.W.

    1977-06-01

    The development of techniques to isolate and purify relatively large quantities of intact vacuoles from mature tissues permits direct biochemical analysis of this ubiquitous mature plant cell organelle. Vacuoles and a fraction enriched in soluble cytoplasmic constituents were quantitatively prepared from Hippeastrum flower petal protoplasts. Vacuolar lysate and soluble cytoplasmic fractions were examined for acid hydrolase activities commonly associated with animal lysosomes, and pH optima were determined. Esterase, protease, carboxypeptidase, ..beta..-galactosidase, ..cap alpha..-glycosidase and ..beta..-glycosidase, not found in the vacuole lysate fraction, were components of the soluble cytoplasmic fraction. Acid phosphatase, RNase and DNase were present in both fractions. Vacuolar enzyme activities were also examined as a function of flower development from bud through senescent stages. The data obtained are not consistent with the concept that the mature plant cell vacuole functions as a generalized lysosome.

  3. Improvement of corn stover bioconversion efficiency by using plant glycoside hydrolase.

    Science.gov (United States)

    Han, Yejun; Chen, Hongzhang

    2011-04-01

    Plant cell wall is the most abundant substrate for bioethanol production, and plants also represent a key resource for glycoside hydrolase (GH). To exploit efficient way for bioethanol production with lower cellulase loading, the potential of plant GH for lignocellulose bioconversion was evaluated. The GH activity for cell wall proteins (CWPs) was detected from fresh corn stover (FCS), and the synergism of which with Trichoderma reesei cellulase was also observed. The properties for the GH of FCS make it a promising enzyme additive for lignocellulose biodegradation. To make use of the plant GH, novel technology for hydrolysis and ethanol fermentation was developed with corn stover as substrate. Taking steam-exploded corn stover as substrate for hydrolysis and ethanol fermentation, compared with T. reesei cellulase loaded alone, the final glucose and ethanol accumulation increased by 60% and 63% respectively with GH of FCS as an addition. Copyright © 2011 Elsevier Ltd. All rights reserved.

  4. Dysregulation of soluble epoxide hydrolase and lipidomic profiles in anorexia nervosa

    KAUST Repository

    Shih, P. B.

    2015-03-31

    Individuals with anorexia nervosa (AN) restrict eating and become emaciated. They tend to have an aversion to foods rich in fat. Because epoxide hydrolase 2 (EPHX2) was identified as a novel AN susceptibility gene, and because its protein product, soluble epoxide hydrolase (sEH), converts bioactive epoxides of polyunsaturated fatty acid (PUFA) to the corresponding diols, lipidomic and metabolomic targets of EPHX2 were assessed to evaluate the biological functions of EPHX2 and their role in AN. Epoxide substrates of sEH and associated oxylipins were measured in ill AN, recovered AN and gender- and race-matched controls. PUFA and oxylipin markers were tested as potential biomarkers for AN. Oxylipin ratios were calculated as proxy markers of in vivo sEH activity. Several free- and total PUFAs were associated with AN diagnosis and with AN recovery. AN displayed elevated n-3 PUFAs and may differ from controls in PUFA elongation and desaturation processes. Cytochrome P450 pathway oxylipins from arachidonic acid, linoleic acid, alpha-linolenic acid and docosahexaenoic acid PUFAs are associated with AN diagnosis. The diol:epoxide ratios suggest the sEH activity is higher in AN compared with controls. Multivariate analysis illustrates normalization of lipidomic profiles in recovered ANs. EPHX2 influences AN risk through in vivo interaction with dietary PUFAs. PUFA composition and concentrations as well as sEH activity may contribute to the pathogenesis and prognosis of AN. Our data support the involvement of EPHX2-associated lipidomic and oxylipin dysregulations in AN, and reveal their potential as biomarkers to assess responsiveness to future intervention or treatment.

  5. Crystal structure of glycoside hydrolase family 127 β-L-arabinofuranosidase from Bifidobacterium longum

    Energy Technology Data Exchange (ETDEWEB)

    Ito, Tasuku; Saikawa, Kyo [Department of Biotechnology, The University of Tokyo, Tokyo (Japan); Kim, Seonah [National Bioenergy Center, National Renewable Energy Laboratory, Golden, CO (United States); Fujita, Kiyotaka [Faculty of Agriculture, Kagoshima University, Korimoto, Kagoshima (Japan); Ishiwata, Akihiro [Synthetic Cellular Chemistry Laboratory, RIKEN (Japan); Kaeothip, Sophon [ERATO Glycotrilogy Project, JST, Wako, Saitama (Japan); Arakawa, Takatoshi; Wakagi, Takayoshi [Department of Biotechnology, The University of Tokyo, Tokyo (Japan); Beckham, Gregg T., E-mail: Gregg.Beckham@nrel.gov [National Bioenergy Center, National Renewable Energy Laboratory, Golden, CO (United States); Ito, Yukishige [Synthetic Cellular Chemistry Laboratory, RIKEN (Japan); ERATO Glycotrilogy Project, JST, Wako, Saitama (Japan); Fushinobu, Shinya, E-mail: asfushi@mail.ecc.u-tokyo.ac.jp [Department of Biotechnology, The University of Tokyo, Tokyo (Japan)

    2014-04-25

    Graphical abstract: - Highlights: • HypBA1 β-L-arabinofuranosidase belongs to glycoside hydrolase family 127. • Crystal structure of HypBA1 was determined. • HypBA1 consists of a catalytic barrel and two additional β-sandwich domains. • The active site contains a Zn{sup 2+} coordinated by glutamate and three cysteines. • A possible reaction mechanism involving cysteine as the nucleophile is proposed. - Abstract: Enzymes acting on β-linked arabinofuranosides have been unknown until recently, in spite of wide distribution of β-L-arabinofuranosyl oligosaccharides in plant cells. Recently, a β-L-arabinofuranosidase from the glycoside hydrolase family 127 (HypBA1) was discovered in the newly characterized degradation system of hydroxyproline-linked β-L-arabinooligosaccharides in the bacterium Bifidobacterium longum. Here, we report the crystal structure of HypBA1 in the ligand-free and β-L-arabinofuranose complex forms. The structure of HypBA1 consists of a catalytic barrel domain and two additional β-sandwich domains, with one β-sandwich domain involved in the formation of a dimer. Interestingly, there is an unprecedented metal-binding motif with Zn{sup 2+} coordinated by glutamate and three cysteines in the active site. The glutamate residue is located far from the anomeric carbon of the β-L-arabinofuranose ligand, but one cysteine residue is appropriately located for nucleophilic attack for glycosidic bond cleavage. The residues around the active site are highly conserved among GH127 members. Based on biochemical experiments and quantum mechanical calculations, a possible reaction mechanism involving cysteine as the nucleophile is proposed.

  6. Limited enzymic degradation of proteins: a new approach in the industrial application of hydrolases

    Energy Technology Data Exchange (ETDEWEB)

    Adler-Nissen, J.

    1982-01-01

    The industrial importance of hydrolases exceeds that of other classes of enzymes. A major application area for hydrolases is for the dissolution of biopolymers such as starch, pectin, cellulose and protein; in many cases it has been the desire to achieve as complete a solubilization as possible. However, with food proteins, it has been demonstrated that a limited controlled hydrolysis may give rise to particularly interesting functional and organoleptic properties. The degree of hydrolysis (DH) is defined as the percentage of peptide bonds cleaved and is used as the controlling indice for the hydrolysis of food proteins. For a given enzyme-substrate system, at least five independent indices can be defined: S(substrate concentration), E/S (enzyme/substrate ratio), pH, T (temperature) and t (time). The advantage of the DH-concept is that of these five variables, four (S,E/S, T, t) can be replaced by DH, i.e. within certain limits of S, E/S, T and t, the properties of a particular protein-enzyme system are solely dependent on DH and pH of the hydrolysis. Empirically, this is demonstrated for soya-protein isolate hydrolyzed with Alcalase and theoretically the same result can be derived from the fact that there is substrate saturation throughout the reaction. These theoretical calculations are the basis for the so-called theta (h)-method, by which the significance of a particular hydrolysis indice can be studied. For each empirically derived hydrolysis curve, the hydrolysis time corresponding to any DH is found. Over a complete DH interval the proportion between the hydrolysis time for each DH is then calculated. If this term, denoted theta (h), is the same for all DH, the properties of the hydrolysates are independent of variations in the hydrolysis indice under study. A statistical procedure must be used to determine if theta (h) is constant or not. (Refs. 20).

  7. Combined cocaine hydrolase gene transfer and anti-cocaine vaccine synergistically block cocaine-induced locomotion.

    Directory of Open Access Journals (Sweden)

    Marilyn E Carroll

    Full Text Available Mice and rats were tested for reduced sensitivity to cocaine-induced hyper-locomotion after pretreatment with anti-cocaine antibody or cocaine hydrolase (CocH derived from human butyrylcholinesterase (BChE. In Balb/c mice, direct i.p. injection of CocH protein (1 mg/kg had no effect on spontaneous locomotion, but it suppressed responses to i.p. cocaine up to 80 mg/kg. When CocH was injected i.p. along with a murine cocaine antiserum that also did not affect spontaneous locomotion, there was no response to any cocaine dose. This suppression of locomotor activity required active enzyme, as it was lost after pretreatment with iso-OMPA, a selective BChE inhibitor. Comparable results were obtained in rats that developed high levels of CocH by gene transfer with helper-dependent adenoviral vector, and/or high levels of anti-cocaine antibody by vaccination with norcocaine hapten conjugated to keyhole limpet hemocyanin (KLH. After these treatments, rats were subjected to a locomotor sensitization paradigm involving a "training phase" with an initial i.p. saline injection on day 1 followed by 8 days of repeated cocaine injections (10 mg/kg, i.p.. A 15-day rest period then ensued, followed by a final "challenge" cocaine injection. As in mice, the individual treatment interventions reduced cocaine-stimulated hyperactivity to a modest extent, while combined treatment produced a greater reduction during all phases of testing compared to control rats (with only saline pretreatment. Overall, the present results strongly support the view that anti-cocaine vaccine and cocaine hydrolase vector treatments together provide enhanced protection against the stimulatory actions of cocaine in rodents. A similar combination therapy in human cocaine users might provide a robust therapy to help maintain abstinence.

  8. Combined Cocaine Hydrolase Gene Transfer and Anti-Cocaine Vaccine Synergistically Block Cocaine-Induced Locomotion

    Science.gov (United States)

    Carroll, Marilyn E.; Zlebnik, Natalie E.; Anker, Justin J.; Kosten, Thomas R.; Orson, Frank M.; Shen, Xiaoyun; Kinsey, Berma; Parks, Robin J.; Gao, Yang; Brimijoin, Stephen

    2012-01-01

    Mice and rats were tested for reduced sensitivity to cocaine-induced hyper-locomotion after pretreatment with anti-cocaine antibody or cocaine hydrolase (CocH) derived from human butyrylcholinesterase (BChE). In Balb/c mice, direct i.p. injection of CocH protein (1 mg/kg) had no effect on spontaneous locomotion, but it suppressed responses to i.p. cocaine up to 80 mg/kg. When CocH was injected i.p. along with a murine cocaine antiserum that also did not affect spontaneous locomotion, there was no response to any cocaine dose. This suppression of locomotor activity required active enzyme, as it was lost after pretreatment with iso-OMPA, a selective BChE inhibitor. Comparable results were obtained in rats that developed high levels of CocH by gene transfer with helper-dependent adenoviral vector, and/or high levels of anti-cocaine antibody by vaccination with norcocaine hapten conjugated to keyhole limpet hemocyanin (KLH). After these treatments, rats were subjected to a locomotor sensitization paradigm involving a “training phase" with an initial i.p. saline injection on day 1 followed by 8 days of repeated cocaine injections (10 mg/kg, i.p.). A 15-day rest period then ensued, followed by a final “challenge" cocaine injection. As in mice, the individual treatment interventions reduced cocaine-stimulated hyperactivity to a modest extent, while combined treatment produced a greater reduction during all phases of testing compared to control rats (with only saline pretreatment). Overall, the present results strongly support the view that anti-cocaine vaccine and cocaine hydrolase vector treatments together provide enhanced protection against the stimulatory actions of cocaine in rodents. A similar combination therapy in human cocaine users might provide a robust therapy to help maintain abstinence. PMID:22912888

  9. Listeria bacteriophage peptidoglycan hydrolases feature high thermoresistance and reveal increased activity after divalent metal cation substitution.

    Science.gov (United States)

    Schmelcher, Mathias; Waldherr, Florian; Loessner, Martin J

    2012-01-01

    The ability of the bacteriophage-encoded peptidoglycan hydrolases (endolysins) to destroy Gram-positive bacteria from without makes these enzymes promising antimicrobials. Recombinant endolysins from Listeria monocytogenes phages have been shown to rapidly lyse and kill the pathogen in all environments. To determine optimum conditions regarding application of recombinant Listeria phage endolysins in food or production equipments, properties of different Listeria endolysins were studied. Optimum NaCl concentration for the amidase HPL511 was 200 nM and 300 mM for the peptidases HPL118, HPL500, and HPLP35. Unlike most other peptidoglycan hydrolases, all four enzymes exhibited highest activity at elevated pH values at around pH 8-9. Lytic activity was abolished by EDTA and could be restored by supplementation with various divalent metal cations, indicating their role in catalytic function. While substitution of the native Zn(2+) by Ca(2+) or Mn(2+) was most effective in case of HPL118, HPL500, and HPLP35, supplementation with Co(2+) and Mn(2+) resulted in an approximately 5-fold increase in HPL511 activity. Interestingly, the glutamate peptidases feature a conserved SxHxxGxAxD zinc-binding motif, which is not present in the amidases, although they also require centrally located divalent metals for activity. The endolysins HPL118, HPL511, and HPLP35 revealed a surprisingly high thermostability, with up to 35% activity remaining after 30 min incubation at 90°C. The available data suggest that denaturation at elevated temperatures is reversible and may be followed by rapid refolding into a functional state.

  10. Altered B Cell Homeostasis in Patients with Major Depressive Disorder and Normalization of CD5 Surface Expression on Regulatory B Cells in Treatment Responders.

    Science.gov (United States)

    Ahmetspahic, Diana; Schwarte, Kathrin; Ambrée, Oliver; Bürger, Christian; Falcone, Vladislava; Seiler, Katharina; Kooybaran, Mehrdad Rahbar; Grosse, Laura; Roos, Fernand; Scheffer, Julia; Jörgens, Silke; Koelkebeck, Katja; Dannlowski, Udo; Arolt, Volker; Scheu, Stefanie; Alferink, Judith

    2018-03-01

    Pro-inflammatory activity and cell-mediated immune responses have been widely observed in patients with major depressive disorder (MDD). Besides their well-known function as antibody-producers, B cells play a key role in inflammatory responses by secreting pro- and anti-inflammatory factors. However, homeostasis of specific B cell subsets has not been comprehensively investigated in MDD. In this study, we characterized circulating B cells of distinct developmental steps including transitional, naïve-mature, antigen-experienced switched, and non-switched memory cells, plasmablasts and regulatory B cells by multi-parameter flow cytometry. In a 6-weeks follow-up, circulating B cells were monitored in a small group of therapy responders and non-responders. Frequencies of naïve lgD + CD27 - B cells, but not lgD + CD27 + memory B cells, were reduced in severely depressed patients as compared to healthy donors (HD) or mildly to moderately depressed patients. Specifically, B cells with immune-regulatory capacities such as CD1d + CD5 + B cells and CD24 + CD38 hi transitional B cells were reduced in MDD. Also Bm1-Bm5 classification in MDD revealed reduced Bm2' cells comprising germinal center founder cells as well as transitional B cells. We further found that reduced CD5 surface expression on transitional B cells was associated with severe depression and normalized exclusively in clinical responders. This study demonstrates a compromised peripheral B cell compartment in MDD with a reduction in B cells exhibiting a regulatory phenotype. Recovery of CD5 surface expression on transitional B cells in clinical response, a molecule involved in activation and down-regulation of B cell responses, further points towards a B cell-dependent process in the pathogenesis of MDD.

  11. Surface expression of Helicobacter pylori HpaA adhesion antigen on Vibrio cholerae, enhanced by co-expressed enterotoxigenic Escherichia coli fimbrial antigens.

    Science.gov (United States)

    Tobias, Joshua; Lebens, Michael; Wai, Sun Nyunt; Holmgren, Jan; Svennerholm, Ann-Mari

    2017-04-01

    Helicobacter pylori infection can cause peptic ulceration and is associated with gastric adenocarcinoma. This study aimed to construct and characterize a non-virulent Vibrio cholerae O1 strain, which grows more rapidly than H. pylori, as vector for H. pylori antigens for possible use as a vaccine strain against H. pylori. This was done by recombinant expression of the H. pylori adhesion antigen HpaA alone or, as a proof of principle, together with different colonization factor (CF) antigens of enterotoxigenic Escherichia coli (ETEC) which may enhance immune responses against HpaA. A recombinant V. cholerae strain co-expressing HpaA and a fimbrial CF antigens CFA/I or CS5, but not the non-fimbrial CF protein CS6, was shown to express larger amounts of HpaA on the surface when compared with the same V. cholerae strain expressing HpaA alone. Mutations in the CFA/I operon showed that the chaperon, possibly together with the usher, was involved in enhancing the surface expression of HpaA. Oral immunization of mice with formaldehyde-inactivated recombinant V. cholerae expressing HpaA alone or together with CFA/I induced significantly higher serum antibody responses against HpaA than mice similarly immunized with inactivated HpaA-expressing H. pylori bacteria. Our results demonstrate that a non-virulent V. cholerae strain can be engineered to allow strong surface expression of HpaA, and that the expression can be further increased by co-expressing it with ETEC fimbrial antigens. Such recombinant V. cholerae strains expressing HpaA, and possibly also other H. pylori antigens, may have the potential as oral inactivated vaccine candidates against H. pylori. Copyright © 2017 Elsevier Ltd. All rights reserved.

  12. Restricted Cell Surface Expression of Receptor Tyrosine Kinase ROR1 in Pediatric B-Lineage Acute Lymphoblastic Leukemia Suggests Targetability with Therapeutic Monoclonal Antibodies

    Science.gov (United States)

    Dave, Hema; Anver, Miriam R.; Butcher, Donna O.; Brown, Patrick; Khan, Javed; Wayne, Alan S.; Baskar, Sivasubramanian; Rader, Christoph

    2012-01-01

    Background Despite high cure rates for pediatric B-lineage acute lymphoblastic leukemia (B-ALL), short-term and long-term toxicities and chemoresistance are shortcomings of standard chemotherapy. Immunotherapy and chemoimmunotherapy based on monoclonal antibodies (mAbs) that target cell surface antigens with restricted expression in pediatric B-ALL may offer the potential to reduce toxicities and prevent or overcome chemoresistance. The receptor tyrosine kinase ROR1 has emerged as a candidate for mAb targeting in select B-cell malignancies. Methodology and Principal Findings Using flow cytometry, Western blotting, immunohistochemistry, and confocal immunofluorescence microscopy, we analyzed the cell surface expression of ROR1 across major pediatric ALL subtypes represented by 14 cell lines and 56 primary blasts at diagnosis or relapse as well as in normal adult and pediatric tissues. Cell surface ROR1 expression was found in 45% of pediatric ALL patients, all of which were B-ALL, and was not limited to any particular genotype. All cell lines and primary blasts with E2A-PBX1 translocation and a portion of patients with other high risk genotypes, such as MLL rearrangement, expressed cell surface ROR1. Importantly, cell surface ROR1 expression was found in many of the pediatric B-ALL patients with multiply relapsed and refractory disease and normal karyotype or low risk cytogenetics, such as hyperdiploidy. Notably, cell surface ROR1 was virtually absent in normal adult and pediatric tissues. Conclusions and Significance Collectively, this study suggests that ROR1 merits preclinical and clinical investigations as a novel target for mAb-based therapies in pediatric B-ALL. We propose cell surface expression of ROR1 detected by flow cytometry as primary inclusion criterion for pediatric B-ALL patients in future clinical trials of ROR1-targeted therapies. PMID:23285131

  13. Restricted cell surface expression of receptor tyrosine kinase ROR1 in pediatric B-lineage acute lymphoblastic leukemia suggests targetability with therapeutic monoclonal antibodies.

    Directory of Open Access Journals (Sweden)

    Hema Dave

    Full Text Available Despite high cure rates for pediatric B-lineage acute lymphoblastic leukemia (B-ALL, short-term and long-term toxicities and chemoresistance are shortcomings of standard chemotherapy. Immunotherapy and chemoimmunotherapy based on monoclonal antibodies (mAbs that target cell surface antigens with restricted expression in pediatric B-ALL may offer the potential to reduce toxicities and prevent or overcome chemoresistance. The receptor tyrosine kinase ROR1 has emerged as a candidate for mAb targeting in select B-cell malignancies.Using flow cytometry, Western blotting, immunohistochemistry, and confocal immunofluorescence microscopy, we analyzed the cell surface expression of ROR1 across major pediatric ALL subtypes represented by 14 cell lines and 56 primary blasts at diagnosis or relapse as well as in normal adult and pediatric tissues. Cell surface ROR1 expression was found in 45% of pediatric ALL patients, all of which were B-ALL, and was not limited to any particular genotype. All cell lines and primary blasts with E2A-PBX1 translocation and a portion of patients with other high risk genotypes, such as MLL rearrangement, expressed cell surface ROR1. Importantly, cell surface ROR1 expression was found in many of the pediatric B-ALL patients with multiply relapsed and refractory disease and normal karyotype or low risk cytogenetics, such as hyperdiploidy. Notably, cell surface ROR1 was virtually absent in normal adult and pediatric tissues.Collectively, this study suggests that ROR1 merits preclinical and clinical investigations as a novel target for mAb-based therapies in pediatric B-ALL. We propose cell surface expression of ROR1 detected by flow cytometry as primary inclusion criterion for pediatric B-ALL patients in future clinical trials of ROR1-targeted therapies.

  14. Post-translational Modification of the NKG2D Ligand RAET1G Leads to Cell Surface Expression of a Glycosylphosphatidylinositol-linked Isoform*

    Science.gov (United States)

    Ohashi, Maki; Eagle, Robert A.; Trowsdale, John

    2010-01-01

    NKG2D is an important activating receptor on lymphocytes. In human, it interacts with two groups of ligands: the major histocompatibility complex class I chain-related A/B (MICA/B) family and the UL-16 binding protein (ULBP) family, also known as retinoic acid early transcript (RAET1). MIC proteins are membrane-anchored, but all of the ULBP/RAET1 proteins, except for RAET1E and RAET1G, are glycosylphosphatidylinositol (GPI)-anchored. To address the reason for these differences we studied the association of RAET1G with the membrane. Using epitope-tagged RAET1G protein in conjunction with antibodies to different parts of the molecule and in pulse-chase experiments, we showed that the C terminus of the protein was cleaved soon after protein synthesis. Endoglycosidase H and peptide N-glycosidase treatment and cell surface immunoprecipitation indicated that most of the protein stayed in the endoplasmic reticulum, but some of the cleaved form was modified in the Golgi and transported to the cell surface. We examined the possibility of GPI anchoring of the protein in three ways: (i) Phosphatidylinositol (PI)-specific phospholipase C released the PI-linked form of the protein. (ii) The surface expression pattern of RAET1G decreased in cells defective in GPI anchoring through mutant GPI-amidase. (iii) Site-directed mutagenesis, to disrupt residues predicted to facilitate GPI-anchoring, resulted in diminished surface expression of RAET1G. Thus, a form of RAET1G is GPI-anchored, in line with most other ULBP/RAET1 family proteins. The cytoplasmic tail and transmembrane domains appear to result from gene duplication and frameshift mutation. Together with our previous results, our data suggest that RAET1G is regulated post-translationally to produce a GPI-anchored isoform. PMID:20304922

  15. Post-translational modification of the NKG2D ligand RAET1G leads to cell surface expression of a glycosylphosphatidylinositol-linked isoform.

    Science.gov (United States)

    Ohashi, Maki; Eagle, Robert A; Trowsdale, John

    2010-05-28

    NKG2D is an important activating receptor on lymphocytes. In human, it interacts with two groups of ligands: the major histocompatibility complex class I chain-related A/B (MICA/B) family and the UL-16 binding protein (ULBP) family, also known as retinoic acid early transcript (RAET1). MIC proteins are membrane-anchored, but all of the ULBP/RAET1 proteins, except for RAET1E and RAET1G, are glycosylphosphatidylinositol (GPI)-anchored. To address the reason for these differences we studied the association of RAET1G with the membrane. Using epitope-tagged RAET1G protein in conjunction with antibodies to different parts of the molecule and in pulse-chase experiments, we showed that the C terminus of the protein was cleaved soon after protein synthesis. Endoglycosidase H and peptide N-glycosidase treatment and cell surface immunoprecipitation indicated that most of the protein stayed in the endoplasmic reticulum, but some of the cleaved form was modified in the Golgi and transported to the cell surface. We examined the possibility of GPI anchoring of the protein in three ways: (i) Phosphatidylinositol (PI)-specific phospholipase C released the PI-linked form of the protein. (ii) The surface expression pattern of RAET1G decreased in cells defective in GPI anchoring through mutant GPI-amidase. (iii) Site-directed mutagenesis, to disrupt residues predicted to facilitate GPI-anchoring, resulted in diminished surface expression of RAET1G. Thus, a form of RAET1G is GPI-anchored, in line with most other ULBP/RAET1 family proteins. The cytoplasmic tail and transmembrane domains appear to result from gene duplication and frameshift mutation. Together with our previous results, our data suggest that RAET1G is regulated post-translationally to produce a GPI-anchored isoform.

  16. Divalent cations and the protein surface co-ordinate the intensity of human platelet adhesion and P-selectin surface expression.

    Science.gov (United States)

    Whiss, P A; Andersson, R G G

    2002-07-01

    At sites of blood vessel injury, platelets adhere to exposed vessel components, such as collagen, or immobilized fibrinogen derived from plasma or activated platelets. The divalent cations Mg(2+) and Ca(2+) are essential for platelet adhesion and activation, but Mg(2+) can also inhibit platelet activation. The present study evaluates, by an enzymatic method, the effects of various divalent cations on the adhesion of isolated human platelets to collagen, fibrinogen, albumin or plastic in vitro. By enzyme-linked immunosorbent assay, platelet surface expression of P-selectin was measured to estimate the state of activation on adherence. Mg(2+) increased platelet adhesion exclusively to collagen and fibrinogen at physiologically relevant concentrations. At higher concentrations, the adhesion declined. Ca(2+) induced a weak adhesion only to fibrinogen at physiological doses and a peak of increased adhesion to all protein-coated surfaces at 10 mmol/l. Mn(2+) elicited dose-dependent adhesion only to collagen and fibrinogen. Zn(2+), Ni(2+) and Cu(2+) increased the adhesion of platelets independently of the surface. Ca(2+) dose-dependently inhibited adhesion elicited by Mg(2+) to collagen and fibrinogen. No other combination of divalent cations elicited such an effect. Mg(2+)-dependent platelet adhesion to collagen and Ca(2+)-dependent adhesion to fibrinogen increased P-selectin expression. Thus, the present study shows that the outcome of the platelet adhesion depends on the surface and the access of divalent cations, which co-ordinate the intensity of platelet adhesion and P-selectin surface expression.

  17. Transmembrane and ubiquitin-like domain-containing protein 1 (Tmub1/HOPS facilitates surface expression of GluR2-containing AMPA receptors.

    Directory of Open Access Journals (Sweden)

    Hyunjeong Yang

    Full Text Available Some ubiquitin-like (UBL domain-containing proteins are known to play roles in receptor trafficking. Alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid receptors (AMPARs undergo constitutive cycling between the intracellular compartment and the cell surface in the central nervous system. However, the function of UBL domain-containing proteins in the recycling of the AMPARs to the synaptic surface has not yet been reported.Here, we report that the Transmembrane and ubiquitin-like domain-containing 1 (Tmub1 protein, formerly known as the Hepatocyte Odd Protein Shuttling (HOPS protein, which is abundantly expressed in the brain and which exists in a synaptosomal membrane fraction, facilitates the recycling of the AMPAR subunit GluR2 to the cell surface. Neurons transfected with Tmub1/HOPS-RNAi plasmids showed a significant reduction in the AMPAR current as compared to their control neurons. Consistently, the synaptic surface expression of GluR2, but not of GluR1, was significantly decreased in the neurons transfected with the Tmub1/HOPS-RNAi and increased in the neurons overexpressing EGFP-Tmub1/HOPS. The altered surface expression of GluR2 was speculated to be due to the altered surface-recycling of the internalized GluR2 in our recycling assay. Eventually, we found that GluR2 and glutamate receptor interacting protein (GRIP were coimmunoprecipitated by the anti-Tmub1/HOPS antibody from the mouse brain. Taken together, these observations show that the Tmub1/HOPS plays a role in regulating basal synaptic transmission; it contributes to maintain the synaptic surface number of the GluR2-containing AMPARs by facilitating the recycling of GluR2 to the plasma membrane.

  18. Sol-gel niobia sorbent with a positively charged octadecyl ligand providing enhanced enrichment of nucleotides and organophosphorus pesticides in capillary microextraction for online HPLC analysis.

    Science.gov (United States)

    Kesani, Sheshanka; Malik, Abdul

    2018-04-01

    A niobia-based sol-gel organic-inorganic hybrid sorbent carrying a positively charged C 18 ligand (Nb 2 O 5 -C 18 (+ve)) was synthesized to achieve enhanced enrichment capability in capillary microextraction of organophosphorus compounds (which include organophosphorus pesticides and nucleotides) before their online analysis by high-performance liquid chromatography. The sorbent was designed to simultaneously provide three different types of molecular level interactions: electrostatic, Lewis acid-base, and van der Waals interactions. To understand relative contributions of various molecular level analyte-sorbent interactions in the extraction process, two other sol-gel niobia sorbents were also created: (a) a purely inorganic sol-gel niobia sorbent (Nb 2 O 5 ) and (b) an organic-inorganic hybrid sol-gel niobia sorbent carrying an electrically neutral-bonded octadecyl ligand (Nb 2 O 5 -C 18 ). The extraction efficiency of the created sol-gel niobia sorbent (Nb 2 O 5 -C 18 (+ve)) was compared with that of analogously designed and synthesized titania-based sol-gel sorbent (TiO 2 -C 18 (+ve)), taking into consideration that titania-based sorbents present state-of-the-art extraction media for organophosphorus compounds. In capillary microextraction with high-performance liquid chromatography analysis, Nb 2 O 5 -C 18 (+ve) had shown 40-50% higher specific extraction values (a measure of extraction efficiency) over that of TiO 2 -C 18 (+ve). Compared to TiO 2 -C 18 (+ve), Nb 2 O 5 -C 18 (+ve) also provided superior analyte desorption efficiency (96 vs. 90%) during the online release of the extracted organophosphorus pesticides from the sorbent coating in the capillary microextraction capillary to the chromatographic column using reversed-phase high-performance liquid chromatography mobile phase. © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  19. Comparison between GC-MS-SIM and GC-ECD for the determination of residues of organochlorine and organophosphorus pesticides in Brazilian citrus essential oils

    OpenAIRE

    Alves, Andréa A. R.; Rezende, Michelle J. C.; Hovell, Ana M. C.; Bizzo, Humberto R.; Oliveira, Ana Carolina L.; Rodrigues, Silvana V.; Rezende, Claudia M.

    2012-01-01

    Brazilian citrus essential oils (mandarin, orange, lemon, bergamot and tangerine) were analyzed for the determination of pesticides residues of organochlorine (aldrin, chlordane, dieldrin and dicofol) and organophosphorus (methyl chlorpyrifos, dimethoate, methidathion and methyl parathion) . A comparative study between gas chromatography-mass spectrometry in selective ion monitoring mode (GC-MS-SIM) and gas chromatography-electron capture detector (GC-ECD) was performed using solid phase extr...

  20. Interspecies differences in the enantioselectivity of epoxide hydrolases in Cryptococcus laurentii (Kufferath) C.E. Skinner and Cryptococcus podzolicus (Bab'jeva & Reshetova) Golubev

    CSIR Research Space (South Africa)

    Botes, AL

    2005-01-01

    Full Text Available Isolates representing Cryptococcus laurentii and Cryptococcus podzolicus, originating from soil of a heath land indigenous to South Africa, were screened for the presence of enantioselective epoxide hydrolases for 2, 2-disubstituted epoxides...

  1. Epoxide hydrolase-catalyzed enantioselective conversion of trans-stilbene oxide: Insights into the reaction mechanism from steady-state and pre-steady-state enzyme kinetics

    Czech Academy of Sciences Publication Activity Database

    Archelas, A.; Zhao, W.; Faure, B.; Iacazio, G.; Kotík, Michael

    2016-01-01

    Roč. 591, FEB 2016 (2016), s. 66-75 ISSN 0003-9861 Institutional support: RVO:61388971 Keywords : Catalytic mechanism * Epoxide hydrolase * Electrophilic catalysis Subject RIV: CE - Biochemistry Impact factor: 3.165, year: 2016

  2. [Feasibility investigation of hydrogen instead of helium as carrier gas in the determination of five organophosphorus pesticides by gas chromatography-mass spectrometry].

    Science.gov (United States)

    Liu, Zhenxue; Zhou, Shixue

    2015-01-01

    Helium is almost the only choosable carrier gas used in gas chromatography-mass spectrometry (GC-MS). A mixed standard solution of five organophosphorus pesticides was analyzed by using GC-MS, and hydrogen or helium as carrier gas, so as to study the feasibility of hydrogen instead of helium as carrier gas for the determination of organophosphorus pesticides. Combining a mass spectrum database built by ourselves, the results were deconvolved and identified by Automated Mass Spectral Deconvolution & Identification System (AMDIS32), a software belonging to the workstation of the instrument. Then, the statistical software, IBM SPSS Statistics 19.0 was used for the clustering analysis of the data. The results indicated that when hydrogen was used as carrier gas, the peaks of the pesticides detected were slightly earlier than those when helium used as carrier gas, but the resolutions of the chromatographic peaks were lower, and the fraction good indices (Frac. Good) were lower, too. When hydrogen was used as carrier gas, the signals of the pesticides were unstable, the measuring accuracies of the pesticides were reduced too, and even more, some compounds were undetectable. Therefore, considering the measuring accuracy, the signal stability, and the safety, etc., hydrogen should be cautiously used as carrier gas in the determination of organophosphorus pesticides by GC-MS.

  3. Use of selective silver impregnation of neuronal degeneration as a biomarker for assessing organophosphorus-induced neuropathy in the central nervous system. A review

    Energy Technology Data Exchange (ETDEWEB)

    Tanaka, D. Jr.; Bursian, S.J. [Michigan State Univ., East Lansing, MI (United States)

    1998-12-31

    Exposure to selected groups of organophosphorus compounds has been shown to result in neuronal degeneration in both avian and mammalian nervous systems. Previous investigators, using hematoxylin and eosin stains, concluded that the degeneration resulting from exposure to these compounds was restricted to peripheral nerves and to long fiber tracts of the spinal cord and lower brainstem. The authors re-examined the results of exposure to the organophosphorus compounds tri-Q-tolyl phosphate, diisopropylphosphorofluoridate, and triphenyl phosphite using a variant of the Fink-Heimer silver impregnation method. This method selectively stains degenerating axions and axon terminals. Chickens, Japanese quail, rats, and mature and immature ferrets were used in these studies because of their differential clinical susceptibility to organophosphorus compounds. Their results indicate that exposure to each compound may result in a variable amount of degeneration within selected nuclei and tracts in the brain and spinal cord. The amount of degeneration seen in each instance corresponds well with the severity of the resultant clinical signs. In addition, the locus and severity of neuronal degeneration is dependent on the particular compound used, the species of animal exposed, and the age of the animal at the time of exposure.

  4. Comparison of the Efficiency of Simultaneous Application of UV/O3 for the Removal of Organophosphorus and Carbamat Pesticides in Aqueous Solutions

    Directory of Open Access Journals (Sweden)

    Mohammad Taghi Samadi

    2010-03-01

    Full Text Available A vast variety of pesticides are used for agricultural pests in Iran. The release of these persistent organic pollutants. into water supplies leaves adverse effects on both the environment and public health. Advanced oxidation processes have been used recently for pesticide removal. In this research, the combined UV/O3 process has been investigated for the removal of organophosphorus pesticides (Diazinon, Chlorpyrifos, Carbamate pesticides (carbaryl. In this survey, samples have been prepared by adding given concentration (1, 5, 10, 15, 20 mg/L of the pesticides to deionized water. The samples at separation periods were exposed to the combined UV/O3 (UV=50-200 Wm-2 and O3 = 1g hr-1in a bath  reactor at different pH levels (6, 7, 9 and for different contact times (0.5,1,1.5,2 hr and the removal efficiencies were determined. Residual concentrations were determined using GC/MS/MS and HPLC.  Based on the results, increasing pH reduced pesticide concentration and increased contact time had a direct effect on enhancing removal efficiency. The combined UV/O3 process was found to have a high efficiency (>80% in degrading both halogenated Organophosphorus(Chlorpyrifos and non- halogenated Organophosphorus (Diazinon pesticides. Its removal efficiency for degrading carbamate pesticide (Carbari was found to be >90%. Based on our results, this method may be suggested for the removal of pesticides from aqueous solutions.

  5. Genetic variants in microsomal epoxide hydrolase and N-acetyltransferase 2 in susceptibility of IBD in the Danish population

    DEFF Research Database (Denmark)

    Ernst, Anja; Andersen, Vibeke; Østergaard, Mette

    , or severity of disease measured either as need for surgery or azathioprine treatment. Smoking was found to be a risk factor of CD (OR=1.8(1.4; 2.3) Pfactor regarding UC (0.7 (0.5-0.9) P=0.02) which is in agreement with previous findings in other study...... induce or sustain an immune response. Changes in detoxification of substances that causes epithelial damage may confer susceptibility to IBD. Hence, polymorphic enzymes involved in the detoxification processes may be risk factors of IBD. Methods. The two biotransformation enzymes microsomal epoxide...... hydrolase and N-acetyltransferase 2 were genotyped using TaqMan based Real-Time PCR in 388 patients with Crohn's disease (CD), 565 patients with ulcerative colitis (UC) and 796 healthy Danish controls. Results. No association was found between low microsomal epoxide hydrolase activity or slow N...

  6. Colloid-based multiplexed method for screening plant biomass-degrading glycoside hydrolase activities in microbial communities

    Energy Technology Data Exchange (ETDEWEB)

    Reindl, W.; Deng, K.; Gladden, J.M.; Cheng, G.; Wong, A.; Singer, S.W.; Singh, S.; Lee, J.-C.; Yao, J.-S.; Hazen, T.C.; Singh, A.K; Simmons, B.A.; Adams, P.D.; Northen, T.R.

    2011-05-01

    The enzymatic hydrolysis of long-chain polysaccharides is a crucial step in the conversion of biomass to lignocellulosic biofuels. The identification and characterization of optimal glycoside hydrolases is dependent on enzyme activity assays, however existing methods are limited in terms of compatibility with a broad range of reaction conditions, sample complexity, and especially multiplexity. The method we present is a multiplexed approach based on Nanostructure-Initiator Mass Spectrometry (NIMS) that allowed studying several glycolytic activities in parallel under diverse assay conditions. Although the substrate analogs carried a highly hydrophobic perfluorinated tag, assays could be performed in aqueous solutions due colloid formation of the substrate molecules. We first validated our method by analyzing known {beta}-glucosidase and {beta}-xylosidase activities in single and parallel assay setups, followed by the identification and characterization of yet unknown glycoside hydrolase activities in microbial communities.

  7. Simultaneous hydrolysis of carbaryl and chlorpyrifos by Stenotrophomonas sp. strain YC-1 with surface-displayed carbaryl hydrolase

    OpenAIRE

    Yang, Chao; Xu, Xiaoqing; Liu, Yanping; Jiang, Hong; Wu, Yunbo; Xu, Ping; Liu, Ruihua

    2017-01-01

    Many sites are often co-contaminated with multiple pesticides. To date, there are no reports on simultaneous degradation of different classes of pesticides by a natural microorganism. In this work, we aim at constructing a live biocatalyst able to simultaneously hydrolyze carbaryl and chlorpyrifos. For this purpose, carbaryl hydrolase (CH) was displayed on the cell surface of a chlorpyrifos-degrading bacterium Stenotrophomonas sp. strain YC-1 using N- and C-terminal domain of ice nucleation p...

  8. A Missense Mutation (Q279R) in the Fumarylacetoacetate Hydrolase Gene, Responsible for Hereditary Tyrosinemia, Acts as a Splicing Mutation

    OpenAIRE

    Dreumont, Natacha; Poudrier, Jacques A; Bergeron, Anne; Baklouti, Faouzi; Tanguay, Robert M; Levy, Harvey Louis

    2001-01-01

    Abstract Background Tyrosinemia type I, the most severe disease of the tyrosine catabolic pathway is caused by a deficiency in fumarylacetoacetate hydrolase (FAH). A patient showing few of the symptoms associated with the disease, was found to be a compound heterozygote for a splice mutation, IVS6-1g->t, and a putative missense mutation, Q279R. Analysis of FAH expression in liver sections obtained after resection for hepatocellular carcinoma revealed a mosaic pattern of expression. No FAH was...

  9. The Natural Product Acivicin as a Tool for ABPP and the Activity of Serine Hydrolases in Uterine Fibroids

    OpenAIRE

    Kreuzer, Johannes

    2015-01-01

    The target proteins of acivicin and structure derived probes in tumor cells were identified using activity-based protein profiling. The target proteins were further characterized and their relation to the antitumor activity of acivicin pointed out. In a further project, the activity of serine hydrolases in myoma and myometrium was examined from tissue samples. This revealed a different activity of mast cell proteases. Mittels Activity-based Protein Profiling wurde eine Identifikation der Z...

  10. How does (E-2-(acetamidomethylenesuccinate bind to its hydrolase? From the binding process to the final result.

    Directory of Open Access Journals (Sweden)

    Ji-Long Zhang

    Full Text Available The binding of (E-2-(acetamidomethylenesuccinate (E-2AMS to E-2AMS hydrolase is crucial for biological function of the enzyme and the last step reaction of vitamin B(6 biological degradation. In the present study, several molecular simulation methods, including molecular docking, conventional molecular dynamics (MD, steered MD (SMD, and free energy calculation methods, were properly integrated to investigate the detailed binding process of E-2AMS to its hydrolase and to assign the optimal enzyme-substrate complex conformation. It was demonstrated that the substrate binding conformation with trans-form amide bond is energetically preferred conformation, in which E-2AMS's pose not only ensures hydrogen bond formation of its amide oxygen atom with the vicinal oxyanion hole but also provides probability of the hydrophobic interaction between its methyl moiety and the related enzyme's hydrophobic cavity. Several key residues, Arg146, Arg167, Tyr168, Arg179, and Tyr259, orientate the E-2AMS's pose and stabilize its conformation in the active site via the hydrogen bond interaction with E-2AMS. Sequentially, the binding process of E-2AMS to E-2AMS hydrolase was studied by SMD simulation, which shows the surprising conformational reversal of E-2AMS. Several important intermediate structures and some significant residues were identified in the simulation. It is stressed that Arg146 and Arg167 are two pivotal residues responsible for the conformational reversal of E-2AMS in the binding or unbinding. Our research has shed light onto the full binding process of the substrate to E-2AMS hydrolase, which could provide more penetrating insight into the interaction of E-2AMS with the enzyme and would help in the further exploration on the catalysis mechanism.

  11. Parabens inhibit fatty acid amide hydrolase: A potential role in paraben-enhanced 3T3-L1 adipocyte differentiation

    OpenAIRE

    Kodani, Sean D.; Overby, Haley B.; Morisseau, Christophe; Chen, Jiangang; Zhao, Ling; Hammock, Bruce D.

    2016-01-01

    Parabens are a class of small molecules that are regularly used as preservatives in a variety of personal care products. Several parabens, including butylparaben and benzylparaben, have been found to interfere with endocrine signaling and to stimulate adipocyte differentiation. We hypothesized these biological effects could be due to interference with the endocannabinoid system and identified fatty acid amide hydrolase (FAAH) as the direct molecular target of parabens. FAAH inhibition by para...

  12. 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.

  13. Leishmania donovani Nucleoside Hydrolase terminal domains in cross-protective immunotherapy against Leishmania amazonensis murine infection

    Directory of Open Access Journals (Sweden)

    Dirlei eNico

    2014-06-01

    Full Text Available Nucleoside hydrolases of the Leishmania genus are vital enzymes for the replication of the DNA and conserved phylogenetic markers of the parasites. Leishmania donovani Nucleoside hydrolase (NH36 induced a main CD4+ T cell driven protective response against Leishmania chagasi infection in mice which is directed against its C-terminal domain. In this study, we used the three recombinant domains of NH36: N-terminal domain (F1, amino acids 1-103, central domain (F2 aminoacids 104-198 and C-terminal domain (F3 amino acids 199-314 in combination with saponin and assayed their immunotherapeutic effect on Balb/c mice previously infected with L. amazonensis. We identified that the F1 and F3 peptides determined strong cross-immunotherapeutic effects, reducing the size of footpad lesions to 48% and 64%, and the parasite load in footpads to 82.6% and 81%, respectively. The F3 peptide induced the strongest anti-NH36 antibody response and intradermal response (IDR against L. amazonenis and a high secretion of IFN-γ and TNF-α with reduced levels of IL-10. The F1 vaccine, induced similar increases of IgG2b antibodies and IFN-γ and TNF-α levels, but no IDR and no reduction of IL-10. The multiparameter flow cytometry analysis was used to assess the immune response after immunotherapy and disclosed that the degree of the immunotherapeutic effect is predicted by the frequencies of the CD4+ and CD8+ T cells producing IL-2 or TNF-α or both. Total frequencies and frequencies of double-cytokine CD4 T cell producers were enhanced by F1 and F3 vaccines. Collectively, our multifunctional analysis disclosed that immunotherapeutic protection improved as the CD4 responses progressed from 1+ to 2+, in the case of the F1 and F3 vaccines, and as the CD8 responses changed qualitatively from 1+ to 3+, mainly in the case of the F1 vaccine, providing new correlates of immunotherapeutic protection against cutaneous leishmaniasis in mice based on T-helper TH1 and CD8+ mediated

  14. Modulation of redox homeostasis under suboptimal conditions by Arabidopsis nudix hydrolase 7

    Directory of Open Access Journals (Sweden)

    Jambunathan Niranjani

    2010-08-01

    Full Text Available Abstract Background Nudix hydrolases play a key role in maintaining cellular homeostasis by hydrolyzing various nuceloside diphosphate derivatives and capped mRNAs. Several independent studies have demonstrated that Arabidopsis nudix hydrolase 7 (AtNUDT7 hydrolyzes NADH and ADP-ribose. Loss of function Atnudt7-1 mutant plants (SALK_046441 exhibit stunted growth, higher levels of reactive oxygen species, enhanced resistance to pathogens. However, using the same T-DNA line, two other groups reported that mutant plants do not exhibit any visible phenotypes. In this study we analyze plausible factors that account for differences in the observed phenotypes in Atnudt7. Secondly, we evaluate the biochemical and molecular consequences of increased NADH levels due to loss of function of AtNUDT7 in Arabidopsis. Results We identified a novel conditional phenotype of Atnudt7-1 knockout plants that was contingent upon nutrient composition of potting mix. In nutrient-rich Metro-Mix, there were no phenotypic differences between mutant and wild-type (WT plants. In the nutrient-poor mix (12 parts vermiculite: 3 parts Redi-earth and 1 part sand, mutant plants showed the characteristic stunted phenotype. Compared with WT plants, levels of glutathione, NAD+, NADH, and in turn NADH:NAD+ ratio were higher in Atnudt7-1 plants growing in 12:3:1 potting mix. Infiltrating NADH and ADP-ribose into WT leaves was sufficient to induce AtNUDT7 protein. Constitutive over-expression of AtNudt7 did not alter NADH levels or resistance to pathogens. Transcriptome analysis identified nearly 700 genes differentially expressed in the Atnudt7-1 mutant compared to WT plants grown in 12:3:1 potting mix. In the Atnudt7-1 mutant, genes associated with defense response, proteolytic activities, and systemic acquired resistance were upregulated, while gene ontologies for transcription and phytohormone signaling were downregulated. Conclusions Based on these observations, we conclude that the

  15. Determination of frequencies of alleles, associated with the pseudodeficiency of lysosomal hydrolases, in population of Ukraine.

    Science.gov (United States)

    Olkhovych, N V; Gorovenko, N G

    2016-01-01

    The pseudodeficiency of lysosomal hydrolases described as a significant reduction in enzyme activi­ty in vitro in clinically healthy individuals, can lead to diagnostic errors in the process of biochemical analysis of lysosomal storage disease in case of its combination with pathology of another origin. Pseudodeficiency is mostly caused by some non-pathogenic changes in the corresponding gene. These changes lead to the in vitro lability of the enzyme molecule, whereas in vivo the enzyme retains its functional activity. To assess the prevalence of the most common lysosomal hydrolases pseudodeficiency alleles in Ukraine, we have determined the frequency of alleles c.1055A>G and c.* 96A>G in the ARSA gene, substitutions c.739C>T (R247W) and c.745C>T (R249W) in the HEXA gene, c.1726G>A (G576S) and c.2065G>A (E689K) in the GAA gene, c.937G>T (D313Y) in the GLA1 gene and c.898G>A (A300T) in the IDUA gene in a group of 117 healthy individuals from different regions of the country and 14 heterozygous carriers of pathogenic mutations in the HEXA gene (parents of children with confirmed diagnosis of Tay-Sachs disease). The total frequency of haplotypes, associated with arylsulfatase A pseudodeficiency, in healthy people in Ukraine (c.1055G/c.*96G and c.1055G/c.*96A haplotypes) was 10.3%. The frequency of c.739C>T (R247W) allele, associated with hexo­saminidase A pseudodeficiency, among Tay-Sachs carriers from Ukraine was 7.1%. The total frequency of α-glucosidase pseudodeficiency haplotypes in healthy individuals in Ukraine (c.1726A/c.2065A and c.1726G/c.2065A haplotypes) was 2.6%. No person among examined individuals with the substitution c.937G>T (D313Y) in the GLA1 gene and c.898G>A (A300T) in the IDUA gene was found. The differential diagnostics of lysosomal storage diseases requires obligatory determination of the presence of the pseudodeficiency alleles, particularly the ones with high incidence in the total population. Ignoring phenomenon of pseudodeficiency may

  16. Determination of frequencies of alleles, associated with the pseudodeficiency of lysosomal hydrolases, in population of Ukraine

    Directory of Open Access Journals (Sweden)

    N. V. Olkhovych

    2016-10-01

    Full Text Available The pseudodeficiency of lysosomal hydrolases described as a significant reduction in enzyme activi­ty in vitro in clinically healthy individuals, can lead to diagnostic errors in the process of biochemical analysis of lysosomal storage disease in case of its combination with pathology of another origin. Pseudodeficiency is mostly caused by some non-pathogenic changes in the corresponding gene. These changes lead to the in vitro lability of the enzyme molecule, whereas in vivo the enzyme retains its functional activity. To assess the prevalence of the most common lysosomal hydrolases pseudodeficiency alleles in Ukraine, we have determined the frequency of alleles c.1055A>G and c.* 96A>G in the ARSA gene, substitutions c.739C>T (R247W and c.745C>T (R249W in the HEXA gene, c.1726G>A (G576S and c.2065G>A (E689K in the GAA gene, c.937G>T (D313Y in the GLA1 gene and c.898G>A (A300T in the IDUA gene in a group of 117 healthy individuals from different regions of the country and 14 heterozygous carriers of pathogenic mutations in the HEXA gene (parents of children with confirmed diagnosis of Tay-Sachs disease. The total frequency of haplotypes, associated with arylsulfatase A pseudodeficiency, in healthy people in Ukraine (c.1055G/c.*96G and c.1055G/c.*96A haplotypes was 10.3%. The frequency of c.739C>T (R247W allele, associated with hexo­saminidase A pseudodeficiency, among Tay-Sachs carriers from Ukraine was 7.1%. The total frequency of α-glucosidase pseudodeficiency haplotypes in healthy individuals in Ukraine (c.1726A/c.2065A and c.1726G/c.2065A haplotypes was 2.6%. No person among examined individuals with the substitution c.937G>T (D313Y in the GLA1 gene and c.898G>A (A300T in the IDUA gene was found. The differential diagnostics of lysosomal storage diseases requires obligatory determination of the presence of the pseudodeficiency alleles, particularly the ones with high incidence in the total population. Ignoring phenomenon of

  17. Lactase-phlorizin hydrolase and aminopeptidase N are differentially regulated in the small intestine of the pig

    DEFF Research Database (Denmark)

    Torp, Niels; Rossi, M; Troelsen, J T

    1993-01-01

    The longitudinal expression of two brush-border enzymes, lactase-phlorizin hydrolase (EC 3.2.1.23/62) and aminopeptidase N (EC 3.4.11.2), was studied in the small intestine of the post-weaned pig. Whereas the level of mRNA, encoding aminopeptidase N (relative to that of beta-actin), only varied...... moderately from the duodenum to the terminal ileum, the amount of lactase-phlorizin hydrolase mRNA exhibited a sharp maximum in the proximal jejunum. For both enzymes, the level of protein synthesis, studied in cultured mucosal explants, correlated well with the level of mRNA, and no major variation in post...... in the gut lumen of pancreatic proteases. In neonatal animals, the level of mRNA for lactase-phlorizin hydrolase in both proximal and distal regions of the intestine was of the same magnitude as in the proximal jejunum of the post-weaned pigs. Our results point to two mechanisms that affect the expression...

  18. An appraisal of eighteen commonly consumed edible plants as functional food based on their antioxidant and starch hydrolase inhibitory activities.

    Science.gov (United States)

    Lee, Yian Hoon; Choo, Candy; Watawana, Mindani I; Jayawardena, Nilakshi; Waisundara, Viduranga Y

    2015-11-01

    Eighteen edible plants were assessed for their antioxidant potential based on oxygen radical absorbance capacity (ORAC), 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging activity, total phenolics, vitamin C content and various lipophilic antioxidants. The inhibitory activities of the plant extracts against the enzymatic activities of α-amylase and α-glucosidase were also evaluated. The antioxidant and starch hydrolase activities of the plants varied widely across a single batch of analysis. The ORAC and DPPH radical scavenging EC50 values varied between 298 and 1984 Trolox equivalents g(-1) fresh weight and between 91 and 533 mg kg(-1) fresh weight, respectively. The total phenolics and vitamin C contents varied between 32 and 125 mg gallic acid equivalents g(-1) fresh weight and between 96 and 285 µg g(-1) fresh weight, respectively. All the plants contained neoxanthin, violaxanthin, and α- and β-carotene in varying amounts. Coccinia grandis, Asparagus racemosus, Costus speciosus, Amaranthus viridis and Annona muricata displayed the highest inhibitory activities against starch hydrolases. They were the most efficient against the breakdown of seven starches exposed to the two enzymes as well. Overall, the edible plants were observed to display a high antioxidant potential with starch hydrolase inhibitory properties, which were beneficial in their being recognized as functional food. © 2014 Society of Chemical Industry.

  19. Skin dryness in apparently healthy human skin is associated with decreased expression of bleomycin hydrolase in the stratum corneum.

    Science.gov (United States)

    Son, E D; Kim, Y; Joo, K M; Kim, H J; Lee, E; Nam, G W; Cho, E G; Noh, M; Chung, J H; Byun, S Y; Lee, T R

    2015-04-01

    Maintenance of water balance in the stratum corneum (SC) is determined by the content of intercellular lipids and natural moisturizing factors (NMFs) in corneocytes. To investigate the association between the NMFs and (pro)filaggrin and the proteases responsible for the processing of (pro)filaggrin to NMFs in the SC of hydrated and dry skin areas of healthy human subjects. The SC hydration state and the transepidermal water loss (TEWL) were measured using a Corneometer and a Tewameter, respectively. Proteases, (pro)filaggrin and NMFs were extracted from SC samples obtained by tape-stripping of the tested skin. Expression levels of (pro)filaggrin were determined by dot blotting and western blotting, and total NMFs by ultra-high performance liquid chromatography. Expression of the proteases caspase-14, calpain-1 and bleomycin hydrolase was measured by western blotting. The levels of (pro)filaggrin were not significantly different between hydrated and dry skin, whereas the level of total NMFs was significantly reduced in dry skin. A negative correlation between (pro)filaggrin and NMFs was found in dry skin (Pearson correlation coefficient r = - 0.57, *P < 0.05). Bleomycin hydrolase expression was significantly decreased in the SC of dry skin. These results suggest that the low hydration state of dry skin may be due to the reduction in (pro)filaggrin degradation caused by decreased bleomycin hydrolase expression. © 2014 British Association of Dermatologists.

  20. Identification and Characterization of a New Alkaline SGNH Hydrolase from a Thermophilic Bacterium Bacillus sp. K91.

    Science.gov (United States)

    Yu, Tingting; Ding, Junmei; Zheng, Qingxia; Han, Nanyu; Yu, Jialin; Yang, Yunjuan; Li, Junjun; Mu, Yuelin; Wu, Qian; Huang, Zunxi

    2016-04-28

    est19 is a gene from Bacillus sp. K91 that encodes a new esterase. A comparison of the amino acid sequence showed that Est19 has typical Ser-Gly-Asn-His (SGNH) family motifs and could be grouped into the SGNH hydrolase family. The Est19 protein was functionally cloned, and expressed and purified from Escherichia coli BL21(DE3). The enzyme activity was optimal at 60°C and pH 9.0, and displayed esterase activity towards esters with short-chain acyl esters (C₂-C₆). A structural model of Est19 was constructed using phospholipase A1 from Streptomyces albidoflavus NA297 as a template. The structure showed an α/β-hydrolase fold and indicated the presence of the typical catalytic triad Ser49-Asp227-His230, which were further investigated by site-directed mutagenesis. To the best of our knowledge, Est19 is a new member of the SGNH hydrolase family identified from thermophiles, which may be applicable in the industrial production of semisynthetic β-lactam antibiotics after modification.