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Sample records for active site serine

  1. Identification of the active site serine in pancreatic cholesterol esterase by chemical modification and site-specific mutagenesis.

    Science.gov (United States)

    DiPersio, L P; Fontaine, R N; Hui, D Y

    1990-10-05

    Chemical modification and site-specific mutagenesis approaches were used in this study to identify the active site serine residue of pancreatic cholesterol esterase. In the first approach, purified porcine pancreatic cholesterol esterase was covalently modified by incubation with [3H]diisopropylfluorophosphate (DFP). The radiolabeled cholesterol esterase was digested with CNBr, and the peptides were separated by high performance liquid chromatography. A single 3H-containing peptide was obtained for sequence determination. The results revealed the binding of DFP to a serine residue within the serine esterase homologous domain of the protein. Furthermore, the DFP-labeled serine was shown to correspond to serine residue 194 of rat cholesterol esterase (Kissel, J. A., Fontaine, R. N., Turck, C. W., Brockman, H. L., and Hui, D. Y. (1989) Biochim. Biophys. Acta 1006, 227-236). The codon for serine 194 in rat cholesterol esterase cDNA was then mutagenized to ACT or GCT to yield mutagenized cholesterol esterase with either threonine or alanine, instead of serine, at position 194. Expression of the mutagenized cDNA in COS-1 cells demonstrated that substitution of serine 194 with threonine or alanine abolished enzyme activity in hydrolyzing the water-soluble substrate, p-nitrophenyl butyrate, and the lipid substrates cholesteryl [14C]oleate and [14C] lysophosphatidylcholine. These studies definitively identified serine 194 in the catalytic site of pancreatic cholesterol esterase.

  2. Site-SpecificCu Labeling of the Serine Protease, Active Site Inhibited Factor Seven Azide (FVIIai-N), Using Copper Free Click Chemistry

    DEFF Research Database (Denmark)

    Jeppesen, Troels E; Kristensen, Lotte K; Nielsen, Carsten H

    2018-01-01

    A method for site-specific radiolabeling of the serine protease active site inhibited factor seven (FVIIai) with64Cu has been applied using a biorthogonal click reaction. FVIIai binds to tissue factor (TF), a trans-membrane protein involved in hemostasis, angiogenesis, proliferation, cell migrati...

  3. Active serine involved in the stabilization of the active site loop in the Humicola lanuginosa lipase

    DEFF Research Database (Denmark)

    Peters, Günther H.j.; Svendsen, A.; Langberg, H.

    1998-01-01

    , whereas only small changes are observed for I-Ill suggesting that the active site Lid in the latter opens more easily and hence more lipase molecules are bound to the liposomes. These observations are in agreement with molecular dynamics simulations and subsequent essential dynamics analyses. The results...

  4. A Camelid-derived Antibody Fragment Targeting the Active Site of a Serine Protease Balances between Inhibitor and Substrate Behavior*

    Science.gov (United States)

    Kromann-Hansen, Tobias; Oldenburg, Emil; Yung, Kristen Wing Yu; Ghassabeh, Gholamreza H.; Muyldermans, Serge; Declerck, Paul J.; Huang, Mingdong; Andreasen, Peter A.; Ngo, Jacky Chi Ki

    2016-01-01

    A peptide segment that binds the active site of a serine protease in a substrate-like manner may behave like an inhibitor or a substrate. However, there is sparse information on which factors determine the behavior a particular peptide segment will exhibit. Here, we describe the first x-ray crystal structure of a nanobody in complex with a serine protease. The nanobody displays a new type of interaction between an antibody and a serine protease as it inserts its complementary determining region-H3 loop into the active site of the protease in a substrate-like manner. The unique binding mechanism causes the nanobody to behave as a strong inhibitor as well as a poor substrate. Intriguingly, its substrate behavior is incomplete, as 30–40% of the nanobody remained intact and inhibitory after prolonged incubation with the protease. Biochemical analysis reveals that an intra-loop interaction network within the complementary determining region-H3 of the nanobody balances its inhibitor versus substrate behavior. Collectively, our results unveil molecular factors, which may be a general mechanism to determine the substrate versus inhibitor behavior of other protease inhibitors. PMID:27226628

  5. Serine deprivation enhances antineoplastic activity of biguanides.

    Science.gov (United States)

    Gravel, Simon-Pierre; Hulea, Laura; Toban, Nader; Birman, Elena; Blouin, Marie-José; Zakikhani, Mahvash; Zhao, Yunhua; Topisirovic, Ivan; St-Pierre, Julie; Pollak, Michael

    2014-12-15

    Metformin, a biguanide widely used in the treatment of type II diabetes, clearly exhibits antineoplastic activity in experimental models and has been reported to reduce cancer incidence in diabetics. There are ongoing clinical trials to evaluate its antitumor properties, which may relate to its fundamental activity as an inhibitor of oxidative phosphorylation. Here, we show that serine withdrawal increases the antineoplastic effects of phenformin (a potent biguanide structurally related to metformin). Serine synthesis was not inhibited by biguanides. Instead, metabolic studies indicated a requirement for serine to allow cells to compensate for biguanide-induced decrease in oxidative phosphorylation by upregulating glycolysis. Furthermore, serine deprivation modified the impact of metformin on the relative abundance of metabolites within the citric acid cycle. In mice, a serine-deficient diet reduced serine levels in tumors and significantly enhanced the tumor growth-inhibitory actions of biguanide treatment. Our results define a dietary manipulation that can enhance the efficacy of biguanides as antineoplastic agents that target cancer cell energy metabolism. ©2014 American Association for Cancer Research.

  6. Serine integrase chimeras with activity in E. coli and HeLa cells

    Directory of Open Access Journals (Sweden)

    Alfonso P. Farruggio

    2014-09-01

    Full Text Available In recent years, application of serine integrases for genomic engineering has increased in popularity. The factor-independence and unidirectionality of these large serine recombinases makes them well suited for reactions such as site-directed vector integration and cassette exchange in a wide variety of organisms. In order to generate information that might be useful for altering the specificity of serine integrases and to improve their efficiency, we tested a hybridization strategy that has been successful with several small serine recombinases. We created chimeras derived from three characterized members of the serine integrase family, phiC31, phiBT1, and TG1 integrases, by joining their amino- and carboxy-terminal portions. We found that several phiBT1-phiC31 (BC and phiC31-TG1 (CT hybrid integrases are active in E. coli. BC chimeras function on native att-sites and on att-sites that are hybrids between those of the two donor enzymes, while CT chimeras only act on the latter att-sites. A BC hybrid, BC{−1}, was also active in human HeLa cells. Our work is the first to demonstrate chimeric serine integrase activity. This analysis sheds light on integrase structure and function, and establishes a potentially tractable means to probe the specificity of the thousands of putative large serine recombinases that have been revealed by bioinformatics studies.

  7. Fibrin(ogen)olytic activity of bumblebee venom serine protease

    International Nuclear Information System (INIS)

    Qiu Yuling; Choo, Young Moo; Yoon, Hyung Joo; Jia Jingming; Cui Zheng; Wang Dong; Kim, Doh Hoon; Sohn, Hung Dae; Jin, Byung Rae

    2011-01-01

    Bee venom is a rich source of pharmacologically active components; it has been used as an immunotherapy to treat bee venom hypersensitivity, and venom therapy has been applied as an alternative medicine. Here, we present evidence that the serine protease found in bumblebee venom exhibits fibrin(ogen)olytic activity. Compared to honeybee venom, bumblebee venom contains a higher content of serine protease, which is one of its major components. Venom serine proteases from bumblebees did not cross-react with antibodies against the honeybee venom serine protease. We provide functional evidence indicating that bumblebee (Bombus terrestris) venom serine protease (Bt-VSP) acts as a fibrin(ogen)olytic enzyme. Bt-VSP activates prothrombin and directly degrades fibrinogen into fibrin degradation products. However, Bt-VSP is not a plasminogen activator, and its fibrinolytic activity is less than that of plasmin. Taken together, our results define roles for Bt-VSP as a prothrombin activator, a thrombin-like protease, and a plasmin-like protease. These findings offer significant insight into the allergic reaction sequence that is initiated by bee venom serine protease and its potential usefulness as a clinical agent in the field of hemostasis and thrombosis. - Graphical abstract: Display Omitted Highlights: → Bumblebee venom serine protease (Bt-VSP) is a fibrin(ogen)olytic enzyme. → Bt-VSP activates prothrombin. → Bt-VSP directly degrades fibrinogen into fibrin degradation products. → Bt-VSP is a hemostatically active protein that is a potent clinical agent.

  8. ACTIVATION OF A CRYPTIC D-SERINE DEAMINASE (DSD) GENE FROM PSEUDOMONAS CEPACIA 17616

    Science.gov (United States)

    D-serine inhibits growth of P. cepacia 17616; however, resistant mutants able to express an ordinarily cryptic D-serine deaminase (dsd) gene were isolated readily. The resistant strains formed high levels of a D-serine deaminase active on D-threonine as well as D-serine. IS eleme...

  9. Snake venom serine proteinases specificity mapping by proteomic identification of cleavage sites.

    Science.gov (United States)

    Zelanis, André; Huesgen, Pitter F; Oliveira, Ana Karina; Tashima, Alexandre K; Serrano, Solange M T; Overall, Christopher M

    2015-01-15

    Many snake venom toxins are serine proteases but their specific in vivo targets are mostly unknown. Various act on components of the coagulation cascade, and fibrinolytic and kallikrein-kinin systems to trigger various pathological effects observed in the envenomation. Despite showing high similarity in terms of primary structure snake venom serine proteinases (SVSPs) show exquisite specificity towards macromolecular substrates. Therefore, the characterization of their peptide bond specificity is important for understanding the active site preference associated with effective proteolysis as well as for the design of peptide substrates and inhibitors. Bothrops jararaca contains various SVSPs among which Bothrops protease A is a specific fibrinogenolytic agent and PA-BJ is a platelet-activating enzyme. In this study we used proteome derived peptide libraries in the Proteomic Identification of protease Cleavage Sites (PICS) approach to explore the peptide bond specificity of Bothrops protease A and PA-BJ in order to determine their individual peptide cleavage sequences. A total of 371 cleavage sites (208 for Bothrops protease A and 163 for PA-BJ) were detected and both proteinases displayed a clear preference for arginine at the P1 position. Moreover, the analysis of the specificity profiles of Bothrops protease A and PA-BJ revealed subtle differences in the preferences along P6-P6', despite a common yet unusual preference for Pro at P2. Taken together, these results map the subsite specificity of both SVSPs and shed light in the functional differences between these proteinases. Proteolysis is key to various pathological effects observed upon envenomation by viperid snakes. The use of the Proteomic Identification of protease Cleavage Sites (PICS) approach for the easy mapping of proteinase subsite preferences at both the prime- and non-prime sides concurrently gives rise to a fresh understanding of the interaction of the snake venom serine proteinases with peptide and

  10. Antibacterial activity of silver nanoparticles synthesized from serine

    Energy Technology Data Exchange (ETDEWEB)

    Jayaprakash, N. [Catalysis and Nanomaterials Research Laboratory, Department of Chemistry, Loyola College, Chennai 600 034 (India); SRM Valliammai Engineering College, Department of Chemistry, Chennai 603 203 (India); Judith Vijaya, J., E-mail: jjvijayaloyola@yahoo.co.in [Catalysis and Nanomaterials Research Laboratory, Department of Chemistry, Loyola College, Chennai 600 034 (India); John Kennedy, L. [Materials Division, School of Advanced Sciences, VIT University, Chennai Campus, Chennai 600 048 (India); Priadharsini, K.; Palani, P. [Department of Center for Advanced Study in Botany, University of Madras, Guindy Campus, Chennai 600 025 (India)

    2015-04-01

    Silver nanoparticles (Ag NPs) were synthesized by a simple microwave irradiation method using polyvinyl pyrrolidone (PVP) as a capping agent and serine as a reducing agent. UV–Visible spectra were used to confirm the formation of Ag NPs by observing the surface plasmon resonance (SPR) band at 443 nm. The emission spectrum of Ag NPs showed an emission band at 484 nm. In the presence of microwave radiation, serine acts as a reducing agent, which was confirmed by Fourier transformed infrared (FT-IR) spectrum. High-resolution transmission electron microscopy (HR-TEM) and high-resolution scanning electron microscopy (HR-SEM) were used to investigate the morphology of the synthesized sample. These images showed the sphere-like morphology. The elemental composition of the sample was determined by the energy dispersive X-ray analysis (EDX). Selected area electron diffraction (SAED) was used to find the crystalline nature of the Ag NPs. The electrochemical behavior of the synthesized Ag NPs was analyzed by the cyclic voltammetry (CV). Antibacterial experiments showed that the prepared Ag NPs showed relatively similar antibacterial activities, when compared with AgNO{sub 3} against Gram-positive and Gram-negative bacteria. - Highlights: • Microwave irradiation method is used to synthesize silver nanoparticles. • Highly stable silver nanoparticles are produced from serine. • A detailed study of antibacterial activities is discussed. • Formation mechanism of silver microspheres has been proposed.

  11. Modulation of glycine sites enhances social memory in rats using PQQ combined with d-serine.

    Science.gov (United States)

    Zhou, Xingqin; Liu, Dong; Zhang, Rongjun; Peng, Ying; Qin, Xiaofeng; Mao, Shishi

    2016-07-15

    The aim of study was to investigate the effects of pyrroloquinoline quinone (PQQ) combined with d-serine on the modulation of glycine sites in the brain of rats using social recognition test. Rats were divided into seven groups (n=10) and given repeated intraperitoneal (ip) injections of saline, MK-801 (0.5mg/kg), clozapine (1mg/kg), haloperidol (0.1mg/kg), d-serine (0.8g/kg), PQQ (2.0μg/kg), or d-serine (0.4g/kg) combined with PQQ (1.0μg/kg) for seven days. A social recognition test, including assessment of time-dependent memory impairment, was performed. A non-competitive NMDA receptor antagonist, MK-801, significantly impaired social memory, and this impairment was significantly repaired with an atypical antipsychotic (clozapine) but not with a typical antipsychotic (haloperidol). Likewise, d-serine combined with PQQ significantly improved MK-801-disrupted cognition in naïve rats, whereas haloperidol was ineffective. The present results show that the co-agonist NMDA receptor treated with PQQ and d-serine enhances social memory and may be an effective approach for treating the cognitive dysfunction observed in schizophrenic patients. PQQ stimulates glycine modulatory sites by which it may antagonize indirectly by removing glycine from the synaptic cleft or by binding the unsaturated site with d-serine in the brain, providing the insights into future research of central nervous system and drug discovery. Copyright © 2016 Elsevier B.V. All rights reserved.

  12. The Membrane-anchored Serine Protease Prostasin (CAP1/PRSS8) Supports Epidermal Development and Postnatal Homeostasis Independent of Its Enzymatic Activity

    DEFF Research Database (Denmark)

    Peters, Diane E; Szabo, Roman; Friis, Stine

    2014-01-01

    . Prostasin null (Prss8(-/-)) mice lack barrier formation and display fatal postnatal dehydration. In sharp contrast, mice homozygous for a point mutation in the Prss8 gene, which causes the substitution of the active site serine within the catalytic histidine-aspartate-serine triad with alanine and renders...

  13. Serine carboxypeptidases of Triatoma brasiliensis (Hemiptera, Reduviidae): Sequence characterization, expression pattern and activity localization.

    Science.gov (United States)

    Waniek, Peter J; Araújo, Catarina A C; Momoli, Marisa M; Azambuja, Patricia; Jansen, Ana M; Genta, Fernando A

    2014-04-01

    Using specific oligonucleotides, 5'- and 3'-RACE and sequencing, two cDNAs encoding serine carboxypeptidases (tbscp-1 and tbscp-2) from the midgut of the blood sucking heteropteran Triatoma brasiliensis were identified. Both cDNAs with an open reading frame of 1389bp, encode serine carboxypeptidase precursors of 463 amino acid residues, which possess a signal peptide cleavage site after Ala19. Analysis of tbscp-1 and tbscp-2 genomic DNA showed an absence of introns in both sequences and the presence of a further intron-free SCP encoding gene (tbscp-2b). By reverse transcription polymerase chain reaction (RT-PCR), tbscp-1 and tbscp-2 transcript abundance was found similarly in fifth instar nymphs at different days after feeding (daf), high in the posterior midgut (small intestine), lower in the anterior midgut (stomach) and fat body and almost undetectable in the salivary glands. In the anterior, middle and posterior regions of the small intestine at 5daf the transcript abundance of both genes was almost identical. Also in adult female and male insects at 5daf both genes showed the strongest signal in the posterior midgut. Molecular modeling suggested that TBSCP-1 has carboxypeptidase D activity; activities against Hippuryl-Phenylalanine and Hippuryl-Arginine were also located at the posterior midgut, both were induced after blood feeding. Treatment of the posterior midgut extracts with the serine protease inhibitor PMSF strongly reduced carboxypeptidase activity. These findings suggest that triatomines might use serine carboxypeptidases, which are usually found in lysosomes, as digestive enzymes in the posterior midgut lumen, from which TBSCP-1 and TBSCP-2 are possible candidates to fulfill this function. Copyright © 2014 Elsevier Ltd. All rights reserved.

  14. The alteration of serine transporter activity in a cell line model of amyotrophic lateral sclerosis (ALS).

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    Lee, Na-Young; Kim, Yunha; Ryu, Hoon; Kang, Young-Sook

    2017-01-29

    The alteration of d-serine levels is associated with the pathogenesis of sporadic ALS and mutant SOD1 (G93A) animal model of ALS. However, the exact mechanism of d-serine transport is not known in ALS. To better understand the distribution of d-serine in ALS, we determined the activity and the expression of serine transporter in a motor neuronal cell line model of ALS (NSC-34/hSOD1 G93A cells). The uptake of [ 3 H]d-serine was significantly lower in NSC-34/hSOD1 G93A cells than in control NSC-34 and NSC-34/hSOD1 wt cells. In contrast, the uptake of [ 3 H]l-serine, precursor of d-serine, was markedly increased in NSC-34/hSOD1 G93A cells compared to control NSC-34 and NSC-34/hSOD1 wt cells. Both [ 3 H]d-serine and [ 3 H]l-serine uptake were saturable in these cells. The estimated Michaelis-Menten constant, K m , for d-serine uptakes was higher in NSC-34/hSOD1 G93A cells than in NSC-34/hSOD1 wt cells while the K m for l-serine uptake was 2 fold lower in NSC-34/hSOD1 G93A cells than in control cells. [ 3 H]d-serine and [ 3 H]l-serine uptakes took place in a Na + -dependent manner, and both uptakes were significantly inhibited by system ASC (alanine-serine-cysteine) substrates. As a result of small interfering RNA experiments, we found that ASCT2 (SLC1A5) and ASCT1 (SLC1A4) are involved in [ 3 H]d-serine and [ 3 H]l-serine uptake in NSC-34/hSOD1 G93A cells, respectively. The level of SLC1A4 mRNA was significantly increased in NSC-34/hSOD1 G93A compared to NSC-34 and NSC-34/hSOD1 wt cells. In contrast, the level of SLC7A10 mRNA was relatively lower in NSC-34/hSOD1 G93A cells than the control cells. Together, these data suggest that the pathological alteration of d- and l-serine uptakes in ALS is driven by the affinity change of d-and l-serine uptake system. Copyright © 2016 Elsevier Inc. All rights reserved.

  15. Serine Proteolytic Pathway Activation Reveals an Expanded Ensemble of Wound Response Genes in Drosophila

    Science.gov (United States)

    Patterson, Rachel A.; Juarez, Michelle T.; Hermann, Anita; Sasik, Roman; Hardiman, Gary; McGinnis, William

    2013-01-01

    After injury to the animal epidermis, a variety of genes are transcriptionally activated in nearby cells to regenerate the missing cells and facilitate barrier repair. The range and types of diffusible wound signals that are produced by damaged epidermis and function to activate repair genes during epidermal regeneration remains a subject of very active study in many animals. In Drosophila embryos, we have discovered that serine protease function is locally activated around wound sites, and is also required for localized activation of epidermal repair genes. The serine protease trypsin is sufficient to induce a striking global epidermal wound response without inflicting cell death or compromising the integrity of the epithelial barrier. We developed a trypsin wounding treatment as an amplification tool to more fully understand the changes in the Drosophila transcriptome that occur after epidermal injury. By comparing our array results with similar results on mammalian skin wounding we can see which evolutionarily conserved pathways are activated after epidermal wounding in very diverse animals. Our innovative serine protease-mediated wounding protocol allowed us to identify 8 additional genes that are activated in epidermal cells in the immediate vicinity of puncture wounds, and the functions of many of these genes suggest novel genetic pathways that may control epidermal wound repair. Additionally, our data augments the evidence that clean puncture wounding can mount a powerful innate immune transcriptional response, with different innate immune genes being activated in an interesting variety of ways. These include puncture-induced activation only in epidermal cells in the immediate vicinity of wounds, or in all epidermal cells, or specifically in the fat body, or in multiple tissues. PMID:23637905

  16. Serine proteolytic pathway activation reveals an expanded ensemble of wound response genes in Drosophila.

    Directory of Open Access Journals (Sweden)

    Rachel A Patterson

    Full Text Available After injury to the animal epidermis, a variety of genes are transcriptionally activated in nearby cells to regenerate the missing cells and facilitate barrier repair. The range and types of diffusible wound signals that are produced by damaged epidermis and function to activate repair genes during epidermal regeneration remains a subject of very active study in many animals. In Drosophila embryos, we have discovered that serine protease function is locally activated around wound sites, and is also required for localized activation of epidermal repair genes. The serine protease trypsin is sufficient to induce a striking global epidermal wound response without inflicting cell death or compromising the integrity of the epithelial barrier. We developed a trypsin wounding treatment as an amplification tool to more fully understand the changes in the Drosophila transcriptome that occur after epidermal injury. By comparing our array results with similar results on mammalian skin wounding we can see which evolutionarily conserved pathways are activated after epidermal wounding in very diverse animals. Our innovative serine protease-mediated wounding protocol allowed us to identify 8 additional genes that are activated in epidermal cells in the immediate vicinity of puncture wounds, and the functions of many of these genes suggest novel genetic pathways that may control epidermal wound repair. Additionally, our data augments the evidence that clean puncture wounding can mount a powerful innate immune transcriptional response, with different innate immune genes being activated in an interesting variety of ways. These include puncture-induced activation only in epidermal cells in the immediate vicinity of wounds, or in all epidermal cells, or specifically in the fat body, or in multiple tissues.

  17. Stromal serine protein kinase activity in spinach chloroplasts

    International Nuclear Information System (INIS)

    Cortez, N.; Lucero, H.A.; Vallejos, R.H.

    1987-01-01

    At least twelve 32 P-labeled stromal proteins were detected by electrophoresis under denaturing conditions when intact chloroplasts were incubated with 32 Pi, in the light but only three were detected in the presence of 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU) or in the dark. Incubation of isolated stroma with [gamma- 32 P]ATP resulted in the preferential phosphorylation of one of them, a 70-kDa polypeptide, in serine residues. Thylakoid membranes in the dark promoted the phosphorylation of two additional stromal polypeptides of 55 and 40 kDa. Illumination during the phosphorylation of stroma in the presence of thylakoids stimulated severalfold the labeling of the 40-kDa polypeptide but not when DCMU was added. The protein kinase activity present in isolated stroma phosphorylated exogenous substrates like histone III, phosvitin, histone II, and casein with specific activities of 3, 1.8, 0.7, and 0.2 pmol X mg-1 X min-1. Histone III polypeptides were phosphorylated differently by stroma and by thylakoids in the dark. Moreover, histone III phosphorylated by thylakoids in the dark yielded a pattern of phosphopeptides after V8 protease treatment that was different from the pattern obtained when histone III was phosphorylated by stroma

  18. Novel Serine 176 Phosphorylation of YBX1 Activates NF-κB in Colon Cancer.

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    Martin, Matthew; Hua, Laiqing; Wang, Benlian; Wei, Han; Prabhu, Lakshmi; Hartley, Antja-Voy; Jiang, Guanglong; Liu, Yunlong; Lu, Tao

    2017-02-24

    Y box protein 1 (YBX1) is a well known oncoprotein that has tumor-promoting functions. YBX1 is widely considered to be an attractive therapeutic target in cancer. To develop novel therapeutics to target YBX1, it is of great importance to understand how YBX1 is finely regulated in cancer. Previously, we have shown that YBX1 could function as a tumor promoter through phosphorylation of its Ser-165 residue, leading to the activation of the NF-κB signaling pathway (1). In this study, using mass spectrometry analysis, we discovered a distinct phosphorylation site, Ser-176, on YBX1. Overexpression of the YBX1-S176A (serine-to-alanine) mutant in either HEK293 cells or colon cancer HT29 cells showed dramatically reduced NF-κB-activating ability compared with that of WT-YBX1, confirming that Ser-176 phosphorylation is critical for the activation of NF-κB by YBX1. Importantly, the mutant of Ser-176 and the previously reported Ser-165 sites regulate distinct groups of NF-κB target genes, suggesting the unique and irreplaceable function of each of these two phosphorylated serine residues. Our important findings could provide a novel cancer therapy strategy by blocking either Ser-176 or Ser-165 phosphorylation or both of YBX1 in colon cancer. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

  19. 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.

  20. Site-specific and synergistic stimulation of methylation on the bacterial chemotaxis receptor Tsr by serine and CheW

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    Weis Robert M

    2005-03-01

    Full Text Available Abstract Background Specific glutamates in the methyl-accepting chemotaxis proteins (MCPs of Escherichia coli are modified during sensory adaptation. Attractants that bind to MCPs are known to increase the rate of receptor modification, as with serine and the serine receptor (Tsr, which contributes to an increase in the steady-state (adapted methylation level. However, MCPs form ternary complexes with two cytoplasmic signaling proteins, the kinase (CheA and an adaptor protein (CheW, but their influences on receptor methylation are unknown. Here, the influence of CheW on the rate of Tsr methylation has been studied to identify contributions to the process of adaptation. Results Methyl group incorporation was measured in a series of membrane samples in which the Tsr molecules were engineered to have one available methyl-accepting glutamate residue (297, 304, 311 or 493. The relative rates at these sites (0.14, 0.05, 0.05 and 1, respectively differed from those found previously for the aspartate receptor (Tar, which was in part due to sequence differences between Tar and Tsr near site four. The addition of CheW generated unexpectedly large and site-specific rate increases, equal to or larger than the increases produced by serine. The increases produced by serine and CheW (added separately were the largest at site one, ~3 and 6-fold, respectively, and the least at site four, no change and ~2-fold, respectively. The rate increases were even larger when serine and CheW were added together, larger than the sums of the increases produced by serine and CheW added separately (except site four. This resulted in substantially larger serine-stimulated increases when CheW was present. Also, CheW enhanced methylation rates when either two or all four sites were available. Conclusion The increase in the rate of receptor methylation upon CheW binding contributes significantly to the ligand specificity and kinetics of sensory adaptation. The synergistic effect of

  1. Endothelin-1 stimulates catalase activity through the PKCδ mediated phosphorylation of Serine 167

    Science.gov (United States)

    Rafikov, Ruslan; Kumar, Sanjiv; Aggarwal, Saurabh; Hou, Yali; Kangath, Archana; Pardo, Daniel; Fineman, Jeffrey R.; Black, Stephen M.

    2013-01-01

    Our previous studies have shown that endothelin-1 (ET-1) stimulates catalase activity in endothelial cells and lambs with acute increases in pulmonary blood flow (PBF), without altering gene expression. The purpose of this study was to investigate the molecular mechanism by which this occurs. Exposing pulmonary arterial endothelial cells (PAEC) to ET-1 increased catalase activity and decreased cellular hydrogen peroxide (H2O2) levels. These changes correlated with an increase in serine phosphorylated catalase. Using the inhibitory peptide δV1.1, this phosphorylation was shown to be PKCδ dependent. Mass spectrometry identified serine167 as the phosphorylation site. Site-directed mutagenesis was used to generate a phospho-mimic (S167D) catalase. Activity assays using recombinant protein purified from E.coli or transiently transfected COS-7 cells, demonstrated that S167D-catalase had an increased ability to degrade H2O2 compared to the wildtype enzyme. Using a phospho-specific antibody, we were able to verify that pS167 catalase levels are modulated in lambs with acute increases in PBF in the presence and absence of the ET receptor antagonist, tezosentan. S167 is being located on the dimeric interface suggesting it could be involved in regulating the formation of catalase tetramers. To evaluate this possibility we utilized analytical gel-filtration to examine the multimeric structure of recombinant wildtype- and S167D-catalase. We found that recombinant wildtype catalase was present as a mixture of monomers and dimers while S167D catalase was primarily tetrameric. Further, the incubation of wildtype catalase with PKCδ was sufficient to convert wildtype catalase into a tetrameric structure. In conclusion, this is the first report indicating that the phosphorylation of catalase regulates its multimeric structure and activity. PMID:24211614

  2. [3H] D-serine labels strychnine-insensitive glycine recognition sites of rat central nervous system

    International Nuclear Information System (INIS)

    Danysz, W.; Fadda, E.; Wroblewski, J.T.; Costa, E.

    1990-01-01

    In the central nervous system, glycine binds to two recognition sites; one of them (G 2 ), associated with the glutamate receptor, is insensitive to strychnine. Strychnine-insensitive sites were predominant in the forebrain areas and bound D-serine and D-alanine better than the respective L stereoisomers. [ 3 H] D-serine was a more selective radioligand than [ 3 H]glycine for the strychnine-insensitive sites. In the forebain, the binding of both ligands was inhibited by the putative G 2 receptor antagonists, 7-chlorokynurenate and 3-amino-1-hydroxy-2-pyrrolidone, while in pons and in spinal cord only the latter drug was effective. This may indicate the heterogeneity of strychnine-insensitive glycine recognition sites

  3. Horseshoe crab coagulation factor B. A unique serine protease zymogen activated by cleavage of an Ile-Ile bond.

    Science.gov (United States)

    Muta, T; Oda, T; Iwanaga, S

    1993-10-05

    Horseshoe crab factor B is an intracellular serine protease zymogen involved in the bacterial endotoxin-responsive hemolymph coagulation cascade. cDNAs for factor B were isolated utilizing a polymerase chain reaction product using two primers derived from the partial amino acid sequence. The cloned cDNA of 1928 base pairs encoded 400 amino acid residues of factor B precursor. The first 23 amino acid residues constitute a presumed prepropeptide that may be processed by both a signal peptidase and a processing protease, similar to mammalian vitamin K-dependent protease precursors. The mature protein consists of 377 amino acids with a calculated molecular mass of 40,570 Da. The overall structure is highly homologous to that of limulus proclotting enzyme (35.9% identity), the substrate for active factor B in the cascade. Like the proclotting enzyme, mature factor B is composed of an amino-terminal "clip"-like domain and a carboxyl-terminal serine protease domain homologous to that of human plasma prekallikrein (36.5%). Internal sequences encode a unique activation peptide. Surprisingly, the cleavage sites of the zymogen factor B for activation by limulus active factor C were found to be an Arg-Ser and an Ile-Ile bond, the latter of which has not been found in any other protease zymogens. These cleavages result in the release of the activation peptide, which consists of 21 residues with a carboxyl-terminal isoleucine. These results indicate that the intracellular clotting system of the limulus hemocyte, like mammalian plasma clotting cascade, proceeds with the sequential activation of three serine protease zymogens: factor C, factor B, and proclotting enzyme.

  4. Alternaria-derived serine protease activity drives IL-33–mediated asthma exacerbations

    Science.gov (United States)

    Snelgrove, Robert J.; Gregory, Lisa G.; Peiró, Teresa; Akthar, Samia; Campbell, Gaynor A.; Walker, Simone A.; Lloyd, Clare M.

    2014-01-01

    Background The fungal allergen Alternaria alternata is implicated in severe asthma and rapid onset life-threatening exacerbations of disease. However, the mechanisms that underlie this severe pathogenicity remain unclear. Objective We sought to investigate the mechanism whereby Alternaria was capable of initiating severe, rapid onset allergic inflammation. Methods IL-33 levels were quantified in wild-type and ST2−/− mice that lacked the IL-33 receptor given inhaled house dust mite, cat dander, or Alternaria, and the effect of inhibiting allergen-specific protease activities on IL-33 levels was assessed. An exacerbation model of allergic airway disease was established whereby mice were sensitized with house dust mite before subsequently being challenged with Alternaria (with or without serine protease activity), and inflammation, remodeling, and lung function assessed 24 hours later. Results Alternaria, but not other common aeroallergens, possessed intrinsic serine protease activity that elicited the rapid release of IL-33 into the airways of mice through a mechanism that was dependent upon the activation of protease activated receptor-2 and adenosine triphosphate signaling. The unique capacity of Alternaria to drive this early IL-33 release resulted in a greater pulmonary inflammation by 24 hours after challenge relative to the common aeroallergen house dust mite. Furthermore, this Alternaria serine protease–IL-33 axis triggered a rapid, augmented inflammation, mucus release, and loss of lung function in our exacerbation model. Conclusion Alternaria-specific serine protease activity causes rapid IL-33 release, which underlies the development of a robust TH2 inflammation and exacerbation of allergic airway disease. PMID:24636086

  5. Alternaria-derived serine protease activity drives IL-33-mediated asthma exacerbations.

    Science.gov (United States)

    Snelgrove, Robert J; Gregory, Lisa G; Peiró, Teresa; Akthar, Samia; Campbell, Gaynor A; Walker, Simone A; Lloyd, Clare M

    2014-09-01

    The fungal allergen Alternaria alternata is implicated in severe asthma and rapid onset life-threatening exacerbations of disease. However, the mechanisms that underlie this severe pathogenicity remain unclear. We sought to investigate the mechanism whereby Alternaria was capable of initiating severe, rapid onset allergic inflammation. IL-33 levels were quantified in wild-type and ST2(-/-) mice that lacked the IL-33 receptor given inhaled house dust mite, cat dander, or Alternaria, and the effect of inhibiting allergen-specific protease activities on IL-33 levels was assessed. An exacerbation model of allergic airway disease was established whereby mice were sensitized with house dust mite before subsequently being challenged with Alternaria (with or without serine protease activity), and inflammation, remodeling, and lung function assessed 24 hours later. Alternaria, but not other common aeroallergens, possessed intrinsic serine protease activity that elicited the rapid release of IL-33 into the airways of mice through a mechanism that was dependent upon the activation of protease activated receptor-2 and adenosine triphosphate signaling. The unique capacity of Alternaria to drive this early IL-33 release resulted in a greater pulmonary inflammation by 24 hours after challenge relative to the common aeroallergen house dust mite. Furthermore, this Alternaria serine protease-IL-33 axis triggered a rapid, augmented inflammation, mucus release, and loss of lung function in our exacerbation model. Alternaria-specific serine protease activity causes rapid IL-33 release, which underlies the development of a robust TH2 inflammation and exacerbation of allergic airway disease. Copyright © 2014 The Authors. Published by Elsevier Inc. All rights reserved.

  6. Fluorescently labeled inhibitors detect localized serine protease activities in Drosophila melanogaster pole cells, embryos, and ovarian egg chambers

    DEFF Research Database (Denmark)

    Jakobsen, Rasmus Kragh; Ono, S.; Powers, J. C.

    2005-01-01

    processes that they mediate. Until only recently, the tools to conveniently address the question of where and when serine proteases are active within complex tissues have been lacking. In order to detect spatially restricted serine protease activities in Drosophila embryos and ovaries we introduce...... activity localized to the oocyte-somatic follicle cell interface of the developing egg chamber. Our results suggest that this technique holds promise to identify new spatially restricted activities in adult Drosophila tissues and developing embryos....

  7. Artemether Exhibits Amoebicidal Activity against Acanthamoeba castellanii through Inhibition of the Serine Biosynthesis Pathway.

    Science.gov (United States)

    Deng, Yihong; Ran, Wei; Man, Suqin; Li, Xueping; Gao, Hongjian; Tang, Wei; Tachibana, Hiroshi; Cheng, Xunjia

    2015-08-01

    Acanthamoeba sp. parasites are the causative agents of Acanthamoeba keratitis, fatal granulomatous amoebic encephalitis, and cutaneous infections. However, there are currently no effective drugs for these organisms. Here, we evaluated the activity of the antimalarial agent artemether against Acanthamoeba castellanii trophozoites and identified potential targets of this agent through a proteomic approach. Artemether exhibited in vitro amoebicidal activity in a time- and dose-dependent manner and induced ultrastructural modification and cell apoptosis. The iTRAQ quantitative proteomic analysis identified 707 proteins that were differentially expressed after artemether treatment. We focused on phosphoglycerate dehydrogenase and phosphoserine aminotransferase in the serine biosynthesis pathway because of their importance to the growth and proliferation of protozoan and cancer cells. The expression of these proteins in Acanthamoeba was validated using quantitative real-time PCR and Western blotting after artemether treatment. The changes in the expression levels of phosphoserine aminotransferase were consistent with those of phosphoglycerate dehydrogenase. Therefore, the downregulation of phosphoserine aminotransferase may be due to the downregulation of phosphoglycerate dehydrogenase. Furthermore, exogenous serine might antagonize the activity of artemether against Acanthamoeba trophozoites. These results indicate that the serine biosynthesis pathway is important to amoeba survival and that targeting these enzymes would improve the treatment of Acanthamoeba infections. Artemether may be used as a phosphoglycerate dehydrogenase inhibitor to control or block Acanthamoeba infections. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

  8. Raf-1 forms a stable complex with Mek1 and activates Mek1 by serine phosphorylation.

    OpenAIRE

    Huang, W; Alessandrini, A; Crews, C M; Erikson, R L

    1993-01-01

    Recombinant Mek1 and Raf-1 proteins produced in Sf9 cells undergo a tight association both in vivo and in vitro, which apparently does not depend on additional factors or the kinase activity of Mek1 or Raf-1. The complex can be disrupted by two polyclonal antibodies raised against Raf-1 peptides. Coinfection with Raf-1 activates Mek1 > 150-fold, and coinfection with Raf-1 and Mek1 activates Erk1 approximately 90-fold. The activation of Mek1 by Raf-1 involves only serine phosphorylation, which...

  9. A 25-kDa serine peptidase with keratinolytic activity secreted by Coccidioides immitis.

    Science.gov (United States)

    Lopes, Bárbara Gabriela Brum; Santos, André Luis Souza Dos; Bezerra, Cláudia de Carvalho Falci; Wanke, Bodo; Dos Santos Lazéra, Márcia; Nishikawa, Marília Martins; Mazotto, Ana Maria; Kussumi, Vânia Monteiro; Haido, Rosa Maria Tavares; Vermelho, Alane Beatriz

    2008-07-01

    Coccidioides immitis is the causative agent of coccidioidomycosis, a systemic mycosis that attacks humans and a wide variety of animals. In the present study, we showed that the C. immitis mycelial form is able to release proteolytic enzyme into the extracellular environment. Under chemically defined growth conditions, mycelia secreted seven distinct polypeptides ranging from 15 to 65 kDa and an extracellular peptidase of 25 kDa. This enzyme had its activity fully inhibited by phenylmethylsulphonyl fluoride, a serine peptidase inhibitor. Conversely, metallo, cysteine, and aspartyl peptidase inhibitors did not alter the 25-kDa enzyme behavior. This extracellular serine peptidase was able to degrade keratin, a fibrous protein that composes human epidermis. Additionally, this peptidase cleaved different protein substrates, including gelatin, casein, hemoglobin, and albumin. Curiously, an 18-kDa serine peptidase activity was evidenced solely when casein was used as the co-polymerized protein substrate into the gel. The existence of different secreted peptidases could be advantageous for the adaptation of C. immitis to distinct environments during its complex life cycle.

  10. A novel effect of MARCKS phosphorylation by activated PKC: the dephosphorylation of its serine 25 in chick neuroblasts.

    Directory of Open Access Journals (Sweden)

    Andrea Toledo

    Full Text Available MARCKS (Myristoylated Alanine-Rich C Kinase Substrate is a peripheral membrane protein, especially abundant in the nervous system, and functionally related to actin organization and Ca-calmodulin regulation depending on its phosphorylation by PKC. However, MARCKS is susceptible to be phosphorylated by several different kinases and the possible interactions between these phosphorylations have not been fully studied in intact cells. In differentiating neuroblasts, as well as some neurons, there is at least one cell-type specific phosphorylation site: serine 25 (S25 in the chick. We demonstrate here that S25 is included in a highly conserved protein sequence which is a Cdk phosphorylatable region, located far away from the PKC phosphorylation domain. S25 phosphorylation was inhibited by olomoucine and roscovitine in neuroblasts undergoing various states of cell differentiation in vitro. These results, considered in the known context of Cdks activity in neuroblasts, suggest that Cdk5 is the enzyme responsible for this phosphorylation. We find that the phosphorylation by PKC at the effector domain does not occur in the same molecules that are phosphorylated at serine 25. The in situ analysis of the subcellular distribution of these two phosphorylated MARCKS variants revealed that they are also segregated in different protein clusters. In addition, we find that a sustained stimulation of PKC by phorbol-12-myristate-13-acetate (PMA provokes the progressive disappearance of phosphorylation at serine 25. Cells treated with PMA, but in the presence of several Ser/Thr phosphatase (PP1, PP2A and PP2B inhibitors indicated that this dephosphorylation is achieved via a phosphatase 2A (PP2A form. These results provide new evidence regarding the existence of a novel consequence of PKC stimulation upon the phosphorylated state of MARCKS in neural cells, and propose a link between PKC and PP2A activity on MARCKS.

  11. Purification and biochemical characterization of a serine alkaline ...

    African Journals Online (AJOL)

    USER

    2010-08-02

    , which sulfonated the essential serine residue in the active site and resulted in the complete loss of its activity. However, the enzyme was resistant to EDTA. The high activity of TC4 in the presence of EDTA was advantageous.

  12. Retinol activates tyrosine hydroxylase acutely by increasing the phosphorylation of serine40 and then serine31 in bovine adrenal chromaffin cells.

    Science.gov (United States)

    Gelain, Daniel P; Moreira, Jose C F; Bevilaqua, Lia R M; Dickson, Phillip W; Dunkley, Peter R

    2007-12-01

    Tyrosine hydroxylase is the rate-limiting enzyme in the biosynthesis of the catecholamines. It has been reported that retinol (vitamin A) modulates tyrosine hydroxylase activity by increasing its expression through the activation of the nuclear retinoid receptors. In this study, we observed that retinol also leads to an acute activation of tyrosine hydroxylase in bovine adrenal chromaffin cells and this was shown to occur via two distinct non-genomic mechanisms. In the first mechanism, retinol induced an influx in extracellular calcium, activation of protein kinase C and serine40 phosphorylation, leading to tyrosine hydroxylase activation within 15 min. This effect then declined over time. The retinol-induced rise in intracellular calcium then led to a second slower mechanism; this involved an increase in reactive oxygen species, activation of extracellular signal-regulated kinase 1/2 and serine31 phosphorylation and the maintenance of tyrosine hydroxylase activation for up to 2 h. No effects were observed with retinoic acid. These results show that retinol activates tyrosine hydroxylase via two sequential non-genomic mechanisms, which have not previously been characterized. These mechanisms are likely to operate in vivo to facilitate the stress response, especially when vitamin supplements are taken or when retinol is used as a therapeutic agent.

  13. 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.

  14. Serine and alanine racemase activities of VanT: a protein necessary for vancomycin resistance in Enterococcus gallinarum BM4174.

    Science.gov (United States)

    Arias, C A; Weisner, J; Blackburn, J M; Reynolds, P E

    2000-07-01

    Vancomycin resistance in Enterococcus gallinarum results from the production of UDP-MurNAc-pentapeptide[D-Ser]. VanT, a membrane-bound serine racemase, is one of three proteins essential for this resistance. To investigate the selectivity of racemization of L-Ser or L-Ala by VanT, a strain of Escherichia coli TKL-10 that requires D-Ala for growth at 42 degrees C was used as host for transformation experiments using plasmids containing the full-length vanT from Ent. gallinarum or the alanine racemase gene (alr) of Bacillus stearothermophilus: both plasmids were able to complement E. coli TKL-10 at 42 degrees C. No alanine or serine racemase activities were detected in the host strain E. coli TKL-10 grown at 30, 34 or 37 degrees C. Serine and alanine racemase activities were found almost exclusively (96%) in the membrane fraction of E. coli TKL-10/pCA4(vanT): the alanine racemase activity of VanT was 14% of the serine racemase activity in both E. coli TKL-10/pCA4(vanT) and E. coli XL-1 Blue/pCA4(vanT). Alanine racemase activity was present mainly (95%) in the cytoplasmic fraction of E. coli TKL-10/pJW40(alr), with a trace (1.6%) of serine racemase activity. Additionally, DNA encoding the soluble domain of VanT was cloned and expressed in E. coli M15 as a His-tagged polypeptide and purified: this polypeptide also exhibited both serine and alanine racemase activities; the latter was approximately 18% of the serine racemase activity, similar to that of the full-length, membrane-bound enzyme. N-terminal sequencing of the purified His-tagged polypeptide revealed a single amino acid sequence, indicating that the formation of heterodimers between subunits of His-tagged C-VanT and endogenous alanine racemases from E. coli was unlikely. The authors conclude that the membrane-bound serine racemase VanT also has alanine racemase activity but is able to racemize serine more efficiently than alanine, and that the cytoplasmic domain is responsible for the racemase activity.

  15. Conservative Mechanisms of Extracellular Trap Formation by Annelida Eisenia andrei: Serine Protease Activity Requirement.

    Directory of Open Access Journals (Sweden)

    Joanna Homa

    Full Text Available Formation of extracellular traps (ETs capturing and immobilizing pathogens is now a well-established defense mechanism added to the repertoire of vertebrate phagocytes. These ETs are composed of extracellular DNA (extDNA, histones and antimicrobial proteins. Formation of mouse and human ETs depends on enzymes (i facilitating decondensation of chromatin by citrullination of histones, and (ii serine proteases degrading histones. In invertebrates, initial reports revealed existence of ETs composed of extDNA and histones, and here we document for the first time that also coelomocytes, immunocompetent cells of an earthworm Eisenia andrei, cast ETs which successfully trap bacteria in a reactive oxygen species (ROS-dependent and -independent manner. Importantly, the formation of ETs was observed not only when coelomocytes were studied ex vivo, but also in vivo, directly in the earthworm coelom. These ETs were composed of extDNA, heat shock proteins (HSP27 and H3 histones. Furthermore, the formation of E. andrei ETs depended on activity of serine proteases, including elastase-like activity. Moreover, ETs interconnected and hold together aggregating coelomocytes, a processes proceeding encapsulation. In conclusion, the study confirms ET formation by earthworms, and unravels mechanisms leading to ET formation and encapsulation in invertebrates.

  16. Application of Asian pumpkin (Cucurbita ficifolia) serine proteinase for production of biologically active peptides from casein.

    Science.gov (United States)

    Dąbrowska, Anna; Szołtysik, Marek; Babij, Konrad; Pokora, Marta; Zambrowicz, Aleksandra; Chrzanowska, Józefa

    2013-01-01

    The main objective of this study was to determine potential application of a serine proteinase derived from Asian pumpkin for obtaining biologically active peptides from casein. The course of casein hydrolysis by three doses of the enzyme (50, 150, 300 U/mg of protein) was monitored for 24 hours by the determinations of: hydrolysis degree DH (%), free amino group content (μmole Gly/g), RP HPLC peptide profiles and by polyacrylamide gel electrophoresis. In all hydrolyzates analyzed antioxidant activities were determined using three tests: the ability to reduce iron ions in FRAP test, the ability to scavenge free radicals in DPPH test, and Fe(2+) chelating activity. The antimicrobial activity of obtained peptide fractions was determined as the ability to inhibit the growth of Escherichia coli, Bacillus cereus and Pseudomonas fluorescens in a diffusion plate test. The deepest degradation, expressed as the DH [%] and the free amino group content (67% and 7528 µmole Gly/mg, respectively), was noted in samples hydrolyzed with 300 U/ml of enzyme for 24 hours, while in other samples the determined values were about three and two times lower. The results were in agreement with the peptide profiles obtained by RP HPLC. The highest antioxidative activities determined in all tests were seen for the casein hydrolysate obtained with 300 U/mg protein of serine proteinase after 24 h of reaction (2.15 µM Trolox/mg, 96.15 µg Fe(3+)/mg, 814.97 µg Fe(2+)/mg). Antimicrobial activity was presented in three preparations. In other samples no antimicrobial activity was detected.

  17. Crystal Structure of Serine Racemase that Produces Neurotransmitter d-Serine for Stimulation of the NMDA Receptor

    Science.gov (United States)

    Goto, Masaru

    d-Serine is an endogenous coagonist for the N-methyl-d-aspartate receptor and is involved in excitatory neurotransmission in the brain. Mammalian pyridoxal 5’-phosphate-dependent serine racemase, which is localized in the mammalian brain, catalyzes the racemization of l-serine to yield d-serine and vice versa. We have determined the structures of three forms of the mammalian enzyme homolog from Schizosaccharomyces pombe. Lys57 and Ser82 located on the protein and solvent sides, respectively, with respect to the cofactor plane, are acid-base catalysts that shuttle protons to the substrate. The modified enzyme, which has a unique lysino-d-alanyl residue at the active site, also binds the substrate serine in the active site, suggesting that the lysino-d-alanyl residue acts as a catalytic base in the same manner as Lys57 of the wild type enzyme.

  18. Random mutagenesis of human serine racemase reveals residues important for the enzymatic activity

    Czech Academy of Sciences Publication Activity Database

    Hoffman, Hillary Elizabeth; Jirásková, Jana; Zvelebil, M.; Konvalinka, Jan

    2010-01-01

    Roč. 75, č. 1 (2010), s. 59-79 ISSN 0010-0765 R&D Projects: GA MŠk 1M0508 Institutional research plan: CEZ:AV0Z40550506 Keywords : D-serine * serine racemase * random mutagenesis Subject RIV: CE - Biochemistry Impact factor: 0.853, year: 2010

  19. Manipulating the fragmentation patterns of phosphopeptides via gas-phase boron derivatization: determining phosphorylation sites in peptides with multiple serines.

    Science.gov (United States)

    Gronert, Scott; Li, Kathy H; Horiuchi, Mizue

    2005-12-01

    Trivalent boron species readily react with protonated phosphopeptides to give addition products with the loss of boron ligands. In the present study, trimethoxyborane (TMB), diisopropoxymethylborane (DIPM), and diethylmethoxyborane (DEMB) were allowed to react with four phosphopeptides, VsSF, LSsF, LsGASA, and VSGAsA (lower-case s indicates phosphoserine). Each of the phosphopeptides contains one serine that is phosphorylated and one that is not. Under collision-activated dissociation (CAD) conditions, the boron-derivatized peptides give fragmentation patterns that differ significantly from that of the protonated phosphopeptide. The patterns vary, depending on the number of labile (i.e., alkoxy) ligands on the boron. In general, boron derivatization increases the yield of phosphate-containing sequence ions, but dramatic effects are only seen with certain reagent/peptide combinations. However, the suite of reagents provides a means of altering and increasing the information content of phosphopeptide CAD spectra.

  20. CHANGES IN LEVELS OF ACTIVITY OF SERINE PROTEASES ACCOMPANY THE EXPOSURE OF COMMON BEAN (PHASEOLUS VULGARIS L. TO WATER DEFICIT

    Directory of Open Access Journals (Sweden)

    M. Budič

    2008-09-01

    Full Text Available A wide variety of proteolytic enzymes exist in plants. On their levels depends protein turnover, a fundamental component in plant development and adaptation to environmental conditions. Cysteine proteases have frequently been reported to be influenced by drought, but only a few serine proteases (SP, among them the trypsin-like enzyme and two aminopeptidases from bean leaves (Bartels and Sunkar, 2005; Hieng et al., 2004. Our starting point was to identify proteolytic activities assigned to SPs that change with drought and then to characterize the corresponding proteases. A quantitative, analytical one-step method was used to separate endopeptidases and aminopeptidases active against a range of substrates in leaf extracts of plants grown in the field (FC. The influence of drought was determined for those of these activities which were confirmed as SPs, based on their inhibition by specific inhibitors. Under water deficit in plants grown under controlled conditions (CC their levels changed in different ways. The levels of SP activities in FC plants, observed during a period of relative drought, were similar to those measured in mildly stressed CC plants. The partial characterisations of some of these SPs will be presented. Our results point to a number of roles for different SPs in the plant response to water stress, which could range from enhanced protein turnover to limited proteolysis at specific sites.

  1. 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...

  2. Anti-fibrinolytic and anti-microbial activities of a serine protease inhibitor from honeybee (Apis cerana) venom.

    Science.gov (United States)

    Yang, Jie; Lee, Kwang Sik; Kim, Bo Yeon; Choi, Yong Soo; Yoon, Hyung Joo; Jia, Jingming; Jin, Byung Rae

    2017-10-01

    Bee venom contains a variety of peptide constituents, including low-molecular-weight protease inhibitors. While the putative low-molecular-weight serine protease inhibitor Api m 6 containing a trypsin inhibitor-like cysteine-rich domain was identified from honeybee (Apis mellifera) venom, no anti-fibrinolytic or anti-microbial roles for this inhibitor have been elucidated. In this study, we identified an Asiatic honeybee (A. cerana) venom serine protease inhibitor (AcVSPI) that was shown to act as a microbial serine protease inhibitor and plasmin inhibitor. AcVSPI was found to consist of a trypsin inhibitor-like domain that displays ten cysteine residues. Interestingly, the AcVSPI peptide sequence exhibited high similarity to the putative low-molecular-weight serine protease inhibitor Api m 6, which suggests that AcVSPI is an allergen Api m 6-like peptide. Recombinant AcVSPI was expressed in baculovirus-infected insect cells, and it demonstrated inhibitory activity against trypsin, but not chymotrypsin. Additionally, AcVSPI has inhibitory effects against plasmin and microbial serine proteases; however, it does not have any detectable inhibitory effects on thrombin or elastase. Consistent with these inhibitory effects, AcVSPI inhibited the plasmin-mediated degradation of fibrin to fibrin degradation products. AcVSPI also bound to bacterial and fungal surfaces and exhibited anti-microbial activity against fungi as well as gram-positive and gram-negative bacteria. These findings demonstrate the anti-fibrinolytic and anti-microbial roles of AcVSPI as a serine protease inhibitor. Copyright © 2017 Elsevier Inc. All rights reserved.

  3. Identification of the site of human mannan-binding lectin involved in the interaction with its partner serine proteases: the essential role of Lys55

    DEFF Research Database (Denmark)

    Teillet, F; Lacroix, M; Thiel, Steffen

    2007-01-01

    Mannan-binding lectin (MBL) is an oligomeric lectin that binds neutral carbohydrates on pathogens, forms complexes with MBL-associated serine proteases (MASP)-1, -2, and -3 and 19-kDa MBL-associated protein (MAp19), and triggers the complement lectin pathway through activation of MASP-2. To ident...... centered on residue Lys(55), which may form an ionic bond representing the major component of the MBL-MASP interaction. The binding sites for MASP-2/MAp19 and MASP-1/3 have common features but are not strictly identical........ To identify the MASP binding site(s) of human MBL, point mutants targeting residues C-terminal to the hinge region were produced and tested for their interaction with the MASPs and MAp19 using surface plasmon resonance and functional assays. Mutation Lys(55)Ala abolished interaction with the MASPs and MAp19...... and prevented formation of functional MBL-MASP-2 complexes. Mutations Lys(55)Gln and Lys(55)Glu abolished binding to MASP-1 and -3 and strongly inhibited interaction with MAp19. Conversely, mutation Lys(55)Arg abolished interaction with MASP-2 and MAp19, but only weakened interaction with MASP-1 and -3...

  4. Induction of serine racemase expression and D-serine release from microglia by amyloid β-peptide

    Directory of Open Access Journals (Sweden)

    Griffin W Sue T

    2004-04-01

    Full Text Available Abstract Background Roles for excitotoxicity and inflammation in Alzheimer's disease have been hypothesized. Proinflammatory stimuli, including amyloid β-peptide (Aβ, elicit a release of glutamate from microglia. We tested the possibility that a coagonist at the NMDA class of glutamate receptors, D-serine, could respond similarly. Methods Cultured microglial cells were exposed to Aβ. The culture medium was assayed for levels of D-serine by HPLC and for effects on calcium and survival on primary cultures of rat hippocampal neurons. Microglial cell lysates were examined for the levels of mRNA and protein for serine racemase, the enzyme that forms D-serine from L-serine. The racemase mRNA was also assayed in Alzheimer hippocampus and age-matched controls. A microglial cell line was transfected with a luciferase reporter construct driven by the putative regulatory region of human serine racemase. Results Conditioned medium from Aβ-treated microglia contained elevated levels of D-serine. Bioassays of hippocampal neurons with the microglia-conditioned medium indicated that Aβ elevated a NMDA receptor agonist that was sensitive to an antagonist of the D-serine/glycine site (5,7-dicholorokynurenic acid; DCKA and to enzymatic degradation of D-amino acids by D-amino acid oxidase (DAAOx. In the microglia, Aβ elevated steady-state levels of dimeric serine racemase, the apparent active form of the enzyme. Promoter-reporter and mRNA analyses suggest that serine racemase is transcriptionally induced by Aβ. Finally, the levels of serine racemase mRNA were elevated in Alzheimer's disease hippocampus, relative to age-matched controls. Conclusions These data suggest that Aβ could contribute to neurodegeneration through stimulating microglia to release cooperative excitatory amino acids, including D-serine.

  5. MST2 phosphorylation at serine 385 in mitosis inhibits its tumor suppressing activity.

    Science.gov (United States)

    Chen, Xingcheng; Chen, Yuanhong; Dong, Jixin

    2016-12-01

    Mammalian sterile 20-like kinase 1/2 (MST1/2) are core tumor suppressors in the Hippo signaling pathway. MST1/2 have been shown to regulate mitotic progression. Here, we report a novel mechanism for phospho-regulation of MST2 in mitosis and its biological significance in cancer. We found that the mitotic kinase cyclin-dependent kinase 1 (CDK1) phosphorylates MST2 in vitro and in vivo at serine 385 during antimitotic drug-induced G2/M phase arrest. This phosphorylation occurs transiently during unperturbed mitosis. Mitotic phosphorylation of MST2 does not affect its kinase activity or Hippo-YAP signaling. We further showed that mitotic phosphorylation-deficient mutant MST2-S385A possesses higher activity in suppressing cell proliferation and anchorage-independent growth in vitro and tumorigenesis in vivo. Together, our findings reveal a novel layer of regulation for MST2 in mitosis and its role in tumorigenesis. Copyright © 2016 Elsevier Inc. All rights reserved.

  6. In vivo and in vitro characterization of site-specific recombination of a novel serine integrase from the temperate phage EFC-1

    Energy Technology Data Exchange (ETDEWEB)

    Yoon, Bohyun; Kim, Inki; Nam, Ja-Ae [Asan Institute for Life Sciences, Asan Medical Center, College of Medicine, University of Ulsan, 86 Asanbyeoungwon-gil, Songpa-gu, Seoul 138-736 (Korea, Republic of); Chang, Hyo-Ihl [College of Life Sciences & Biotechnology, Korea University, 5-1 Anam-dong, Seongbuk-gu, Seoul 136-701 (Korea, Republic of); Ha, Chang Hoon, E-mail: chhoonha@amc.seoul.kr [Asan Institute for Life Sciences, Asan Medical Center, College of Medicine, University of Ulsan, 86 Asanbyeoungwon-gil, Songpa-gu, Seoul 138-736 (Korea, Republic of)

    2016-04-22

    EFC-1 integrase is a site-specific recombinase that belongs to the large family of serine recombinase. In previously study, we isolated the temperate phage EFC-1, and characterized its genomic sequence. Within its genome, Orf28 was predicted encode a 464 amino acid of a putative integrase gene. In this study, EFC-1 integrase was characterized in vitro and in vivo. In vitro assay was performed using purified His-tag fusion integrase. Also, to identify which serine is involved in the catalytic domain, we used site-directed mutagenesis and analyzed by a recombination assay in vitro. In vivo assay involved PCR and confocal microscopy in HEK293 cells, and determined the minimal lengths of attP and attB sites. According to our results, the EFC-1 integrase-mediated recombination was site-specific and unidirectional system in vitro and in vivo. Serine 21 of EFC-1 integrase plays a major role in the catalytic domain, and minimal sizes of attB and attP was defined 48 and 54 bp. Our finding may help develop a useful tool for gene therapy and gene delivery system. - Highlights: • EFC-1 integrase-mediated recombination was site-specific and unidirectional system. • Serine 21 of EFC-1 integrase plays a major role in the catalytic domain. • The functional minimal sizes of attB and attP was defined 48 and 54 bp.

  7. Insight into the intermolecular recognition mechanism involved in complement component 4 activation through serine protease-trypsin.

    Science.gov (United States)

    Sinha, Vikrant Kumar; Sharma, Om Prakash; Kumar, Muthuvel Suresh

    2018-02-01

    Serine protease cleaved-complement component 4 (C4) at sessile loop, which is significant for completion of lectin and classical complement pathways at the time of infections. The co-crystalized structure of C4 with Mannose-binding protein-associated serine protease 2 (MASP2) provided the structural and functional aspects of its interaction and underlined the C4 activation by MASP2. The same study also revealed the significance of complement control protein (CCP) domain through mutational study, where mutated CCP domain led to the inhibition of C4 activation. However, the interaction of trypsin serine domain with C4α sessile loop revealed another aspect of C4 activation. The human C4 cleavage by Trypsin (Tryp) in a control manner was explored but not yet revealed the identification of cleaved fragments. Hence, the present study investigated the Tryp mediated C4 activation using computational approach (protein-protein docking and molecular dynamics simulation) by comparing with the co-crystalized structure of C4-MASP2. Docking result identified the crucial interacting residues Gly219, Gln178, and Asn102 of Tryp catalytic pocket which were interacting with Arg756 and Glu759 (sessile loop) of α-Chain (C4) in a similar manner to C4-MASP2 co-crystallized complex. Moreover, MD simulation results and mutational study underlined the conformational rearrangements in the C4 due to the Tryp interaction. Comparative analysis of C4 alone, C4-Tryp, and C4-MASP2 revealed the impact of Tryp on C4 was similar as MASP2. These studies designate the role of sessile loop in the interaction with serine domain, which could be useful to understand the various interactions of C4 with other complement components.

  8. Epigenetic Activation of ASCT2 in the Hippocampus Contributes to Depression-Like Behavior by Regulating D-Serine in Mice

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    Jiesi Wang

    2017-05-01

    Full Text Available The roles of D-serine in depression are raised concerned recently as an intrinsic co-agonist for the NMDA receptor. However, the mechanisms underlying its regulation are not fully elucidated. ASCT2 is a Na+-dependent D-serine transporter. We found that decreased D-serine and increased hippocampal ASCT2 levels correlated with chronic social defeat stress (CSDS in mice. Lentivirus-mediated shRNA-mediated knockdown of ASCT2 and the administration of exogenous D-serine in the hippocampus alleviated CSDS-induced social avoidance and immobility. In vivo and in vitro experiments revealed that upregulation of ASCT2 expression in CSDS was regulated through histone hyper-acetylation, not DNA methylation in its promoter region. Immunohistochemistry demonstrated the co-localization of ASCT2 and D-serine. Uptake of D-serine by ASCT2 was demonstrated by in vivo and in vitro experiments. Our results indicate that CSDS induces ASCT2 expression through epigenetic activation and decreases hippocampal D-serine levels, leading to social avoidance, and immobility. Thus, targeting D-serine transport represents an attractive new strategy for treating depression.

  9. Modulation of Escherichia coli serine acetyltransferase catalytic activity in the cysteine synthase complex

    Czech Academy of Sciences Publication Activity Database

    Benoni, Roberto; De Bei, O.; Paredi, G.; Hayes, C. S.; Franko, N.; Mozzarelli, A.; Bettati, S.; Campanini, B.

    2017-01-01

    Roč. 591, č. 9 (2017), s. 1212-1224 ISSN 0014-5793 Institutional support: RVO:61388963 Keywords : cysteine synthase * protein-protein interaction * serine acetyltransferase Subject RIV: CE - Biochemistry OBOR OECD: Biochemistry and molecular biology Impact factor: 3.623, year: 2016

  10. Fractalkine (CX3CL1) enhances hippocampal N-methyl-d-aspartate receptor (NMDAR) function via d-serine and adenosine receptor type A2 (A2AR) activity

    Science.gov (United States)

    2013-01-01

    Background N-Methyl-d-aspartate receptors (NMDARs) play fundamental roles in basic brain functions such as excitatory neurotransmission and learning and memory processes. Their function is largely regulated by factors released by glial cells, including the coagonist d-serine. We investigated whether the activation of microglial CX3CR1 induces the release of factors that modulate NMDAR functions. Methods We recorded the NMDAR component of the field excitatory postsynaptic potentials (NMDA-fEPSPs) elicited in the CA1 stratum radiatum of mouse hippocampal slices by Shaffer collateral stimulation and evaluated d-serine content in the extracellular medium of glial primary cultures by mass spectrometry analysis. Results We demonstrated that CX3CL1 increases NMDA-fEPSPs by a mechanism involving the activity of the adenosine receptor type A2 (A2AR) and the release of the NMDAR coagonist d-serine. Specifically (1) the selective A2AR blocker 7-(2-phenylethyl)-5-amino-2-(2-furyl)-pyrazolo-[4,3-e]-1,2,4-triazolo[1,5-c]pyrimidine (SCH58261) and the genetic ablation of A2AR prevent CX3CL1 action while the A2AR agonist 5-(6-amino-2-(phenethylthio)-9H-purin-9-yl)-N-ethyl-3,4-dihydroxytetrahydrofuran-2-carboxamide (VT7) mimics CX3CL1 effect, and (2) the selective blocking of the NMDAR glycine (and d-serine) site by 5,7-dicholorokynurenic acid (DCKA), the enzymatic degradation of d-serine by d-amino acid oxidase (DAAO) and the saturation of the coagonist site by d-serine, all block the CX3CL1 effect. In addition, mass spectrometry analysis demonstrates that stimulation of microglia and astrocytes with CX3CL1 or VT7 increases d-serine release in the extracellular medium. Conclusions CX3CL1 transiently potentiates NMDAR function though mechanisms involving A2AR activity and the release of d-serine. PMID:23981568

  11. Purification and characterization of a serine protease and chitinases from Paecilomyces lilacinus and detection of chitinase activity on 2D gels.

    Science.gov (United States)

    Khan, Alamgir; Williams, Keith; Molloy, Mark P; Nevalainen, Helena

    2003-12-01

    The filamentous fungus Paecilomyces lilacinus is currently developed as a biocontrol agent against plant parasitic nematodes. Nematode eggs and cuticles are the infection sites for biocontrol agents that penetrate by the production of lytic enzymes. P. lilacinus was cultured in liquid media and proteases and chitinases were induced by the introduction of egg yolk and chitin, respectively. A serine protease was purified from a culture medium using Sepharose-bacitracin affinity column. The protease occurred in three forms, two of which were C-terminally truncated. Chitinase activity was also observed in the culture supernatant, and after separation by isoelectric focusing six proteins were detected that showed activity. Chitinase activity was further confirmed on non-denaturing one-dimensional (1D) and two-dimensional (2D) gels using a sandwich assay with glycol chitin as a substrate. Two of the proteins had similarities with endochitinases as shown by their N-terminal amino acid sequences.

  12. Dual function of a bee venom serine protease: prophenoloxidase-activating factor in arthropods and fibrin(ogen)olytic enzyme in mammals.

    Science.gov (United States)

    Choo, Young Moo; Lee, Kwang Sik; Yoon, Hyung Joo; Kim, Bo Yeon; Sohn, Mi Ri; Roh, Jong Yul; Je, Yeon Ho; Kim, Nam Jung; Kim, Iksoo; Woo, Soo Dong; Sohn, Hung Dae; Jin, Byung Rae

    2010-05-03

    Bee venom contains a variety of peptides and enzymes, including serine proteases. While the presence of serine proteases in bee venom has been demonstrated, the role of these proteins in bee venom has not been elucidated. Furthermore, there is currently no information available regarding the melanization response or the fibrin(ogen)olytic activity of bee venom serine protease, and the molecular mechanism of its action remains unknown. Here we show that bee venom serine protease (Bi-VSP) is a multifunctional enzyme. In insects, Bi-VSP acts as an arthropod prophenoloxidase (proPO)-activating factor (PPAF), thereby triggering the phenoloxidase (PO) cascade. Bi-VSP injected through the stinger induces a lethal melanization response in target insects by modulating the innate immune response. In mammals, Bi-VSP acts similarly to snake venom serine protease, which exhibits fibrin(ogen)olytic activity. Bi-VSP activates prothrombin and directly degrades fibrinogen into fibrin degradation products, defining roles for Bi-VSP as a prothrombin activator, a thrombin-like protease, and a plasmin-like protease. These findings provide a novel view of the mechanism of bee venom in which the bee venom serine protease kills target insects via a melanization strategy and exhibits fibrin(ogen)olytic activity.

  13. Proteolytic profiling and comparative analyses of active trypsin-like serine peptidases in preimaginal stages of Culex quinquefasciatus

    Directory of Open Access Journals (Sweden)

    Borges-Veloso Andre

    2012-06-01

    Full Text Available Abstract Background The mosquito Culex quinquefasciatu s, a widespread insect in tropical and sub-tropical regions of the world, is a vector of multiple arboviruses and parasites, and is considered an important risk to human and veterinary health. Proteolytic enzymes play crucial roles in the insect physiology including the modulation of embryonic development and food digestion. Therefore, these enzymes represent important targets for the development of new control strategies. This study presents zymographic characterization and comparative analysis of the proteolytic activity found in eggs, larval instars and pupae of Culex quinquefasciatus. Methods The proteolytic profiles of eggs, larvae and pupa of Cx. quinquefasciatus were characterized by SDS-PAGE co-polymerized with 0.1% gelatin, according to the pH, temperature and peptidase inhibitor sensitivity. In addition, the proteolytic activities were characterized in solution using 100 μM of the fluorogenic substrate Z-Phe-Arg-AMC. Results Comparison of the proteolytic profiles by substrate-SDS-PAGE from all preimaginal stages of the insect revealed qualitative and quantitative differences in the peptidase expression among eggs, larvae and pupae. Use of specific inhibitors revealed that the proteolytic activity from preimaginal stages is mostly due to trypsin-like serine peptidases that display optimal activity at alkaline pH. In-solution, proteolytic assays of the four larval instars using the fluorogenic substrate Z-Phe-Arg-AMC in the presence or absence of a trypsin-like serine peptidase inhibitor confirmed the results obtained by substrate-SDS-PAGE analysis. The trypsin-like serine peptidases of the four larval instars were functional over a wide range of temperatures, showing activities at 25°C and 65°C, with an optimal activity between 37°C and 50°C. Conclusion The combined use of zymography and in-solution assays, as performed in this study, allowed for a more detailed analysis of the

  14. Purification and physicochemical characterization of a serine protease with fibrinolytic activity from latex of a medicinal herb Euphorbia hirta.

    Science.gov (United States)

    Patel, Girijesh Kumar; Kawale, Ashish Ashok; Sharma, Ashwani Kumar

    2012-03-01

    A 34 kDa serine protease, designated as hirtin, with fibrinolytic activity was purified to homogeneity from the latex of Euphorbia hirta by the combination of ion exchange and gel filtration chromatography. The N-terminal sequence of hirtin was found to be YAVYIGLILETAA/NNE. Hirtin exhibited esterase and amidase activities along with azocaseinolytic, gelatinolytic, fibrinogenolytic and fibrinolytic activities. It preferentially hydrolyzed Aα and α-chains, followed by Bβ and β, and γ and γ-γ chains of fibrinogen and fibrin clot respectively. The optimum pH and temperature for enzyme activity was found to be pH 7.2 and 50 °C respectively. Enzymatic activity of hirtin was significantly inhibited by PMSF and AEBSF. It showed higher specificity for synthetic substrate p-tos-GPRNA for thrombin. The CD spectra of hirtin showed a high content of β-sheets as compared to α-helix. The results indicate that hirtin is a thrombin-like serine protease and may have potential industrial and therapeutic applications. Copyright © 2011 Elsevier Masson SAS. All rights reserved.

  15. Matriptase-2, a membrane-bound mosaic serine proteinase predominantly expressed in human liver and showing degrading activity against extracellular matrix proteins.

    Science.gov (United States)

    Velasco, Gloria; Cal, Santiago; Quesada, Victor; Sánchez, Luis M; López-Otín, Carlos

    2002-10-04

    We have identified and cloned a fetal liver cDNA encoding a new serine proteinase that has been called matriptase-2. This protein exhibits a domain organization similar to other members of an emerging family of membrane-bound serine proteinases known as type II transmembrane serine proteinases. Matriptase-2 contains a short cytoplasmic domain, a type II transmembrane sequence, a central region with several modular structural domains including two CUB (complement factor C1s/C1r, urchin embryonic growth factor, bone morphogenetic protein) domains and three low density lipoprotein receptor tandem repeats, and finally, a C-terminal catalytic domain with all typical features of serine proteinases. The human matriptase-2 gene maps to 22q12-q13, a location that differs from all type II transmembrane serine proteinase genes mapped to date. Immunofluorescence and Western blot analysis of COS-7 cells transfected with the isolated cDNA confirmed that matriptase-2 is anchored to the cell surface. Matriptase-2 was expressed in Escherichia coli, and the purified recombinant protein hydrolyzed synthetic substrates used for assaying serine proteinases and endogenous proteins such as type I collagen, fibronectin, and fibrinogen. Matriptase-2 could also activate single-chain urokinase plasminogen activator, albeit with low efficiency. These activities were abolished by inhibitors of serine proteinases but not by inhibitors of other classes of proteolytic enzymes. Northern blot analysis demonstrated that matriptase-2 transcripts are only detected at significant levels in both fetal and adult liver, suggesting that this novel serine proteinase may play a specialized role in matrix remodeling processes taking place in this tissue during development or in adult tissues.

  16. Involvement of neuronal and glial activities in control of the extracellular d-serine concentrations by the AMPA glutamate receptor in the mouse medial prefrontal cortex.

    Science.gov (United States)

    Ishiwata, Sayuri; Umino, Asami; Nishikawa, Toru

    2017-09-28

    It has been well accepted that d-serine may be an exclusive endogenous coagonist for the N-methyl-d-aspartate (NMDA)-type glutamate receptor in mammalian forebrain regions. We have recently found by using an in vivo dialysis method that an intra-medial prefrontal cortex infusion of S-α-amino-3-hydroxyl-5-methyl-4-isoxazole-propionate (S-AMPA), a selective AMPA-type glutamate receptor agonist, causes a reduction in the extracellular levels of d-serine in a calcium-permeable AMPA receptor antagonist-sensitive manner. The inhibitory influence by the AMPA receptor on the extracellular d-serine, however, contradicts the data obtained from in vitro experiments that the AMPA receptor stimulation leads to facilitation of the d-serine liberation. This discrepancy appears to be due to the different cell setups between the in vivo and in vitro preparations. From the viewpoints of the previous reports indicating (1) the neuronal presence of d-serine synthesizing enzyme, serine racemase, and d-serine-like immunoreactivity and (2) the same high tissue concentrations of d-serine in the glia-enriched white matter and in the neuron-enriched gray matter of the mammalian neocortex, we have now investigated in the mouse medial prefrontal cortex, the effects of attenuation of neuronal and glial activities, by tetrodotoxin or fluorocitrate, respectively, on the S-AMPA-induced downregulation of the extracellular d-serine contents. In vivo dialysis studies revealed that a local infusion of tetrodotoxin or fluorocitrate eliminated the ability of S-AMPA given intra-cortically to cause a significant decrease in the dialysate concentrations of d-serine without affecting the elevating effects of S-AMPA on those of glycine, another intrinsic coagonist for the NMDA receptor. These findings suggest that the control by the AMPA receptor of the extracellular d-serine levels could be modulated by the neuronal and glial activities in the prefrontal cortex. It cannot be excluded that

  17. A serine protease isolated from the bristles of the Amazonic caterpillar, Premolis semirufa, is a potent complement system activator.

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    Isadora Maria Villas Boas

    Full Text Available The caterpillar of the moth Premolis semirufa, commonly named pararama, is found in the Brazilian Amazon region. Accidental contact with the caterpillar bristles causes an intense itching sensation, followed by symptoms of an acute inflammation, which last for three to seven days after the first incident. After multiple accidents a chronic inflammatory reaction, called "Pararamose", characterized by articular synovial membrane thickening with joint deformities common to chronic synovitis, frequently occurs. Although complement mediated inflammation may aid the host defense, inappropriate or excessive activation of the complement system and generation of anaphylatoxins can lead to inflammatory disorder and pathologies. The aim of the present study was to evaluate, in vitro, whether the Premolis semirufa's bristles extract could interfere with the human complement system.The bristles extract was able to inhibit the haemolytic activity of the alternative pathway, as well as the activation of the lectin pathway, but had no effect on the classical pathway, and this inhibition seemed to be caused by activation and consumption of complement components. The extract induced the production of significant amounts of all three anaphylatoxins, C3a, C4a and C5a, promoted direct cleavage of C3, C4 and C5 and induced a significant generation of terminal complement complexes in normal human serum. By using molecular exclusion chromatography, a serine protease of 82 kDa, which activates complement, was isolated from P. semirufa bristles extract. The protease, named here as Ps82, reduced the haemolytic activity of the alternative and classical pathways and inhibited the lectin pathway. In addition, Ps82 induced the cleavage of C3, C4 and C5 and the generation of C3a and C4a in normal human serum and it was capable to cleave human purified C5 and generate C5a. The use of Phenanthroline, metalloprotease inhibitor, in the reactions did not significantly interfere

  18. Activation of the bacterial thermosensor DesK involves a serine zipper dimerization motif that is modulated by bilayer thickness.

    Science.gov (United States)

    Cybulski, Larisa Estefanía; Ballering, Joost; Moussatova, Anastassiia; Inda, Maria Eugenia; Vazquez, Daniela B; Wassenaar, Tsjerk A; de Mendoza, Diego; Tieleman, D Peter; Killian, J Antoinette

    2015-05-19

    DesK is a bacterial thermosensor protein involved in maintaining membrane fluidity in response to changes in environmental temperature. Most likely, the protein is activated by changes in membrane thickness, but the molecular mechanism of sensing and signaling is still poorly understood. Here we aimed to elucidate the mode of action of DesK by studying the so-called "minimal sensor DesK" (MS-DesK), in which sensing and signaling are captured in a single transmembrane segment. This simplified version of the sensor allows investigation of membrane thickness-dependent protein-lipid interactions simply by using synthetic peptides, corresponding to the membrane-spanning parts of functional and nonfunctional mutants of MS-DesK incorporated in lipid bilayers with varying thicknesses. The lipid-dependent behavior of the peptides was investigated by circular dichroism, tryptophan fluorescence, and molecular modeling. These experiments were complemented with in vivo functional studies on MS-DesK mutants. Based on the results, we constructed a model that suggests a new mechanism for sensing in which the protein is present as a dimer and responds to an increase in bilayer thickness by membrane incorporation of a C-terminal hydrophilic motif. This results in exposure of three serines on the same side of the transmembrane helices of MS-DesK, triggering a switching of the dimerization interface to allow the formation of a serine zipper. The final result is activation of the kinase state of MS-DesK.

  19. Active site of tripeptidyl peptidase II from human erythrocytes is of the subtilisin type

    International Nuclear Information System (INIS)

    Tomkinson, B.; Wernstedt, C.; Hellman, U.; Zetterqvist, Oe.

    1987-01-01

    The present report presents evidence that the amino acid sequence around the serine of the active site of human tripeptidyl peptidase II is of the subtilisin type. The enzyme from human erythrocytes was covalently labeled at its active site with [ 3 H]diisopropyl fluorophosphate, and the protein was subsequently reduced, alkylated, and digested with trypsin. The labeled tryptic peptides were purified by gel filtration and repeated reversed-phase HPLC, and their amino-terminal sequences were determined. Residue 9 contained the radioactive label and was, therefore, considered to be the active serine residue. The primary structure of the part of the active site (residues 1-10) containing this residue was concluded to be Xaa-Thr-Gln-Leu-Met-Asx-Gly-Thr-Ser-Met. This amino acid sequence is homologous to the sequence surrounding the active serine of the microbial peptidases subtilisin and thermitase. These data demonstrate that human tripeptidyl peptidase II represents a potentially distinct class of human peptidases and raise the question of an evolutionary relationship between the active site of a mammalian peptidase and that of the subtilisin family of serine peptidases

  20. Serine Hydroxymethyltransferase from the Cold Adapted Microorganism Psychromonas ingrahamii: A Low Temperature Active Enzyme with Broad Substrate Specificity

    Directory of Open Access Journals (Sweden)

    Stefano Pascarella

    2012-01-01

    Full Text Available Serine hydroxymethyltransferase from the psychrophilic microorganism Psychromonas ingrahamii was expressed in Escherichia coli and purified as a His-tag fusion protein. The enzyme was characterized with respect to its spectroscopic, catalytic, and thermodynamic properties. The properties of the psychrophilic enzyme have been contrasted with the characteristics of the homologous counterpart from E. coli, which has been structurally and functionally characterized in depth and with which it shares 75% sequence identity. Spectroscopic measures confirmed that the psychrophilic enzyme displays structural properties almost identical to those of the mesophilic counterpart. At variance, the P. ingrahamii enzyme showed decreased thermostability and high specific activity at low temperature, both of which are typical features of cold adapted enzymes. Furthermore, it was a more efficient biocatalyst compared to E. coli serine hydroxymethyltransferase (SHMT particularly for side reactions. Many β-hydroxy-α-amino acids are SHMT substrates and represent important compounds in the synthesis of pharmaceuticals, agrochemicals and food additives. Thanks to these attractive properties, this enzyme could have a significant potential for biotechnological applications.

  1. Isolation and characterization of a serine proteinase with thrombin-like activity from the venom of the snake Bothrops asper

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    A.V Pérez

    2008-01-01

    Full Text Available A serine proteinase with thrombin-like activity was isolated from the venom of the Central American pit viper Bothrops asper. Isolation was performed by a combination of affinity chromatography on aminobenzamidine-Sepharose and ion-exchange chromatography on DEAE-Sepharose. The enzyme accounts for approximately 0.13% of the venom dry weight and has a molecular mass of 32 kDa as determined by SDS-PAGE, and of 27 kDa as determined by MALDI-TOF mass spectrometry. Its partial amino acid sequence shows high identity with snake venom serine proteinases and a complete identity with a cDNA clone previously sequenced from this species. The N-terminal sequence of the enzyme is VIGGDECNINEHRSLVVLFXSSGFL CAGTLVQDEWVLTAANCDSKNFQ. The enzyme induces clotting of plasma (minimum coagulant dose = 4.1 µg and fibrinogen (minimum coagulant dose = 4.2 µg in vitro, and promotes defibrin(ogenation in vivo (minimum defibrin(ogenating dose = 1.0 µg. In addition, when injected intravenously in mice at doses of 5 and 10 µg, it induces a series of behavioral changes, i.e., loss of the righting reflex, opisthotonus, and intermittent rotations over the long axis of the body, which closely resemble the `gyroxin-like' effect induced by other thrombin-like enzymes from snake venoms.

  2. Isolation and characterization of a serine proteinase with thrombin-like activity from the venom of the snake Bothrops asper.

    Science.gov (United States)

    Pérez, A V; Rucavado, A; Sanz, L; Calvete, J J; Gutiérrez, J M

    2008-01-01

    A serine proteinase with thrombin-like activity was isolated from the venom of the Central American pit viper Bothrops asper. Isolation was performed by a combination of affinity chromatography on aminobenzamidine-Sepharose and ion-exchange chromatography on DEAE-Sepharose. The enzyme accounts for approximately 0.13% of the venom dry weight and has a molecular mass of 32 kDa as determined by SDS-PAGE, and of 27 kDa as determined by MALDI-TOF mass spectrometry. Its partial amino acid sequence shows high identity with snake venom serine proteinases and a complete identity with a cDNA clone previously sequenced from this species. The N-terminal sequence of the enzyme is VIGGDECNINEHRSLVVLFXSSGFL CAGTLVQDEWVLTAANCDSKNFQ. The enzyme induces clotting of plasma (minimum coagulant dose = 4.1 microg) and fibrinogen (minimum coagulant dose = 4.2 microg) in vitro, and promotes defibrin(ogen)ation in vivo (minimum defibrin(ogen)ating dose = 1.0 microg). In addition, when injected intravenously in mice at doses of 5 and 10 microg, it induces a series of behavioral changes, i.e., loss of the righting reflex, opisthotonus, and intermittent rotations over the long axis of the body, which closely resemble the ;gyroxin-like' effect induced by other thrombin-like enzymes from snake venoms.

  3. Serine hydroxymethyltransferase from the cold adapted microorganism Psychromonas ingrahamii: a low temperature active enzyme with broad substrate specificity.

    Science.gov (United States)

    Angelaccio, Sebastiana; Florio, Rita; Consalvi, Valerio; Festa, Guido; Pascarella, Stefano

    2012-01-01

    Serine hydroxymethyltransferase from the psychrophilic microorganism Psychromonas ingrahamii was expressed in Escherichia coli and purified as a His-tag fusion protein. The enzyme was characterized with respect to its spectroscopic, catalytic, and thermodynamic properties. The properties of the psychrophilic enzyme have been contrasted with the characteristics of the homologous counterpart from E. coli, which has been structurally and functionally characterized in depth and with which it shares 75% sequence identity. Spectroscopic measures confirmed that the psychrophilic enzyme displays structural properties almost identical to those of the mesophilic counterpart. At variance, the P. ingrahamii enzyme showed decreased thermostability and high specific activity at low temperature, both of which are typical features of cold adapted enzymes. Furthermore, it was a more efficient biocatalyst compared to E. coli serine hydroxymethyltransferase (SHMT) particularly for side reactions. Many β-hydroxy-α-amino acids are SHMT substrates and represent important compounds in the synthesis of pharmaceuticals, agrochemicals and food additives. Thanks to these attractive properties, this enzyme could have a significant potential for biotechnological applications.

  4. DOE site performance assessment activities

    International Nuclear Information System (INIS)

    1990-07-01

    Information on performance assessment capabilities and activities was collected from eight DOE sites. All eight sites either currently dispose of low-level radioactive waste (LLW) or plan to dispose of LLW in the near future. A survey questionnaire was developed and sent to key individuals involved in DOE Order 5820.2A performance assessment activities at each site. The sites surveyed included: Hanford Site (Hanford), Idaho National Engineering Laboratory (INEL), Los Alamos National Laboratory (LANL), Nevada Test Site (NTS), Oak Ridge National Laboratory (ORNL), Paducah Gaseous Diffusion Plant (Paducah), Portsmouth Gaseous Diffusion Plant (Portsmouth), and Savannah River Site (SRS). The questionnaire addressed all aspects of the performance assessment process; from waste source term to dose conversion factors. This report presents the information developed from the site questionnaire and provides a comparison of site-specific performance assessment approaches, data needs, and ongoing and planned activities. All sites are engaged in completing the radioactive waste disposal facility performance assessment required by DOE Order 5820.2A. Each site has achieved various degrees of progress and have identified a set of critical needs. Within several areas, however, the sites identified common needs and questions

  5. The pro-coagulant fibrinogenolytic serine protease isoenzymes purified from Daboia russelii russelii venom coagulate the blood through factor V activation: role of glycosylation on enzymatic activity.

    Directory of Open Access Journals (Sweden)

    Ashis K Mukherjee

    Full Text Available Proteases from Russell's viper venom (RVV induce a variety of toxic effects in victim. Therefore, four new RVV protease isoenzymes of molecular mass 32901.044 Da, 333631.179 Da, 333571.472 Da, and 34594.776 Da, were characterized in this study. The first 10 N-terminal residues of these serine protease isoenzymes showed significant sequence homology with N-terminal sequences of snake venom thrombin-like and factor V-activating serine proteases, which was reconfirmed by peptide mass fingerprinting analysis. These proteases were found to be different from previously reported factor V activators isolated from snake venoms. These proteases showed significantly different fibrinogenolytic, BAEE-esterase and plasma clotting activities but no fibrinolytic, TAME-esterase or amidolytic activity against the chromogenic substrate for trypsin, thrombin, plasmin and factor Xa. Their Km and Vmax values towards fibrinogen were determined in the range of 6.6 to 10.5 µM and 111.0 to 125.5 units/mg protein, respectively. On the basis of fibrinogen degradation pattern, they may be classified as A/B serine proteases isolated from snake venom. These proteases contain ∼ 42% to 44% of N-linked carbohydrates by mass whereas partially deglycosylated enzymes showed significantly less catalytic activity as compared to native enzymes. In vitro these protease isoenzymes induce blood coagulation through factor V activation, whereas in vivo they provoke dose-dependent defibrinogenation and anticoagulant activity in the mouse model. At a dose of 5 mg/kg, none of these protease isoenzymes were found to be lethal in mice or house geckos, suggesting therapeutic application of these anticoagulant peptides for the prevention of thrombosis.

  6. Cathelicidin, kallikrein 5, and serine protease activity is inhibited during treatment of rosacea with azelaic acid 15% gel.

    Science.gov (United States)

    Coda, Alvin B; Hata, Tissa; Miller, Jeremiah; Audish, David; Kotol, Paul; Two, Aimee; Shafiq, Faiza; Yamasaki, Kenshi; Harper, Julie C; Del Rosso, James Q; Gallo, Richard L

    2013-10-01

    Excess cathelicidin and kallikrein 5 (KLK5) have been hypothesized to play a role in the pathophysiology of rosacea. We sought to evaluate the effects of azelaic acid (AzA) on these elements of the innate immune system. Gene expression and protease activity were measured in laboratory models and patients with rosacea during a 16-week multicenter, prospective, open-label study of 15% AzA gel. AzA directly inhibited KLK5 in cultured keratinocytes and gene expression of KLK5, Toll-like receptor-2, and cathelicidin in mouse skin. Patients with rosacea showed reduction in cathelicidin and KLK5 messenger RNA after treatment with AzA gel. Subjects without rosacea had lower serine protease activity (SPA) than patients with rosacea. Distinct subsets of patients with rosacea who had high and low baseline SPA were identified, and patients with high baseline exhibited a statistically significant reduction of SPA with 15% AzA gel treatment. Study size was insufficient to predict clinical efficacy based on the innate immune response to AzA. These results show that cathelicidin and KLK5 decrease in association with AZA exposure. Our observations suggest a new mechanism of action for AzA and that SPA may be a useful biomarker for disease activity. Copyright © 2013 American Academy of Dermatology, Inc. Published by Mosby, Inc. All rights reserved.

  7. Cathelicidin, kallikrein 5, and serine protease activity is inhibited during treatment of rosacea with azelaic acid 15% gel

    Science.gov (United States)

    Coda, Alvin B.; Hata, Tissa; Miller, Jeremiah; Audish, David; Kotol, Paul; Two, Aimee; Shafiq, Faiza; Yamasaki, Kenshi; Harper, Julie C.; Del Rosso, James Q.; Gallo, Richard L.

    2014-01-01

    Background Excess cathelicidin and kallikrein 5 (KLK5) have been hypothesized to play a role in the pathophysiology of rosacea. Objective We sought to evaluate the effects of azelaic acid (AzA) on these elements of the innate immune system. Methods Gene expression and protease activity were measured in laboratory models and patients with rosacea during a 16-week multicenter, prospective, open-label study of 15% AzA gel. Results AzA directly inhibited KLK5 in cultured keratinocytes and gene expression of KLK5, Toll-like receptor-2, and cathelicidin in mouse skin. Patients with rosacea showed reduction in cathelicidin and KLK5 messenger RNA after treatment with AzA gel. Subjects without rosacea had lower serine protease activity (SPA) than patients with rosacea. Distinct subsets of patients with rosacea who had high and low baseline SPA were identified, and patients with high baseline exhibited a statistically significant reduction of SPA with 15% AzA gel treatment. Limitations Study size was insufficient to predict clinical efficacy based on the innate immune response to AzA. Conclusions These results show that cathelicidin and KLK5 decrease in association with AZA exposure. Our observations suggest a new mechanism of action for AzA and that SPA may be a useful biomarker for disease activity. PMID:23871720

  8. Inhibition of plasmin, urokinase, tissue plasminogen activator, and C1S by a myxoma virus serine proteinase inhibitor.

    Science.gov (United States)

    Lomas, D A; Evans, D L; Upton, C; McFadden, G; Carrell, R W

    1993-01-05

    The myxoma and malignant rabbit fibroma poxviruses are lethal tumorigenic viruses of rabbits whose virulence is modulated by the production of a virus-encoded secreted serine proteinase inhibitor, SERP-1. This viral protein was detected in medium harvested from myxoma and malignant rabbit fibroma virus-infected cells, and its inhibitory profile has been characterized by gel and kinetic analysis. SERP-1 forms complexes with and inhibits the human fibrinolytic enzymes plasmin, urokinase, and two-chain tissue-type plasminogen activator (association rate constants 3.4 x 10(4), 4.3 x 10(4), and 3.6 x 10(4) M-1 s-1 respectively). It is also able to inhibit C1S, the first enzyme in the complement cascade with an association rate constant which was unaffected by the addition of heparin (1.3 x 10(3) M-1 s-1). SERP-1 acts as a substrate for and is cleaved by thrombin, porcine trypsin, human neutrophil elastase, porcine pancreatic elastase, thermolysin, subtilisin, bovine alpha-chymotrypsin, and factor Xa. Incubation with kallikrein and cathepsin G had no effect. The structure of SERP-1 has been modeled on other members of the serpin family which revealed the characteristic serpin architecture apart from the absence of the D-helix. Structural analysis and kinetic assays demonstrate that the absence of this region does not prevent inhibitory activity and furthermore allow the identification of cysteine residues involved in internal and intermolecular disulfide bonding.

  9. Pdx1 is post-translationally modified in vivo and serine 61 is the principal site of phosphorylation

    DEFF Research Database (Denmark)

    Frogne, Thomas; Sylvestersen, Kathrine Beck; Kubicek, Stefan

    2012-01-01

    signaling. Several studies have shown that post-translational modifications are regulating Pdx1 activity through intracellular localization and binding to co-factors. Understanding the signaling cues converging on Pdx1 and modulating its activity is therefore an attractive approach in diabetes treatment. We......Maintaining sufficient levels of Pdx1 activity is a prerequisite for proper regulation of blood glucose homeostasis and beta cell function. Mice that are haploinsufficient for Pdx1 display impaired glucose tolerance and lack the ability to increase beta cell mass in response to decreased insulin...

  10. A novel serine protease secreted by medicinal maggots enhances plasminogen activator-induced fibrinolysis

    DEFF Research Database (Denmark)

    van der Plas, Mariena J A; Andersen, Anders S; Nazir, Sheresma

    2014-01-01

    . Recombinant Sericase degraded plasminogen leading amongst others to the formation of the mini-plasminogen like fragment Val454-plasminogen. In addition, the presence of a non-proteolytic cofactor in secretions was discovered, which plays a role in the enhancement of plasminogen activator-induced fibrinolysis...

  11. D-serine and serine racemase are associated with PSD-95 and glutamatergic synapse stability

    Directory of Open Access Journals (Sweden)

    Hong eLin

    2016-02-01

    Full Text Available D-serine is an endogenous coagonist at the glycine site of synaptic NMDA receptors (NMDARs, synthesized by serine racemase (SR through conversion of L-serine. It is crucial for synaptic plasticity and is implicated in schizophrenia. Our previous studies demonstrated specific loss of SR, D-serine-responsive synaptic NMDARs, and glutamatergic synapses in cortical neurons lacking alpha7 nicotinic acetylcholine receptors, which promotes glutamatergic synapse formation and maturation during development. We thus hypothesize that D-serine and SR (D-serine/SR are associated with glutamatergic synaptic development. Using morphological and molecular studies in cortical neuronal cultures, we demonstrate that D-serine/SR are associated with PSD-95 and NMDARs in postsynaptic neurons and with glutamatergic synapse stability during synaptic development. Endogenous D-serine and SR colocalize with PSD-95, but not presynaptic vesicular glutamate transporter 1 (VGLUT1, in glutamatergic synapses of cultured cortical neurons. Low-density astrocytes in cortical neuronal cultures lack SR expression but contain enriched D-serine in large vesicle-like structures, suggesting possible synthesis of D-serine in postsynaptic neurons and storage in astrocytes. More interestingly, endogenous D-serine and SR colocalize with PSD-95 in the postsynaptic terminals of glutamatergic synapses during early and late synaptic development, implicating involvement of D-serine/SR in glutamatergic synaptic development. Exogenous application of D-serine enhances the interactions of SR with PSD-95 and NR1, and increases the number of VGLUT1- and PSD-95-positive glutamatergic synapses, suggesting that exogenous D-serine enhances postsynaptic SR/PSD-95 signaling and stabilizes glutamatergic synapses during cortical synaptic development. This is blocked by NMDAR antagonist 2-amino-5-phosphonopentanoic acid (AP5 and 7-chlorokynurenic acid (7-CK, a specific antagonist at the glycine site of NMDARs

  12. Regulation of Serine-Threonine Kinase Akt Activation by NAD+-Dependent Deacetylase SIRT7

    Directory of Open Access Journals (Sweden)

    Jia Yu

    2017-01-01

    Full Text Available The Akt pathway is a central regulator that promotes cell survival in response to extracellular signals. Depletion of SIRT7, an NAD+-dependent deacetylase that is the least-studied sirtuin, is known to significantly increase Akt activity in mice through unknown mechanisms. In this study, we demonstrate that SIRT7 depletion in breast cancer cells results in Akt hyper-phosphorylation and increases cell survival following genotoxic stress. Mechanistically, SIRT7 specifically interacts with and deacetylates FKBP51 at residue lysines 28 and 155 (K28 and K155, resulting in enhanced interactions among FKBP51, Akt, and PHLPP, as well as Akt dephosphorylation. Mutating both lysines to arginines abolishes the effect of SIRT7 on Akt activity through FKBP51 deacetylation. Finally, energy stress strengthens SIRT7-mediated effects on Akt dephosphorylation through FKBP51 and thus sensitizes cancer cells to cytotoxic agents. These results reveal a direct role of SIRT7 in Akt regulation and raise the possibility of using the glucose analog 2-deoxy-D-glucose (2DG as a chemo-sensitizing agent.

  13. A two disulfide bridge Kazal domain from Phytophthora exhibits stable inhibitory activity against serine proteases of the subtilisin family

    Directory of Open Access Journals (Sweden)

    Kamoun Sophien

    2005-08-01

    Full Text Available Abstract Background Kazal-like serine protease inhibitors are defined by a conserved sequence motif. A typical Kazal domain contains six cysteine residues leading to three disulfide bonds with a 1–5/2–4/3–6 pattern. Most Kazal domains described so far belong to this class. However, a novel class of Kazal domains with two disulfide bridges resulting from the absence of the third and sixth cysteines have been found in biologically important molecules, such as human LEKTI, a 15-domain inhibitor associated with the severe congenital disease Netherton syndrome. These domains are referred to as atypical Kazal domains. Previously, EPI1, a Kazal-like protease inhibitor from the oomycete plant pathogen Phytophthora infestans, was shown to be a tight-binding inhibitor of subtilisin A. EPI1 also inhibits and interacts with the pathogenesis-related P69B subtilase of the host plant tomato, suggesting a role in virulence. EPI1 is composed of two Kazal domains, the four-cysteine atypical domain EPI1a and the typical domain EPI1b. Results In this study, we predicted the inhibition constants of EPI1a and EPI1b to subtilisin A using the additivity-based sequence to reactivity algorithm (Laskowski algorithm. The atypical domain EPI1a, but not the typical domain EPI1b, was predicted to have strong inhibitory activity against subtilisin A. Inhibition assays and coimmunoprecipitation experiments showed that recombinant domain EPI1a exhibited stable inhibitory activity against subilisin A and was solely responsible for inhibition and interaction with tomato P69B subtilase. Conclusion The finding that the two disulfide bridge atypical Kazal domain EPI1a is a stable inhibitor indicates that the missing two cysteines and their corresponding disulfide bond are not essential for inhibitor reactivity and stability. This report also suggests that the Laskowski algorithm originally developed and validated with typical Kazal domains might operate accurately for atypical

  14. The roles of serine protease, intracellular and extracellular phenoloxidase in activation of prophenoloxidase system, and characterization of phenoloxidase from shrimp haemocytes induced by lipopolysaccharide or dopamine

    Science.gov (United States)

    Xie, Peng; Pan, Luqing; Xu, Wujie; Yue, Feng

    2013-09-01

    We investigated the effects of lipopolysaccharide (LPS) and dopamine (DA) on the activation of the prophenoloxidase (proPO) system of Litopenaeus vannamei. LPS and DA were shown with a negative dose-dependent effect on hyalne cells (HC), semi-granular cells (SGC), large granular cells (LGC), and total haemocyte count (THC). When haemocytes were treated with LPS or DA, serine proteinase activity and intracellular phenoloxidase (PO) activity were significantly reduced, but extracellular PO activity increased significantly. These findings indicated that the reduction in haemocyte counts was mainly because of the degranulation and activation of the proPO system from semi-granule and large granule cells. The PKC inhibitor, chelerythrine, and the TPK inhibitor, genistein, had an inhibitory effect on extracellular PO activity, while serine proteinase and intracellular PO activity increased. This suggests that the LPS and DA induce the activation of proPO in haemocytes via PKC and TPK-related signaling pathways, but serine proteinase may be activated only by PKC, as the genistein effects were not statistically significant. Electrophoresis analysis revealed that POs induced by LPS or DA have the same molecular mass and high diphenolase activity. Two PO bands at 526 kDa and 272 kDa were observed in PAGE, while in the haemocyte lysate supernatant (HLS), only a 272-kDa band was observed. This band was resolved after SDS-PAGE under non-reducing and reducing conditions into two groups of POs, 166 kDa and 126 kDa, and 78.1 kDa and 73.6 kDa, respectively, suggesting that PO in L. vannamei is an oligomer, which may have different compositions intra- and extracellularly.

  15. Serine Hydroxymethyltransferase from the Cold Adapted Microorganism Psychromonas ingrahamii: A Low Temperature Active Enzyme with Broad Substrate Specificity

    OpenAIRE

    Angelaccio, Sebastiana; Florio, Rita; Consalvi, Valerio; Festa, Guido; Pascarella, Stefano

    2012-01-01

    Serine hydroxymethyltransferase from the psychrophilic microorganism Psychromonas ingrahamii was expressed in Escherichia coli and purified as a His-tag fusion protein. The enzyme was characterized with respect to its spectroscopic, catalytic, and thermodynamic properties. The properties of the psychrophilic enzyme have been contrasted with the characteristics of the homologous counterpart from E. coli, which has been structurally and functionally characterized in depth and with which it shar...

  16. A novel phosphorylation site, Serine 199, in the C-terminus of cardiac troponin I regulates calcium sensitivity and susceptibility to calpain-induced proteolysis

    NARCIS (Netherlands)

    Wijnker, P.J.M.; Li, Y.; Zhang, P.; Foster, D.B.; dos Remedios, C.; van Eyk, J.E.; Stienen, G.J.M.; Murphy, A.M.; van der Velden, J.

    2015-01-01

    Phosphorylation of cardiac troponin I (cTnI) by protein kinase C (PKC) is implicated in cardiac dysfunction. Recently, Serine 199 (Ser199) was identified as a target for PKC phosphorylation and increased Ser199 phosphorylation occurs in end-stage failing compared with non-failing human myocardium.

  17. Apical serine protease activity is necessary for assembly of a high-resistance renal collecting duct epithelium

    DEFF Research Database (Denmark)

    Steensgaard, Mette; Svenningsen, Per; Tinning, Anne R

    2010-01-01

    Abstract AIM: We hypothesized that the serine protease prostasin is necessary for differentiation of a high resistance renal collecting duct epithelium governed by glucocorticoid. METHODS: Postnatal rat kidney and adult human kidney was used to study expression and localization of prostasin...... 21) and was detected in collecting ducts. Immunoreactive prostasin was associated with collecting ducts and loops of Henle in human kidney. In rat, adrenalectomy at day 10 had no effect on prostasin mRNA level in kidney at day 20. Cultured M-1 cells exhibited parallel increases in prostasin m...

  18. Short hydrogen bonds in the catalytic mechanism of serine proteases

    Directory of Open Access Journals (Sweden)

    VLADIMIR LESKOVAC

    2008-04-01

    Full Text Available The survey of crystallographic data from the Protein Data Bank for 37 structures of trypsin and other serine proteases at a resolution of 0.78–1.28 Å revealed the presence of hydrogen bonds in the active site of the enzymes, which are formed between the catalytic histidine and aspartate residues and are on average 2.7 Å long. This is the typical bond length for normal hydrogen bonds. The geometric properties of the hydrogen bonds in the active site indicate that the H atom is not centered between the heteroatoms of the catalytic histidine and aspartate residues in the active site. Taken together, these findings exclude the possibility that short “low-barrier” hydrogen bonds are formed in the ground state structure of the active sites examined in this work. Some time ago, it was suggested by Cleland that the “low-barrier hydrogen bond” hypothesis is operative in the catalytic mechanism of serine proteases, and requires the presence of short hydrogen bonds around 2.4 Å long in the active site, with the H atom centered between the catalytic heteroatoms. The conclusions drawn from this work do not exclude the validity of the “low-barrier hydrogen bond” hypothesis at all, but they merely do not support it in this particular case, with this particular class of enzymes.

  19. Contribution of the D-Serine-dependent pathway to the cellular mechanisms underlying cognitive aging

    Directory of Open Access Journals (Sweden)

    Emilie Rouaud

    2010-02-01

    Full Text Available An association between age-related memory impairments and changes in functional plasticity in the aging brain has been under intense study within the last decade. In this article, we show that an impaired activation of the strychnine-insensitive glycine site of N-Methyl-D-Aspartate receptors (NMDA-R by its agonist D-serine contributes to deficits of synaptic plasticity in the hippocampus of memory-impaired aged rats. Supplementation with exogenous D-serine prevents the age-related deficits of isolated NMDA-R-dependent synaptic potentials as well as those of theta-burst-induced long-term potentiation and synaptic depotentiation. Endogenous levels of D-serine are reduced in the hippocampus with aging, that correlates with a weaker expression of serine racemase synthesizing the amino acid. On the contrary, the affinity of D-serine binding to NMDA-R is not affected by aging. These results point to a critical role for the D-serine-dependent pathway in the functional alterations of the brain underlying memory impairment and provide key information in the search for new therapeutic strategies for the treatment of memory deficits in the elderly.

  20. Serine biosynthesis and transport defects.

    Science.gov (United States)

    El-Hattab, Ayman W

    2016-07-01

    l-serine is a non-essential amino acid that is biosynthesized via the enzymes phosphoglycerate dehydrogenase (PGDH), phosphoserine aminotransferase (PSAT), and phosphoserine phosphatase (PSP). Besides its role in protein synthesis, l-serine is a potent neurotrophic factor and a precursor of a number of essential compounds including phosphatidylserine, sphingomyelin, glycine, and d-serine. Serine biosynthesis defects result from impairments of PGDH, PSAT, or PSP leading to systemic serine deficiency. Serine biosynthesis defects present in a broad phenotypic spectrum that includes, at the severe end, Neu-Laxova syndrome, a lethal multiple congenital anomaly disease, intermediately, infantile serine biosynthesis defects with severe neurological manifestations and growth deficiency, and at the mild end, the childhood disease with intellectual disability. A serine transport defect resulting from deficiency of the ASCT1, the main transporter for serine in the central nervous system, has been recently described in children with neurological manifestations that overlap with those observed in serine biosynthesis defects. l-serine therapy may be beneficial in preventing or ameliorating symptoms in serine biosynthesis and transport defects, if started before neurological damage occurs. Herein, we review serine metabolism and transport, the clinical, biochemical, and molecular aspects of serine biosynthesis and transport defects, the mechanisms of these diseases, and the potential role of serine therapy. Copyright © 2016 Elsevier Inc. All rights reserved.

  1. Loss of MYC confers resistance to doxorubicin-induced apoptosis by preventing the activation of multiple serine protease- and caspase-mediated pathways

    DEFF Research Database (Denmark)

    Grassilli, Emanuela; Ballabeni, Andrea; Maellaro, Emilia

    2004-01-01

    c-Myc plays an essential role in proliferation, differentiation, and apoptosis. Because of its relevance to cancer, most studies have focused on the cellular consequences of c-Myc overexpression. Here, we address the role of physiological levels of c-Myc in drug-induced apoptosis. By using c-MYC...... null cells we confirm and extend recent reports showing a c-Myc requirement for the induction of apoptosis by a number of anticancer agents. In particular, we show that c-Myc is required for the induction of apoptosis by doxorubicin and etoposide, whereas it is not required for camptothecin......-3 activation. Finally, a complete rescue from doxorubicin-induced apoptosis is obtained only when serine proteases, caspase-3, and mitochondrial activation are inhibited simultaneously. Interestingly, doxorubicin requires c-Myc for the activation of all of these pathways. Our findings therefore...

  2. Identification of covalent active site inhibitors of dengue virus protease

    Directory of Open Access Journals (Sweden)

    Koh-Stenta X

    2015-12-01

    Full Text Available Xiaoying Koh-Stenta,1 Joma Joy,1 Si Fang Wang,1 Perlyn Zekui Kwek,1 John Liang Kuan Wee,1 Kah Fei Wan,2 Shovanlal Gayen,1 Angela Shuyi Chen,1 CongBao Kang,1 May Ann Lee,1 Anders Poulsen,1 Subhash G Vasudevan,3 Jeffrey Hill,1 Kassoum Nacro11Experimental Therapeutics Centre, Agency for Science, Technology and Research (A*STAR, Singapore; 2Novartis Institute for Tropical Diseases, Singapore; 3Program in Emerging Infectious Diseases, Duke-NUS Graduate Medical School, SingaporeAbstract: Dengue virus (DENV protease is an attractive target for drug development; however, no compounds have reached clinical development to date. In this study, we utilized a potent West Nile virus protease inhibitor of the pyrazole ester derivative class as a chemical starting point for DENV protease drug development. Compound potency and selectivity for DENV protease were improved through structure-guided small molecule optimization, and protease-inhibitor binding interactions were validated biophysically using nuclear magnetic resonance. Our work strongly suggests that this class of compounds inhibits flavivirus protease through targeted covalent modification of active site serine, contrary to an allosteric binding mechanism as previously described.Keywords: flavivirus protease, small molecule optimization, covalent inhibitor, active site binding, pyrazole ester derivatives

  3. On-site and off-site activities

    International Nuclear Information System (INIS)

    Martin, H.D.

    1986-01-01

    Design principles for NPP training programs. Effects of NPP contracts. Effects of domestic industrial activities. The role of international bodies. Requirements for on-site training. Training abroad, technical, financial and social aspects. Training center on-site, an evaluation. (orig.)

  4. Purification and characterization of tenerplasminin-1, a serine peptidase inhibitor with antiplasmin activity from the coral snake (Micrurus tener tener) venom.

    Science.gov (United States)

    Vivas, Jeilyn; Ibarra, Carlos; Salazar, Ana M; Neves-Ferreira, Ana G C; Sánchez, Elda E; Perales, Jonás; Rodríguez-Acosta, Alexis; Guerrero, Belsy

    2016-01-01

    A plasmin inhibitor, named tenerplasminin-1 (TP1), was isolated from Micrurus tener tener (Mtt) venom. It showed a molecular mass of 6542Da, similarly to Kunitz-type serine peptidase inhibitors. The amidolytic activity of plasmin (0.5nM) on synthetic substrate S-2251 was inhibited by 91% following the incubation with TP1 (1nM). Aprotinin (2nM) used as the positive control of inhibition, reduced the plasmin amidolytic activity by 71%. Plasmin fibrinolytic activity (0.05nM) was inhibited by 67% following incubation with TP1 (0.1nM). The degradation of fibrinogen chains induced by plasmin, trypsin or elastase was inhibited by TP1 at a 1:2, 1:4 and 1:20 enzyme:inhibitor ratio, respectively. On the other hand, the proteolytic activity of crude Mtt venom on fibrinogen chains, previously attributed to metallopeptidases, was not abolished by TP1. The tPA-clot lysis assay showed that TP1 (0.2nM) acts like aprotinin (0.4nM) inducing a delay in lysis time and lysis rate which may be associated with the inhibition of plasmin generated from the endogenous plasminogen activation. TP1 is the first serine protease plasmin-like inhibitor isolated from Mtt snake venom which has been characterized in relation to its mechanism of action, formation of a plasmin:TP1 complex and therapeutic potential as anti-fibrinolytic agent, a biological characteristic of great interest in the field of biomedical research. They could be used to regulate the fibrinolytic system in pathologies such as metastatic cancer, parasitic infections, hemophilia and other hemorrhagic syndromes, in which an intense fibrinolytic activity is observed. Copyright © 2015 Elsevier Inc. All rights reserved.

  5. A cytotoxic serine proteinase isolated from mouse submandibular gland.

    Science.gov (United States)

    Shimamura, T; Nagumo, N; Ikigai, H; Murakami, K; Okubo, S; Toda, M; Ohnishi, R; Tomita, M

    1989-08-01

    We have isolated a novel cytotoxic factor from the submandibular glands of male BALB/c mice by Sephadex G-50 gel filtration chromatography and reverse-phase HPLC. The cytotoxic factor is a serine proteinase, which belongs to the mouse glandular kallikrein (mGK) family, with an Mr of approximately 27,000. The purified serine proteinase showed cytotoxic activity against mouse thymocytes in a dose-dependent manner, and a serine proteinase inhibitor, diisopropyl fluorophosphate, blocked its cytotoxic activity.

  6. The chaperone and potential mannan-binding lectin (MBL) co-receptor calreticulin interacts with MBL through the binding site for MBL-associated serine proteases

    DEFF Research Database (Denmark)

    Pagh, Rasmus; Duus, Karen; Laursen, Inga

    2008-01-01

    was immobilized on a solid surface or bound to mannan on a surface. The binding was non-covalent and biphasic with an initial salt-sensitive phase followed by a more stable salt-insensitive interaction. For plasma-derived MBL, known to be complexed with MBL-associated serine proteases (MASPs), no binding...... with calreticulin. Comparative analysis of MBL with complement component C1q, its counterpart of the classical pathway, revealed that they display similar binding characteristics for calreticulin, providing further indication that calreticulin is a common co-receptor/chaperone for both proteins. In conclusion......The chaperone calreticulin has been suggested to function as a C1q and collectin receptor. The interaction of calreticulin with mannan-binding lectin (MBL) was investigated by solid-phase binding assays. Calreticulin showed saturable and time-dependent binding to recombinant MBL, provided that MBL...

  7. Exploiting subsite S1 of trypsin-like serine proteases for selectivity: potent and selective inhibitors of urokinase-type plasminogen activator.

    Science.gov (United States)

    Mackman, R L; Katz, B A; Breitenbucher, J G; Hui, H C; Verner, E; Luong, C; Liu, L; Sprengeler, P A

    2001-11-08

    A nonselective inhibitor of trypsin-like serine proteases, 2-(2-hydroxybiphenyl-3-yl)-1H-indole-5-carboxamidine (1) (Verner, E.; Katz, B. A.; Spencer, J.; Allen, D.; Hataye, J.; Hruzewicz, W.; Hui, H. C.; Kolesnikov, A.; Li, Y.; Luong, C.; Martelli, A.; Radika. K.; Rai, R.; She, M.; Shrader, W.; Sprengeler, P. A.; Trapp, S.; Wang, J.; Young, W. B.; Mackman, R. L. J. Med. Chem. 2001, 44, 2753-2771) has been optimized through minor structural changes on the S1 binding group to afford remarkably selective and potent inhibitors of urokinase-type plasminogen activator (uPA). The trypsin-like serine proteases(1) that comprise drug targets can be broadly categorized into two subfamilies, those with Ser190 and those with Ala190. A single-atom modification, for example, replacement of hydrogen for chlorine at the 6-position of the 5-amidinoindole P1 group on 1, generated up to 6700-fold selectivity toward the Ser190 enzymes and against the Ala190 enzymes. The larger chlorine atom displaces a water molecule (H(2)O1(S1)) that binds near residue 190 in all the complexes of 1, and related inhibitors, in uPA, thrombin, and trypsin. The water molecule, H(2)O1(S1), in both the Ser190 or Ala190 enzymes, hydrogen bonds with the amidine N1 nitrogen of the inhibitor. When it is displaced, a reduction in affinity toward the Ala190 enzymes is observed due to the amidine N1 nitrogen of the bound inhibitor being deprived of a key hydrogen-bonding partner. In the Ser190 enzymes the affinity is maintained since the serine hydroxyl oxygen O gamma(Ser190) compensates for the displaced water molecule. High-resolution crystallography provided evidence for the displacement of the water molecule and validated the design rationale. In summation, a novel and powerful method for engineering selectivity toward Ser190 proteases and against Ala190 proteases without substantially increasing molecular weight is described.

  8. D-Serine/N-methyl-D-aspartate receptor signaling decreases DNA-binding activity of the transcriptional repressor DREAM in Müller glia from the retina.

    Science.gov (United States)

    Chavira-Suárez, Erika; Ramírez, Mónica; Lamas, Mónica

    2008-02-20

    In the adult retina, N-methyl-D-aspartate (NMDA) neurotoxicity induces Müller cell reactive gliosis which is characterized by changes in gene expression that lead to proliferation and affect retinal physiology. The amino acid D-serine is synthesized in Müller cells and modulates these processes acting as a coagonist of NMDA receptors. We have found that the transcription factor DREAM (downstream regulatory element antagonist modulator), which acts as a transcriptional repressor by binding as a tetramer to regulatory elements located in the promoter region of target genes, is expressed in these cells and that its DNA-binding activity is modulated by NMDA receptor activation. Consistently, immunocytochemical analysis demonstrates that NMDA receptor activation induces changes in the nuclear localization of this transcription factor. DREAM is a pleiotropic transcription factor capable to repress and activate genes involved in several physiological events in different tissues. These results link, for the first time, this transcription factor with NMDA-receptor activation. Given the relevance of glutamatergic transmission in the retina and the remarkable functional plasticity of Müller cells, these findings support the notion that the NMDA receptor-dependent modulation of DREAM activity could play a role in relevant physiological processes ranging from retinal response to injury to differentiation capacity of retinal progenitor cells.

  9. Inhibition of serine proteases by peptidyl fluoromethyl ketones

    International Nuclear Information System (INIS)

    Imperiali, B.; Abeles, R.H.

    1986-01-01

    Peptidyl fluoromethyl ketones that are specific inhibitors of the serine proteases α-chymotrypsin and porcine pancreatic elastase were synthesized. By analogy with the corresponding aldehydes it is assumed that the fluoromethyl ketones react with the γ-OH group of the active site serine to form a stable hemiacetal. 19 F NMR studies of the chymotrypsin-bound trifluoromethyl ketone inhibitors Ac-Leu-ambo-Phe-CF 3 1 and Ac-ambo-Phe-CF 3 clearly indicate that the carbonyl carbon is tetrahedral at the active site of the enzyme. The inhibitor is bound as either the stable hydrat or the hemiacetal, involving the active site serine. The effect of varying the number of amino acid residues in the peptidyl portion of the inhibitor and the number of fluorines in the fluoromethyl ketone moiety is examined. In the series of trifluoromethyl ketone elastase inhibitors, the lowering of K/sub i/ concomitant with the change from a dipeptide analogue to a tetrapeptide analogue correlates well with the variation in V/K for hydrolysis of the corresponding amide substrates. This trend is indicative of the inhibitors acting as transition-state analogues. In addition to chain length, the number of fluorine substituents also affects the K/sub i/. In the case of chymotrypsin, the K/sub i/ for Ac-Leu-ambo-Phe-CF 3 is 30-fold lower than that for Ac-Leu-ambo-Phe-CF 2 H. With elastase this trend is not as profound. In all cases, however, the difluoro- and trifluoromethyl ketones are better inhibitors than the monofluoromethyl and nonfluorinated analogues. This improvement must be associated with both the degree of hydration of the fluoromethyl ketones and the significant effect that fluorine substitution has on lowering the first pK/sub a/ of the hemiacetal hydroxyl. The monofluoromethyl ketone inhibitor of chymotrypsin, Ac-Leu-ambo-Phe-CFH 2 , is a weak competitive inhibitor

  10. Molecular dynamics simulations suggest changes in electrostatic interactions as a potential mechanism through which serine phosphorylation inhibits DNA Polymerase β's activity.

    Science.gov (United States)

    Homouz, Dirar; Joyce-Tan, Kwee Hong; Shahir Shamsir, Mohd; Moustafa, Ibrahim M; Idriss, Haitham

    2018-01-01

    DNA polymerase β is a 39kDa enzyme that is a major component of Base Excision Repair in human cells. The enzyme comprises two major domains, a 31kDa domain responsible for the polymerase activity and an 8kDa domain, which bind ssDNA and has a deoxyribose phosphate (dRP) lyase activity. DNA polymerase β was shown to be phosphorylated in vitro with protein kinase C (PKC) at serines 44 and 55 (S44 and S55), resulting in loss of its polymerase enzymic activity, but not its ability to bind ssDNA. In this study, we investigate the potential phosphorylation-induced structural changes for DNA polymerase β using molecular dynamics. The simulations show drastic conformational changes of the polymerase structure as a result of S44 phosphorylation. Phosphorylation-induced conformational changes transform the closed (active) enzyme structure into an open one. Further analysis of the results points to a key hydrogen bond and newly formed salt bridges as potential drivers of these structural fluctuations. The changes observed with S44/55 and S55 phosphorylation were less dramatic than S44 and the integrity of the H-bond was not compromised. Thus the phosphorylation of S44 is likely the major contributor to structural fluctuations that lead to loss of enzymatic activity. Copyright © 2017. Published by Elsevier Inc.

  11. Timing-dependent reduction in ethanol sedation and drinking preference by NMDA receptor co-agonist d-serine.

    Science.gov (United States)

    Lockridge, Amber; Romero, Gabriel; Harrington, Justin; Newland, Brett; Gong, Zi; Cameron, Andrew; Yuan, Li-Lian

    2012-06-01

    NMDA receptors become a major contributor to acute ethanol intoxication effects at high concentrations as ethanol binds to a unique site on the receptor and inhibits glutamatergic activity in multiple brain areas. Although a convincing body of literature exists on the ability of NMDA receptor antagonists to mimic and worsen cellular and behavioral ethanol effects, receptor agonists have been less well-studied. In addition to a primary agonist site for glutamate, the NMDA receptor contains a separate co-agonist site that responds to endogenous amino acids glycine and d-serine. d-serine is both selective for this co-agonist site and potent in boosting NMDA dependent activity even after systemic administration. In this study, we hypothesized that exogenous d-serine might ameliorate some acute ethanol behaviors by opposing NMDA receptor inhibition. We injected adult male C57 mice with a high concentration of d-serine at various time windows relative to ethanol administration and monitored sedation, motor coordination and voluntary ethanol drinking. d-serine (2.7 g/kg, ip) prolonged latency to a loss of righting reflex (LoRR) and shortened LoRR duration when given 15 min before ethanol (3 g/kg) but not when it was injected with or shortly after ethanol. Blood samples taken at sedative recovery and at fixed time intervals revealed no effect of d-serine on ethanol concentration but an ethanol-induced decrease in l-serine and glycine content was prevented by acute d-serine pre-administration. d-serine had no effect on ethanol-induced (2 g/kg) rotarod deficits in young adult animals but independently and interactively degraded motor performance in a subset of older mice. Finally, a week-long series of daily ip injections resulted in a 50% decrease in free choice ethanol preference for d-serine treated animals compared to saline-injected controls in a two-bottle choice experiment. Copyright © 2012 Elsevier Inc. All rights reserved.

  12. Identification, recombinant production and partial biochemical characterization of an extracellular cold-active serine-metalloprotease from an Antarctic Pseudomonas isolate

    Directory of Open Access Journals (Sweden)

    Natalia Fullana

    2017-08-01

    Full Text Available Cold-adapted enzymes are generally derived from psychrophilic microorganisms and have features that make them very attractive for industrial and biotechnological purposes. In this work, we identified a 50 kDa extracellular protease (MP10 from the Antarctic isolate Pseudomonas sp. AU10. The enzyme was produced by recombinant DNA technology, purified using immobilized metal affinity chromatography and partially characterized. MP10 is an alkaline thermosensitive serine-metallo protease with optimal activity at pH 8.0 and 40 ℃, in the presence of 1.5 mM Ca2+. MP10 showed 100% residual activity and stability (up to 60 min when incubated with 7% of non-ionic surfactants (Triton X-100, Tween-80 and Tween-20 and 1.5% of the oxidizing agent hydrogen peroxide. The 3D MP10 structure was predicted and compared with the crystal structure of mesophilic homologous protease produced by Pseudomonas aeruginosa PA01 (reference strain and other proteases, showing similarity in surface area and volume of proteins, but a significantly higher surface pocket area and volume of MP10. The observed differences presumably may explain the enhanced activity of MP10 for substrate binding at low temperatures. These results give insight to the potential use of MP10 in developing new biotechnologically processes active at low to moderate temperatures, probably with focus in the detergent industry.

  13. Efficient oxygen electrocatalysis on special active sites

    DEFF Research Database (Denmark)

    Halck, Niels Bendtsen

    Oxygen electrocatalysis will be pivotal in future independent of fossil fuels. Renewable energy production will rely heavily on oxygen electrocatalysis as a method for storing energy from intermittent energy sources such as the wind and sun in the form of chemical bonds and to release the energy...... throughout this thesis to understand these local structure effects and their influence on surface reactions. The concept of these special active sites is used to explain how oxygen evolution reaction (OER) catalysts can have activities beyond the limits of what was previously thought possible. The concept...... functional theory calculation provides an insight into the how the activity is increased at these special active sites and proposes a modified reaction mechanism for the oxygen evolution reaction on these sites. Another type of special active site can explain the production of hydrogen peroxide on nickel...

  14. Catalysis: Elusive active site in focus

    Science.gov (United States)

    Labinger, Jay A.

    2016-08-01

    The identification of the active site of an iron-containing catalyst raises hopes of designing practically useful catalysts for the room-temperature conversion of methane to methanol, a potential fuel for vehicles. See Letter p.317

  15. Planar integrated optical waveguide used as a transducer to yield chemical information: detection of the activity of proteolytic enzymes e.g. serine-proteases

    Science.gov (United States)

    Zhylyak, Gleb; Ramoz-Perez, Victor; Linnhoff, Michael; Hug, Thomas; Citterio, Daniel; Spichiger-Keller, Ursula E.

    2005-03-01

    The paper shows the very first results of a feasibility study where the activity of proteolytic enzymes towards dye-labelled artificial substrates immobilized on the surface of planar optical Ta2O5 waveguide was investigated. Within this project, a chromophore label was developed, synthesized and attached to the carboxy-terminus of specific tripeptides. The goal was to develop a highly sensitive optical assay in order to monitor the activity of serine-proteases by cleavage of the amide bond between peptide and chromophore. On the one hand, a strategy was developed to immobilize the labeled tripeptide unto integrated planar waveguides. On the other hand, an instrument, the so-called "chip-reader" was developed to detect the biological process on the surface of the integrated planar optical waveguide. Surface characteristics were analyzed by XPS, TOF-SIMS and contact angle measurements. A comparison between the effectivity of ATR-photometry on chip using TE0 mode and photometry in transmission mode is discussed.

  16. Genetic influences on mannan-binding lectin (MBL) and mannan-binding lectin associated serine protease-2 (MASP-2) activity

    DEFF Research Database (Denmark)

    Sørensen, Grith Lykke; Petersen, Inge; Thiel, Steffen

    2007-01-01

    The lectin pathway of the complement system is activated when Mannan-binding lectin (MBL) in complex with MASP-2 binds microorganisms. Polymorphisms in both genes are responsible for low serum levels, which associate with increased risk of infection and autoimmune disease. The present study...... includes 1215 MBL measurements and 1214 MASP-2 activity measurements in healthy Danish adult twins. Total MASP-2 activity was estimated by C4 cleaving activity of samples diluted in an excess of MBL. Twin-twin correlations were higher in monozygotic (MZ) than in dizygotic (DZ) twins for both traits...

  17. Phosphorylation of mouse serine racemase regulates D-serine synthesis

    DEFF Research Database (Denmark)

    Foltyn, Veronika N; Zehl, Martin; Dikopoltsev, Elena

    2010-01-01

    Serine racemase (SR) catalyses the synthesis of the transmitter/neuromodulator D-serine, which plays a major role in synaptic plasticity and N-methyl D-aspartate receptor neurotoxicity. We now report that SR is phosphorylated at Thr71 and Thr227 as revealed by mass spectrometric analysis...

  18. Activation of the bacterial thermosensor DesK involves a serine zipper dimerization motif that is modulated by bilayer thickness

    NARCIS (Netherlands)

    Cybulski, Larisa; Ballering, Joost|info:eu-repo/dai/nl/325784876; Moussatova, Anastassiia; Inda, Maria Eugenia; Vazquez, Daniela B; Wassenaar, Tsjerk A; de Mendoza, Diego; Tieleman, D Peter; Killian, J Antoinette|info:eu-repo/dai/nl/071792317

    2015-01-01

    DesK is a bacterial thermosensor protein involved in maintaining membrane fluidity in response to changes in environmental temperature. Most likely, the protein is activated by changes in membrane thickness, but the molecular mechanism of sensing and signaling is still poorly understood. Here we

  19. Genetic influences on mannan-binding lectin (MBL) and mannan-binding lectin associated serine protease-2 (MASP-2) activity

    DEFF Research Database (Denmark)

    Sørensen, GL; Petersen, I; Thiel, Steffen

    2007-01-01

    The lectin pathway of the complement system is activated when Mannan-binding lectin (MBL) in complex with MASP-2 binds microorganisms. Polymorphisms in both genes are responsible for low serum levels, which associate with increased risk of infection and autoimmune disease. The present study...

  20. Genetic influences on Mannan-binding lectin (MBL) and Mannan-binding lectin associated serine protease-2 (MASP-2) activity

    DEFF Research Database (Denmark)

    Sorensen, Grith L; Petersen, Inge; Thiel, Steffen

    2007-01-01

    The lectin pathway of the complement system is activated when Mannan-binding lectin (MBL) in complex with MASP-2 binds microorganisms. Polymorphisms in both genes are responsible for low serum levels, which associate with increased risk of infection and autoimmune disease. The present study...

  1. Bioinsecticidal activity of Talisia esculenta reserve protein on growth and serine digestive enzymes during larval development of Anticarsia gemmatalis.

    Science.gov (United States)

    Macedo, Maria Lígia R; Freire, Maria das Graças M; Kubo, Carlos Eduardo G; Parra, José Roberto P

    2011-01-01

    Plants synthesize a variety of molecules to defend themselves against an attack by insects. Talisin is a reserve protein from Talisia esculenta seeds, the first to be characterized from the family Sapindaceae. In this study, the insecticidal activity of Talisin was tested by incorporating the reserve protein into an artificial diet fed to the velvetbean caterpillar Anticarsia gemmatalis, the major pest of soybean crops in Brazil. At 1.5% (w/w) of the dietary protein, Talisin affected larval growth, pupal weight, development and mortality, adult fertility and longevity, and produced malformations in pupae and adult insects. Talisin inhibited the trypsin-like activity of larval midgut homogenates. The trypsin activity in Talisin-fed larvae was sensitive to Talisin, indicating that no novel protease-resistant to Talisin was induced in Talisin-fed larvae. Affinity chromatography showed that Talisin bound to midgut proteinases of the insect A. gemmatalis, but was resistant to enzymatic digestion by these larval proteinases. The transformation of genes coding for this reserve protein could be useful for developing insect resistant crops. Copyright © 2010 Elsevier Inc. All rights reserved.

  2. Oxidative stress impairs cGMP-dependent protein kinase activation and vasodilator-stimulated phosphoprotein serine-phosphorylation.

    Science.gov (United States)

    Banday, Anees A; Lokhandwala, Mustafa F

    2018-02-09

    Reactive oxygen species induce vascular dysfunction and hypertension by directly interacting with nitric oxide (NO) which leads to NO inactivation. In addition to a decrease in NO bioavailability, there is evidence that oxidative stress can also modulate NO signaling during hypertension. Here, we investigated the effect of oxidative stress on NO signaling molecules cGMP-dependent protein kinase (PKG) and vasodilator-stimulated phosphoprotein (VASP) which are known to mediate vasodilatory actions of NO. Male Sprague Dawley (SD) rats were provided with tap water (control), 30 mM L-buthionine sulfoximine (BSO, a pro-oxidant), 1 mM tempol (T, an antioxidant) and BSO + T for 3 wks. BSO-treated rats exhibited high blood pressure and oxidative stress. Incubation of mesenteric arterial rings with NO donors caused concentration-dependent relaxation in control rats. However, the response to NO donors was significantly lower in BSO-treated rats with a marked decrease in pD2. In control rats, NO donors activated mesenteric PKG, increased VASP phosphorylation and its interaction with transient receptor potential channels 4 (TRPC4) and inhibited store-operated Ca 2+ influx. NO failed to activate these signaling molecules in mesenteric arteries from BSO-treated rats. Supplementation of BSO-treated rats with tempol reduced oxidative stress and blood pressure and normalized the NO signaling. These data suggest that oxidative stress can reduce NO-mediated PKG activation and VASP-TRPC4 interaction which leads to failure of NO to reduce Ca 2+ influx in smooth muscle cells. The increase in intracellular Ca 2+ contributes to sustained vasoconstriction and subsequent hypertension. Antioxidant supplementation decreases oxidative stress, normalizes NO signaling and reduces blood pressure.

  3. Managing Siting Activities for Nuclear Power Plants

    International Nuclear Information System (INIS)

    2012-01-01

    One of the IAEA's statutory objectives is to ''seek to accelerate and enlarge the contribution of atomic energy to peace, health and prosperity throughout the world''. One way this objective is achieved is through the publication of a range of technical series. Two of these are the IAEA Nuclear Energy Series and the IAEA Safety Standards Series. According to Article III.A.6 of the IAEA Statute, the safety standards establish 'standards of safety for protection of health and minimization of danger to life and property.' The safety standards include the Safety Fundamentals, Safety Requirements and Safety Guides. These standards are written primarily in a regulatory style, and are binding on the IAEA for its own programmes. The principal users are the regulatory bodies in Member States and other national authorities. The IAEA Nuclear Energy Series comprises reports designed to encourage and assist R and D on, and application of, nuclear energy for peaceful uses. This includes practical examples to be used by owners and operators of utilities in Member States, implementing organizations, academia, and government officials, among others. This information is presented in guides, reports on technology status and advances, and best practices for peaceful uses of nuclear energy based on inputs from international experts. The IAEA Nuclear Energy Series complements the IAEA Safety Standards Series. The introduction of nuclear power brings new challenges to States - one of them being the selection of appropriates sites. It is a project that needs to begin early, be well managed, and deploy good communications with all stakeholders; including regulators. This is important, not just for those States introducing nuclear power for the first time, but for any State looking to build a new nuclear power plant. The purpose of the siting activities goes beyond choosing a suitable site and acquiring a licence. A large part of the project is about producing and maintaining a validated

  4. Serine 77 in the PDZ domain of PICK1 is a protein kinase Cα phosphorylation site regulated by lipid membrane binding

    DEFF Research Database (Denmark)

    Ammendrup-Johnsen, Ina; Thorsen, Thor Seneca; Gether, Ulrik

    2012-01-01

    PICK1 (protein interacting with C kinase 1) contains an N-terminal protein binding PDZ domain and a C-terminal lipid binding BAR domain. PICK1 plays a key role in several physiological processes, including synaptic plasticity. However, little is known about the cellular mechanisms governing...... site for PKCα. Mutation of Ser77 reduced the level of PKCα-mediated phosphorylation ~50%, whereas no reduction was observed upon mutation of seven other predicted sites. Addition of lipid vesicles increased the level of phosphorylation of Ser77 10-fold, indicating that lipid binding is critical...... lipid binding and/or polymerization capacity. We propose that PICK1 is phosphorylated at Ser77 by PKCα preferentially when bound to membrane vesicles and that this phosphorylation in turn modulates its cellular distribution....

  5. Characterization and modelling of VanT: a novel, membrane-bound, serine racemase from vancomycin-resistant Enterococcus gallinarum BM4174.

    Science.gov (United States)

    Arias, C A; Martín-Martinez, M; Blundell, T L; Arthur, M; Courvalin, P; Reynolds, P E

    1999-03-01

    Sequence determination of a region downstream from the vanXYc gene in Enterococcus gallinarum BM4174 revealed an open reading frame, designated vanT, that encodes a 698-amino-acid polypeptide with an amino-terminal domain containing 10 predicted transmembrane segments. The protein contained a highly conserved pyridoxal phosphate attachment site in the C-terminal domain, typical of alanine racemases. The protein was overexpressed in Escherichia coli, and serine racemase activity was detected in the membrane but not in the cytoplasmic fraction after centrifugation of sonicated cells, whereas alanine racemase activity was located almost exclusively in the cytoplasm. When the protein was overexpressed as a polypeptide lacking the predicted transmembrane domain, serine racemase activity was detected in the cytoplasm. The serine racemase activity was partially (64%) inhibited by D-cycloserine, whereas host alanine racemase activity was almost totally inhibited (97%). Serine racemase activity was also detected in membrane preparations of constitutively vancomycin-resistant E. gallinarum BM4174 but not in BM4175, in which insertional inactivation of the vanC-1 D-Ala:D-Ser ligase gene probably had a polar effect on expression of the vanXYc and vanT genes. Comparative modelling of the deduced C-terminal domain was based on the alignment of VanT with the Air alanine racemase from Bacillus stearothermophilus. The model revealed that almost all critical amino acids in the active site of Air were conserved in VanT, indicating that the C-terminal domain of VanT is likely to adopt a three-dimensional structure similar to that of Air and that the protein could exist as a dimer. These results indicate that the source of D-serine for peptidoglycan synthesis in vancomycin-resistant enterococci expressing the VanC phenotype involves racemization of L- to D-serine by a membrane-bound serine racemase.

  6. Phosphorylation of transglutaminase 2 (TG2 at serine-216 has a role in TG2 mediated activation of nuclear factor-kappa B and in the downregulation of PTEN

    Directory of Open Access Journals (Sweden)

    Wang Yi

    2012-07-01

    Full Text Available Abstract Background Transglutaminase 2 (TG2 and its phosphorylation have been consistently found to be upregulated in a number of cancer cell types. At the molecular level, TG2 has been associated with the activation of nuclear factor-kappa B (NF-κB, protein kinase B (PKB/Akt and in the downregulation of phosphatase and tensin homologue deleted on chromosome 10 (PTEN. However, the underlying mechanism involved is not known. We have reported that protein kinase A (PKA induced phosphorylation of TG2 at serine-216 (Ser216 regulates TG2 function and facilitates protein-protein interaction. However, the role of TG2 phosphorylation in the modulation of NF-κB, Akt and PTEN is not explored. Methods In this study we have investigated the effect of TG2 phosphorylation on NF-κB, Akt and PTEN using embryonic fibroblasts derived from TG2 null mice (MEFtg2-/- overexpressing native TG2 or mutant-TG2 (m-TG2 lacking Ser216 phosphorylation site with and without dibutyryl cyclic-AMP (db-cAMP stimulation. Functional consequences on cell cycle and cell motility were determined by fluorescence activated cell sorting (FACS analysis and cell migration assay respectively. Results PKA activation in TG2 overexpressing MEFtg2-/- cells resulted in an increased activation of NF-κB and Akt phosphorylation in comparison to empty vector transfected control cells as determined by the reporter-gene assay and immunoblot analysis respectively. These effects were not observed in MEFtg2-/- cells overexpressing m-TG2. Similarly, a significant downregulation of PTEN at both, the mRNA and protein levels were found in cells overexpressing TG2 in comparison to empty vector control and m-TG2 transfected cells. Furthermore, Akt activation correlated with the simultaneous activation of NF-κB and a decrease in PTEN suggesting that the facilitatory effect of TG2 on Akt activation occurs in a PTEN-dependent manner. Similar results were found with MCF-7 and T-47D breast cancer cells

  7. Phosphorylation of transglutaminase 2 (TG2) at serine-216 has a role in TG2 mediated activation of nuclear factor-kappa B and in the downregulation of PTEN

    International Nuclear Information System (INIS)

    Wang, Yi; Ande, Sudharsana R; Mishra, Suresh

    2012-01-01

    Transglutaminase 2 (TG2) and its phosphorylation have been consistently found to be upregulated in a number of cancer cell types. At the molecular level, TG2 has been associated with the activation of nuclear factor-kappa B (NF-κB), protein kinase B (PKB/Akt) and in the downregulation of phosphatase and tensin homologue deleted on chromosome 10 (PTEN). However, the underlying mechanism involved is not known. We have reported that protein kinase A (PKA) induced phosphorylation of TG2 at serine-216 (Ser 216 ) regulates TG2 function and facilitates protein-protein interaction. However, the role of TG2 phosphorylation in the modulation of NF-κB, Akt and PTEN is not explored. In this study we have investigated the effect of TG2 phosphorylation on NF-κB, Akt and PTEN using embryonic fibroblasts derived from TG2 null mice (MEF tg2-/- ) overexpressing native TG2 or mutant-TG2 (m-TG2) lacking Ser 216 phosphorylation site with and without dibutyryl cyclic-AMP (db-cAMP) stimulation. Functional consequences on cell cycle and cell motility were determined by fluorescence activated cell sorting (FACS) analysis and cell migration assay respectively. PKA activation in TG2 overexpressing MEF tg2-/- cells resulted in an increased activation of NF-κB and Akt phosphorylation in comparison to empty vector transfected control cells as determined by the reporter-gene assay and immunoblot analysis respectively. These effects were not observed in MEF tg2-/- cells overexpressing m-TG2. Similarly, a significant downregulation of PTEN at both, the mRNA and protein levels were found in cells overexpressing TG2 in comparison to empty vector control and m-TG2 transfected cells. Furthermore, Akt activation correlated with the simultaneous activation of NF-κB and a decrease in PTEN suggesting that the facilitatory effect of TG2 on Akt activation occurs in a PTEN-dependent manner. Similar results were found with MCF-7 and T-47D breast cancer cells overexpressing TG2 and m-TG2 further

  8. D-Serine Is a Substrate for Neutral Amino Acid Transporters ASCT1/SLC1A4 and ASCT2/SLC1A5, and Is Transported by Both Subtypes in Rat Hippocampal Astrocyte Cultures

    OpenAIRE

    Foster, Alan C.; Farnsworth, Jill; Lind, Genevieve E.; Li, Yong-Xin; Yang, Jia-Ying; Dang, Van; Penjwini, Mahmud; Viswanath, Veena; Staubli, Ursula; Kavanaugh, Michael P.

    2016-01-01

    N-methyl-D-aspartate (NMDA) receptors play critical roles in synaptic transmission and plasticity. Activation of NMDA receptors by synaptically released L-glutamate also requires occupancy of co-agonist binding sites in the tetrameric receptor by either glycine or D-serine. Although D-serine appears to be the predominant co-agonist at synaptic NMDA receptors, the transport mechanisms involved in D-serine homeostasis in brain are poorly understood. In this work we show that the SLC1 amino acid...

  9. Lectin pathway effector enzyme mannan-binding lectin-associated serine protease-2 can activate native complement C3 in absence of C4 and/or C2

    DEFF Research Database (Denmark)

    Yaseen, Sadam; Demopulos, Gregory; Dudler, Thomas

    2017-01-01

    All 3 activation pathways of complement-the classic pathway (CP), the alternative pathway, and the lectin pathway (LP)- converge into a common central event: the cleavage and activation of the abundant third complement component, C3,viaformation of C3-activating enzymes (C3 convertases). The four......., Sacks, S., Garred, P., Andrew, P., Sim, R. B., Lachmann, P. J., Wallis, R., Lynch, N., Schwaeble, W. J. Lectin pathway effector enzyme mannan-binding lectin-associated serine protease-2 can activate native complement C3 in absence of C4 and/or C2....

  10. Modeling and structural analysis of evolutionarily diverse S8 family serine proteases.

    Science.gov (United States)

    Laskar, Aparna; Rodger, Euan James; Chatterjee, Aniruddha; Mandal, Chhabinath

    2011-01-01

    Serine proteases are an abundant class of enzymes that are involved in a wide range of physiological processes and are classified into clans sharing structural homology. The active site of the subtilisin-like clan contains a catalytic triad in the order Asp, His, Ser (S8 family) or a catalytic tetrad in the order Glu, Asp and Ser (S53 family). The core structure and active site geometry of these proteases is of interest for many applications. The aim of this study was to investigate the structural properties of different S8 family serine proteases from a diverse range of taxa using molecular modeling techniques. In conjunction with 12 experimentally determined three-dimensional structures of S8 family members, our predicted structures from an archaeon, protozoan and a plant were used for analysis of the catalytic core. Amino acid sequences were obtained from the MEROPS database and submitted to the LOOPP server for threading based structure prediction. The predicted structures were refined and validated using PROCHECK, SCRWL and MODELYN. Investigation of secondary structures and electrostatic surface potential was performed using MOLMOL. Encompassing a wide range of taxa, our structural analysis provides an evolutionary perspective on S8 family serine proteases. Focusing on the common core containing the catalytic site of the enzyme, the analysis presented here is beneficial for future molecular modeling strategies and structure-based rational drug design.

  11. Preferred stereoselective brain uptake of D-serine - a modulator of glutamatergic neurotransmission

    International Nuclear Information System (INIS)

    Bauer, Dagmar; Hamacher, Kurt; Broeer, Stefan; Pauleit, Dirk; Palm, Christoph; Zilles, Karl; Coenen, Heinz H.; Langen, Karl-Josef

    2005-01-01

    Although it has long been presumed that D-amino acids are uncommon in mammalians, substantial amounts of free D-serine have been detected in the mammalian brain. D-Serine has been demonstrated to be an important modulator of glutamatergic neurotransmission and acts as an agonist at the strychnine-insensitive glycine site of N-methyl-D-aspartate receptors. The blood-to-brain transfer of D-serine is thought to be extremely low, and it is assumed that D-serine is generated by isomerization of L-serine in the brain. Stimulated by the observation of a preferred transport of the D-isomer of proline at the blood-brain barrier, we investigated the differential uptake of [ 3 H]-D-serine and [ 3 H]-L-serine in the rat brain 1 h after intravenous injection using quantitative autoradiography. Surprisingly, brain uptake of [ 3 H]-D-serine was significantly higher than that of [ 3 H]-L-serine, indicating a preferred transport of the D-enantiomer of serine at the blood-brain barrier. This finding indicates that exogenous D-serine may have a direct influence on glutamatergic neurotransmission and associated diseases

  12. LRRK2 Kinase Activity and Biology are Not Uniformly Predicted by its Autophosphorylation and Cellular Phosphorylation Site Status

    Directory of Open Access Journals (Sweden)

    April eReynolds

    2014-06-01

    Full Text Available Missense mutations in the Leucine Rich Repeat protein Kinase 2 (LRRK2 gene are the most common genetic predisposition to develop Parkinson’s disease (PD LRRK2 is a large multi-domain phosphoprotein with a GTPase domain and a serine/threonine protein kinase domain whose activity is implicated in neuronal toxicity; however the precise mechanism is unknown. LRRK2 autophosphorylates on several serine/threonine residues across the enzyme and is found constitutively phosphorylated on Ser910, Ser935, Ser955 and Ser973, which are proposed to be regulated by upstream kinases. Here we investigate the phosphoregulation at these sites by analyzing the effects of disease-associated mutations Arg1441Cys, Arg1441Gly, Ala1442Pro, Tyr1699Cys, Ile2012Thr, Gly2019Ser, and Ile2020Thr. We also studied alanine substitutions of phosphosite serines 910, 935, 955 and 973 and specific LRRK2 inhibition on autophosphorylation of LRRK2 Ser1292, Thr1491, Thr2483 and phosphorylation at the cellular sites. We found that mutants in the Roc-COR domains, including Arg1441Cys, Arg1441His, Ala1442Pro and Tyr1699Cys, can positively enhance LRRK2 kinase activity while concomitantly inducing the dephosphorylation of the cellular sites. Mutation of the cellular sites individually did not affect LRRK2 intrinsic kinase activity; however, Ser910/935/955/973Ala mutations trended toward increased kinase activity of LRRK2. Increased cAMP levels did not lead to increased LRRK2 cellular site phosphorylation, 14-3-3 binding or kinase activity. In cells, inhibition of LRRK2 kinase activity leads to dephosphorylation of Ser1292 by Calyculin A and okadaic acid sensitive phosphatases, while the cellular sites are dephosphorylated by Calyculin A sensitive phosphatases. These findings indicate that comparative analysis of both Ser1292 and Ser910/935/955/973 phosphorylation sites will provide important and distinct measures of LRRK2 kinase and biological activity in vitro and in vivo.

  13. Characterization of active sites in zeolite catalysts

    Energy Technology Data Exchange (ETDEWEB)

    Eckert, J. [Los Alamos National Lab., NM (United States); Bug, A. [Swarthmore Coll., PA (United States); Nicol, J.M. [MOLTECH (United States)] [and others

    1997-11-01

    This is the final report of a three-year, Laboratory Directed Research and Development (LDRD) project at the Los Alamos National Laboratory (LANL). Atomic-level details of the interaction of adsorbed molecules with active sites in catalysts are urgently needed to facilitate development of more effective and/or environmentally benign catalysts. To this end the authors have carried out neutron scattering studies combined with theoretical calculations of the dynamics of small molecules inside the cavities of zeolite catalysts. The authors have developed the use of H{sub 2} as a probe of adsorption sites by observing the hindered rotations of the adsorbed H{sub 2} molecule, and they were able to show that an area near the four-rings is the most likely adsorption site for H{sub 2} in zeolite A while adsorption of H{sub 2} near cations located on six-ring sites decreases in strength as Ni {approximately} Co > Ca > Zn {approximately} Na. Vibrational and rotational motions of ethylene and cyclopropane adsorption complexes were used as a measure for zeolite-adsorbate interactions. Preliminary studies of the binding of water, ammonia, and methylamines were carried out in a number of related guest-host materials.

  14. Disulfide bond assignment in human interleukin-7 by matrix-assisted laser desorption/ionization mass spectroscopy and site-directed cysteine to serine mutational analysis.

    Science.gov (United States)

    Cosenza, L; Sweeney, E; Murphy, J R

    1997-12-26

    Interleukin-7 (IL-7) is a proteinaceous biological response modifier that has a bioactive tertiary structure dependent on disulfide bond formation. Disulfide bond assignments in human (h)IL-7 are based upon the results of matrix-assisted laser desorption/ionization (MALDI) mass spectroscopy and Cys to Ser mutational analyses. A gene encoding the hIL-7 was synthesized incorporating Escherichia coli codon usage bias and was used to express biologically active protein as determined by stimulation of precursor B-cell proliferation. MALDI mass spectroscopic analysis of trypsin-digested hIL-7 was performed and compared with the anticipated results of a simulated tryptic digestion. Many of the anticipated hIL-7 tryptic fragments were detected including one with a molecular mass equivalent to the sum of two polypeptides linked through a disulfide bond formed from Cys residues (Cys3 and Cys142). Subsequently, Cys to Ser substitution mutational analyses were performed. A hIL-7 variant with all six Cys substituted with Ser was found to be biologically inactive (EC50 > 1 x 10(-7) M). In contrast, a family of single disulfide bond-forming variants of hIL-7 were constructed by reintroducing Cys pairs (Cys3-Cys142, Cys35-Cys130, and Cys48-Cys93), and each could stimulate cell proliferation with an EC50 of 4 x 10(-9), 2 x 10(-8), and 2 x 10(-9) M, respectively. In single disulfide bond-forming mutants of hIL-7, the ability to stimulate cell proliferation was abolished in the presence of 2 mM dithiothreitol. The results presented strongly suggest that only a single disulfide bond is required for hIL-7 to form a tertiary structure capable of stimulating precursor B-cell proliferation.

  15. Novel serine-based gemini surfactants as chemical permeation enhancers of local anesthetics: A comprehensive study on structure-activity relationships, molecular dynamics and dermal delivery.

    Science.gov (United States)

    Teixeira, Raquel S; Cova, Tânia F G G; Silva, Sérgio M C; Oliveira, Rita; do Vale, M Luísa C; Marques, Eduardo F; Pais, Alberto A C C; Veiga, Francisco J B

    2015-06-01

    This work aims at studying the efficacy of a series of novel biocompatible, serine-based surfactants as chemical permeation enhancers for two different local anesthetics, tetracaine and ropivacaine, combining an experimental and computational approach. The surfactants consist of gemini molecules structurally related, but with variations in headgroup charge (nonionic vs. cationic) and in the hydrocarbon chain lengths (main and spacer chains). In vitro permeation and molecular dynamics studies combined with cytotoxicity profiles were performed to investigate the permeation of both drugs, probe skin integrity, and rationalize the interactions at molecular level. Results show that these enhancers do not have significant deleterious effects on the skin structure and do not cause relevant changes on cell viability. Permeation across the skin is clearly improved using some of the selected serine-based gemini surfactants, namely the cationic ones with long alkyl chains and shorter spacer. This is noteworthy in the case of ropivacaine hydrochloride, which is not easily administered through the stratum corneum. Molecular dynamics results provide a mechanistic view of the surfactant action on lipid membranes that essentially corroborate the experimental observations. Overall, this study suggests the viability of these serine-based surfactants as suitable and promising delivery agents in pharmaceutical formulations. Copyright © 2015. Published by Elsevier B.V.

  16. Interferon-alpha signalling in bovine adrenal chromaffin cells: involvement of signal-transducer and activator of transcription 1 and 2, extracellular signal-regulated protein kinases 1/2 and serine 31 phosphorylation of tyrosine hydroxylase.

    Science.gov (United States)

    Douglas, S A; Bunn, S J

    2009-03-01

    Adrenal medullary chromaffin cells are an integral part of the neuroendocrine system, playing an important role in the physiological adaptation to stress. In response to a wide variety of stimuli, including acetylcholine released from the splanchnic nerve, hormones such as angiotensin II or paracrine signals such as prostaglandins, chromaffin cells synthesise and secrete catecholamines and a number of biologically active peptides. This adrenal medullary output mediates a complex and diverse stress response. We report that chromaffin cells also respond both acutely and chronically to interferon (IFN)-alpha, thus providing a mechanism of interaction between the immune system and the stress response. Incubation of isolated bovine chromaffin cells maintained in culture, with IFN-alpha resulted in a rapid, transient activation of the extracellular signal-regulated protein kinase (ERK)1/2, which was maximal after 5 min. IFN-alpha mediated activation of ERK1/2 appeared to be responsible for the increased phosphorylation of tyrosine hydroxylase, the rate-limiting enzyme in catecholamine synthesis. This tyrosine hydroxylase phosphorylation was exclusively on serine 31, with no change in the phosphorylation of serine 19 or 40. This increase in the serine 31 phosphorylation of tyrosine hydroxylase was prevented by inhibition of protein kinase C or ERK1/2 activation. Incubation with IFN-alpha also resulted in a time- and concentration-dependent phosphorylation and nuclear translocation of signal transducer and activator of transcription proteins (STAT)1 and 2. This response was maximal after approximately 60 min. Prolonged treatment with IFN-alpha (12-48 h) resulted in increased expression of STAT1 and, to a lesser extent, STAT2. Thus, these findings demonstrate that adrenal medullary chromaffin cells are responsive to IFN-alpha and provide a possible cellular mechanism by which this immune-derived signal can potentially influence and integrate with the stress response.

  17. D-Serine Is a Substrate for Neutral Amino Acid Transporters ASCT1/SLC1A4 and ASCT2/SLC1A5, and Is Transported by Both Subtypes in Rat Hippocampal Astrocyte Cultures.

    Directory of Open Access Journals (Sweden)

    Alan C Foster

    Full Text Available N-methyl-D-aspartate (NMDA receptors play critical roles in synaptic transmission and plasticity. Activation of NMDA receptors by synaptically released L-glutamate also requires occupancy of co-agonist binding sites in the tetrameric receptor by either glycine or D-serine. Although D-serine appears to be the predominant co-agonist at synaptic NMDA receptors, the transport mechanisms involved in D-serine homeostasis in brain are poorly understood. In this work we show that the SLC1 amino acid transporter family members SLC1A4 (ASCT1 and SLC1A5 (ASCT2 mediate homo- and hetero-exchange of D-serine with physiologically relevant kinetic parameters. In addition, the selectivity profile of D-serine uptake in cultured rat hippocampal astrocytes is consistent with uptake mediated by both ASCT1 and ASCT2. Together these data suggest that SLC1A4 (ASCT1 may represent an important route of Na-dependent D-serine flux in the brain that has the ability to regulate extracellular D-serine and thereby NMDA receptor activity.

  18. D-Serine Is a Substrate for Neutral Amino Acid Transporters ASCT1/SLC1A4 and ASCT2/SLC1A5, and Is Transported by Both Subtypes in Rat Hippocampal Astrocyte Cultures.

    Science.gov (United States)

    Foster, Alan C; Farnsworth, Jill; Lind, Genevieve E; Li, Yong-Xin; Yang, Jia-Ying; Dang, Van; Penjwini, Mahmud; Viswanath, Veena; Staubli, Ursula; Kavanaugh, Michael P

    2016-01-01

    N-methyl-D-aspartate (NMDA) receptors play critical roles in synaptic transmission and plasticity. Activation of NMDA receptors by synaptically released L-glutamate also requires occupancy of co-agonist binding sites in the tetrameric receptor by either glycine or D-serine. Although D-serine appears to be the predominant co-agonist at synaptic NMDA receptors, the transport mechanisms involved in D-serine homeostasis in brain are poorly understood. In this work we show that the SLC1 amino acid transporter family members SLC1A4 (ASCT1) and SLC1A5 (ASCT2) mediate homo- and hetero-exchange of D-serine with physiologically relevant kinetic parameters. In addition, the selectivity profile of D-serine uptake in cultured rat hippocampal astrocytes is consistent with uptake mediated by both ASCT1 and ASCT2. Together these data suggest that SLC1A4 (ASCT1) may represent an important route of Na-dependent D-serine flux in the brain that has the ability to regulate extracellular D-serine and thereby NMDA receptor activity.

  19. FBXO22 Protein Is Required for Optimal Synthesis of the N-Methyl-d-Aspartate (NMDA) Receptor Coagonist d-Serine

    DEFF Research Database (Denmark)

    Dikopoltsev, Elena; Foltyn, Veronika N; Zehl, Martin

    2014-01-01

    d-Serine is a physiological activator of NMDA receptors (NMDARs) in the nervous system that mediates several NMDAR-mediated processes ranging from normal neurotransmission to neurodegeneration. d-Serine is synthesized from l-serine by serine racemase (SR), a brain-enriched enzyme. However, little...

  20. Astrocytes in primary cultures express serine racemase, synthesize d-serine and acquire A1 reactive astrocyte features.

    Science.gov (United States)

    Li, Suyan; Uno, Yota; Rudolph, Uwe; Cobb, Johanna; Liu, Jing; Anderson, Thea; Levy, Deborah; Balu, Darrick T; Coyle, Joseph T

    2018-01-03

    d-Serine is a co-agonist at forebrain N-methyl-d-aspartate receptors (NMDAR) and is synthesized by serine racemase (SR). Although d-serine and SR were originally reported to be localized to glia, recent studies have provided compelling evidence that under healthy physiologic conditions both are localized primarily in neurons. However, in pathologic conditions, reactive astrocytes can also express SR and synthesize d-serine. Since cultured astrocytes exhibit features of reactive astrocytes, we have characterized d-serine synthesis and the expression of enzymes involved in its disposition in primary glial cultures. The levels of SR were quite low early in culture and increased markedly in all astrocytes with the duration in vitro. The concentration of d-serine in the culture medium increased in parallel with SR expression in the astrocytes. Microglia, identified by robust expression of Iba1, did not express SR. While the levels of glial fibrillary acidic protein (GFAP), glycine decarboxylase (GLDC) and phosphoglycerate dehydrogenase (PHGDH), the initial enzyme in the pathway converting glycine to l-serine, remained constant in culture, the expression of lipocalin-2, a marker for pan-reactive astrocytes, increased several-fold. The cultured astrocytes also expressed Complement-3a, a marker for a subpopulation of reactive astrocytes (A1). Astrocytes grown from mice with a copy number variant associated with psychosis, which have four copies of the GLDC gene, showed a more rapid production of d-serine and a reduction in glycine in the culture medium. These results substantiate the conclusion that A1 reactive astrocytes express SR and release d-serine under pathologic conditions, which may contribute to their neurotoxic effects by activating extra-synaptic NMDARs. Copyright © 2018 Elsevier Inc. All rights reserved.

  1. A central role for CK1 in catalysing phosphorylation of the P53 transactivation domain at serine 20 after HHV-6B viral infection

    DEFF Research Database (Denmark)

    Maclaine, NJ; Øster, Bodil; Bundgaard, Bettina

    2008-01-01

    The tumour suppressor protein p53 is activated by distinct cellular stresses including radiation, hypoxia, type-I interferon, and DNA/RNA virus infection. The transactivation domain of p53 contains a phosphorylation site at serine 20 (Ser20) whose modification stabilises the binding of the transc...

  2. High throughput protease profiling comprehensively defines active site specificity for thrombin and ADAMTS13.

    Science.gov (United States)

    Kretz, Colin A; Tomberg, Kärt; Van Esbroeck, Alexander; Yee, Andrew; Ginsburg, David

    2018-02-12

    We have combined random 6 amino acid substrate phage display with high throughput sequencing to comprehensively define the active site specificity of the serine protease thrombin and the metalloprotease ADAMTS13. The substrate motif for thrombin was determined by >6,700 cleaved peptides, and was highly concordant with previous studies. In contrast, ADAMTS13 cleaved only 96 peptides (out of >10 7 sequences), with no apparent consensus motif. However, when the hexapeptide library was substituted into the P3-P3' interval of VWF73, an exosite-engaging substrate of ADAMTS13, 1670 unique peptides were cleaved. ADAMTS13 exhibited a general preference for aliphatic amino acids throughout the P3-P3' interval, except at P2 where Arg was tolerated. The cleaved peptides assembled into a motif dominated by P3 Leu, and bulky aliphatic residues at P1 and P1'. Overall, the P3-P2' amino acid sequence of von Willebrand Factor appears optimally evolved for ADAMTS13 recognition. These data confirm the critical role of exosite engagement for substrates to gain access to the active site of ADAMTS13, and define the substrate recognition motif for ADAMTS13. Combining substrate phage display with high throughput sequencing is a powerful approach for comprehensively defining the active site specificity of proteases.

  3. MBL-associated serine protease-3 circulates in high serum concentrations predominantly in complex with Ficolin-3 and regulates Ficolin-3 mediated complement activation

    DEFF Research Database (Denmark)

    Skjoedt, Mikkel-ole; Palarasah, Yaseelan; Munthe-fog, Lea

    2010-01-01

    The human lectin complement pathway (LCP) involves circulating complexes consisting of mannose-binding lectin (MBL) or ficolins in association with serine proteases named MASP-1, -2 and -3 and a non-enzymatic protein, sMAP. MASP-3 originates from the MASP1 gene through differential splicing...... and little is known about its biological characteristics. For this reason we expressed recombinant MASP-3 and generated specific monoclonal antibodies to establish biochemical characteristics and to determine the serum levels, the interactions with the LCP recognition molecules and the influence...

  4. Substrate specificity and catalysis by the editing active site of alanyl-tRNA synthetase from Escherichia coli†

    Science.gov (United States)

    Pasman, Zvi; Robey-Bond, Susan; Mirando, Adam C.; Smith, Gregory J.; Lague, Astrid; Francklyn, Christopher S.

    2011-01-01

    Aminoacyl-tRNA synthetases (ARSs) enhance the fidelity of protein synthesis through multiple mechanisms, including hydrolysis of the adenylate and cleavage of misacylated tRNA. Alanyl-tRNA synthetase (AlaRS) limits misacylation with glycine and serine by use of a dedicated editing domain, and a mutation in this activity has been genetically linked to a mouse model of a progressive neurodegenerative disease. Using the free standing P. horikoshii AlaX editing domain complexed with serine as a model and both Ser-tRNAAla and Ala-tRNAAla as substrates, the deacylation activities of the wild type and five different E. coli AlaRS editing site substitution mutants were characterized. The wild type AlaRS editing domain deacylated Ser-tRNAAla with a kcat/KM of 6.6 × 105 M−1 s−1, equivalent to a rate enhancement of 6000 over the rate of enzyme-independent deacylation, but only 12.2-fold greater than the rate with Ala-tRNAAla. While the E664A and T567G substitutions only minimally decreased kcat/KM, Q584H, I667E, and C666A AlaRS were more compromised in activity, with decreases in kcat/KM in the range of 6-, 7.3-, and 15-fold. C666A AlaRS was 1.4-fold more active on Ala-tRNAAla relative to Ser-tRNAAla, providing the only example of a true reversal of substrate specificity and highlighting a potential role of the coordinated zinc in editing substrate specificity. Along with the potentially serious physiological consequences of serine mis-incorporation, the relatively modest specificity of the AlaRS editing domain may provide a rationale for the widespread phylogenetic distribution of AlaX free standing editing domains, thereby contributing a further mechanism to lower concentrations of misacylated tRNAAla. PMID:21241052

  5. Structure and Reactivity of the Phosphotriesterase Active Site

    National Research Council Canada - National Science Library

    Krauss, Morris

    2002-01-01

    ... wide range of theoretical structures can be optimized for metal substituted and mutant active sites. The structural behavior of the active site is modeled using a new effective potential for representing the protein molecular environment...

  6. Purification and biochemical characterization of a serine alkaline ...

    African Journals Online (AJOL)

    USER

    2010-08-02

    Aug 2, 2010 ... The enzyme was inactivated by diisopropyl fluorophosphate and phenylmethylsulfonyl fluoride, suggesting that it is a serine protease. The protease was stable in 0.5% SDS and retained 70.3% of its initial activity after 1 h of incubation. It was active in the presence of 3% Triton X-100 with 100% activity.

  7. An update on serine deficiency disorders

    NARCIS (Netherlands)

    van der Crabben, S. N.; Verhoeven-Duif, N. M.; Brilstra, E. H.; Van Maldergem, L.; Coskun, T.; Rubio-Gozalbo, E.; Berger, R.; de Koning, T. J.

    Serine deficiency disorders are caused by a defect in one of the three synthesising enzymes of the L-serine biosynthesis pathway. Serine deficiency disorders give rise to a neurological phenotype with psychomotor retardation, microcephaly and seizures in newborns and children or progressive

  8. Dosage compensation of serine-4 transfer RNA in Drosophila melanogaster

    International Nuclear Information System (INIS)

    Birchler, J.A.; Owenby, R.K.; Jacobson, K.B.

    1982-01-01

    A dosage series of the X chromosome site for serine-4 transfer RNA consisting of one of three copies in females and one to two in males was constructed to test whether transfer RNA expression is governed by dosage compensation. A dosage effect on the level of the serine-4 isoacceptor was observed in both females and males when the structural locus was varied. However, in males, each dose had a relatively greater expression so the normal one dose was slightly greater than the total female value and the duplicated male had the highest relative expression of all the types examined. Serine-4 levels in males and females from an isogenic Oregon-R stock were similar. Thus the transfer RNA levels conform to the expectations of dosage compensation

  9. Promoter proximal polyadenylation sites reduce transcription activity

    DEFF Research Database (Denmark)

    Andersen, Pia Kjølhede; Lykke-Andersen, Søren; Jensen, Torben Heick

    2012-01-01

    , which in turn causes reduced transcription. Functional depletion of U1 snRNP in the context of the wild-type SD triggers the same CpA event accompanied by decreased RNA levels. Thus, in accordance with recent findings, U1 snRNP can shield premature pA sites. The negative impact of unshielded pA sites...

  10. Crystal Structure of a Novel Viral Protease with a Serine/Lysine Catalytic Dyad Mechanism

    Energy Technology Data Exchange (ETDEWEB)

    Feldman,A.; Lee, J.; Delmas, B.; Paetzel, M.

    2006-01-01

    The blotched snakehead virus (BSNV), an aquatic birnavirus, encodes a polyprotein (NH2-pVP2-X-VP4-VP3-COOH) that is processed through the proteolytic activity of its own protease (VP4) to liberate itself and the viral proteins pVP2, X and VP3. The protein pVP2 is further processed by VP4 to give rise to the capsid protein VP2 and four structural peptides. We report here the crystal structure of a VP4 protease from BSNV, which displays a catalytic serine/lysine dyad in its active site. This is the first crystal structure of a birnavirus protease and the first crystal structure of a viral protease that utilizes a lysine general base in its catalytic mechanism. The topology of the VP4 substrate binding site is consistent with the enzymes substrate specificity and a nucleophilic attack from the si-face of the substrates scissile bond. Despite low levels of sequence identity, VP4 shows similarities in its active site to other characterized Ser/Lys proteases such as signal peptidase, LexA protease and Lon protease. Together, the structure of VP4 provides insights into the mechanism of a recently characterized clan of serine proteases that utilize a lysine general base and reveals the structure of potential targets for antiviral therapy, especially for other related and economically important viruses, such as infectious bursal disease virus in poultry and infectious pancreatic necrosis virus in aquaculture.

  11. The binding mechanism of a peptidic cyclic serine protease inhibitor

    DEFF Research Database (Denmark)

    Jiang, Longguang; Svane, Anna Sigrid P.; Sørensen, Hans Peter

    2011-01-01

    Serine proteases are classical objects for studies of catalytic and inhibitory mechanisms as well as interesting as therapeutic targets. Since small-molecule serine protease inhibitors generally suffer from specificity problems, peptidic inhibitors, isolated from phage-displayed peptide libraries......, have attracted considerable attention. Here, we have investigated the mechanism of binding of peptidic inhibitors to serine protease targets. Our model is upain-1 (CSWRGLENHRMC), a disulfide-bond-constrained competitive inhibitor of human urokinase-type plasminogen activator with a noncanonical...... inhibitory mechanism and an unusually high specificity. Using a number of modified variants of upain-1, we characterised the upain-1-urokinase-type plasminogen activator complex using X-ray crystal structure analysis, determined a model of the peptide in solution by NMR spectroscopy, and analysed binding...

  12. Use of degenerate primers and heat-soaked polymerase chain reaction (PCR) to clone a serine protease antigen from Dermatophilus congolensis.

    Science.gov (United States)

    Mine, O M; Carnegie, P R

    1997-10-01

    Serine proteases are thought to be involved in the initial attack on sheep skin by Dermatophilus congolensis and are obvious antigens for inclusion in a vaccine to prevent lumpy wool disease (dermatophilosis). Degenerate primers were designed after alignment of seven bacterial serine proteases. Inosine was incorporated into the primers at positions of three- and four-base redundancy, and this reduced the complexity of the primer mixtures from several thousand to sixteen different sequences for each primer. The primers were validated by production and sequencing of amplicons from serine protease genes in Bacillus subtilis and Serratia marcescens. The primers were used with heat-soaked polymerase chain reaction (PCR) to produce amplicons from two D. congolensis strains, AG and MB. In the amplicon codons for arginine, rather than the expected serine, were found where inosine was used for both the first and third positions for a codon in the primer. A search with the deduced amino acid sequences of the amplicons showed significant similarity to a keratinase and other serine proteases from various organisms. Similarity was most apparent around the active site residues and other essential secondary structural elements.

  13. Activities on the site during construction phase

    International Nuclear Information System (INIS)

    Fickel, O.F.

    1977-01-01

    A survey is given of the work done on the site from site-opening till turn over of the plant to the client. After a short introduction to time schedules, manpower on site, site facilities and civil work and constructions, the commissioning and trial operation phase is discussed in detail. This phase begins with finishing the assembly of individual systems and components and ends with the trial operation and the acceptance measurement. During this period the subsystems are started-up in a useful sequence, first from cold, then from hot conditions and are finally operated as a total with nuclear energy. The single steps are: a) commissioning of indivudal systems; b) hot functional test 1 (without fuels) c) baseline inspection at the reactor pressure vessel; d) hot functional test 2 (with fuels); e) preparation for first criticality; f) postcriticality test program; g) trial operation: h) acceptance measurement. (HP) [de

  14. Inhibition of serine palmitoyltransferase in vitro and long-chain base biosynthesis in intact Chinese hamster ovary cells by β-chloroalanine

    International Nuclear Information System (INIS)

    Medlock, K.A.; Merrill, A.H. Jr.

    1988-01-01

    The effects of β-chloroalanine (β-Cl-alanine) on the serine palmitoyltransferase activity and the de novo biosynthesis of sphinganine and sphingenine were investigated in vitro with rat liver microsomes and in vivo with intact Chinese hamster ovary (CHO) cells. The inhibition in vitro was rapid, irreversible, and concentration and time dependent and apparently involved the active site because inactivation only occurred with β-Cl-L-alanine and was blocked by L-serine. These are characteristics of mechanism-based (suicide) inhibition. Serine palmitoyltransferase (SPT) was also inhibited when intact CHO cells were incubated with β-Cl-alanine and this treatment inhibited [ 14 C]serine incorporation into long-chain bases by intact cells. The concentration dependence of the loss of SPT activity and of long-chain base synthesis was identical. The effects of β-Cl-alanine appeared to occur with little perturbation of other cell functions: the cells exhibited no loss in cell viability, [ 14 C]serine uptake was not blocked, total lipid biosynthesis from [ 14 C]acetic acid was not decreased (nor was the appearance of radiolabel in cholesterol and phosphatidylcholine), and [ 3 H]thymidine incorporation into DNA was not affected. There appeared to be little effect on protein synthesis based on the incorporation of [ 3 H]leucine, which was only decreased by 14%. Although β-Cl-L-alanine is known to inhibit other pyridoxal 5'-phosphate dependent enzymes, alanine and aspartate transaminases were not inhibited under these conditions. These results establish the close association between the activity of serine palmitoyltransferase and the cellular rate of long-chain base formation and indicate that β-Cl-alanine and other mechanism-based inhibitors might be useful to study alterations in cellular long-chain base synthesis

  15. Host perception and signal transduction studies in wild-type Blumeria graminis f. sp. hordei and a quinoxyfen-resistant mutant implicate quinoxyfen in the inhibition of serine esterase activity.

    Science.gov (United States)

    Lee, Susannah; Gustafson, Gary; Skamnioti, Pari; Baloch, Roobina; Gurr, Sarah

    2008-05-01

    Quinoxyfen is a potent and effective fungicide, hitherto considered to control powdery mildew disease by perturbing signal transduction during early germling differentiation. The aim of this paper is to understand the mode of action of quinoxyfen by comparing the perception of host-derived signals and signal relay in a wild-type Blumeria graminis f. sp. hordei EM Marchal (Bgh) (WT/IM82) and a quinoxyfen-resistant field isolate (QR/2B11). QR/2B11 germinates more promiscuously on host-like and artificial surfaces than the quinoxyfen-sensitive WT/IM82. The pivotal role of host cuticle deprivation in the formation of hooked appressorial germ tubes (hAGTs) in WT/IM82 and a dramatic drop in germling differentiation in the presence of the mildewicide are demonstrated. QR/2B11 strain shows a dependence on host cuticle-like features for hAGT formation but no significant difference between germling differentiation in the presence or absence of quinoxyfen. PKC-inhibitor Ro 318220 induces morphological changes similar to those seen in quinoxyfen-treated germlings. PKC1 transcript accumulation is equivalently upregulated by quinoxyfen in QR/2B11 and WT/IM82 strains, but Bgh cutinase CUT1 transcript is 8 times more abundant in QR/2B11 conidia than in WT/IM82 conidia. Quinoxyfen inhibits serine esterase activity in WT/IM82, but not in QR/2B11. Collectively, these data suggest that quinoxyfen interferes with the perception of host-derived signals required for full germling differentiation, and that QR/2B11 bypasses the need for such signals. Moreover, quinoxyfen appears to target serine esterase activity, with a downstream perturbation in signal transduction; this represents the first demonstrable biochemical difference between the quinoxyfen-resistant and -sensitive isolates. Copyright (c) 2008 Society of Chemical Industry.

  16. Thromboxane receptor hyper-responsiveness in hypoxic pulmonary hypertension requires serine 324.

    Science.gov (United States)

    Santhosh, K T; Sikarwar, A S; Hinton, M; Chelikani, P; Dakshinamurti, S

    2014-02-01

    Dysregulation of the thromboxane A₂ (TP) receptor, resulting in agonist hypersensitivity and hyper-responsiveness, contributes to exaggerated vasoconstriction in the hypoxic pulmonary artery in neonatal persistent pulmonary hypertension. We previously reported that hypoxia inhibits TP receptor phosphorylation, causing desensitization. Hence, we examined the role of PKA-accessible serine residues in determining TP receptor affinity, using site-directed mutational analysis. Vasoconstriction to a thromboxane mimetic and phosphorylation of TP receptor serine was examined in pulmonary arteries from neonatal swine with persistent pulmonary hypertension and controls. Effects of hypoxia were determined in porcine and human TP receptors. Human TPα serines at positions 324, 329 and 331 (C-terminal tail) were mutated to alanine and transiently expressed in HEK293T cells. Saturation binding and displacement kinetics of a TP antagonist and agonist were determined in porcine TP, wild-type human TPα and all TP mutants. Agonist-elicited calcium mobilization was determined for each TP mutant, in the presence of a PKA activator or inhibitor, and in hypoxic and normoxic conditions. The Ser324A mutant was insensitive to PKA activation and hypoxia, had a high affinity for agonist and increased agonist-induced calcium mobilization. Ser329A was no different from wild-type TP receptors. Ser331A was insensitive to hypoxia and PKA with a decreased agonist-mediated response. In hypoxic pulmonary hypertension, loss of site-specific phosphorylation of the TP receptor causes agonist hyper-responsiveness. Ser324 is the primary residue phosphorylated by PKA, which regulates TP receptor-agonist interactions. Ser331 mutation confers loss of TP receptor-agonist interaction, regardless of PKA activity. © 2013 The British Pharmacological Society.

  17. Astrocytes are involved in trigeminal dynamic mechanical allodynia: potential role of D-serine.

    Science.gov (United States)

    Dieb, W; Hafidi, A

    2013-09-01

    Trigeminal neuropathic pain affects millions of people worldwide. Despite decades of study on the neuronal processing of pain, mechanisms underlying enhanced pain states after injury remain unclear. N-methyl-D-aspartate (NMDA) receptor-dependent changes play a critical role in triggering central sensitization in neuropathic pain. These receptors are regulated at the glycine site through a mandatory endogenous co-agonist D-serine, which is synthesized by astrocytes. Therefore, the present study was carried out to determine whether astrocytes are involved, through D-serine secretion, in dynamic mechanical allodynia (DMA) obtained after chronic constriction of the infraorbital nerve (CCI-IoN) in rats. Two weeks after CCI-IoN, an important reaction of astrocytes was present in the medullary dorsal horn (MDH), as revealed by an up-regulation of glial fibrillary acidic protein (GFAP) in allodynic rats. In parallel, an increase in D-serine synthesis, which co-localized with its synthesis enzyme serine racemase, was strictly observed in astrocytes. Blocking astrocyte metabolism by intracisternal delivery of fluorocitrate alleviated DMA. Furthermore, the administration of D-amino-acid oxidase (DAAO), a D-serine-degrading enzyme, or that of L-serine O-sulfate (LSOS), a serine racemase inhibitor, significantly decreased pain behavior in allodynic rats. These results demonstrate that astrocytes are involved in the modulation of orofacial post-traumatic neuropathic pain via the release of the gliotransmitter D-serine.

  18. Improvement in regional CBF by L-serine contributes to its neuroprotective effect in rats after focal cerebral ischemia.

    Directory of Open Access Journals (Sweden)

    Tao-Jie Ren

    Full Text Available To investigate the mechanisms underlying the neuroprotective effect of L-serine, permanent focal cerebral ischemia was induced by occlusion of the middle cerebral artery while monitoring cerebral blood flow (CBF. Rats were divided into control and L-serine-treated groups after middle cerebral artery occlusion. The neurological deficit score and brain infarct volume were assessed. Nissl staining was used to quantify the cortical injury. L-serine and D-serine levels in the ischemic cortex were analyzed with high performance liquid chromatography. We found that L-serine treatment: 1 reduced the neurological deficit score, infarct volume and cortical neuron loss in a dose-dependent manner; 2 improved CBF in the cortex, and this effect was inhibited in the presence of apamin plus charybdotoxin while the alleviation of both neurological deficit score and infarct volume was blocked; and 3 increased the amount of L-serine and D-serine in the cortex, and inhibition of the conversion of L-serine into D-serine by aminooxyacetic acid did not affect the reduction of neurological deficit score and infarct volume by L-serine. In conclusion, improvement in regional CBF by L-serine may contribute to its neuroprotective effect on the ischemic brain, potentially through vasodilation which is mediated by the small- and intermediate-conductance Ca(2+-activated K(+ channels on the cerebral blood vessel endothelium.

  19. Savannah River Site prioritization of transition activities

    International Nuclear Information System (INIS)

    Finley, R.H.

    1993-11-01

    Effective management of SRS conversion from primarily a production facility to other missions (or Decontamination and Decommissioning (D ampersand D)) requires a systematic and consistent method of prioritizing the transition activities. This report discusses the design of a prioritizing method developed to achieve systematic and consistent methods of prioritizing these activities

  20. Safety Oversight of Decommissioning Activities at DOE Nuclear Sites

    International Nuclear Information System (INIS)

    Zull, Lawrence M.; Yeniscavich, William

    2008-01-01

    The Defense Nuclear Facilities Safety Board (Board) is an independent federal agency established by Congress in 1988 to provide nuclear safety oversight of activities at U.S. Department of Energy (DOE) defense nuclear facilities. The activities under the Board's jurisdiction include the design, construction, startup, operation, and decommissioning of defense nuclear facilities at DOE sites. This paper reviews the Board's safety oversight of decommissioning activities at DOE sites, identifies the safety problems observed, and discusses Board initiatives to improve the safety of decommissioning activities at DOE sites. The decommissioning of former defense nuclear facilities has reduced the risk of radioactive material contamination and exposure to the public and site workers. In general, efforts to perform decommissioning work at DOE defense nuclear sites have been successful, and contractors performing decommissioning work have a good safety record. Decommissioning activities have recently been completed at sites identified for closure, including the Rocky Flats Environmental Technology Site, the Fernald Closure Project, and the Miamisburg Closure Project (the Mound site). The Rocky Flats and Fernald sites, which produced plutonium parts and uranium materials for defense needs (respectively), have been turned into wildlife refuges. The Mound site, which performed R and D activities on nuclear materials, has been converted into an industrial and technology park called the Mound Advanced Technology Center. The DOE Office of Legacy Management is responsible for the long term stewardship of these former EM sites. The Board has reviewed many decommissioning activities, and noted that there are valuable lessons learned that can benefit both DOE and the contractor. As part of its ongoing safety oversight responsibilities, the Board and its staff will continue to review the safety of DOE and contractor decommissioning activities at DOE defense nuclear sites

  1. Characterization of a membrane-associated serine protease in Escherichia coli

    International Nuclear Information System (INIS)

    Palmer, S.M.; St John, A.C.

    1987-01-01

    Three membrane-associated proteolytic activities in Escherichia coli were resolved by DEAE-cellulose chromatography from detergent extracts of the total envelope fraction. On the basis of substrate specificity for the hydrolysis of chromogenic amino acid ester substrates, the first two eluting activities were determined previously to be protease V and protease IV, respectively. The third proteolytic activity eluting from the DEAE-cellulose column was further purified by affinity chromatography on benzamidine-Sepharose 6B. They termed this enzyme protease VI. Protease VI did not hydrolyze any of the chromogenic substrates used in the detection of protease IV and protease V. However, all three enzymes generated acid-soluble fragments from a mixture of E. coli membrane proteins which were biosynthetically labeled with radioactive amino acids. The activity of protease VI was sensitive to serine protease inhibitors. Using [ 3 H]diisopropylfluorophosphate as an active-site labeling reagent, they determined that protease VI has an apparent molecular weight of 43,000 in polyacrylamide gels. All three membrane-associated serine proteases were insensitive to inhibition by Ecotin, an endogenous, periplasmic inhibitor of trypsin

  2. A functional SUMO-motif in the active site of PIM1 promotes its degradation via RNF4, and stimulates protein kinase activity.

    Science.gov (United States)

    Iyer, R Sumanth; Chatham, Lynsey; Sleigh, Roger; Meek, David W

    2017-06-15

    The PIM1 serine/threonine protein kinase mediates growth factor and survival signalling, and cooperates potently with c-MYC during tumorigenesis. PIM1 is overexpressed in many human cancers and is a promising target for drug development. PIM1 levels are regulated mainly through cytokine-induced transcription and protein degradation, but mechanisms regulating its activity and levels remain largely unexplored. Here, we show that PIM1 is modified in vitro and in cultured cells by the Small ubiquitin-like modifier (SUMO) on two independent sites: K169, within a consensus SUMOylation motif (IK 169 DE 171 ) in the active site of PIM1, and also at a second promiscuous site. Alanine substitution of E171 (within the consensus motif) abolished SUMOylation, significantly increased the half-life of PIM1, and markedly reduced its ubiquitylation. Mechanistically, SUMOylation promoted ubiquitin-mediated degradation of PIM1 via recruitment of the SUMO-targeted ubiquitin ligase, RNF4. Additionally, SUMOylated PIM1 showed enhanced protein kinase activity in vitro. Interestingly, the E171A mutant was active in vitro but displayed altered substrate specificity in cultured cells, consistent with the idea that SUMOylation may govern PIM1 substrate specificity under certain contexts. Taken together, these data demonstrate that the protein kinase activity and levels of PIM1 can be regulated by a covalent post-translational modification.

  3. Potent and Selective Peptidyl Boronic Acid Inhibitors of the Serine Protease Prostate-Specific Antigen

    Science.gov (United States)

    LeBeau, Aaron M.; Singh, Pratap; Isaacs, John T.; Denmeade, Samuel R.

    2012-01-01

    SUMMARY Prostate cancer cells produce high (microgram to milligram/milliliter) levels of the serine protease Prostate-Specific Antigen (PSA). PSA is enzymatically active in the extracellular fluid surrounding prostate cancers but is found at 1,000- to 10,000-fold lower concentrations in the circulation, where it is inactivated due to binding to abundant serum protease inhibitors. The exclusive presence of high levels of active PSA within prostate cancer sites makes PSA an attractive candidate for targeted imaging and therapeutics. A synthetic approach based on a peptide substrate identified first peptide aldehyde and then boronic acid inhibitors of PSA. The best of these had the sequence Cbz-Ser-Ser-Lys-Leu-(boro)Leu, with a Ki for PSA of 65 nM. The inhibitor had a 60-fold higher Ki for chymotrypsin. A validated model of PSA’s catalytic site confirmed the critical interactions between the inhibitor and residues within the PSA enzyme. PMID:18635003

  4. N-methyl-d-aspartate receptor coagonist d-serine suppresses intake of high-preference food.

    Science.gov (United States)

    Sasaki, Tsutomu; Kinoshita, Yoshihiro; Matsui, Sho; Kakuta, Shigeru; Yokota-Hashimoto, Hiromi; Kinoshita, Kuni; Iwasaki, Yusaku; Kinoshita, Toshio; Yada, Toshihiko; Amano, Naoji; Kitamura, Tadahiro

    2015-09-01

    d-Serine is abundant in the forebrain and physiologically important for modulating excitatory glutamatergic neurotransmission as a coagonist of synaptic N-methyl-d-aspartate (NMDA) receptor. NMDA signaling has been implicated in the control of food intake. However, the role of d-serine on appetite regulation is unknown. To clarify the effects of d-serine on appetite, we investigated the effect of oral d-serine ingestion on food intake in three different feeding paradigms (one-food access, two-food choice, and refeeding after 24-h fasting) using three different strains of male mice (C57Bl/6J, BKS, and ICR). The effect of d-serine was also tested in leptin signaling-deficient db/db mice and sensory-deafferented (capsaicin-treated) mice. The expression of orexigenic neuropeptides [neuropeptide Y (Npy) and agouti-related protein (Agrp)] in the hypothalamus was compared in fast/refed experiments. Conditioned taste aversion for high-fat diet (HFD) was tested in the d-serine-treated mice. Under the one-food-access paradigm, some of the d-serine-treated mice showed starvation, but not when fed normal chow. HFD feeding with d-serine ingestion did not cause aversion. Under the two-food-choice paradigm, d-serine suppressed the intake of high-preference food but not normal chow. d-Serine also effectively suppressed HFD intake but not normal chow in db/db mice and sensory-deafferented mice. In addition, d-serine suppressed normal chow intake after 24-h fasting despite higher orexigenic gene expression in the hypothalamus. d-Serine failed to suppress HFD intake in the presence of L-701,324, the selective and full antagonist at the glycine-binding site of the NMDA receptor. Therefore, d-serine suppresses the intake of high-preference food through coagonism toward NMDA receptors. Copyright © 2015 the American Physiological Society.

  5. L-serine in disease and development

    NARCIS (Netherlands)

    de Koning, Tom J.; Snell, Keith; Duran, Marinus; Berger, Ruud; Poll-The, Bwee-Tien; Surtees, Robert

    2003-01-01

    The amino acid L-serine, one of the so-called non-essential amino acids, plays a central role in cellular proliferation. L-Serine is the predominant source of one-carbon groups for the de novo synthesis of purine nucleotides and deoxythymidine monophosphate. It has long been recognized that, in cell

  6. Robotics at Savannah River site: activity report

    International Nuclear Information System (INIS)

    Byrd, J.S.

    1984-09-01

    The objectives of the Robotics Technology Group at the Savannah River Laboratory are to employ modern industrial robots and to develop unique automation and robotic systems to enhance process operations at the Savannah River site (SRP and SRL). The incentives are to improve safety, reduce personnel radiation exposure, improve product quality and productivity, and to reduce operating costs. During the past year robotic systems have been installed to fill chemical dilution vials in a SRP laboratory at 772-F and remove radioactive waste materials in the SRL Californium Production Facility at 773-A. A robotic system to lubricate an extrusion press has been developed and demonstrated in the SRL robotics laboratory and is scheduled for installation at the 321-M fuel fabrication area. A mobile robot was employed by SRP for a radiation monitoring task at a waste tank top in H-Area. Several other robots are installed in the SRL robotics laboratories and application development programs are underway. The status of these applications is presented in this report

  7. Identification of a highly reactive threonine residue at the active site of γ-glutamyl transpeptidase

    International Nuclear Information System (INIS)

    Stole, E.; Seddon, A.P.; Wellner, D.; Meister, A.

    1990-01-01

    γ-Glutamyl transpeptidase an enzyme of major importance in glutathione metabolism, was inactivated by treating it with L-(αS,5S)-α-amino-3-chloro-4,5-dihydro-5-[3- 14 C]isoxazoleacetic acid. This selective reagent binds stoichiometrically to the enzyme; more than 90% of the label was bound to its light subunit. Enzymatic digestion of the light subunit gave a 14 C-labeled peptide that corresponds to amino acid residues 517-527 of the enzyme and two incomplete digestion products that contain this labeled peptide moiety. The radioactivity associated with this peptide was released with threonine-523 during sequencing by the automated gas-phase Edman method. The light subunit contains 14 other threonine residues and a total of 19 serine residues; these were not labeled. Threonine-523 is situated in the enzyme in an environment that greatly increases its reactivity, indicating that other amino acid residues of the enzyme must also participate in the active-site chemistry of the enzyme

  8. Insulin Induces Phosphorylation of Serine Residues of Translationally Controlled Tumor Protein in 293T Cells

    Directory of Open Access Journals (Sweden)

    Jeehye Maeng

    2015-04-01

    Full Text Available Insulin induces the activation of Na,K-ATPase while translationally controlled tumor protein (TCTP inhibits this enzyme and the associated pump activity. Because binding of insulin with its membrane receptor is known to mediate the phosphorylation of multiple intracellular proteins, phosphorylation of TCTP by insulin might be related to the sodium pump regulation. We therefore examined whether insulin induces TCTP phosphorylation in embryonic kidney 293T cells. Using immunoprecipitation and Western blotting, we found that insulin phosphorylates serine (Ser residues of TCTP. Following fractionation of the insulin-treated cells into cytosol and membrane fractions, phosphorylated TCTP at its Ser residue (p-Ser-TCTP was detected exclusively in the cytosolic part and not in the membrane fraction. Phosphorylation of TCTP reached maximum in about 10 min after insulin treatment in 293T cells. In studies of cell-type specificity of insulin-mediated phosphorylation of TCTP, insulin did not phosphorylate TCTP in HeLa cells. Computational prediction and immunoprecipitation using several constructs having Ser to Ala mutation at potential p-Ser sites of TCTP revealed that insulin phosphorylated the serine-9 and -15 residues of TCTP. Elucidations of how insulin-mediated TCTP phosphorylation promotes Na,K-ATPase activation, may offer potential therapeutic approaches to diseases associated with vascular activity and sodium pump dysregulation.

  9. Continuing education in neurometabolic disorders--serine deficiency disorders

    NARCIS (Netherlands)

    de Koning, T. J.; Poll-The, B. T.; Jaeken, J.

    1999-01-01

    Serine deficiency disorders comprise a new group of inborn errors of serine metabolism. Patients affected with these disorders present with major neurological symptoms including congenital microcephaly, seizures, psychomotor retardation or polyneuropathy. The diagnosis of serine deficiency is based

  10. Active site - a site of binding of affinity inhibitors in baker's yeast inorganic pyrophosphatase

    International Nuclear Information System (INIS)

    Svyato, I.E.; Sklyankina, V.A.; Avaeva, S.M.

    1986-01-01

    The interaction of the enzyme-substrate complex with methyl phosphate, O-phosphoethanolamine, O-phosphopropanolamine, N-acetylphosphoserine, and phosphoglyolic acid, as well as pyrophosphatase, modified by monoesters of phosphoric acid, with pyrophosphate and tripolyphosphate, was investigated. It was shown that the enzyme containing the substrate in the active site does not react with monophosphates, but modified pyrophosphatase entirely retains the ability to bind polyanions to the regulatory site. It is concluded that the inactivation of baker's yeast inorganic pyrophosphatase by monoesters of phosphoric acid, which are affinity inhibitors of it, is the result of modification of the active site of the enzyme

  11. Protein kinase D regulates the human cardiac L-type voltage-gated calcium channel through serine 1884.

    Science.gov (United States)

    Aita, Yusuke; Kurebayashi, Nagomi; Hirose, Shigehisa; Maturana, Andrés D

    2011-12-15

    Protein kinase D (PKD) regulates the activity of the L-type calcium channel in rat ventricular cardiomyocytes. However, the functional target residues of PKD on the L-type calcium channel remain to be identified. Our aim was to identify the functional phosphorylation sites of PKD on the human L-type calcium channel. The pore subunit of the human CaV1.2 (hCaV1.2) was stably expressed in HEK293 cells. Both the expression of a dominant-negative mutant of PKD and the mutation of serine 1884 but not serine 1930, putative targets of PKD, strongly reduced L-type calcium currents and single channel activity without affecting the channel's expression at the plasma membrane. Our results suggest that serine 1884 is essential for the regulation of hCaV1.2 by PKD. Copyright © 2011 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

  12. Crystal Structure of the Catalytic Domain of a Serine Threonine Protein Phosphatase

    Science.gov (United States)

    Swinglel, Mark; Honkanel, Richard; Ciszak, Ewa

    2003-01-01

    Reversible phosphorylation of serine and threonine residues is a well-recognized mechanism in eukaryotic cells for the regulation of cell-cycle progression, cell growth and metabolism. Human serine/threonine phosphatases can be placed into two major families, PPP and PPM. To date the structure on one PPP family member (PPl) has been determined. Here we present the structure of a 323-residue catalytic domain of a second phosphatase belonging to the PPP family of enzyme. catalytic domain of the enzyme has been determined to 1.60Angstrom resolution and refined to R=17.5 and Rfree = 20.8%. The catalytic domain possesses a unique fold consisting of a largely monolithic structure, divisible into closely-associated helical and sheet regions. The catalytic site contains two manganese ions that are involved in substrate binding and catalysis. The enzyme crystallizes as a dimer that completely buries catalytic surfaces of both monomers, Also, the structure shows evidence of some flexibility around the active site cleft that may be related to substrate specificity of this enzyme.

  13. RNA polymerase II phosphorylation at serine 2 and histone H3 tri-methylation at lysine 36 are key steps for thyroid hormone receptor β gene activation by thyroid hormone in Rana catesbeiana tadpole liver.

    Science.gov (United States)

    Mochizuki, Kazuki; Ishihara, Akinori; Goda, Toshinao; Yamauchi, Kiyoshi

    2012-01-20

    Thyroid hormone (TH) is essential for amphibian metamorphosis, during which the expression of many genes is controlled directly or indirectly through TH receptors (TRs). Thyroid hormone binding to TRs induces coregulator switching on regulatory regions of TH-inducible genes: corepressors complexed with unliganded TRs are replaced by coactivators complexed with liganded TR resulting in transcriptionally active states. The coregulator switching is linked to histone acetylation. In our study, we have investigated the acetylation and methylation states of histones H3 and H4 using chromatin immunoprecipitation (ChIP) assays on the 5' coding region of the TRβ gene, a primary TH-response gene, in the liver from Rana catesbeiana tadpoles either treated with or not treated with 3,3',5-triiodothyronine (T3). 3,3',5-Triiodothyronine treatment for 3 days increased the amount of TRβ transcript by 19-fold. This increase was associated with increases in the acetylation of histone H4 and lysine 9 in histone H3 (H3-K9), and tri-methylation of lysine 36 in histone H3 (H3-K36). In addition, the amounts of RNA polymerase II (PolII) and serine 2 phosphorylation in PolII (PolII-S2) increased. These results suggest that T3 treatment enhances the elongation activity of PolII on the TRβ gene in the liver by increasing histone H3-K36 tri-methylation through PolII-S2 phosphorylation. Copyright © 2011 Elsevier Inc. All rights reserved.

  14. The Evaluation of Dipeptidyl Peptidase (DPP)-IV, α-Glucosidase and Angiotensin Converting Enzyme (ACE) Inhibitory Activities of Whey Proteins Hydrolyzed with Serine Protease Isolated from Asian Pumpkin (Cucurbita ficifolia).

    Science.gov (United States)

    Konrad, Babij; Anna, Dąbrowska; Marek, Szołtysik; Marta, Pokora; Aleksandra, Zambrowicz; Józefa, Chrzanowska

    2014-01-01

    In the present study, whey protein concentrate (WPC-80) and β-lactoglobulin were hydrolyzed with a noncommercial serine protease isolated from Asian pumpkin ( Cucurbita ficifolia ). Hydrolysates were further fractionated by ultrafiltration using membranes with cut-offs equal 3 and 10 kDa. Peptide fractions of molecular weight lower than 3 and 3-10 kDa were further subjected to the RP-HPLC. Separated preparations were investigated for their potential as the natural inhibitors of dipeptidyl peptidase (DPP-IV), α-glucosidase and angiotensin converting enzyme (ACE). WPC-80 hydrolysate showed higher inhibitory activities against the three tested enzymes than β-lactoglobulin hydrolysate. Especially high biological activities were exhibited by peptide fractions of molecular weight lower than 3 kDa, with ACE IC50 <0.64 mg/mL and DPP-IV IC50 <0.55 mg/mL. This study suggests that peptides generated from whey proteins may support postprandial glycemia regulation and blood pressure maintenance, and could be used as functional food ingredients in the diet of patients with type 2 diabetes.

  15. Resolving the Structure of Active Sites on Platinum Catalytic Nanoparticles

    DEFF Research Database (Denmark)

    Chang, Lan Yun; Barnard, Amanda S.; Gontard, Lionel Cervera

    2010-01-01

    Accurate understanding of the structure of active sites is fundamentally important in predicting catalytic properties of heterogeneous nanocatalysts. We present an accurate determination of both experimental and theoretical atomic structures of surface monatomic steps on industrial platinum nanop...

  16. A Conserved Active Site Tyrosine Residue of Proline Dehydrogenase Helps Enforce the Preference for Proline over Hydroxyproline as the Substrate

    Energy Technology Data Exchange (ETDEWEB)

    Ostrander, E.L.; Larson, J.D.; Schuermann, J.P.; Tanner, J.J.; (Cornell); (Missouri)

    2009-03-02

    Proline dehydrogenase (PRODH) catalyzes the oxidation of L-proline to {Delta}-1-pyrroline-5-carboxylate. PRODHs exhibit a pronounced preference for proline over hydroxyproline (trans-4-hydroxy-L-proline) as the substrate, but the basis for specificity is unknown. The goal of this study, therefore, is to gain insight into the structural determinants of substrate specificity of this class of enzyme, with a focus on understanding how PRODHs discriminate between the two closely related molecules, proline and hydroxyproline. Two site-directed mutants of the PRODH domain of Escherichia coli PutA were created: Y540A and Y540S. Kinetics measurements were performed with both mutants. Crystal structures of Y540S complexed with hydroxyproline, proline, and the proline analogue L-tetrahydro-2-furoic acid were determined at resolutions of 1.75, 1.90, and 1.85 {angstrom}, respectively. Mutation of Tyr540 increases the catalytic efficiency for hydroxyproline 3-fold and decreases the specificity for proline by factors of 20 (Y540S) and 50 (Y540A). The structures show that removal of the large phenol side chain increases the volume of the substrate-binding pocket, allowing sufficient room for the 4-hydroxyl of hydroxyproline. Furthermore, the introduced serine residue participates in recognition of hydroxyproline by forming a hydrogen bond with the 4-hydroxyl. This result has implications for understanding the substrate specificity of the related enzyme human hydroxyproline dehydrogenase, which has serine in place of tyrosine at this key active site position. The kinetic and structural results suggest that Tyr540 is an important determinant of specificity. Structurally, it serves as a negative filter for hydroxyproline by clashing with the 4-hydroxyl group of this potential substrate.

  17. Glycine inhibitory dysfunction turns touch into pain through astrocyte-derived D-serine.

    Science.gov (United States)

    Miraucourt, Loïs S; Peirs, Cédric; Dallel, Radhouane; Voisin, Daniel L

    2011-06-01

    Glycine inhibitory dysfunction provides a useful experimental model for studying the mechanism of dynamic mechanical allodynia, a widespread and intractable symptom of neuropathic pain. In this model, allodynia expression relies on N-methyl-d-aspartate receptors (NMDARs), and it has been shown that astrocytes can regulate their activation through the release of the NMDAR coagonist d-serine. Recent studies also suggest that astrocytes potentially contribute to neuropathic pain. However, the involvement of astrocytes in dynamic mechanical allodynia remains unknown. Here, we show that after blockade of glycine inhibition, orofacial tactile stimuli activated medullary dorsal horn (MDH) astrocytes, but not microglia. Accordingly, the glia inhibitor fluorocitrate, but not the microglia inhibitor minocycline, prevented allodynia. Fluorocitrate also impeded activation of astrocytes and blocked activation of the superficial MDH neural circuit underlying allodynia, as revealed by study of Fos expression. MDH astrocytes are thus required for allodynia. They may also produce d-serine because astrocytic processes were selectively immunolabeled for serine racemase, the d-serine synthesizing enzyme. Accordingly, selective degradation of d-serine with d-amino acid oxidase applied in vivo prevented allodynia and activation of the underlying neural circuit. Conversely, allodynia blockade by fluorocitrate was reversed by exogenous d-serine. These results suggest the following scenario: removal of glycine inhibition makes tactile stimuli able to activate astrocytes; activated astrocytes may provide d-serine to enable NMDAR activation and thus allodynia. Such a contribution of astrocytes to pathological pain fuels the emerging concept that astrocytes are critical players in pain signaling. Glycine disinhibition makes tactile stimuli able to activate astrocytes, which may provide d-serine to enable NMDA receptor activation and thus allodynia. Copyright © 2011 International Association

  18. Klipperaas study site. Scope of activities and main results

    International Nuclear Information System (INIS)

    Ahlbom, K.; Andersson, J.E.; Andersson, Peter; Ittner, T.; Tiren, S.; Ljunggren, C.

    1992-09-01

    During the period from 1977 - 1986 SKB (Swedish Nuclear Fuel and Waste Management Co.) performed surface and borehole investigations of 14 study sites for the purpose of assessing their suitability for a repository of spent nuclear fuel. The next phase in the SKB site selection rpogramme will be to perform detailed characterisation, including characterization from shafts and/or tunnels, of two or three sites. The detailed investigations will continue over several years to provide all the data needed for a licensing application to build a repository. Such an application is foreseen to be given to the authorities around the year 2003. It is presently not clear if any of the study sites will be selected as a site for detailed characterization. Other sites with geological and/or socio-economical characteristics judged more favorable may very well be the ones selected. However, as a part of the background documentation needed for the site selection studies to come, summary reports will be prepared for most study sites. These reports will include scope of activities, main results, uncertainties and need of complementary investigations. This report concern the Klipperaas study site. The main topics are the scope of activities, geologic model, geohydrological model, groundwater chemistry, assessment of solute transport, and rock mechanics

  19. Neurospora tryptophan synthase: N-terminal analysis and the sequence of the pyridoxal phosphate active site peptide

    International Nuclear Information System (INIS)

    Pratt, M.L.; Hsu, P.Y.; DeMoss, J.A.

    1986-01-01

    Tryptophan synthase (TS), which catalyzes the final step of tryptophan biosynthesis, is a multifunctional protein requiring pyridoxal phosphate (B6P) for two of its three distinct enzyme activities. TS from Neurospora has a blocked N-terminal, is a homodimer of 150 KDa and binds one mole of B6P per mole of subunit. The authors shown the N-terminal residue to be acyl-serine. The B6P-active site of holoenzyme was labelled by reduction of the B6P-Schiff base with [ 3 H]-NaBH 4 , and resulted in a proportionate loss of activity in the two B6P-requiring reactions. SDS-polyacrylamide gel electrophoresis of CNBr-generated peptides showed the labelled, active site peptide to be 6 KDa. The sequence of this peptide, purified to apparent homogeneity by a combination of C-18 reversed phase and TSK gel filtration HPLC is: gly-arg-pro-gly-gln-leu-his-lys-ala-glu-arg-leu-thr-glu-tyr-ala-gly-gly-ala-gln-ile-xxx-leu-lys-arg-glu-asp-leu-asn-his-xxx-gly-xxx-his-/sub ***/-ile-asn-asn-ala-leu. Although four residues (xxx, /sub ***/) are unidentified, this peptide is minimally 78% homologous with the corresponding peptide from yeast TS, in which residue (/sub ***/) is the lysine that binds B6P

  20. Effects of two serine proteases from Bothrops pirajai snake venom on the complement system and the inflammatory response.

    Science.gov (United States)

    Menaldo, Danilo L; Bernardes, Carolina P; Pereira, Juliana C; Silveira, Denise S C; Mamede, Carla C N; Stanziola, Leonilda; Oliveira, Fábio de; Pereira-Crott, Luciana S; Faccioli, Lúcia H; Sampaio, Suely V

    2013-04-01

    The present study aimed to evaluate the effects of two serine proteases from Bothrops pirajai snake venom, named BpirSP27 and BpirSP41, on the complement system and the inflammatory response. The effects of these enzymes on the human complement system were assessed by kinetic hemolytic assays, evaluating the hemolysis promoted by the classical/lectin (CP/LP) and alternative (AP) pathways after incubation of normal human serum with the serine proteases. The results suggested that these enzymes were able to induce modulation of CP/LP and AP at different levels: BpirSP41 showed higher inhibitory effects on the hemolytic activity of CP/LP than BpirSP27, with inhibition values close to 40% and 20%, respectively, for the highest concentration assayed. Regarding AP, both enzymes showed percentages of inhibition of the hemolytic activity around 20% for the highest concentrations tested, indicating similar effects on this complement pathway. The proinflammatory effects of B. pirajai serine proteases were evaluated regarding their ability to induce paw edema, variations in the pain threshold and leukocyte recruitment at the site of injection. Both showed mild effects on these inflammatory processes, leading to low levels of increase of paw volumes and decrease in pain thresholds in rats up to 6 h after injection, and inducing neutrophil recruitment without significant increases in the total number of leukocytes in the inflammatory exudates after 6 and 24 h of administration into mice peritoneal cavity. These results suggest that serine proteases must present a minor role in the inflammation caused by B. pirajai snake venom. Copyright © 2013 Elsevier B.V. All rights reserved.

  1. Structure Determination of Mycobacterium tuberculosis Serine Protease Hip1 (Rv2224c)

    Energy Technology Data Exchange (ETDEWEB)

    Naffin-Olivos, Jacqueline L.; Daab, Andrew; White, Andre; Goldfarb, Nathan E.; Milne, Amy C.; Liu, Dali; Baikovitz, Jacqueline; Dunn, Ben M.; Rengarajan, Jyothi; Petsko, Gregory A.; Ringe, Dagmar

    2017-04-07

    The Mycobacterium tuberculosis (Mtb) serine protease Hip1 (hydrolase important for pathogenesis; Rv2224c) promotes tuberculosis (TB) pathogenesis by impairing host immune responses through proteolysis of a protein substrate, Mtb GroEL2. The cell surface localization of Hip1 and its immunomodulatory functions make Hip1 a good drug target for new adjunctive immune therapies for TB. Here, we report the crystal structure of Hip1 to a resolution of 2.6 Å and the kinetic studies of the enzyme against model substrates and the protein GroEL2. The structure shows a two-domain protein, one of which contains the catalytic residues that are the signature of a serine protease. Surprisingly, a threonine is located within the active site close enough to hydrogen bond with the catalytic residues Asp463 and His490. Mutation of this residue, Thr466, to alanine established its importance for function. Our studies provide insights into the structure of a member of a novel family of proteases. Knowledge of the Hip1 structure will aid in designing inhibitors that could block Hip1 activity

  2. Urinary serine proteases and activation of ENaC in kidney-implications for physiological renal salt handling and hypertensive disorders with albuminuria

    DEFF Research Database (Denmark)

    Svenningsen, Per; Andersen, Henrik; Nielsen, Lise H

    2015-01-01

    and diabetic nephropathy; blood pressure is salt-sensitive in conditions with microalbuminuria/proteinuria; and extracellular volume is expanded, plasma atrial natriuretic peptide (ANP) concentration is increased, and diuretics, like amiloride and spironolactone, are effective blood pressure-reducing add......-ons. Active plasmin in urine has been demonstrated in diabetes, preeclampsia, and nephrosis. Urine from these patients activates, plasmin-dependently, amiloride-sensitive inward current in vitro. The concept predicts that patients with albuminuria may benefit particularly from reduced salt intake with RAS...

  3. Dashboard applications to monitor experiment activities at sites

    International Nuclear Information System (INIS)

    Andreeva, Julia; Gaidioz, Benjamin; Grigoras, Costin; Kokoszkiewicz, Lukasz; Lanciotti, Elisa; Rocha, Ricardo; Saiz, Pablo; Santinelli, Roberto; Sidorova, Irina; Sciaba, Andrea; Belforte, Stefano; Boehm, Max; Casajus, Adrian; Flix, Josep; Tsaregorodtsev, Andrei

    2010-01-01

    In the framework of a distributed computing environment, such as WLCG, monitoring has a key role in order to keep under control activities going on in sites located in different countries and involving people based in many different sites. To be able to cope with such a large scale heterogeneous infrastructure, it is necessary to have monitoring tools providing a complete and reliable view of the overall performance of the sites. Moreover, the structure of a monitoring system critically depends on the object to monitor and on the users it is addressed to. In this article we will describe two different monitoring systems both aimed to monitor activities and services provided in the WLCG framework, but designed in order to meet the requirements of different users: Site Status Board has an overall view of the services available in all the sites supporting an experiment, whereas Siteview provides a complete view of all the activities going on at a site, for all the experiments supported by the site.

  4. In vivo [(123)I]CNS-1261 binding to D-serine-activated and MK801-blocked NMDA receptors: A storage phosphor imaging study in rats

    NARCIS (Netherlands)

    Knol, Remco J. J.; de Bruin, Kora; van Eck-Smit, Berthe L. F.; Pimlott, Sally; Wyper, David J.; Booij, Jan

    2009-01-01

    Disturbances of activity of the glutamatergic neurotransmitter system in the brain are present in many neuropsychiatric disorders. The N-methyl-D-aspartate (NMDA) receptor is the most abundant receptor of the glutamatergic system. In the neurodegenerative events of Alzheimer's disease, excessive

  5. The proteolytic activation of (H3N2) influenza A virus hemagglutinin is facilitated by different type II transmembrane serine proteases

    NARCIS (Netherlands)

    N. Kühn (Nora); S. Bergmann (Silke); N. Kösterke (Nadine); R.L.O. Lambertz (Ruth L.O.); A. Keppner (Anna); J.M.A. van den Brand (Judith); S. Pöhlmann (Stefan); S. Weiß (Siegfried); E. Hummler (Edith); B. Hatesuer (Bastian); K. Schughart (Klaus)

    2016-01-01

    textabstractCleavage of influenza virus hemagglutinin (HA) by host cell proteases is necessary for viral activation and infectivity. In humans and mice, members of the type II transmembrane protease family (TTSP), e.g., TMPRSS2, TMPRSS4, and TMPRSS11d (HAT), have been shown to cleave influenza virus

  6. Inhibition of serine beta-lactamases by vanadate-catechol complexes.

    Science.gov (United States)

    Adediran, S A; Pratt, R F

    2008-09-09

    All three classes of serine beta-lactamases are inhibited at micromolar levels by 1:1 complexes of catechols with vanadate. Vanadate reacts with catechols at submillimolar concentrations in aqueous buffer at neutral pH in several steps, initially forming 1:1, 1:2, and, possibly, 1:3 complexes. Formation of these complexes is followed by the slower reduction of vanadate (V (V)) to vanadyl (V (IV)) and oxidation of the catechol. Vanadyl-catechol complexes, however, do not inhibit the beta-lactamases. Rate and equilibrium constants of formation of the 1:1 and 1:2 complexes of vanadate with catechol itself and with 2,3-dihydroxynaphthalene were measured by stopped-flow spectrophotometry. Typical examples of all three classes of serine beta-lactamases (the class A TEM-2, class C P99, and class D OXA-1 enzymes) were competitively inhibited by the 1:1 vanadate-catechol complexes. The inhibition was modestly enhanced by hydrophobic substituents on the catechol. The 1:1 vanadate complexes are considerably better inhibitors of the P99 beta-lactamase than 1:1 complexes of catechol with boric acid and are likely to contain penta- or hexacoordinated vanadium rather than tetracooordinated. Molecular modeling showed that a pentacoordinated 1:1 vanadate-catechol complex readily fits into the class C beta-lactamase active site with coordination to the nucleophilic serine hydroxyl oxygen. Such complexes may resemble the pentacoordinated transition states of phosphyl transfer, a reaction also catalyzed by beta-lactamases.

  7. Active sites environmental monitoring program FY 1997 annual report

    International Nuclear Information System (INIS)

    Morrissey, C.M.; Marshall, D.S.; Cunningham, G.R.

    1998-03-01

    This report summarizes the activities conducted by the Active Sites Environmental Monitoring Program (ASEMP) from October 1996 through September 1997. The purpose of the program is to provide early detection and performance monitoring at active low-level waste (LLW) disposal sites in Solid Waste Storage Area (SWSA) 6 and transuranic (TRU) waste storage sites in SWSA 5 North. This report continues a series of annual and semiannual reports that present the results of ASEMP monitoring activities. This report details monitoring results for fiscal year (FY) 1997 from SWSA 6, including the Interim Waste Management Facility (IWMF) and the Hillcut Disposal Test Facility (HDTF), and (2) TRU-waste storage areas in SWSA 5 N. This report presents a summary of the methodology used to gather data for each major area along with the FY 1997 results. Figures referenced in the text are found in Appendix A and data tables are presented in Appendix B

  8. D-Serine exposure resulted in gene expression changes implicated in neurodegenerative disorders and neuronal dysfunction in male Fischer 344 rats.

    Science.gov (United States)

    Davidson, Molly E; Kerepesi, Laura A; Soto, Armando; Chan, Victor T

    2009-08-01

    D-Serine, an endogenous amino acid, is involved in many physiological processes through its interaction with the glycine binding site of the N-methyl-D-aspartate (NMDA) receptor. It has important roles in development, learning, and cell death signaling. Recent evidence suggests that decreased function of the NMDA receptor is related to the etiology of schizophrenia, and the use of D-serine as add-on therapy is beneficial in alleviating the symptoms of treatment-refractory schizophrenia. The NMDA receptor also plays a major role in neuronal cell death and neurodegeneration mediated by excitatory amino acid toxicity in ischemia, epilepsy, and trauma. Due to its co-activator function, D-serine can markedly potentiate NMDA-mediated excitotoxicity. To investigate potential adverse effects of D-serine treatment, we investigated gene expression changes in the forebrain of male F-344 rats treated with a single intraperitoneal injection of D-serine (5, 20, 50, 200, or 500 mg/kg) at 96 h post-treatment. Gene expression profiling using Affymetrix Rat Genome 230 2.0 arrays revealed that D-serine treatment resulted in up- and down-regulation of 134 and 52 genes, respectively, based on the common genes identified using three statistical methods, i.e. t test (p Self organized map (SOM) clustering analysis of the differentially expressed genes showed two clusters, one with all 134 up-regulated probe sets and the other with all 52 down-regulated probe sets. The dose-response pattern of the down-regulated cluster showed nearly a perfect mirror image of that of the up-regulated one. Gene ontology analysis revealed that pathways implicated in neuronal functions and/or neurodegenerative disorders are over-represented among the differentially expressed genes. Specifically, genes involved in vesicle-mediated transport, endocytosis, ubiquitin conjugation pathway, regulation of actin filament polymerization/depolymerization, focal adhesion, Wnt signaling, and insulin signaling were up

  9. Downregulation of PERK activity and eIF2α serine 51 phosphorylation by mTOR complex 1 elicits pro-oxidant and pro-death effects in tuberous sclerosis-deficient cells.

    Science.gov (United States)

    Krishnamoorthy, Jothilatha; Tenkerian, Clara; Gupta, Jyotsana; Ghaddar, Nour; Wang, Shuo; Darini, Cedric; Staschke, Kirk A; Ghosh, Abhishek; Gandin, Valentina; Topisirovic, Ivan; Kristof, Arnold S; Hatzoglou, Maria; Simos, George; Koromilas, Antonis E

    2018-02-15

    Oxidative stress determines cell fate through several mechanisms, among which regulation of mRNA translation by the phosphorylation of the alpha (α) subunit of the translation initiation factor eIF2α at serine 51 (eIF2αP) plays a prominent role. Increased eIF2αP can contribute to tumor progression as well as tumor suppression. While eIF2αP is increased in most cells to promote survival and adaptation to different forms of stress, we demonstrate that eIF2αP is reduced in tuberous sclerosis complex 2 (TSC2)-deficient cells subjected to oxidative insults. Decreased eIF2αP in TSC2-deficient cells depends on reactive oxygen species (ROS) production and is associated with a reduced activity of the endoplasmic reticulum (ER)-resident kinase PERK owing to the hyper-activation of the mammalian target of rapamycin complex 1 (mTORC1). Downregulation of PERK activity and eIF2αP is accompanied by increased ROS production and enhanced susceptibility of TSC2-deficient cells to extrinsic pro-oxidant stress. The decreased levels of eIF2αP delay tumor formation of TSC2-deficient cells in immune deficient mice, an effect that is significantly alleviated in mice subjected to an anti-oxidant diet. Our findings reveal a previously unidentified connection between mTORC1 and eIF2αP in TSC2-deficient cells with potential implications in tumor suppression in response to oxidative insults.

  10. Scabies mite inactive serine proteases are potent inhibitors of the human complement lectin pathway.

    Directory of Open Access Journals (Sweden)

    Simone L Reynolds

    2014-05-01

    Full Text Available Scabies is an infectious skin disease caused by the mite Sarcoptes scabiei and has been classified as one of the six most prevalent epidermal parasitic skin diseases infecting populations living in poverty by the World Health Organisation. The role of the complement system, a pivotal component of human innate immunity, as an important defence against invading pathogens has been well documented and many parasites have an arsenal of anti-complement defences. We previously reported on a family of scabies mite proteolytically inactive serine protease paralogues (SMIPP-Ss thought to be implicated in host defence evasion. We have since shown that two family members, SMIPP-S D1 and I1 have the ability to bind the human complement components C1q, mannose binding lectin (MBL and properdin and are capable of inhibiting all three human complement pathways. This investigation focused on inhibition of the lectin pathway of complement activation as it is likely to be the primary pathway affecting scabies mites. Activation of the lectin pathway relies on the activation of MBL, and as SMIPP-S D1 and I1 have previously been shown to bind MBL, the nature of this interaction was examined using binding and mutagenesis studies. SMIPP-S D1 bound MBL in complex with MBL-associated serine proteases (MASPs and released the MASP-2 enzyme from the complex. SMIPP-S I1 was also able to bind MBL in complex with MASPs, but MASP-1 and MASP-2 remained in the complex. Despite these differences in mechanism, both molecules inhibited activation of complement components downstream of MBL. Mutagenesis studies revealed that both SMIPP-Ss used an alternative site of the molecule from the residual active site region to inhibit the lectin pathway. We propose that SMIPP-Ss are potent lectin pathway inhibitors and that this mechanism represents an important tool in the immune evasion repertoire of the parasitic mite and a potential target for therapeutics.

  11. Structural Mechanisms of Inactivation in Scabies Mite Serine Protease Paralogues

    Energy Technology Data Exchange (ETDEWEB)

    Fischer, Katja; Langendorf, Christopher G.; Irving, James A.; Reynolds, Simone; Willis, Charlene; Beckham, Simone; Law, Ruby H.P.; Yang, Sundy; Bashtannyk-Puhalovich, Tanya A.; McGowan, Sheena; Whisstock, James C.; Pike, Robert N.; Kemp, David J.; Buckle, Ashley M.; (Monash); (Queensland Inst. of Med. Rsrch.)

    2009-08-07

    The scabies mite (Sarcoptes scabiei) is a parasite responsible for major morbidity in disadvantaged communities and immuno-compromised patients worldwide. In addition to the physical discomfort caused by the disease, scabies infestations facilitate infection by Streptococcal species via skin lesions, resulting in a high prevalence of rheumatic fever/heart disease in affected communities. The scabies mite produces 33 proteins that are closely related to those in the dust mite group 3 allergen and belong to the S1-like protease family (chymotrypsin-like). However, all but one of these molecules contain mutations in the conserved active-site catalytic triad that are predicted to render them catalytically inactive. These molecules are thus termed scabies mite inactivated protease paralogues (SMIPPs). The precise function of SMIPPs is unclear; however, it has been suggested that these proteins might function by binding and protecting target substrates from cleavage by host immune proteases, thus preventing the host from mounting an effective immune challenge. In order to begin to understand the structural basis for SMIPP function, we solved the crystal structures of SMIPP-S-I1 and SMIPP-S-D1 at 1.85 {angstrom} and 2.0 {angstrom} resolution, respectively. Both structures adopt the characteristic serine protease fold, albeit with large structural variations over much of the molecule. In both structures, mutations in the catalytic triad together with occlusion of the S1 subsite by a conserved Tyr200 residue is predicted to block substrate ingress. Accordingly, we show that both proteases lack catalytic function. Attempts to restore function (via site-directed mutagenesis of catalytic residues as well as Tyr200) were unsuccessful. Taken together, these data suggest that SMIPPs have lost the ability to bind substrates in a classical 'canonical' fashion, and instead have evolved alternative functions in the lifecycle of the scabies mite.

  12. Phospholipid metabolism of serine in Plasmodium-infected erythrocytes involves phosphatidylserine and direct serine decarboxylation.

    Science.gov (United States)

    Elabbadi, N; Ancelin, M L; Vial, H J

    1997-01-01

    Erythrocytes infected with Plasmodium falciparum or Plasmodium knowlesi efficiently incorporated radioactive serine into phosphatidylserine (PtdSer), phosphatidylethanolamine (PtdEtn) and phosphatidylcholine (PtdCho). Serine was also metabolized into ethanolamine (Etn) and phosphorylethanolamine (P-Etn) via direct serine decarboxylation; this is a major phenomenon since together these metabolites represent 60% of total radioactive water-soluble metabolites. They were identified by reverse-phase HPLC and two TLC-type analyses and confirmed by alkaline phosphatase treatment, which depleted the radioactive P-Etn peak completely with a concomitant increase in that of Etn. In the presence of 5 microM labelled serine, radioactivity appeared in Etn and P-Etn after a 25 min lag period, and isotopic equilibrium was reached at 40 and 95 min respectively. There was a similar lag period for PtdEtn formation, which accumulated steadily for at least 180 min. Incorporation of serine into phospholipids and water-soluble metabolites increased in the presence of up to 500 microM external serine. An apparent plateau was then reached for all metabolites except intracellular serine and Etn. Exogenous Etn (at 20 microM) induced a concomitant dramatic decrease in serine incorporation into P-Etn and all phospholipids, but not into Etn. Increasing exogenous serine to 100 microM decreased the incorporation of radioactive Etn into PtdEtn by only 30%, and the PtdCho level was not affected. 2-Hydroxyethylhydrazine significantly decreased serine incorporation into P-Etn and PtdEtn, whereas Etn was accumulated. No concomitant inhibition of PtdSer or PtdCho labelling from serine occurred, even when PtdEtn formation was decreased by 95%. This indicates that the PtdEtn pool derived from direct serine decarboxylation differed from that derived from PtdSer decarboxylation, and the latter appeared to be preferentially used for PtdCho biosynthesis. Hydroxylamine also inhibited phosphorylation of serine

  13. D-serine increases adult hippocampal neurogenesis

    Directory of Open Access Journals (Sweden)

    Sebastien eSultan

    2013-08-01

    Full Text Available Adult hippocampal neurogenesis results in the continuous formation of new neurons and is a process of brain plasticity involved in learning and memory. The neurogenic niche regulates the stem cell proliferation and the differentiation and survival of new neurons and a major contributor to the neurogenic niche are astrocytes. Among the molecules secreted by astrocytes, D-serine is an important gliotransmitter and is a co-agonist of the glutamate, N-methyl-D-aspartate (NMDA receptor. D-serine has been shown to enhance the proliferation of neural stem cells in vitro, but its effect on adult neurogenesis in vivo is unknown. Here, we tested the effect of exogenous administration of D-serine on adult neurogenesis in the mouse dentate gyrus. We found that 1 week of treatment with D-serine increased cell proliferation in vivo and in vitro and increased the density of neural stem cells and transit amplifying progenitors. Furthermore, D-serine increased the survival of newborn neurons. Together, these results indicate that D-serine treatment resulted in the improvement of several steps of adult neurogenesis in vivo.

  14. Cloning and chromosomal assignment of a human cDNA encoding a T cell- and natural killer cell-specific trypsin-like serine protease

    International Nuclear Information System (INIS)

    Gershenfeld, H.K.; Hershberger, R.J.; Shows, T.B.; Weissman, I.L.

    1988-01-01

    A cDNA clone encoding a human T cell- and natural killer cell-specific serine protease was obtained by screening a phage λgt10 cDNA library from phytohemagglutinin-stimulated human peripheral blood lymphocytes with the mouse Hanukah factor cDNA clone. In an RNA blot-hybridization analysis, this human Hanukah factor cDNA hybridized with a 1.3-kilobase band in allogeneic-stimulated cytotoxic T cells and the Jurkat cell line, but this transcript was not detectable in normal muscle, liver, tonsil, or thymus. By dot-blot hybridization, this cDNA hybridized with RNA from three cytolytic T-cell clones and three noncytolytic T-cell clones grown in vitro as well as with purified CD16 + natural killer cells and CD3 + , CD16 - T-cell large granular lymphocytes from peripheral blood lymphocytes (CD = cluster designation). The nucleotide sequence of this cDNA clone encodes a predicted serine protease of 262 amino acids. The active enzyme is 71% and 77% similar to the mouse sequence at the amino acid and DNA level, respectively. The human and mouse sequences conserve the active site residues of serine proteases--the trypsin-specific Asp-189 and all 10 cysteine residues. The gene for the human Hanukah factor serine protease is located on human chromosome 5. The authors propose that this trypsin-like serine protease may function as a common component necessary for lysis of target cells by cytotoxic T lymphocytes and natural killer cells

  15. Active chemisorption sites in functionalized ionic liquids for carbon capture.

    Science.gov (United States)

    Cui, Guokai; Wang, Jianji; Zhang, Suojiang

    2016-07-25

    Development of novel technologies for the efficient and reversible capture of CO2 is highly desired. In the last decade, CO2 capture using ionic liquids has attracted intensive attention from both academia and industry, and has been recognized as a very promising technology. Recently, a new approach has been developed for highly efficient capture of CO2 by site-containing ionic liquids through chemical interaction. This perspective review focuses on the recent advances in the chemical absorption of CO2 using site-containing ionic liquids, such as amino-based ionic liquids, azolate ionic liquids, phenolate ionic liquids, dual-functionalized ionic liquids, pyridine-containing ionic liquids and so on. Other site-containing liquid absorbents such as amine-based solutions, switchable solvents, and functionalized ionic liquid-amine blends are also investigated. Strategies have been discussed for how to activate the existent reactive sites and develop novel reactive sites by physical and chemical methods to enhance CO2 absorption capacity and reduce absorption enthalpy. The carbon capture mechanisms of these site-containing liquid absorbents are also presented. Particular attention has been paid to the latest progress in CO2 capture in multiple-site interactions by amino-free anion-functionalized ionic liquids. In the last section, future directions and prospects for carbon capture by site-containing ionic liquids are outlined.

  16. Active Sites Environmental Monitoring Program: Mid-FY 1991 report

    Energy Technology Data Exchange (ETDEWEB)

    Ashwood, T.L.; Wickliff, D.S.; Morrissey, C.M.

    1991-10-01

    This report summarizes the activities of the Active Sites Environmental Monitoring Program (ASEMP) from October 1990 through March 1991. The ASEMP was established in 1989 by Solid Waste Operations and the Environmental Sciences Division to provide early detection and performance monitoring at active low-level radioactive waste (LLW) disposal sites in Solid Waste Storage Area (SWSA) 6 and transuranic (TRU) waste storage sites in SWSA 5 as required by chapters II and III of US Department of Energy Order 5820.2A. Monitoring results continue to demonstrate the no LLW is being leached from the storage vaults on the tumulus pads. Loading of vaults on Tumulus II began during this reporting period and 115 vaults had been loaded by the end of March 1991.

  17. Heterologous activation of protein kinase C stimulates phosphorylation of delta-opioid receptor at serine 344, resulting in beta-arrestin- and clathrin-mediated receptor internalization

    DEFF Research Database (Denmark)

    Xiang, B; Yu, G H; Guo, J

    2001-01-01

    The purpose of the current study is to investigate the effect of opioid-independent, heterologous activation of protein kinase C (PKC) on the responsiveness of opioid receptor and the underlying molecular mechanisms. Our result showed that removing the C terminus of delta opioid receptor (DOR......) containing six Ser/Thr residues abolished both DPDPE- and phorbol 12-myristate 13-acetate (PMA)-induced DOR phosphorylation. The phosphorylation levels of DOR mutants T352A, T353A, and T358A/T361A/S363S were comparable to that of the wild-type DOR, whereas S344G substitution blocked PMA-induced receptor......, and ionomycin resulted in DOR internalization that required phosphorylation of Ser-344. Expression of dominant negative beta-arrestin and hypertonic sucrose treatment blocked PMA-induced DOR internalization, suggesting that PKC mediates DOR internalization via a beta-arrestin- and clathrin-dependent mechanism...

  18. Oxygen reduction and evolution at single-metal active sites

    DEFF Research Database (Denmark)

    Calle-Vallejo, F.; Martínez, J.I.; García Lastra, Juan Maria

    2013-01-01

    overpotentials and is made of precious materials. A possible solution is the use of non-noble electrocatalysts with single-metal active sites. Here, on the basis of DFT calculations of adsorbed intermediates and a thermodynamic analysis, we compare the oxygen reduction (ORR) and evolution (OER) activities...... of functionalized graphitic materials and gas-phase porphyrins with late transition metals. We find that both kinds of materials follow approximately the same activity trends, and active sites with transition metals from groups 7 to 9 may be good ORR and OER electrocatalysts. However, spin analyses show more...... flexibility in the possible oxidation states of the metal atoms in solid electrocatalysts, while in porphyrins they must be +2. These observations reveal that the catalytic activity of these materials is mainly due to nearest-neighbor interactions. Based on this, we propose that this class of electrocatalysts...

  19. Stepwise Versus Concerted Mechanisms in General-Base Catalysis by Serine Proteases.

    Science.gov (United States)

    Uritsky, Neta; Shokhen, Michael; Albeck, Amnon

    2016-01-26

    General-base catalysis in serine proteases still poses mechanistic challenges despite decades of research. Whether proton transfer from the catalytic Ser to His and nucleophilic attack on the substrate are concerted or stepwise is still under debate, even for the classical Asp-His-Ser catalytic triad. To address these key catalytic steps, the transformation of the Michaelis complex to tetrahedral complex in the covalent inhibition of two prototype serine proteases was studied: chymotrypsin (with the catalytic triad) inhibition by a peptidyl trifluoromethane and GlpG rhomboid (with Ser-His dyad) inhibition by an isocoumarin derivative. The sampled MD trajectories of averaged pKa  values of catalytic residues were QM calculated by the MD-QM/SCRF(VS) method on molecular clusters simulating the active site. Differences between concerted and stepwise mechanisms are controlled by the dynamically changing pKa  values of the catalytic residues as a function of their progressively reduced water exposure, caused by the incoming ligand. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  20. Synthesis of Isomeric Phosphoubiquitin Chains Reveals that Phosphorylation Controls Deubiquitinase Activity and Specificity

    Directory of Open Access Journals (Sweden)

    Nicolas Huguenin-Dezot

    2016-07-01

    Full Text Available Ubiquitin is post-translationally modified by phosphorylation at several sites, but the consequences of these modifications are largely unknown. Here, we synthesize multi-milligram quantities of ubiquitin phosphorylated at serine 20, serine 57, and serine 65 via genetic code expansion. We use these phosphoubiquitins for the enzymatic assembly of 20 isomeric phosphoubiquitin dimers, with different sites of isopeptide linkage and/or phosphorylation. We discover that phosphorylation of serine 20 on ubiquitin converts UBE3C from a dual-specificity E3 ligase into a ligase that primarily synthesizes K48 chains. We profile the activity of 31 deubiquitinases on the isomeric phosphoubiquitin dimers in 837 reactions, and we discover that phosphorylation at distinct sites in ubiquitin can activate or repress cleavage of a particular linkage by deubiquitinases and that phosphorylation at a single site in ubiquitin can control the specificity of deubiquitinases for distinct ubiquitin linkages.

  1. Genetically modified microorganisms having improved tolerance towards l-serine

    DEFF Research Database (Denmark)

    2016-01-01

    The present invention generally relates to the microbiological industry, and specifically to the production of L-serine or L-serine derivatives using genetically modified bacteria. The present invention provides genetically modified microorganisms, such as bacteria, wherein the expression of genes...... tolerant towards higher concentrations of serine. The present invention also provides methods for the production of L-serine or L-serine derivative using such genetically modified microorganisms....

  2. Structural and Functional Adaptation of Vancomycin Resistance VanT Serine Racemases.

    Science.gov (United States)

    Meziane-Cherif, Djalal; Stogios, Peter J; Evdokimova, Elena; Egorova, Olga; Savchenko, Alexei; Courvalin, Patrice

    2015-08-11

    Vancomycin resistance in Gram-positive bacteria results from the replacement of the D-alanyl-D-alanine target of peptidoglycan precursors with D-alanyl-D-lactate or D-alanyl-D-serine (D-Ala-D-Ser), to which vancomycin has low binding affinity. VanT is one of the proteins required for the production of D-Ala-D-Ser-terminating precursors by converting L-Ser to D-Ser. VanT is composed of two domains, an N-terminal membrane-bound domain, likely involved in L-Ser uptake, and a C-terminal cytoplasmic catalytic domain which is related to bacterial alanine racemases. To gain insight into the molecular function of VanT, the crystal structure of the catalytic domain of VanTG from VanG-type resistant Enterococcus faecalis BM4518 was determined. The structure showed significant similarity to type III pyridoxal 5'-phosphate (PLP)-dependent alanine racemases, which are essential for peptidoglycan synthesis. Comparative structural analysis between VanTG and alanine racemases as well as site-directed mutagenesis identified three specific active site positions centered around Asn696 which are responsible for the L-amino acid specificity. This analysis also suggested that VanT racemases evolved from regular alanine racemases by acquiring additional selectivity toward serine while preserving that for alanine. The 4-fold-lower relative catalytic efficiency of VanTG against L-Ser versus L-Ala implied that this enzyme relies on its membrane-bound domain for L-Ser transport to increase the overall rate of d-Ser production. These findings illustrate how vancomycin pressure selected for molecular adaptation of a housekeeping enzyme to a bifunctional enzyme to allow for peptidoglycan remodeling, a strategy increasingly observed in antibiotic-resistant bacteria. Vancomycin is one of the drugs of last resort against Gram-positive antibiotic-resistant pathogens. However, bacteria have evolved a sophisticated mechanism which remodels the drug target, the D-alanine ending precursors in cell wall

  3. Tryptophanase-Catalyzed L-Tryptophan Synthesis from D-Serine in the Presence of Diammonium Hydrogen Phosphate

    Directory of Open Access Journals (Sweden)

    Fujii Noriko

    2009-06-01

    Full Text Available Tryptophanase, an enzyme with extreme absolute stereospecificity for optically active stereoisomers, catalyzes the synthesis of L-tryptophan from L-serine and indole through a β-substitution mechanism of the ping-pong type, and has no activity on D-serine. We previously reported that tryptophanase changed its stereospecificity to degrade D-tryptophan in highly concentrated diammonium hydrogen phosphate, (NH42HPO4 solution. The present study provided the same stereospecific change seen in the D-tryptophan degradation reaction also occurs in tryptophan synthesis from D-serine. Tryptophanase became active to D-serine to synthesize L-tryptophan in the presence of diammonium hydrogen phosphate. This reaction has never been reported before. D-serine seems to undergo β-replacement via an enzyme-bonded α-aminoacylate intermediate to yield L-tryptophan.

  4. Phosphorylation of connexin43 on serine 306 regulates electrical coupling

    DEFF Research Database (Denmark)

    Procida, Kristina; Jørgensen, Lone; Schmitt, Nicole

    2009-01-01

    BACKGROUND: Phosphorylation is a key regulatory event in controlling the function of the cardiac gap junction protein connexin43 (Cx43). Three new phosphorylation sites (S296, S297, S306) have been identified on Cx43; two of these sites (S297 and S306) are dephosphorylated during ischemia....... The functional significance of these new sites is currently unknown. OBJECTIVE: The purpose of this study was to examine the role of S296, S297, and S306 in the regulation of electrical intercellular communication. METHODS: To mimic constitutive dephosphorylation, serine was mutated to alanine at the three sites...... and expressed in HeLa cells. Electrical coupling and single channel measurements were performed by double patch clamp. Protein expression levels were assayed by western blotting, localization of Cx43, and phosphorylation of S306 by immunolabeling. Free hemichannels were assessed by biotinylation. RESULTS...

  5. HIV-mediated phosphatidylinositol 3-kinase/serine-threonine kinase activation in APCs leads to programmed death-1 ligand upregulation and suppression of HIV-specific CD8 T cells.

    Science.gov (United States)

    Muthumani, Karuppiah; Shedlock, Devon J; Choo, Daniel K; Fagone, Paolo; Kawalekar, Omkar U; Goodman, Jonathan; Bian, Chaoran B; Ramanathan, Aarti A; Atman, Parikh; Tebas, Pablo; Chattergoon, Michael A; Choo, Andrew Y; Weiner, David B

    2011-09-15

    Recent evidence demonstrates that HIV-1 infection leads to the attenuation of cellular immune responses, which has been correlated with the increased expression of programmed death (PD)-1 on virus-specific CD8(+) T cells. PD-1 is induced upon T cell activation, and its prolonged expression facilitates CD8(+) T cell inhibitory signals when bound to its B7 family ligands, PD-ligand (L)1/2, which are expressed on APCs. Importantly, early reports demonstrated that blockade of the PD-1/PD-L interaction by Abs may help to counter the development of immune exhaustion driven by HIV viral persistence. To better understand the regulation of the PD-1 pathway during HIV infection, we examined the ability of the virus to induce PD-L expression on macrophages and dendritic cells. We found a direct relationship between the infection of APCs and the expression of PD-L1 in which virus-mediated upregulation induced a state of nonresponsiveness in uninfected HIV-specific T cells. Furthermore, this exhaustion phenotype was revitalized by the blockade of PD-L1, after which T cells regained their capacity for proliferation and the secretion of proinflammatory cytokines IFN-γ, IL-2, and IL-12 upon restimulation. In addition, we identify a critical role for the PI3K/serine-threonine kinase signaling pathway in PD-L1 upregulation of APCs by HIV, because inhibition of these intracellular signal transducer enzymes significantly reduced PD-L1 induction by infection. These data identify a novel mechanism by which HIV exploits the immunosuppressive PD-1 pathway and suggest a new role for virus-infected cells in the local corruption of immune responses required for viral suppression.

  6. Contribution of cutinase serine 42 side chain to the stabilization of the oxyanion transition state.

    Science.gov (United States)

    Nicolas, A; Egmond, M; Verrips, C T; de Vlieg, J; Longhi, S; Cambillau, C; Martinez, C

    1996-01-16

    Cutinase from the fungus Fusarium solani pisi is a lipolytic enzyme able to hydrolyze both aggregated and soluble substrates. It therefore provides a powerful tool for probing the mechanisms underlying lipid hydrolysis. Lipolytic enzymes have a catalytic machinery similar to those present in serine proteinases. It is characterized by the triad Ser, His, and Asp (Glu) residues, by an oxyanion binding site that stabilizes the transition state via hydrogen bonds with two main chain amide groups, and possibly by other determinants. It has been suggested on the basis of a covalently bond inhibitor that the cutinase oxyanion hole may consist not only of two main chain amide groups but also of the Ser42 O gamma side chain. Among the esterases and the serine and the cysteine proteases, only Streptomyces scabies esterase, subtilisin, and papain, respectively, have a side chain residue which is involved in the oxyanion hole formation. The position of the cutinase Ser42 side chain is structurally conserved in Rhizomucor miehei lipase with Ser82 O gamma, in Rhizopus delemar lipase with Thr83 O gamma 1, and in Candida antartica B lipase with Thr40 O gamma 1. To evaluate the increase in the tetrahedral intermediate stability provided by Ser42 O gamma, we mutated Ser42 into Ala. Furthermore, since the proper orientation of Ser42 O gamma is directed by Asn84, we mutated Asn84 into Ala, Leu, Asp, and Trp, respectively, to investigate the contribution of this indirect interaction to the stabilization of the oxyanion hole. The S42A mutation resulted in a drastic decrease in the activity (450-fold) without significantly perturbing the three-dimensional structure. The N84A and N84L mutations had milder kinetic effects and did not disrupt the structure of the active site, whereas the N84W and N84D mutations abolished the enzymatic activity due to drastic steric and electrostatic effects, respectively.

  7. Active Sites Environmental Monitoring Program: FY 1990 annual report

    International Nuclear Information System (INIS)

    Wickliff, D.S.; Morrissey, C.M.; Ashwood, T.L.

    1991-10-01

    Chapter 3 of US Department of Energy (DOE) Order 5820.2A (DOE 1988) sets forth requirements for environmental monitoring of active low-level waste (LLW) disposal sites. Active sites are defined as those LLW facilities that were in use on or after the date of the order (September 1988). The transuranic (TRU) waste storage areas in Solid Waste Storage Area (SWSA) 5 North are covered by Chap. 2 of the order. In both chapters, monitoring is required to provide for early warning of leaks before those leaks pose a threat to human health or the environment. Chapter 3 also requires that monitoring be conducted to evaluate the short- and long-term performance of LLW disposal facilities. In accordance with this order, the Solid Waste Operations Department at Oak Ridge National Laboratory (ORNL) has established an Active Sites Environmental Monitoring Program (ASEMP) that is implemented by staff of the Environmental Sciences Division (ESD) at ORNL. This report summarizes data from ASEMP monitoring activities for the final 6 months of FY 1990. A brief summary of the monitoring methodology for each site is presented also

  8. Active site modeling in copper azurin molecular dynamics simulations

    NARCIS (Netherlands)

    Rizzuti, B; Swart, M; Sportelli, L; Guzzi, R

    Active site modeling in molecular dynamics simulations is investigated for the reduced state of copper azurin. Five simulation runs (5 ns each) were performed at room temperature to study the consequences of a mixed electrostatic/constrained modeling for the coordination between the metal and the

  9. Energy transfer at the active sites of heme proteins

    International Nuclear Information System (INIS)

    Dlott, D.D.; Hill, J.R.

    1995-01-01

    Experiments using a picosecond pump-probe apparatus at the Picosecond Free-electron Laser Center at Stanford University, were performed to investigate the relaxation of carbon monoxide bound to the active sites of heme proteins. The significance of these experiments is two-fold: (1) they provide detailed information about molecular dynamics occurring at the active sites of proteins; and (2) they provide insight into the nature of vibrational relaxation processes in condensed matter. Molecular engineering is used to construct various molecular systems which are studied with the FEL. We have studied native proteins, mainly myoglobin obtained from different species, mutant proteins produced by genetic engineering using recombinant DNA techniques, and a variety of model systems which mimic the structures of the active sites of native proteins, which are produced using molecular synthesis. Use of these different systems permits us to investigate how specific molecular structural changes affect dynamical processes occurring at the active sites. This research provides insight into the problems of how different species needs are fulfilled by heme proteins which have greatly different functionality, which is induced by rather small structural changes

  10. Modelling the active site properties of dopamine b-hydroxylase

    Indian Academy of Sciences (India)

    Administrator

    Dopamine b-hydroxylase (DbH) is a copper-containing glycoprotein that hydroxylates dopamine to norepinephrine 1,2. Based on spectroscopic studies the active site of the metalloenzyme is proposed to have two copper centres. The enzyme in the oxidized dicopper(II) form gets reduced to the dicopper(I) unit by ascorbate ...

  11. The nature of the active site in heterogeneous metal catalysis

    DEFF Research Database (Denmark)

    Nørskov, Jens Kehlet; Bligaard, Thomas; Larsen, Britt Hvolbæk

    2008-01-01

    This tutorial review, of relevance for the surface science and heterogeneous catalysis communities, provides a molecular-level discussion of the nature of the active sites in metal catalysis. Fundamental concepts such as "Bronsted-Evans-Polanyi relations'' and "volcano curves'' are introduced...

  12. Leachate characterization of active and closed dump sites in Port ...

    African Journals Online (AJOL)

    This study characterizes the leachate quality of both active and closed dump sites in Port Harcourt City. Leachates were sampled from the base of the dum psites and analysed, pH, dissolved oxygen (DO), electrical conductivity and total dissolved solids were determined on the samples in-situ. While chloride, sulphate ...

  13. Functional and catalytic active sites prediction and docking analysis ...

    African Journals Online (AJOL)

    Bioinformatics

    2015-07-01

    Jul 1, 2015 ... African Journal of Biotechnology. Full Length Research Paper. Functional and catalytic active sites prediction and docking analysis of azoreductase enzyme in. Pseudomonas putida with a variety of commercially available azodyes. Bikash Thakuria, Chandra J Singha, Premchand Maisnam and Samrat ...

  14. Discovery of an Unexplored Protein Structural Scaffold of Serine Protease from Big Blue Octopus (Octopus cyanea): A New Prospective Lead Molecule.

    Science.gov (United States)

    Panda, Subhamay; Kumari, Leena

    2017-01-01

    Serine proteases are a group of enzymes that hydrolyses the peptide bonds in proteins. In mammals, these enzymes help in the regulation of several major physiological functions such as digestion, blood clotting, responses of immune system, reproductive functions and the complement system. Serine proteases obtained from the venom of Octopodidae family is a relatively unexplored area of research. In the present work, we tried to effectively utilize comparative composite molecular modeling technique. Our key aim was to propose the first molecular model structure of unexplored serine protease 5 derived from big blue octopus. The other objective of this study was to analyze the distribution of negatively and positively charged amino acid over molecular modeled structure, distribution of secondary structural elements, hydrophobicity molecular surface analysis and electrostatic potential analysis with the aid of different bioinformatic tools. In the present study, molecular model has been generated with the help of I-TASSER suite. Afterwards the refined structural model was validated with standard methods. For functional annotation of protein molecule we used Protein Information Resource (PIR) database. Serine protease 5 of big blue octopus was analyzed with different bioinformatical algorithms for the distribution of negatively and positively charged amino acid over molecular modeled structure, distribution of secondary structural elements, hydrophobicity molecular surface analysis and electrostatic potential analysis. The functionally critical amino acids and ligand- binding site (LBS) of the proteins (modeled) were determined using the COACH program. The molecular model data in cooperation to other pertinent post model analysis data put forward molecular insight to proteolytic activity of serine protease 5, which helps in the clear understanding of procoagulant and anticoagulant characteristics of this natural lead molecule. Our approach was to investigate the octopus

  15. Direct instrumental identification of catalytically active surface sites

    Science.gov (United States)

    Pfisterer, Jonas H. K.; Liang, Yunchang; Schneider, Oliver; Bandarenka, Aliaksandr S.

    2017-09-01

    The activity of heterogeneous catalysts—which are involved in some 80 per cent of processes in the chemical and energy industries—is determined by the electronic structure of specific surface sites that offer optimal binding of reaction intermediates. Directly identifying and monitoring these sites during a reaction should therefore provide insight that might aid the targeted development of heterogeneous catalysts and electrocatalysts (those that participate in electrochemical reactions) for practical applications. The invention of the scanning tunnelling microscope (STM) and the electrochemical STM promised to deliver such imaging capabilities, and both have indeed contributed greatly to our atomistic understanding of heterogeneous catalysis. But although the STM has been used to probe and initiate surface reactions, and has even enabled local measurements of reactivity in some systems, it is not generally thought to be suited to the direct identification of catalytically active surface sites under reaction conditions. Here we demonstrate, however, that common STMs can readily map the catalytic activity of surfaces with high spatial resolution: we show that by monitoring relative changes in the tunnelling current noise, active sites can be distinguished in an almost quantitative fashion according to their ability to catalyse the hydrogen-evolution reaction or the oxygen-reduction reaction. These data allow us to evaluate directly the importance and relative contribution to overall catalyst activity of different defects and sites at the boundaries between two materials. With its ability to deliver such information and its ready applicability to different systems, we anticipate that our method will aid the rational design of heterogeneous catalysts.

  16. Pnserpin: A Novel Serine Protease Inhibitor from Extremophile Pyrobaculum neutrophilum

    Directory of Open Access Journals (Sweden)

    Huan Zhang

    2017-01-01

    Full Text Available Serine protease inhibitors (serpins are native inhibitors of serine proteases, constituting a large protein family with members spread over eukaryotes and prokaryotes. However, only very few prokaryotic serpins, especially from extremophiles, have been characterized to date. In this study, Pnserpin, a putative serine protease inhibitor from the thermophile Pyrobaculum neutrophilum, was overexpressed in Escherichia coli for purification and characterization. It irreversibly inhibits chymotrypsin-, trypsin-, elastase-, and subtilisin-like proteases in a temperature range from 20 to 100 °C in a concentration-dependent manner. The stoichiometry of inhibition (SI of Pnserpin for proteases decreases as the temperature increases, indicating that the inhibitory activity of Pnserpin increases with the temperature. SDS-PAGE (sodium dodecyl sulfate polyacrylamide gel electrophoresis showed that Pnserpin inhibits proteases by forming a SDS-resistant covalent complex. Homology modeling and molecular dynamic simulations predicted that Pnserpin can form a stable common serpin fold. Results of the present work will help in understanding the structural and functional characteristics of thermophilic serpin and will broaden the current knowledge about serpins from extremophiles.

  17. Pnserpin: A Novel Serine Protease Inhibitor from Extremophile Pyrobaculum neutrophilum.

    Science.gov (United States)

    Zhang, Huan; Fei, Rui; Xue, Baigong; Yu, Shanshan; Zhang, Zuoming; Zhong, Sheng; Gao, Yuanqi; Zhou, Xiaoli

    2017-01-07

    Serine protease inhibitors (serpins) are native inhibitors of serine proteases, constituting a large protein family with members spread over eukaryotes and prokaryotes. However, only very few prokaryotic serpins, especially from extremophiles, have been characterized to date. In this study, Pnserpin, a putative serine protease inhibitor from the thermophile Pyrobaculum neutrophilum , was overexpressed in Escherichia coli for purification and characterization. It irreversibly inhibits chymotrypsin-, trypsin-, elastase-, and subtilisin-like proteases in a temperature range from 20 to 100 °C in a concentration-dependent manner. The stoichiometry of inhibition (SI) of Pnserpin for proteases decreases as the temperature increases, indicating that the inhibitory activity of Pnserpin increases with the temperature. SDS-PAGE (sodium dodecyl sulfate polyacrylamide gel electrophoresis) showed that Pnserpin inhibits proteases by forming a SDS-resistant covalent complex. Homology modeling and molecular dynamic simulations predicted that Pnserpin can form a stable common serpin fold. Results of the present work will help in understanding the structural and functional characteristics of thermophilic serpin and will broaden the current knowledge about serpins from extremophiles.

  18. The charge density distribution in a model compound of the catalytic triad in serine proteases.

    Science.gov (United States)

    Overgaard, J; Schiøtt, B; Larsen, F K; Iversen, B B

    2001-09-03

    Combined low temperature (28(1) K) X-ray and neutron diffraction measurements were carried out on the co-crystallised complex of betaine, imidazole, and picric acid (1). The experimental charge density was determined and compared with ab initio theoretical calculations at the B3LYP/6-311G(d,p) level of theory. The complex serves as a model for the active site in, for example, the serine protease class of enzymes, the so-called catalytic triad. The crystal contains three short strong N-H...O hydrogen bonds (HBs) with dN...O comparison with low-barrier and single-well hydrogen bonding systems (e.g., benzoylacetone and nitromalonamide) shows that the low-barrier hydrogen bond (LBHB) state is characterized by an enormously increased hydrogen atom source contribution to the bond critical point in the HB. In this context, HB2 can be characterized as intermediate between localized HBs and delocalized LBHBs.

  19. Serine protease inhibitors to treat inflammation: a patent review (2011-2016).

    Science.gov (United States)

    Soualmia, Feryel; El Amri, Chahrazade

    2018-02-01

    Inflammation is a physiological part of the complex biological response of tissues to counteract various harmful signals. This process involves diverse actors such as immune cells, blood vessels, and nerves as sources of mediators for inflammation control. Among them serine proteases are key elements in both physiological and pathological inflammation. Areas covered: Serine protease inhibitors to treat inflammatory diseases are being actively investigated by various industrial and academic institutions. The present review covers patent literature on serine protease inhibitors for the therapy of inflammatory diseases patented between 2011 and 2016. Expert opinion: Serine proteases regulating inflammation are versatile enzymes, usually involved in proinflammatory cytokine production and activation of immune cells. Their dysregulation during inflammation can have devastating consequences, promoting various diseases including skin and lung inflammation, neuroinflammation, and inflammatory arthritis. Several serine proteases were selected for their contribution to inflammatory diseases and significant efforts that are spread to develop inhibitors. Strategies developed for inhibitor identification consist on either peptide-based inhibitor derived from endogenous protein inhibitors or small-organic molecules. It is also worth noting that among the recent patents on serine protease inhibitors related to inflammation a significant number are related to retinal vascular dysfunction and skin diseases.

  20. Crystal structure and characterization of a novel L-serine ammonia-lyase from Rhizomucor miehei

    Energy Technology Data Exchange (ETDEWEB)

    Qin, Zhen [College of Food Science and Nutritional Engineering, Beijing Advanced Innovation Center of Food Nutrition and Human Health, China Agricultural University, Beijing 100083 (China); Yan, Qiaojuan [College of Engineering, China Agricultural University, Beijing 100083 (China); Ma, Qingjun [Key Laboratory of Experimental Marine Biology, Institute of Oceanology, Chinese Academy of Sciences, Qingdao 266071 (China); Jiang, Zhengqiang, E-mail: zhqjiang@cau.edu.cn [College of Food Science and Nutritional Engineering, Beijing Advanced Innovation Center of Food Nutrition and Human Health, China Agricultural University, Beijing 100083 (China)

    2015-10-23

    L-serine ammonia-lyase, as a member of the β-family of pyridoxal-5′-phosphate (PLP) dependent enzymes, catalyzes the conversion of L-serine (L-threonine) to pyruvate (α-ketobutyrate) and ammonia. The crystal structure of L-serine ammonia-lyase from Rhizomucor miehei (RmSDH) was solved at 1.76 Å resolution by X-ray diffraction method. The overall structure of RmSDH had the characteristic β-family PLP dependent enzyme fold. It consisted of two distinct domains, both of which show the typical open twisted α/β structure. A PLP cofactor was located in the crevice between the two domains, which was attached to Lys52 by a Schiff-base linkage. Unique residue substitutions (Gly78, Pro79, Ser146, Ser147 and Thr312) were discovered at the catalytic site of RmSDH by comparison of structures of RmSDH and other reported eukaryotic L-serine ammonia-lyases. Optimal pH and temperature of the purified RmSDH were 7.5 and 40 °C, respectively. It was stable in the pH range of 7.0–9.0 and at temperatures below 40 °C. This is the first crystal structure of a fungal L-serine ammonia-lyase. It will be useful to study the catalytic mechanism of β-elimination enzymes and will provide a basis for further enzyme engineering. - Highlights: • The crystal structure of a fungal L-serine ammonia-lyase (RmSDH) was solved. • Five unique residue substitutions are found at the catalytic site of RmSDH. • RmSDH was expressed in Pichia. pastoris and biochemically characterized. • RmSDH has potential application in splitting D/L-serine.

  1. Mapping the active site of vaccinia virus RNA triphosphatase

    International Nuclear Information System (INIS)

    Gong Chunling; Shuman, Stewart

    2003-01-01

    The RNA triphosphatase component of vaccinia virus mRNA capping enzyme (the product of the viral D1 gene) belongs to a family of metal-dependent phosphohydrolases that includes the RNA triphosphatases of fungi, protozoa, Chlorella virus, and baculoviruses. The family is defined by two glutamate-containing motifs (A and C) that form the metal-binding site. Most of the family members resemble the fungal and Chlorella virus enzymes, which have a complex active site located within the hydrophilic interior of a topologically closed eight-stranded β barrel (the so-called ''triphosphate tunnel''). Here we queried whether vaccinia virus capping enzyme is a member of the tunnel subfamily, via mutational mapping of amino acids required for vaccinia triphosphatase activity. We identified four new essential side chains in vaccinia D1 via alanine scanning and illuminated structure-activity relationships by conservative substitutions. Our results, together with previous mutational data, highlight a constellation of six acidic and three basic amino acids that likely compose the vaccinia triphosphatase active site (Glu37, Glu39, Arg77, Lys107, Glu126, Asp159, Lys161, Glu192, and Glu194). These nine essential residues are conserved in all vertebrate and invertebrate poxvirus RNA capping enzymes. We discerned no pattern of clustering of the catalytic residues of the poxvirus triphosphatase that would suggest structural similarity to the tunnel proteins (exclusive of motifs A and C). We infer that the poxvirus triphosphatases are a distinct lineage within the metal-dependent RNA triphosphatase family. Their unique active site, which is completely different from that of the host cell's capping enzyme, recommends the poxvirus RNA triphosphatase as a molecular target for antipoxviral drug discovery

  2. Increased tolerance towards serine obtained by adaptive laboratory evolution

    DEFF Research Database (Denmark)

    Mundhada, Hemanshu; Seoane, Jose Miguel; Koza, Anna

    2014-01-01

    The amino acid serine has previously been identified as one of the top 30 candidates of value added chemicals, making the production of serine from glucose attractive. Production of serine have previously been attempted in E. coli and C. glutamicum, however, titers sufficient for commercial...... by glyA), the conversion of serine to pyruvate (encoded by sdaA, sdaB and tdcG) was also deleted. As expected, the resulting strain turned out to be susceptible to even low concentrations of serine in the media. In order to improve the tolerance of the strain towards serine, adaptive laboratory evolution...

  3. Decommissioning and decontamination activity, Gnome Site, Eddy County, New Mexico

    International Nuclear Information System (INIS)

    1978-10-01

    The purpose of this assessment is to present a brief description of the proposed activity and its potential impacts on the environment. This assessment will constitute an evaluation as to whether or not a formal Environmental Statement need be prepared. As background to the proposed activity, Project Gnome was an underground nuclear test conducted in December 1961 as part of the PLOWSHARE Program. The project site is located about 25 miles southeast of Carlsbad, New Mexico. By means of an excavated shaft and tunnel, a 3-kiloton nuclear explosive was emplaced and detonated in a salt bed about 1200 feet below the surface. The uncontaminated rock and salt muck from the original excavation and subsequent contaminated muck and minor construction debris from reentry activities into the nuclear cavity is commingled and stored in a pile near the Gnome/Coach Shaft. Other areas on the site are known to have been contaminated. In 1969, a program was conducted to cleanup and dispose of all surface contamination to whatever depth it occurred in excess of 0.1 mR/hr. Contaminated materials and soil were collected and disposed into the Gnome shaft, which was filled and sealed. Since then, NV has proposed to DOE/HQ much lower criteria for residual radioactive contamination for the Gnome Site. These proposed criteria were to collect and dispose of surficial materials which contain more than 2 x 10 -5 microcuries per gram of soil for beta/gamma emitters and 3 x 10 -2 microcuries per milliliter of tritium in soil moisture. According to the latest reconnaissance in 1972, low concentrations of Cs-137, Sr-90 and tritium were present at various locations on the site in excess of these proposed guidelines. Other operational areas within the site are suspected of containing radioactive contamination in much lesser volume, which are to be determined by careful probing and monitoring, as described in the next section

  4. Methodology for contaminated sites of military activity territories restoration

    International Nuclear Information System (INIS)

    Khrushchov, D. P.; Yushchenko, Yu. V.; Shekhunova, S. B.

    2002-01-01

    Major part of Eastern Europe countries meet environmental problems related to sites of military activity. Major part of these sites is characterised with degradation of natural landscapes and contamination of geological environment with toxic and hazardous waste representing actual and potential danger for population and environment. Actual danger is caused with localisation of toxic waste, hazardous materials and waste which are preventing normal land use. Potential danger is related to successive dispersion of contamination in biosphere as well as origin of new derivatives and products having toxic and hazardous properties. The list of such sites and objects comprises bases of land, air and naval forces. These objects include a network of infrastructures: storages of fuels and lubricants (surface, underground), filling stations, pipe lines, reparation stations, garages, decontamination stations, underground storages of different purposes, depots (for ammunition, chemical products), hospitals, constructions, firing grounds (tank, artillery, aircraft bombing etc.) and waste disposal sites. Special programs aimed at military industries and bases contaminated sites remediation have been carrying out in developed countries (USA, United Kingdom, Germany etc.). This experience was used in the frames of joint programs having been founded in several countries of Central and Eastern Europe (Chesh Republic, Slovakia, Lithuania etc.). (author)

  5. Ketamine Metabolites Enantioselectively Decrease Intracellular D-Serine Concentrations in PC-12 Cells.

    Directory of Open Access Journals (Sweden)

    Nagendra S Singh

    Full Text Available D-Serine is an endogenous NMDA receptor co-agonist that activates synaptic NMDA receptors modulating neuronal networks in the cerebral cortex and plays a key role in long-term potentiation of synaptic transmission. D-serine is associated with NMDA receptor neurotoxicity and neurodegeneration and elevated D-serine concentrations have been associated with Alzheimer's and Parkinsons' diseases and amyotrophic lateral sclerosis. Previous studies have demonstrated that the ketamine metabolites (rac-dehydronorketamine and (2S,6S-hydroxynorketamine decrease intracellular D-serine concentrations in a concentration dependent manner in PC-12 cells. In the current study, PC-12 cells were incubated with a series of ketamine metabolites and the IC50 values associated with attenuated intracellular D-serine concentrations were determined. The results demonstrate that structural and stereochemical features of the studied compounds contribute to the magnitude of the inhibitory effect with (2S,6S-hydroxynorketamine and (2R,6R-hydroxynorketamine displaying the most potent inhibition with IC50 values of 0.18 ± 0.04 nM and 0.68 ± 0.09 nM. The data was utilized to construct a preliminary 3D-QSAR/pharmacophore model for use in the design of new and more efficient modulators of D-serine.

  6. Delta Learning Rule for the Active Sites Model

    OpenAIRE

    Lingashetty, Krishna Chaithanya

    2010-01-01

    This paper reports the results on methods of comparing the memory retrieval capacity of the Hebbian neural network which implements the B-Matrix approach, by using the Widrow-Hoff rule of learning. We then, extend the recently proposed Active Sites model by developing a delta rule to increase memory capacity. Also, this paper extends the binary neural network to a multi-level (non-binary) neural network.

  7. Active sites in char gasification: Final technical report

    Energy Technology Data Exchange (ETDEWEB)

    Wojtowicz, M.; Lilly, W.D.; Perkins, M.T.; Hradil, G.; Calo, J.M.; Suuberg, E.M.

    1987-09-01

    Among the key variables in the design of gasifiers and combustors is the reactivity of the chars which must be gasified or combusted. Significant loss of unburned char is unacceptable in virtually any process; the provision of sufficient residence time for complete conversion is essential. A very wide range of reactivities are observed, depending upon the nature of the char in a process. The current work focuses on furthering the understanding of gasification reactivities of chars. It has been well established that the reactivity of char to gasification generally depends upon three principal factors: (1) the concentration of ''active sites'' in the char; (2) mass transfer within the char; and (3) the type and concentration of catalytic impurities in the char. The present study primarily addresses the first factor. The subject of this research is the origin, nature, and fate of active sites in chars derived from parent hydrocarbons with coal-like structure. The nature and number of the active sites and their reactivity towards oxygen are examined in ''model'' chars derived from phenol-formaldehyde type resins. How the active sites are lost by the process of thermal annealing during heat treatment of chars are studied, and actual rate for the annealing process is derived. Since intrinsic char reactivities are of primary interest in the present study, a fair amount of attention was given to the model char synthesis and handling so that the effect of catalytic impurities and oxygen-containing functional groups in the chemical structure of the material were minimized, if not completely eliminated. The project would not be considered complete without comparing characteristic features of synthetic chars with kinetic behavior exhibited by natural chars, including coal chars.

  8. Exploiting Innocuous Activity for Correlating Users Across Sites

    OpenAIRE

    Goga , Oana; Lei , Howard; Parthasarathi , Sree Hari Krishnan; Friedland , Gerald; Sommer , Robin; Teixeira , Renata

    2013-01-01

    International audience; We study how potential attackers can identify accounts on different social network sites that all belong to the same user, exploiting only innocuous activity that inherently comes with posted content. We examine three specific features on Yelp, Flickr, and Twitter: the geo-location attached to a user's posts, the timestamp of posts, and the user's writing style as captured by language models. We show that among these three features the location of posts is the most powe...

  9. Multiple nucleophilic elbows leading to multiple active sites in a single module esterase from Sorangium cellulosum

    DEFF Research Database (Denmark)

    Udatha, D.B.R.K. Gupta; Madsen, Karina Marie; Panagiotou, Gianni

    2015-01-01

    The catalytic residues in carbohydrate esterase enzyme families constitute a highly conserved triad: serine, histidine and aspartic acid. This catalytic triad is generally located in a very sharp turn of the protein backbone structure, called the nucleophilic elbow and identified by the consensus...... sequence GXSXG. An esterase from Sorangium cellulosum Soce56 that contains five nucleophilic elbows was cloned and expressed in Escherichia coli and the function of each nucleophilic elbowed site was characterized. In order to elucidate the function of each nucleophilic elbow, site directed mutagenesis....... To our knowledge, this is the first report presenting the role of multiple nucleophilic elbows in the catalytic promiscuity of an esterase. Further structural analysis at protein unit level indicates the new evolutionary trajectories in emerging promiscuous esterases....

  10. Communication activities for NUMO's site selection process

    International Nuclear Information System (INIS)

    Takeuchi, Mitsuo; Okuyama, Shigeru; Kitayama, Kazumi; Kuba, Michiyoshi

    2004-01-01

    A siting program for geological disposal of high-level radioactive waste (HLW) in Japan has just started and is moving into a new stage of communication with the public. A final repository site will be selected via a stepwise process, as stipulated in the Specified Radioactive Waste Final Disposal Act promulgated in June 2000. Based on the Act, the site selection process of the Nuclear Waste Management Organization of Japan (NUMO, established in October 2000) will be carried out in the three steps: selection of Preliminary Investigation Areas (PIAs), selection of Detailed Investigation Areas (DIAs) and selection of the Repository Site. The Act also defines NUMO's responsibilities in terms of implementing the HLW disposal program in an open and transparent manner. NUMO fully understands the importance of public participation in its activities and is aiming to promote public involvement in the process of site selection based on a fundamental policy, which consists of 'adopting a stepwise approach', 'respecting the initiative of municipalities' and 'ensuring transparency in information disclosure'. This policy is clearly reflected in the adoption of an open solicitation approach for volunteer municipalities for Preliminary Investigation Areas (PIAs). NUMO made the official announcement of the start of its open solicitation program on 19 December 2002. This paper outlines how NUMO's activities are currently carried out with a view to encouraging municipalities to volunteer as PIAs and how public awareness of the safety of the HLW disposal is evaluated at this stage

  11. Seismic activity parameters of the Finnish potential repository sites

    International Nuclear Information System (INIS)

    Saari, J.

    2000-10-01

    Posiva Oy has started a project for estimating the possible earthquake induced rock movements on the deposition holes containing canisters of spent nuclear fuel. These estimates will be made for the four investigation sites, Romuvaara, Kivetty, Olkiluoto and Haestholmen. This study deals with the current and future seismicity associated with the above mentioned sites. Seismic belts that participate the seismic behaviour of the studied sites have been identified and the magnitude-frequency distributions of these belts have been estimated. The seismic activity parameters of the sites have been deduced from the characteristics of the seismic belts in order to forecast the seismicity during the next 100,000 years. The report discusses the possible earthquakes induced by future glaciation. The seismic interpretation seems to indicate that the previous postglacial faults in Finnish Lapland have been generated in compressional environment. The orientation of the rather uniform compression has been NW-SE, which coincide with the current stress field. It seems that, although the impact of postglacial crustal rebound must have been significant, the impact of plate tectonics has been dominant. A major assumption of this study has been that future seismicity will generally resemble the current seismicity. However, when the postglacial seismicity is concerned, the magnitude-frequency distribution is likely different and the expected maximum magnitude will be higher. Maximum magnitudes of future postglacial earthquakes have been approximated by strain release examinations. Seismicity has been examined within the framework of the lineament maps, in order to associate the future significant earthquakes with active fault zones in the vicinity of the potential repository sites. (orig.)

  12. Evaluation of oxidative stress in D-serine induced nephrotoxicity

    International Nuclear Information System (INIS)

    Orozco-Ibarra, Marisol; Medina-Campos, Omar Noel; Sanchez-Gonzalez, Dolores Javier; Martinez-Martinez, Claudia Maria; Floriano-Sanchez, Esau; Santamaria, Abel; Ramirez, Victoria; Bobadilla, Norma A.; Pedraza-Chaverri, Jose

    2007-01-01

    It has been suggested that oxidative stress is involved in D-serine-induced nephrotoxicity. The purpose of this study was to assess if oxidative stress is involved in this experimental model using several approaches including (a) the determination of several markers of oxidative stress and the activity of some antioxidant enzymes in kidney and (b) the use of compounds with antioxidant or prooxidant effects. Rats were sacrificed at several periods of time (from 3 to 24 h) after a single i.p. injection of D-serine (400 mg/kg). Control rats were injected with L-serine (400 mg/kg) and sacrificed 24 h after. The following markers were used to assess the temporal aspects of renal damage: (a) urea nitrogen (BUN) and creatinine in blood serum, (b) kidney injury molecule (KIM-1) mRNA levels, and (c) tubular necrotic damage. In addition, creatinine clearance, proteinuria, and urinary excretion of N-acetyl-β-D-glucosaminidase (NAG) were measured 24 h after D-serine injection. Protein carbonyl content, malondialdehyde (MDA), 4-hydroxy-2-nonenal (4-HNE), fluorescent products of lipid peroxidation, reactive oxygen species (ROS), glutathione (GSH) content, and heme oxygenase-1 (HO-1) expression were measured as markers of oxidative stress in the kidney. Additional experiments were performed using the following compounds with antioxidant or pro-oxidant effects before D-serine injection: (a) α-phenyl-tert-butyl-nitrone (PBN), a spin trapping agent; (b) 5,10,15,20-tetrakis (4-sulfonatophenyl) porphyrinato iron(III) (FeTPPS), a soluble complex able to metabolize peroxynitrite; (c) aminotriazole (ATZ), a catalase (CAT) inhibitor; (d) stannous chloride (SnCl 2 ), an HO-1 inductor; (e) tin mesoporphyrin (SnMP), an HO inhibitor. In the time-course study, serum creatinine and BUN increased significantly on 15-24 and 20-24 h, respectively, and KIM-1 mRNA levels increased significantly on 6-24 h. Histological analyses revealed tubular necrosis at 12 h. The activity of antioxidant enzymes

  13. Phosphorylation of JAK2 at serine 523: a negative regulator of JAK2 that is stimulated by growth hormone and epidermal growth factor

    DEFF Research Database (Denmark)

    Mazurkiewicz-Munoz, Anna M.; Argetsinger, Lawrence S.; Kouadio, Jean-Louis K.

    2006-01-01

    spectrometry identified serine 523 (Ser523) in JAK2 as a site of phosphorylation. A phosphoserine 523 antibody revealed that Ser523 is rapidly but transiently phosphorylated in response to growth hormone (GH). MEK1 inhibitor UO126 suppresses GH-dependent phosphorylation of Ser523, suggesting that extracellular...... signal-regulated kinases (ERKs) 1 and/or 2 or another kinase downstream of MEK1 phosphorylate Ser523 in response to GH. Other ERK activators, phorbol 12-myristate 13-acetate and epidermal growth factor, also stimulate phosphorylation of Ser523. When Ser523 in JAK2 was mutated, JAK2 kinase activity...

  14. Insulin resistance and muscle insulin receptor substrate‐1 serine hyperphosphorylation

    Science.gov (United States)

    Stuart, Charles A.; Howell, Mary E. A.; Cartwright, Brian M.; McCurry, Melanie P.; Lee, Michelle L.; Ramsey, Michael W.; Stone, Michael H.

    2014-01-01

    Abstract Insulin resistance in metabolic syndrome subjects is profound in spite of muscle insulin receptor and insulin‐responsive glucose transporter (GLUT4) expression being nearly normal. Insulin receptor tyrosine kinase phosphorylation of insulin receptor substrate‐1 (IRS‐1) at Tyr896 is a necessary step in insulin stimulation of translocation of GLUT4 to the cell surface. Serine phosphorylation of IRS‐1 by some kinases diminishes insulin action in mice. We evaluated the phosphorylation status of muscle IRS‐1 in 33 subjects with the metabolic syndrome and seventeen lean controls. Each underwent euglycemic insulin clamps and a thigh muscle biopsy before and after 8 weeks of either strength or endurance training. Muscle IRS‐1 phosphorylation at six sites was quantified by immunoblots. Metabolic syndrome muscle IRS‐1 had excess phosphorylation at Ser337 and Ser636 but not at Ser307, Ser789, or Ser1101. Ser337 is a target for phosphorylation by glycogen synthase kinase 3 (GSK3) and Ser636 is phosphorylated by c‐Jun N‐terminal kinase 1 (JNK1). Exercise training without weight loss did not change the IRS‐1 serine phosphorylation. These data suggest that baseline hyperphosphorylation of at least two key serines within muscle IRS‐1 diminishes the transmission of the insulin signal and thereby decreases the insulin‐stimulated translocation of GLUT4. Excess fasting phosphorylation of muscle IRS‐1 at Ser636 may be a major cause of the insulin resistance seen in obesity and might prevent improvement in insulin responsiveness when exercise training is not accompanied by weight loss. PMID:25472611

  15. Cleavage specificity analysis of six type II transmembrane serine proteases (TTSPs using PICS with proteome-derived peptide libraries.

    Directory of Open Access Journals (Sweden)

    Olivier Barré

    Full Text Available Type II transmembrane serine proteases (TTSPs are a family of cell membrane tethered serine proteases with unclear roles as their cleavage site specificities and substrate degradomes have not been fully elucidated. Indeed just 52 cleavage sites are annotated in MEROPS, the database of proteases, their substrates and inhibitors.To profile the active site specificities of the TTSPs, we applied Proteomic Identification of protease Cleavage Sites (PICS. Human proteome-derived database searchable peptide libraries were assayed with six human TTSPs (matriptase, matriptase-2, matriptase-3, HAT, DESC and hepsin to simultaneously determine sequence preferences on the N-terminal non-prime (P and C-terminal prime (P' sides of the scissile bond. Prime-side cleavage products were isolated following biotinylation and identified by tandem mass spectrometry. The corresponding non-prime side sequences were derived from human proteome databases using bioinformatics. Sequencing of 2,405 individual cleaved peptides allowed for the development of the family consensus protease cleavage site specificity revealing a strong specificity for arginine in the P1 position and surprisingly a lysine in P1' position. TTSP cleavage between R↓K was confirmed using synthetic peptides. By parsing through known substrates and known structures of TTSP catalytic domains, and by modeling the remainder, structural explanations for this strong specificity were derived.Degradomics analysis of 2,405 cleavage sites revealed a similar and characteristic TTSP family specificity at the P1 and P1' positions for arginine and lysine in unfolded peptides. The prime side is important for cleavage specificity, thus making these proteases unusual within the tryptic-enzyme class that generally has overriding non-prime side specificity.

  16. Current activities handbook: formerly utilized sites remedial action program

    Energy Technology Data Exchange (ETDEWEB)

    None

    1981-02-27

    This volume is one of a series produced under contract with the DOE, by Politech Corporation to develop a legislative and regulatory data base to assist the FUSRAP management in addressing the institutional and socioeconomic issues involved in carrying out the Formerly Utilized Sites Remedial Action Program. This Information Handbook series contains information about all relevant government agencies at the Federal and state levels, the pertinent programs they administer, each affected state legislature, and current Federal and state legislative and regulatory initiatives. This volume is a compilation of information about the activities each of the thirteen state legislatures potentially affected by the Formerly Utilized Sites Remedial Action Program. It contains a description of the state legislative procedural rules and a schedule of each legislative session; a summary of pending relevant legislation; the name and telephone number of legislative and state agency contacts; and the full text of all bills identified.

  17. Current activities handbook: formerly utilized sites remedial action program

    International Nuclear Information System (INIS)

    1981-01-01

    This volume is one of a series produced under contract with the DOE, by Politech Corporation to develop a legislative and regulatory data base to assist the FUSRAP management in addressing the institutional and socioeconomic issues involved in carrying out the Formerly Utilized Sites Remedial Action Program. This Information Handbook series contains information about all relevant government agencies at the Federal and state levels, the pertinent programs they administer, each affected state legislature, and current Federal and state legislative and regulatory initiatives. This volume is a compilation of information about the activities each of the thirteen state legislatures potentially affected by the Formerly Utilized Sites Remedial Action Program. It contains a description of the state legislative procedural rules and a schedule of each legislative session; a summary of pending relevant legislation; the name and telephone number of legislative and state agency contacts; and the full text of all bills identified

  18. Identification and characterization of fusolisin, the Fusobacterium nucleatum autotransporter serine protease.

    Directory of Open Access Journals (Sweden)

    Lior Doron

    Full Text Available Fusobacterium nucleatum is an oral anaerobe associated with periodontal disease, adverse pregnancy outcomes and colorectal carcinoma. A serine endopeptidase of 61-65 kDa capable of damaging host tissue and of inactivating immune effectors was detected previously in F. nucleatum. Here we describe the identification of this serine protease, named fusolisin, in three oral F. nucleatum sub-species. Gel zymogram revealed fusobacterial proteolytic activity with molecular masses ranging from 55-101 kDa. All of the detected proteases were inhibited by the serine protease inhibitor PMSF. analysis revealed that all of the detected proteases are encoded by genes encoding an open reading frame (ORF with a calculated mass of approximately 115 kDa. Bioinformatics analysis of the identified ORFs demonstrated that they consist of three domains characteristic of autotransporters of the type Va secretion system. Our results suggest that the F. nucleatum fusolisins are derived from a precursor of approximately 115 kDa. After crossing the cytoplasmic membrane and cleavage of the leader sequence, the C-terminal autotransporter domain of the remaining 96-113 kDa protein is embedded in the outer membrane and delivers the N-terminal S8 serine protease passenger domain to the outer cell surface. In most strains the N-terminal catalytic 55-65 kDa domain self cleaves and liberates itself from the autotransporter domain after its transfer across the outer cell membrane. In F. nucleatum ATCC 25586 this autocatalytic activity is less efficient resulting in a full length membrane-anchored serine protease. The mature serine protease was found to cleave after Thr, Gly, Ala and Leu residues at the P1 position. Growth of F. nucleatum in complex medium was inhibited when serine protease inhibitors were used. Additional experiments are needed to determine whether fusolisin might be used as a target for controlling fusobacterial infections.

  19. Identification and characterization of fusolisin, the Fusobacterium nucleatum autotransporter serine protease.

    Science.gov (United States)

    Doron, Lior; Coppenhagen-Glazer, Shunit; Ibrahim, Yara; Eini, Amir; Naor, Ronit; Rosen, Graciela; Bachrach, Gilad

    2014-01-01

    Fusobacterium nucleatum is an oral anaerobe associated with periodontal disease, adverse pregnancy outcomes and colorectal carcinoma. A serine endopeptidase of 61-65 kDa capable of damaging host tissue and of inactivating immune effectors was detected previously in F. nucleatum. Here we describe the identification of this serine protease, named fusolisin, in three oral F. nucleatum sub-species. Gel zymogram revealed fusobacterial proteolytic activity with molecular masses ranging from 55-101 kDa. All of the detected proteases were inhibited by the serine protease inhibitor PMSF. analysis revealed that all of the detected proteases are encoded by genes encoding an open reading frame (ORF) with a calculated mass of approximately 115 kDa. Bioinformatics analysis of the identified ORFs demonstrated that they consist of three domains characteristic of autotransporters of the type Va secretion system. Our results suggest that the F. nucleatum fusolisins are derived from a precursor of approximately 115 kDa. After crossing the cytoplasmic membrane and cleavage of the leader sequence, the C-terminal autotransporter domain of the remaining 96-113 kDa protein is embedded in the outer membrane and delivers the N-terminal S8 serine protease passenger domain to the outer cell surface. In most strains the N-terminal catalytic 55-65 kDa domain self cleaves and liberates itself from the autotransporter domain after its transfer across the outer cell membrane. In F. nucleatum ATCC 25586 this autocatalytic activity is less efficient resulting in a full length membrane-anchored serine protease. The mature serine protease was found to cleave after Thr, Gly, Ala and Leu residues at the P1 position. Growth of F. nucleatum in complex medium was inhibited when serine protease inhibitors were used. Additional experiments are needed to determine whether fusolisin might be used as a target for controlling fusobacterial infections.

  20. D-Serine and Glycine Differentially Control Neurotransmission during Visual Cortex Critical Period.

    Directory of Open Access Journals (Sweden)

    Claire N J Meunier

    Full Text Available N-methyl-D-aspartate receptors (NMDARs play a central role in synaptic plasticity. Their activation requires the binding of both glutamate and d-serine or glycine as co-agonist. The prevalence of either co-agonist on NMDA-receptor function differs between brain regions and remains undetermined in the visual cortex (VC at the critical period of postnatal development. Here, we therefore investigated the regulatory role that d-serine and/or glycine may exert on NMDARs function and on synaptic plasticity in the rat VC layer 5 pyramidal neurons of young rats. Using selective enzymatic depletion of d-serine or glycine, we demonstrate that d-serine and not glycine is the endogenous co-agonist of synaptic NMDARs required for the induction and expression of Long Term Potentiation (LTP at both excitatory and inhibitory synapses. Glycine on the other hand is not involved in synaptic efficacy per se but regulates excitatory and inhibitory neurotransmission by activating strychnine-sensitive glycine receptors, then producing a shunting inhibition that controls neuronal gain and results in a depression of synaptic inputs at the somatic level after dendritic integration. In conclusion, we describe for the first time that in the VC both D-serine and glycine differentially regulate somatic depolarization through the activation of distinct synaptic and extrasynaptic receptors.

  1. Method for the production of l-serine using genetically engineered microorganisms deficient in serine degradation pathways

    DEFF Research Database (Denmark)

    2016-01-01

    The present invention generally relates to the microbiological industry, and specifically to the production of L-serine using genetically modified bacteria. The present invention provides genetically modified microorganisms, such as bacteria, wherein the expression of genes encoding for enzymes...... concentrations of serine. The present invention also provides methods for the production of L-serine or L-serine derivative using such genetically modified microorganisms....

  2. Study the active site of flavonoid applying radiation chemistry

    International Nuclear Information System (INIS)

    Wu Jilan; Sun Gang; Zhang Fugen; He Yongke; Li Jiuqiang

    2000-01-01

    Flavonoid are a large and important class of naturally occurring, low molecular weight benzo-γ-pyrone derivatives which are reported to have a myriad of biological activities, but the study on the active sites of flavonoids is still ambiguous. In this paper, rutin, quercetin and baicalin have been selected as model compounds. It is well known that rutin is used in inhibiting arteriosclerosis and baicalin is antibacterial and antiviral. They have similar basic structure, but their medicinal properties are so different, why? As most flavonoids contain carbonyl group, which can capture electron effectively, we predict that flavonoids can capture electron to form radical anion. The formation of anion radical may have influence on the mitochondrial electron transport chain. The difference in the ability of forming anion radical may cause the difference in their medicinal effects. (author)

  3. Study the active site of flavonoid applying radiation chemistry

    Energy Technology Data Exchange (ETDEWEB)

    Wu Jilan; Sun Gang; Zhang Fugen; He Yongke; Li Jiuqiang [Department of Technical Physics, Peking Univ., Beijing (China)

    2000-03-01

    Flavonoid are a large and important class of naturally occurring, low molecular weight benzo-{gamma}-pyrone derivatives which are reported to have a myriad of biological activities, but the study on the active sites of flavonoids is still ambiguous. In this paper, rutin, quercetin and baicalin have been selected as model compounds. It is well known that rutin is used in inhibiting arteriosclerosis and baicalin is antibacterial and antiviral. They have similar basic structure, but their medicinal properties are so different, why? As most flavonoids contain carbonyl group, which can capture electron effectively, we predict that flavonoids can capture electron to form radical anion. The formation of anion radical may have influence on the mitochondrial electron transport chain. The difference in the ability of forming anion radical may cause the difference in their medicinal effects. (author)

  4. The essential structures of ISP-I that influence serine palmitoyltransferase inhibition in Chinese hamster ovary cells.

    Science.gov (United States)

    Mizukoshi, Koji; Matsumoto, Katsuo; Hirose, Ryoji; Fujita, Tetsuro

    2012-01-01

    We investigated the structure-activity relationship between various ISP-I (myriocin, thermozymocidin) analogous which has sphingosine-like structure and serine palmitoyltransferase (SPT) in Chinese hamster ovary (CHO) cells utilizing sphingolipid production as a marker. Our data suggest that the double bond and/or ketone group within the alkyl chain as well as the alkyl chain are necessary for ISP-I to inhibit SPT. In addition, a serine structure is necessary for SPT inhibitory activity, which confirms previous findings.

  5. Maxey Flats low-level waste disposal site closure activities

    International Nuclear Information System (INIS)

    Haight, C.P.; Mills, D.; Razor, J.E.

    1987-01-01

    The Maxey Flats Radioactive Waste Disposal Facility in Fleming County, Kentucky is in the process of being closed. The facility opened for commercial business in the spring of 1963 and received approximately 4.75 million cubic feet of radioactive waste by the time it was closed in December of 1977. During fourteen years of operation approximately 2.5 million curies of by-product material, 240,000 kilograms of source material, and 430 kilograms of special nuclear material were disposed. The Commonwealth purchased the lease hold estate and rights in May 1978 from the operating company. This action was taken to stabilize the facility and prepare it for closure consisting of passive care and monitoring. To prepare the site for closure, a number of remedial activities had to be performed. The remediation activities implemented have included erosion control, surface drainage modifications, installation of a temporary plastic surface cover, leachate removal, analysis, treatment and evaporation, US DOE funded evaporator concentrates solidification project and their on-site disposal in an improved disposal trench with enhanced cover for use in a humid environment situated in a fractured geology, performance evaluation of a grout injection demonstration, USGS subsurface geologic investigation, development of conceptual closure designs, and finally being added to the US EPA National Priority List for remediation and closure under Superfund. 13 references, 3 figures

  6. Eel calcitonin binding site distribution and antinociceptive activity in rats

    International Nuclear Information System (INIS)

    Guidobono, F.; Netti, C.; Sibilia, V.; Villa, I.; Zamboni, A.; Pecile, A.

    1986-01-01

    The distribution of binding site for [ 125 I]-eel-calcitonin (ECT) to rat central nervous system, studied by an autoradiographic technique, showed concentrations of binding in the diencephalon, the brain stem and the spinal cord. Large accumulations of grains were seen in the hypothalamus, the amygdala, in the fasciculus medialis prosencephali, in the fasciculus longitudinalis medialis, in the ventrolateral part of the periventricular gray matter, in the lemniscus medialis and in the raphe nuclei. The density of grains in the reticular formation and in the nucleus tractus spinalis nervi trigemini was more moderate. In the spinal cord, grains were scattered throughout the dorsal horns. Binding of the ligand was displaced equally by cold ECT and by salmon CT(sCT), indicating that both peptides bind to the same receptors. Human CT was much weaker than sCT in displacing [ 125 I]-ECT binding. The administration of ECT into the brain ventricles of rats dose-dependently induced a significant and long-lasting enhancement of hot-plate latencies comparable with that obtained with sCT. The antinociceptive activity induced by ECT is compatible with the topographical distribution of binding sites for the peptide and is a further indication that fish CTs are active in the mammalian brain

  7. Quantitative serine protease assays based on formation of copper(II)-oligopeptide complexes.

    Science.gov (United States)

    Ding, Xiaokang; Yang, Kun-Lin

    2015-01-07

    A quantitative protease assay based on the formation of a copper-oligopeptide complex is developed. In this assay, when a tripeptide GGH fragment is cleaved from an oligopeptide chain by serine proteases, the tripeptide quickly forms a pink GGH/Cu(2+) complex whose concentration can be determined quantitatively by using UV-Vis spectroscopy. Therefore, activities of serine proteases can be determined from the formation rate of the GGH/Cu(2+) complex. This principle can be used to detect the presence of serine protease in a real-time manner, or measure proteolytic activities of serine protease cleaving different oligopeptide substrates. For example, by using this assay, we demonstrate that trypsin, a model serine protease, is able to cleave two oligopeptides GGGGKGGH () and GGGGRGGH (). However, the specificity constant (kcat/Km) for is higher than that of (6.4 × 10(3) mM(-1) min(-1)vs. 1.3 × 10(3) mM(-1) min(-1)). This result shows that trypsin is more specific toward arginine (R) than lysine (K) in the oligopeptide sequence.

  8. Sol-gel immobilization of serine proteases for application in organic solvents

    NARCIS (Netherlands)

    van Unen, D.J.; Engbersen, Johannes F.J.; Reinhoudt, David

    2001-01-01

    The serine proteases α-chymotrypsin, trypsin, and subtilisin Carlsberg were immobilized in a sol-gel matrix and the effects on the enzyme activity in organic media are evaluated. The percentage of immobilized enzyme is 90% in the case of α-chymotrypsin and the resulting specific enzyme activity in

  9. Enhancer transcripts mark active estrogen receptor binding sites.

    Science.gov (United States)

    Hah, Nasun; Murakami, Shino; Nagari, Anusha; Danko, Charles G; Kraus, W Lee

    2013-08-01

    We have integrated and analyzed a large number of data sets from a variety of genomic assays using a novel computational pipeline to provide a global view of estrogen receptor 1 (ESR1; a.k.a. ERα) enhancers in MCF-7 human breast cancer cells. Using this approach, we have defined a class of primary transcripts (eRNAs) that are transcribed uni- or bidirectionally from estrogen receptor binding sites (ERBSs) with an average transcription unit length of ∼3-5 kb. The majority are up-regulated by short treatments with estradiol (i.e., 10, 25, or 40 min) with kinetics that precede or match the induction of the target genes. The production of eRNAs at ERBSs is strongly correlated with the enrichment of a number of genomic features that are associated with enhancers (e.g., H3K4me1, H3K27ac, EP300/CREBBP, RNA polymerase II, open chromatin architecture), as well as enhancer looping to target gene promoters. In the absence of eRNA production, strong enrichment of these features is not observed, even though ESR1 binding is evident. We find that flavopiridol, a CDK9 inhibitor that blocks transcription elongation, inhibits eRNA production but does not affect other molecular indicators of enhancer activity, suggesting that eRNA production occurs after the assembly of active enhancers. Finally, we show that an enhancer transcription "signature" based on GRO-seq data can be used for de novo enhancer prediction across cell types. Together, our studies shed new light on the activity of ESR1 at its enhancer sites and provide new insights about enhancer function.

  10. Metal active site elasticity linked to activation of homocysteine in methionine synthases

    Energy Technology Data Exchange (ETDEWEB)

    Koutmos, Markos; Pejchal, Robert; Bomer, Theresa M.; Matthews, Rowena G.; Smith, Janet L.; Ludwig, Martha L. (Michigan)

    2008-04-02

    Enzymes possessing catalytic zinc centers perform a variety of fundamental processes in nature, including methyl transfer to thiols. Cobalamin-independent (MetE) and cobalamin-dependent (MetH) methionine synthases are two such enzyme families. Although they perform the same net reaction, transfer of a methyl group from methyltetrahydrofolate to homocysteine (Hcy) to form methionine, they display markedly different catalytic strategies, modular organization, and active site zinc centers. Here we report crystal structures of zinc-replete MetE and MetH, both in the presence and absence of Hcy. Structural investigation of the catalytic zinc sites of these two methyltransferases reveals an unexpected inversion of zinc geometry upon binding of Hcy and displacement of an endogenous ligand in both enzymes. In both cases a significant movement of the zinc relative to the protein scaffold accompanies inversion. These structures provide new information on the activation of thiols by zinc-containing enzymes and have led us to propose a paradigm for the mechanism of action of the catalytic zinc sites in these and related methyltransferases. Specifically, zinc is mobile in the active sites of MetE and MetH, and its dynamic nature helps facilitate the active site conformational changes necessary for thiol activation and methyl transfer.

  11. Differential active site loop conformations mediate promiscuous activities in the lactonase SsoPox.

    Directory of Open Access Journals (Sweden)

    Julien Hiblot

    Full Text Available Enzymes are proficient catalysts that enable fast rates of Michaelis-complex formation, the chemical step and products release. These different steps may require different conformational states of the active site that have distinct binding properties. Moreover, the conformational flexibility of the active site mediates alternative, promiscuous functions. Here we focused on the lactonase SsoPox from Sulfolobus solfataricus. SsoPox is a native lactonase endowed with promiscuous phosphotriesterase activity. We identified a position in the active site loop (W263 that governs its flexibility, and thereby affects the substrate specificity of the enzyme. We isolated two different sets of substitutions at position 263 that induce two distinct conformational sampling of the active loop and characterized the structural and kinetic effects of these substitutions. These sets of mutations selectively and distinctly mediate the improvement of the promiscuous phosphotriesterase and oxo-lactonase activities of SsoPox by increasing active-site loop flexibility. These observations corroborate the idea that conformational diversity governs enzymatic promiscuity and is a key feature of protein evolvability.

  12. STRUCTURAL ASPECTS OF STRONG INHIBITION AND ROLE OF SCAFFOLD FOR SERINE PROTEASE INHIBITORS

    Directory of Open Access Journals (Sweden)

    Jhimli Dasgupta

    2011-12-01

    Full Text Available Canonical serine protease inhibitors inhibit their cognate enzymes by binding tightly at the enzyme active site in a substrate-like manner, being cleaved extremely slowly compared to a true substrate. They interact with cognate enzymes through P3-P2 region of the inhibitory loop while the scaffold hardly makes any contact. Neighbouring scaffolding residues like arginine or asparagine shape-up the inhibitory loop and religate the cleaved scissile bond. The specificity of the inhibitor can be altered by mutating the hyper solvent accessible P1 residue without changing loop-scaffold interactions. To understand the loop-scaffold compatibility, we prepared three chimeric proteins ECIL-WCIS , ETIL-WCIS , and STIL-WCIS , where the inhibitory loops of ECI, ETI, and STI were placed on the scaffold of their homologue WCI. Results showed that although ECIL-WCIS and STIL-WCIS behave like inhibitors, ETIL-WCIS behaves like a substrate. Crystal structure of ETIL-WCIS and its comparison with ETI indicated that three novel scaffolding residues Trp88, Arg74, and Tyr113 in ETI act as barrier to confine the inhibitory loop to canonical conformation. Absence of this barrier in the scaffold of WCI makes the inhibitory loop flexible in ETIL-WCIS leading to a loss of canonical conformation, explaining its substrate-like behaviour. Furthermore, complex structures of the inhibitors with their cognate enzymes indicate that rigidification of the inhibitory loop at the enzyme active site is necessary for efficient inhibition.

  13. Metals in the active site of native protein phosphatase-1.

    Science.gov (United States)

    Heroes, Ewald; Rip, Jens; Beullens, Monique; Van Meervelt, Luc; De Gendt, Stefan; Bollen, Mathieu

    2015-08-01

    Protein phosphatase-1 (PP1) is a major protein Ser/Thr phosphatase in eukaryotic cells. Its activity depends on two metal ions in the catalytic site, which were identified as manganese in the bacterially expressed phosphatase. However, the identity of the metal ions in native PP1 is unknown. In this study, total reflection X-ray fluorescence (TXRF) was used to detect iron and zinc in PP1 that was purified from rabbit skeletal muscle. Metal exchange experiments confirmed that the distinct substrate specificity of recombinant and native PP1 is determined by the nature of their associated metals. We also found that the iron level associated with native PP1 is decreased by incubation with inhibitor-2, consistent with a function of inhibitor-2 as a PP1 chaperone. Copyright © 2015 Elsevier Inc. All rights reserved.

  14. Serine/threonine ligation for the chemical synthesis of proteins.

    Science.gov (United States)

    Lee, Chi Lung; Li, Xuechen

    2014-10-01

    Advances in the development of efficient peptide ligation methods have enabled the total synthesis of complex proteins to be successfully undertaken. Recently, a Ser/Thr ligation has emerged as a new tool in synthetic protein chemistry. The chemoselective reaction between an N-terminal serine or threonine of an unprotected peptide segment and a C-terminal salicylaldehyde ester of another unprotected peptide segment gives rise to an N,O-benzylidene acetal linked product, which upon acidolysis produces a native peptide bond at the site of ligation. Ser/Thr ligation has been used for the synthesis of the human erythrocyte acylphosphatase protein and MUC1 glycopeptide segments, semisynthesis of peptoid/PEG-RNase S protein hybrids, and cyclic peptide synthesis including cyclic tetrapeptides, cyclomontanin B, yunnanin C, mahafacyclin B, and daptomycin. Copyright © 2014 Elsevier Ltd. All rights reserved.

  15. Active site loop conformation regulates promiscuous activity in a lactonase from Geobacillus kaustophilus HTA426.

    Directory of Open Access Journals (Sweden)

    Yu Zhang

    Full Text Available Enzyme promiscuity is a prerequisite for fast divergent evolution of biocatalysts. A phosphotriesterase-like lactonase (PLL from Geobacillus kaustophilus HTA426 (GkaP exhibits main lactonase and promiscuous phosphotriesterase activities. To understand its catalytic and evolutionary mechanisms, we investigated a "hot spot" in the active site by saturation mutagenesis as well as X-ray crystallographic analyses. We found that position 99 in the active site was involved in substrate discrimination. One mutant, Y99L, exhibited 11-fold improvement over wild-type in reactivity (kcat/Km toward the phosphotriesterase substrate ethyl-paraoxon, but showed 15-fold decrease toward the lactonase substrate δ-decanolactone, resulting in a 157-fold inversion of the substrate specificity. Structural analysis of Y99L revealed that the mutation causes a ∼6.6 Å outward shift of adjacent loop 7, which may cause increased flexibility of the active site and facilitate accommodation and/or catalysis of organophosphate substrate. This study provides for the PLL family an example of how the evolutionary route from promiscuity to specificity can derive from very few mutations, which promotes alteration in the conformational adjustment of the active site loops, in turn draws the capacity of substrate binding and activity.

  16. An analysis approach to identify specific functional sites in orthologous proteins using sequence and structural information: application to neuroserpin reveals regions that differentially regulate inhibitory activity.

    Science.gov (United States)

    Lee, Tet Woo; Yang, Annie Shu-Ping; Brittain, Thomas; Birch, Nigel P

    2015-01-01

    The analysis of sequence conservation is commonly used to predict functionally important sites in proteins. We have developed an approach that first identifies highly conserved sites in a set of orthologous sequences using a weighted substitution-matrix-based conservation score and then filters these conserved sites based on the pattern of conservation present in a wider alignment of sequences from the same family and structural information to identify surface-exposed sites. This allows us to detect specific functional sites in the target protein and exclude regions that are likely to be generally important for the structure or function of the wider protein family. We applied our method to two members of the serpin family of serine protease inhibitors. We first confirmed that our method successfully detected the known heparin binding site in antithrombin while excluding residues known to be generally important in the serpin family. We next applied our sequence analysis approach to neuroserpin and used our results to guide site-directed polyalanine mutagenesis experiments. The majority of the mutant neuroserpin proteins were found to fold correctly and could still form inhibitory complexes with tissue plasminogen activator (tPA). Kinetic analysis of tPA inhibition, however, revealed altered inhibitory kinetics in several of the mutant proteins, with some mutants showing decreased association with tPA and others showing more rapid dissociation of the covalent complex. Altogether, these results confirm that our sequence analysis approach is a useful tool that can be used to guide mutagenesis experiments for the detection of specific functional sites in proteins. © 2014 Wiley Periodicals, Inc.

  17. Data on the phosphorylation state of the catalytic serine of enzymes in the α-D-phosphohexomutase superfamily

    Directory of Open Access Journals (Sweden)

    Yingying Lee

    2017-02-01

    Full Text Available Most enzymes in the α-D-phosphohexomutase superfamily catalyze the reversible conversion of 1- to 6-phosphosugars. They play important roles in carbohydrate and sugar nucleotide metabolism, and participate in the biosynthesis of polysaccharides, glycolipids, and other exoproducts. Mutations in genes encoding these enzymes are associated with inherited metabolic diseases in humans, including glycogen storage disease and congenital disorders of glycosylation. Enzymes in the superfamily share a highly conserved active site serine that participates in the multi-step phosphoryl transfer reaction. Here we provide data on the effects of various phosphosugar ligands on the phosphorylation of this serine, as monitored by electrospray ionization mass spectrometry (ESI-MS data on the intact proteins. We also show data on the longevity of the phospho-enzyme under various solution conditions in one member of the superfamily from Pseudomonas aeruginosa, and present inhibition data for several ligands. These data should be useful for the production of homogeneous samples of phosphorylated or unphosphorylated proteins, which are essential for biophysical characterization of these enzymes.

  18. Mechanism of Ribonuclease III Catalytic Regulation by Serine Phosphorylation

    Science.gov (United States)

    Gone, Swapna; Alfonso-Prieto, Mercedes; Paudyal, Samridhdi; Nicholson, Allen W.

    2016-05-01

    Ribonuclease III (RNase III) is a conserved, gene-regulatory bacterial endonuclease that cleaves double-helical structures in diverse coding and noncoding RNAs. RNase III is subject to multiple levels of control, reflective of its global regulatory functions. Escherichia coli (Ec) RNase III catalytic activity is known to increase during bacteriophage T7 infection, reflecting the expression of the phage-encoded protein kinase, T7PK. However, the mechanism of catalytic enhancement is unknown. This study shows that Ec-RNase III is phosphorylated on serine in vitro by purified T7PK, and identifies the targets as Ser33 and Ser34 in the N-terminal catalytic domain. Kinetic experiments reveal a 5-fold increase in kcat and a 1.4-fold decrease in Km following phosphorylation, providing a 7.4-fold increase in catalytic efficiency. Phosphorylation does not change the rate of substrate cleavage under single-turnover conditions, indicating that phosphorylation enhances product release, which also is the rate-limiting step in the steady-state. Molecular dynamics simulations provide a mechanism for facilitated product release, in which the Ser33 phosphomonoester forms a salt bridge with the Arg95 guanidinium group, thereby weakening RNase III engagement of product. The simulations also show why glutamic acid substitution at either serine does not confer enhancement, thus underscoring the specific requirement for a phosphomonoester.

  19. Serine Protease Zymography: Low-Cost, Rapid, and Highly Sensitive RAMA Casein Zymography.

    Science.gov (United States)

    Yasumitsu, Hidetaro

    2017-01-01

    To detect serine protease activity by zymography, casein and CBB stain have been used as a substrate and a detection procedure, respectively. Casein zymography has been using substrate concentration at 1 mg/mL and employing conventional CBB stain. Although ordinary casein zymography provides reproducible results, it has several disadvantages including time-consuming and relative low sensitivity. Improved casein zymography, RAMA casein zymography, is rapid and highly sensitive. RAMA casein zymography completes the detection process within 1 h after incubation and increases the sensitivity at least by tenfold. In addition to serine protease, the method also detects metalloprotease 7 (MMP7, Matrilysin) with high sensitivity.

  20. Active sites monitoring at Oak Ridge National Laboratory

    International Nuclear Information System (INIS)

    Ashwood, T.L.; Wickliff, D.S.; Morrissey, C.M.; Adair, H.L.

    1990-01-01

    Oak Ridge National Laboratory (ORNL) currently disposes of solid low-level radioactive wastes (LLW) in Solid Waste Storage Area (SWSA) 6 and stores transuranic (TRU) wastes in SWSA 5 North. Environmental monitoring of all TRU storage areas and those LLW disposal facilities active on or after September 1988 is required by U.S. Department of Energy (DOE) Order 5820.2A to provide for early detection of contaminant migration and to enable assessment of the performance of these facilities with regard to the exposure limits established in the order. In this paper, the authors develop a cost-effective monitoring program that is consistent with the authors' best understanding of the hydrogeology in the affected areas while complying with the DOE order. Active LLW disposal sites at ORNL consist of: lined auger holes with concrete bottoms, vertical concrete silos in trenches, and tumulus technology facilities (above-grade storage in concrete vaults on concrete pads to be covered by impermeable caps)

  1. Serine proteinases and their inhibitors in fertilization

    Czech Academy of Sciences Publication Activity Database

    Jonáková, Věra; Jelínková-Slavíčková, Petra

    2004-01-01

    Roč. 8, 3,4 (2004), s. 108-110 ISSN 1211-8869. [Central European Conference on Human Tumor Markers /5./. Praha, 01.10.2004-03.10.2004] R&D Projects: GA ČR GA303/02/0433; GA ČR GP303/02/P069; GA ČR GP303/04/P070; GA MZd NJ7463 Institutional research plan: CEZ:AV0Z5052915 Keywords : serine proteinase * proteinase inhibitors * fertilization Subject RIV: CE - Biochemistry

  2. Identification of Active Edge Sites for Electrochemical H2 Evolution from MoS2 Nanocatalysts

    DEFF Research Database (Denmark)

    Jaramillo, Thomas; Jørgensen, Kristina Pilt; Bonde, Jacob

    2007-01-01

    The identification of the active sites in heterogeneous catalysis requires a combination of surface sensitive methods and reactivity studies. We determined the active site for hydrogen evolution, a reaction catalyzed by precious metals, on nanoparticulate molybdenum disulfide (MoS2) by atomically....... Electrocatalytic activity measurements for hydrogen evolution correlate linearly with the number of edge sites on the MoS2 catalyst....

  3. Inhibition of AmpC beta-lactamase through a destabilizing interaction in the active site

    Energy Technology Data Exchange (ETDEWEB)

    Trehan, I.; Beadle, B.M.; Shoichet, B.K. (NWU)

    2010-03-08

    {beta}-Lactamases hydrolyze {beta}-lactam antibiotics, including penicillins and cephalosporins; these enzymes are the most widespread resistance mechanism to these drugs and pose a growing threat to public health. {beta}-Lactams that contain a bulky 6(7){alpha} substituent, such as imipenem and moxalactam, actually inhibit serine {beta}-lactamases and are widely used for this reason. Although mutant serine {beta}-lactamases have arisen that hydrolyze {beta}-lactamase resistant {beta}-lactams (e.g., ceftazidime) or avoid mechanism-based inhibitors (e.g., clavulanate), mutant serine {beta}-lactamases have not yet arisen in the clinic with imipenemase or moxalactamase activity. Structural and thermodynamic studies suggest that the 6(7){alpha} substituents of these inhibitors form destabilizing contacts within the covalent adduct with the conserved Asn152 in class C {beta}-lactamases (Asn132 in class A {beta}-lactamases). This unfavorable interaction may be crucial to inhibition. To test this destabilization hypothesis, we replaced Asn152 with Ala in the class C {beta}-lactamase AmpC from Escherichia coli and examined the mutant enzyme's thermodynamic stability in complex with imipenem and moxalactam. Consistent with the hypothesis, the Asn152 {yields} Ala substitution relieved 0.44 and 1.10 kcal/mol of strain introduced by imipenem and moxalactam, respectively, relative to the wild-type complexes. However, the kinetic efficiency of AmpC N152A was reduced by 6300-fold relative to that of the wild-type enzyme. To further investigate the inhibitor's interaction with the mutant enzyme, the X-ray crystal structure of moxalactam in complex with N152A was determined to a resolution of 1.83 {angstrom}. Moxalactam in the mutant complex is significantly displaced from its orientation in the wild-type complex; however, moxalactam does not adopt an orientation that would restore competence for hydrolysis. Although Asn152 forces {beta}-lactams with 6(7){alpha

  4. Membrane-anchored Serine Protease Matriptase Is a Trigger of Pulmonary Fibrogenesis

    NARCIS (Netherlands)

    Bardou, Olivier; Menou, Awen; François, Charlène; Duitman, Jan Willem; von der Thüsen, Jan H.; Borie, Raphaël; Sales, Katiuchia Uzzun; Mutze, Kathrin; Castier, Yves; Sage, Edouard; Liu, Ligong; Bugge, Thomas H.; Fairlie, David P.; Königshoff, Mélanie; Crestani, Bruno; Borensztajn, Keren S.

    2016-01-01

    Idiopathic pulmonary fibrosis (IPF) is a devastating disease that remains refractory to current therapies. To characterize the expression and activity of the membrane-anchored serine protease matriptase in IPF in humans and unravel its potential role in human and experimental pulmonary fibrogenesis.

  5. Membrane-anchored serine protease matriptase is a trigger of pulmonary fibrogenesis

    NARCIS (Netherlands)

    Bardou, O. (Olivier); Menou, A. (Awen); François, C. (Charlène); J.W. Duitman (Jan Willem); J. von der Thusen (Jan); Borie, R. (Raphaël); Sales, K.U. (Katiuchia Uzzun); Mutze, K. (Kathrin); Y. Castier (Yves); Sage, E. (Edouard); Liu, L. (Ligong); Bugge, T.H. (Thomas H.); Fairlie, D.P. (David P.); Königshoff, M. (Mélanie); B. Crestani (Bruno); K. Borensztajn (Keren)

    2016-01-01

    textabstractRationale: Idiopathic pulmonary fibrosis (IPF) is a devastating disease that remains refractory to current therapies. Objectives: To characterize the expression and activity of the membrane-anchored serine protease matriptase in IPF in humans and unravel its potential role in human and

  6. Protein kinase A phosphorylates serine 267 in the homeodomain of engrailed-2 leading to decreased DNA binding

    DEFF Research Database (Denmark)

    Hjerrild, Majbrit; Stensballe, Allan; Jensen, Ole N

    2004-01-01

    Engrailed-2 (En-2) belongs to an evolutionarily conserved family of DNA binding homeodomain-containing proteins that are expressed in mammalian brain during development. Here, we demonstrate that serine 267 in the homeodomain of En-2 is phosphorylated by protein kinase A (PKA) in forskolin......-treated COS-7 cells. Furthermore, we analyze the physiological function of En-2 phosphorylation by PKA. The nuclear localization of En-2 is not influenced by the phosphorylation of serine 267. However, substitution of serine 267 with alanine resulted in increased binding of En-2 to DNA, while replacing serine...... 267 with glutamic acid resulted in decreased En-2 DNA binding. These results suggest that the transcriptional activity of En-2 is regulated by PKA....

  7. Structural basis of metallo-β-lactamase, serine-β-lactamase and penicillin-binding protein inhibition by cyclic boronates

    Science.gov (United States)

    Brem, Jürgen; Cain, Ricky; Cahill, Samuel; McDonough, Michael A.; Clifton, Ian J.; Jiménez-Castellanos, Juan-Carlos; Avison, Matthew B.; Spencer, James; Fishwick, Colin W. G.; Schofield, Christopher J.

    2016-08-01

    β-Lactamases enable resistance to almost all β-lactam antibiotics. Pioneering work revealed that acyclic boronic acids can act as `transition state analogue' inhibitors of nucleophilic serine enzymes, including serine-β-lactamases. Here we report biochemical and biophysical analyses revealing that cyclic boronates potently inhibit both nucleophilic serine and zinc-dependent β-lactamases by a mechanism involving mimicking of the common tetrahedral intermediate. Cyclic boronates also potently inhibit the non-essential penicillin-binding protein PBP 5 by the same mechanism of action. The results open the way for development of dual action inhibitors effective against both serine- and metallo-β-lactamases, and which could also have antimicrobial activity through inhibition of PBPs.

  8. Conserved water molecules in bacterial serine hydroxymethyltransferases.

    Science.gov (United States)

    Milano, Teresa; Di Salvo, Martino Luigi; Angelaccio, Sebastiana; Pascarella, Stefano

    2015-10-01

    Water molecules occurring in the interior of protein structures often are endowed with key structural and functional roles. We report the results of a systematic analysis of conserved water molecules in bacterial serine hydroxymethyltransferases (SHMTs). SHMTs are an important group of pyridoxal-5'-phosphate-dependent enzymes that catalyze the reversible conversion of l-serine and tetrahydropteroylglutamate to glycine and 5,10-methylenetetrahydropteroylglutamate. The approach utilized in this study relies on two programs, ProACT2 and WatCH. The first software is able to categorize water molecules in a protein crystallographic structure as buried, positioned in clefts or at the surface. The other program finds, in a set of superposed homologous proteins, water molecules that occur approximately in equivalent position in each of the considered structures. These groups of molecules are referred to as 'clusters' and represent structurally conserved water molecules. Several conserved clusters of buried or cleft water molecules were found in the set of 11 bacterial SHMTs we took into account for this work. The majority of these clusters were not described previously. Possible structural and functional roles for the conserved water molecules are envisaged. This work provides a map of the conserved water molecules helpful for deciphering SHMT mechanism and for rational design of molecular engineering experiments. © The Author 2015. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.

  9. Feedback inactivation of D-serine synthesis by NMDA receptor-elicited translocation of serine racemase to the membrane

    DEFF Research Database (Denmark)

    Balan, Livia; Foltyn, Veronika N; Zehl, Martin

    2009-01-01

    translocation of SR to the plasma membrane, which dramatically reduces the enzyme activity. Membrane-bound SR isolated from rat brain is not extracted from the membrane by high detergent and salt concentration, indicating a strong association. Colocalization studies indicate that most membrane-bound SR...... is located at the plasma membrane and dendrites, with much less SR observed in other types of membrane. NMDAR activation promotes translocation of the cytosolic SR to the membrane, resulting in reduced D-serine synthesis, and this effect is averted by blockade of NMDARs. In primary neuronal cultures, SR...... translocation to the membrane is blocked by a palmitoylation inhibitor, indicating that membrane binding is mediated by fatty acid acylation of SR. In agreement, we found that SR is acylated in transfected neuroblastoma cells using [(3)H]palmitate or [(3)H]octanoic acid as precursors. In contrast to classical S...

  10. Neonatal disruption of serine racemase causes schizophrenia-like behavioral abnormalities in adulthood: clinical rescue by d-serine.

    Directory of Open Access Journals (Sweden)

    Hiroko Hagiwara

    Full Text Available D-Serine, an endogenous co-agonist of the N-methyl-D-aspartate (NMDA receptor, is synthesized from L-serine by serine racemase (SRR. Given the role of D-serine in both neurodevelopment and the pathophysiology of schizophrenia, we examined whether neonatal disruption of D-serine synthesis by SRR inhibition could induce behavioral abnormalities relevant to schizophrenia, in later life.Neonatal mice (7-9 days were injected with vehicle or phenazine methosulfate (Met-Phen: 3 mg/kg/day, an SRR inhibitor. Behavioral evaluations, such as spontaneous locomotion, novel object recognition test (NORT, and prepulse inhibition (PPI were performed at juvenile (5-6 weeks old and adult (10-12 weeks old stages. In addition, we tested the effects of D-serine on PPI deficits in adult mice after neonatal Met-Phen exposure. Finally, we assessed whether D-serine could prevent the onset of schizophrenia-like behavior in these mice. Neonatal Met-Phen treatment reduced D-serine levels in the brain, 24 hours after the final dose. Additionally, this treatment caused behavioral abnormalities relevant to prodromal symptoms in juveniles and to schizophrenia in adults. A single dose of D-serine improved PPI deficits in adult mice. Interestingly, chronic administration of D-serine (900 mg/kg/day from P35 to P70 significantly prevented the onset of PPI deficits after neonatal Met-Phen exposure.This study shows that disruption of D-serine synthesis during developmental stages leads to behavioral abnormalities relevant to prodromal symptoms and schizophrenia, in later life. Furthermore, early pharmacological intervention with D-serine may prevent the onset of psychosis in adult.

  11. Kazal-type serine proteinase inhibitors in the midgut of Phlebotomus papatasi

    Directory of Open Access Journals (Sweden)

    Leah Theresa Sigle

    2013-09-01

    Full Text Available Sandflies (Diptera: Psychodidae are important disease vectors of parasites of the genus Leishmania, as well as bacteria and viruses. Following studies of the midgut transcriptome of Phlebotomus papatasi, the principal vector of Leishmania major, two non-classical Kazal-type serine proteinase inhibitors were identified (PpKzl1 and PpKzl2. Analyses of expression profiles indicated that PpKzl1 and PpKzl2 transcripts are both regulated by blood-feeding in the midgut of P. papatasi and are also expressed in males, larva and pupa. We expressed a recombinant PpKzl2 in a mammalian expression system (CHO-S free style cells that was applied to in vitro studies to assess serine proteinase inhibition. Recombinant PpKzl2 inhibited α-chymotrypsin to 9.4% residual activity and also inhibited α-thrombin and trypsin to 33.5% and 63.9% residual activity, suggesting that native PpKzl2 is an active serine proteinase inhibitor and likely involved in regulating digestive enzymes in the midgut. Early stages of Leishmania are susceptible to killing by digestive proteinases in the sandfly midgut. Thus, characterising serine proteinase inhibitors may provide new targets and strategies to prevent transmission of Leishmania.

  12. Induction of viral, 7-methyl-guanosine cap-independent translation and oncolysis by mitogen-activated protein kinase-interacting kinase-mediated effects on the serine/arginine-rich protein kinase.

    Science.gov (United States)

    Brown, Michael C; Bryant, Jeffrey D; Dobrikova, Elena Y; Shveygert, Mayya; Bradrick, Shelton S; Chandramohan, Vidyalakshmi; Bigner, Darell D; Gromeier, Matthias

    2014-11-01

    Protein synthesis, the most energy-consuming process in cells, responds to changing physiologic priorities, e.g., upon mitogen- or stress-induced adaptations signaled through the mitogen-activated protein kinases (MAPKs). The prevailing status of protein synthesis machinery is a viral pathogenesis factor, particularly for plus-strand RNA viruses, where immediate translation of incoming viral RNAs shapes host-virus interactions. In this study, we unraveled signaling pathways centered on the ERK1/2 and p38α MAPK-interacting kinases MNK1/2 and their role in controlling 7-methyl-guanosine (m(7)G) "cap"-independent translation at enterovirus type 1 internal ribosomal entry sites (IRESs). Activation of Raf-MEK-ERK1/2 signals induced viral IRES-mediated translation in a manner dependent on MNK1/2. This effect was not due to MNK's known functions as eukaryotic initiation factor (eIF) 4G binding partner or eIF4E(S209) kinase. Rather, MNK catalytic activity enabled viral IRES-mediated translation/host cell cytotoxicity through negative regulation of the Ser/Arg (SR)-rich protein kinase (SRPK). Our investigations suggest that SRPK activity is a major determinant of type 1 IRES competency, host cell cytotoxicity, and viral proliferation in infected cells. We are targeting unfettered enterovirus IRES activity in cancer with PVSRIPO, the type 1 live-attenuated poliovirus (PV) (Sabin) vaccine containing a human rhinovirus type 2 (HRV2) IRES. A phase I clinical trial of PVSRIPO with intratumoral inoculation in patients with recurrent glioblastoma (GBM) is showing early promise. Viral translation proficiency in infected GBM cells is a core requirement for the antineoplastic efficacy of PVSRIPO. Therefore, it is critically important to understand the mechanisms controlling viral cap-independent translation in infected host cells. Copyright © 2014, American Society for Microbiology. All Rights Reserved.

  13. Robotics and Automation Activities at the Savannah River Site: A Site Report for SUBWOG 39F

    International Nuclear Information System (INIS)

    Teese, G.D.

    1995-01-01

    The Savannah River Site has successfully used robots, teleoperators, and remote video to reduce exposure to ionizing radiation, improve worker safety, and improve the quality of operations. Previous reports have described the use of mobile teleoperators in coping with a high level liquid waste spill, the removal of highly contaminated equipment, and the inspection of nuclear reactor vessels. This report will cover recent applications at the Savannah River, as well as systems which SRS has delivered to other DOE site customers

  14. Site characterization activities at Stripa and other Swedish projects

    International Nuclear Information System (INIS)

    Ahlstroehm, P.E.

    1991-01-01

    The Swedish research programme concerning spent nuclear fuel disposal aims for submitting a siting license application around the year 2000. An important step towards that goal will be the detailed characterization of at least two potential sites in late 1990s. In preparation for such characterization several research projects are conducted. One is the international Stripa Project that includes a site characterization and validation project for a small size granite rock body. The Stripa work also includes further development of instrumentation and measurement techniques. Another project is the Finnsjoen Fracture Zone Project, which is characterizing a subhorizontal zone at depths from 100 to 350 meters. The third project is the new Swedish Hard Rock Laboratory planned at the site of the Oskarshamn nuclear power plant. The preinvestigations and construction of this laboratory include major efforts in development, application and validation of site characterization methodology. (author) 6 figs., 9 refs

  15. Implantation Serine Proteinases heterodimerize and are critical in hatching and implantation

    Directory of Open Access Journals (Sweden)

    Meng Guoliang

    2006-12-01

    Full Text Available Abstract Background We have recently reported the expression of murine Implantation Serine Proteinase genes in pre-implantation embryos (ISP1 and uterus (ISP1 and ISP2. These proteinases belong to the S1 proteinase family and are similar to mast cell tryptases, which function as multimers. Results Here, we report the purification and initial characterization of ISP1 and 2 with respect to their physico-chemical properties and physiological function. In addition to being co-expressed in uterus, we show that ISP1 and ISP2 are also co-expressed in the pre-implantation embryo. Together, they form a heterodimer with an approximate molecular weight of 63 kD. This complex is the active form of the enzyme, which we have further characterized as being trypsin-like, based on substrate and inhibitor specificities. In addition to having a role in embryo hatching and outgrowth, we demonstrate that ISP enzyme is localized to the site of embryo invasion during implantation and that its activity is important for successful implantation in vivo. Conclusion On the basis of similarities in structural, chemical, and functional properties, we suggest that this ISP enzyme complex represents the classical hatching enzyme, strypsin. Our results demonstrate a critical role for ISP in embryo hatching and implantation.

  16. Intramolecular Cooperativity in the Reaction of Diacyl Phosphates with Serine ß-Lactamases

    Science.gov (United States)

    Majumdar, Sudipta; Pratt, R.F.

    2010-01-01

    Asymmetric diaroyl phosphates (ArCOOPO2−OCOAr′, where Ar = Ph, Ar′ = 4-biphenyl, 2-benzothiophenyl and 2-benzofuranyl), have been prepared, evaluated as serine (classes A, C, D) β-lactamase inhibitors, and compared with respect to the latter with their symmetric “parents”, where Ar = Ar′. The asymmetric compounds, in general, were found to react with the β-lactamases in two modes, corresponding to different orientations with respect to the active site, whereby either of the two aroyl groups may acylate the enzyme to form two different inert acyl-enzymes, E-COAr and E-COAr′. In all cases, the asymmetric compounds, in one orientation, react more rapidly with the enzymes tested than one symmetrical “parent” but not both. From comparisons of activation free energy differences, it was found that the changes in free energy on changing from one aryl group to another, in either the acyl group or the leaving group, were not additive, i.e. that the effect of changing one aroyl group to another depended on the leaving group and vice versa. Thus, intramolecular cooperativity between the aroyl groups must exist, arising either from direct interaction between them or from protein-mediated interaction, or from a combination of both. Such cooperativity brings fresh opportunities and challenges to the search for novel ß-lactamase inhibitors. PMID:19678666

  17. Using Carbohydrate Interaction Assays to Reveal Novel Binding Sites in Carbohydrate Active Enzymes

    DEFF Research Database (Denmark)

    Cockburn, Darrell; Wilkens, Casper; Dilokpimol, Adiphol

    2016-01-01

    Carbohydrate active enzymes often contain auxiliary binding sites located either on independent domains termed carbohydrate binding modules (CBMs) or as so-called surface binding sites (SBSs) on the catalytic module at a certain distance from the active site. The SBSs are usually critical...

  18. Overexpression of the methanol dehydrogenase gene mxaF in Methylobacterium sp. MB200 enhances L-serine production.

    Science.gov (United States)

    Chao, H; Wu, B; Shen, P

    2015-10-01

    Increase in L-serine production is of interest for industry. Here, we describe a metabolic engineering approach to increase the production of L-serine in a methylotrophic bacterium. mxaF, the gene encoding the large subunit of a methanol dehydrogenase, was cloned from Methylobacterium sp. MB200 through transposon mutagenesis. Deletion of mxaF gene prevented the strain to grow on methanol, suggesting that mxaF is involved in methanol metabolism. Overexpression of mxaF gene in the strain MB200 resulted in a fivefold increase in methanol dehydrogenase activity compared to the wild-type. Resting cell assays showed that the recombinant strain accumulated 6·6 mg ml(-1) L-serine in 72 h with 30 mg ml(-1) wet cells from 50 mg ml(-1) glycine and 50 mg ml(-1) methanol, representing a 1·5-fold increment for L-serine production in contrast to the wild-type strain. These results demonstrate that the potential for improving the production of L-serine can be achieved by overexpressing mxaF gene in methylotrophic bacteria. The amount of L-serine produced each year worldwide is relatively small compared with the amounts of the other amino acids and hence it is in great demand. Here, we describe a metabolic engineering approach to increase the production of L-serine in a methylotrophic bacterium Methylobacterium sp. MB200. The result demonstrates that raising the output of L-serine can be achieved by overexpressing mxaF gene in methylotrophic bacteria. © 2015 The Society for Applied Microbiology.

  19. Control of Recombination Directionality by the Listeria Phage A118 Protein Gp44 and the Coiled-Coil Motif of Its Serine Integrase.

    Science.gov (United States)

    Mandali, Sridhar; Gupta, Kushol; Dawson, Anthony R; Van Duyne, Gregory D; Johnson, Reid C

    2017-06-01

    The serine integrase of phage A118 catalyzes integrative recombination between attP on the phage and a specific attB locus on the chromosome of Listeria monocytogenes , but it is unable to promote excisive recombination between the hybrid attL and attR sites found on the integrated prophage without assistance by a recombination directionality factor (RDF). We have identified and characterized the phage-encoded RDF Gp44, which activates the A118 integrase for excision and inhibits integration. Gp44 binds to the C-terminal DNA binding domain of integrase, and we have localized the primary binding site to be within the mobile coiled-coil (CC) motif but distinct from the distal tip of the CC that is required for recombination. This interaction is sufficient to inhibit integration, but a second interaction involving the N-terminal end of Gp44 is also required to activate excision. We provide evidence that these two contacts modulate the trajectory of the CC motifs as they extend out from the integrase core in a manner dependent upon the identities of the four att sites. Our results support a model whereby Gp44 shapes the Int-bound complexes to control which att sites can synapse and recombine. IMPORTANCE Serine integrases mediate directional recombination between bacteriophage and bacterial chromosomes. These highly regulated site-specific recombination reactions are integral to the life cycle of temperate phage and, in the case of Listeria monocytogenes lysogenized by A118 family phage, are an essential virulence determinant. Serine integrases are also utilized as tools for genetic engineering and synthetic biology because of their exquisite unidirectional control of the DNA exchange reaction. Here, we identify and characterize the recombination directionality factor (RDF) that activates excision and inhibits integration reactions by the phage A118 integrase. We provide evidence that the A118 RDF binds to and modulates the trajectory of the long coiled-coil motif that

  20. cDNA cloning of glucose-6-phosphate isomerase from crucian carp (Carassius carassius) and expression of the active region as myofibril-bound serine proteinase inhibitor in Escherichia coli.

    Science.gov (United States)

    Han, Long; Cao, Min-Jie; Shi, Chao-lan; Wei, Xiao-Nan; Li, Huan; Du, Cui-Hong

    2014-02-01

    Glucose-6-phosphate isomerase (GPI) (EC 5.3.1.9) can act as a myofibril-bound serine proteinase (MBSP) inhibitor (MBSPI) in fish. In order to better understand the biological information of the GPI and its functional domain for inhibiting MBSP, the cDNA of GPI was cloned from crucian carp (Carassius carassius) with RT-PCR, nested-PCR and 3'-RACE. The result of sequencing showed that the GPI cDNA had an open reading frame of 1662bp encoding 553 amino acid residues. After constructing and comparing the three-dimensional structures of GPI and MBSP, the middle fragment of crucian carp GPI (GPI-M) was predicted as a functional domain for inhibiting MBSP. Then the crucian carp GPI-M gene was cloned and expressed in Escherichia coli. SDS-polyacrylamide gel electrophoresis (SDS-PAGE) showed that the recombinant GPI-M (rGPI-M) with molecular mass of approximately 21kDa in the form of inclusion bodies. The rGPI-M was obtained at an electrophoresis level purity of approximately 95% after denaturation and dialysis renaturation. Copyright © 2013 Elsevier Inc. All rights reserved.

  1. D-serine : The right or wrong isoform?

    NARCIS (Netherlands)

    Fuchs, Sabine A; Berger, Ruud; de Koning, Tom J

    2011-01-01

    Only recently, d-amino acids have been identified in mammals. Of these, d-serine has been most extensively studied. d-Serine was found to play an important role as a neurotransmitter in the human central nervous system (CNS) by binding to the N-methyl-d-aspartate receptor (NMDAr), similar to

  2. Serine protease isoforms in Gloydius intermedius venom: Full sequences, molecular phylogeny and evolutionary implications.

    Science.gov (United States)

    Yang, Zhang-Min; Yu, Hui; Liu, Zhen-Zhen; Pei, Jian-Zhu; Yang, Yu-E; Yan, Su-Xian; Zhang, Cui; Zhao, Wen-Long; Wang, Zhe-Zhi; Wang, Ying-Ming; Tsai, Inn-Ho

    2017-07-05

    Nine distinct venom serine proteases (vSPs) of Gloydius intermedius were studied by transcriptomic, sub-proteomic and phylogenetic analyses. Their complete amino acid sequences were deduced after Expression Sequence Tag (EST) analyses followed by cDNA cloning and sequencing. These vSPs appear to be paralogs and contain the catalytic triads and 1-4 potential N-glycosylation sites. Their relative expression levels evaluated by qPCR were grossly consistent with their EST hit-numbers. The major vSPs were purified by HPLC and their N-terminal sequences matched well to the deduced sequences, while fragments of the minor vSPs were detected by LC-MS/MS identification. Specific amidolytic activities of the fractions from HPLC and anion exchange separation were assayed using four chromogenic substrates, respectively. Molecular phylogenetic tree based on the sequences of these vSPs and their orthologs revealed six major clusters, one of them covered four lineages of plasminogen activator like vSPs. N-glycosylation patterns and variations for the vSPs are discussed. The high sequence similarities between G. intermedius vSPs and their respective orthologs from American pitvipers suggest that most of the isoforms evolved before Asian pitvipers migrated to the New World. Our results also indicate that the neurotoxic venoms contain more kallikrein-like vSPs and hypotensive components than the hemorrhagic venoms. Full sequences and expression levels of nine paralogous serine proteases (designated as GiSPs) of Gloydius intermedius venom have been studied. A kallikrein-like enzyme is most abundant and four isoforms homologous to venom plasminogen-activators are also expressed in this venom. Taken together, the present and previous data demonstrate that the neurotoxic G. intermedius venoms contain more hypotensive vSPs relative to other hemorrhagic pitviper venoms and the pitviper vSPs are highly versatile and diverse. Their structure-function relationships remain to be explored and

  3. Disulfide bond within mu-calpain active site inhibits activity and autolysis.

    Science.gov (United States)

    Lametsch, René; Lonergan, Steven; Huff-Lonergan, Elisabeth

    2008-09-01

    Oxidative processes have the ability to influence mu-calpain activity. In the present study the influence of oxidation on activity and autolysis of mu-calpain was examined. Furthermore, LC-MS/MS analysis was employed to identify and characterize protein modifications caused by oxidation. The results revealed that the activity of mu-calpain is diminished by oxidation with H2O2 in a reversible manner involving cysteine and that the rate of autolysis of mu-calpain concomitantly slowed. The LC-MS/MS analysis of the oxidized mu-calpain revealed that the amino acid residues 105-133 contained a disulfide bond between Cys(108) and Cys(115). The finding that the active site cysteine in mu-calpain is able to form a disulfide bond has, to our knowledge, not been reported before. This could be part of a unique oxidation mechanism for mu-calpain. The results also showed that the formation of the disulfide bond is limited in the control (no oxidant added), and further limited in a concentration-dependent manner when beta-mercaptoethanol is added. However, the disulfide bond is still present to some extent in all conditions indicating that the active site cysteine is potentially highly susceptible to the formation of this intramolecular disulfide bond.

  4. Calpain 3 Is Activated through Autolysis within the Active Site and Lyses Sarcomeric and Sarcolemmal Components

    Science.gov (United States)

    Taveau, Mathieu; Bourg, Nathalie; Sillon, Guillaume; Roudaut, Carinne; Bartoli, Marc; Richard, Isabelle

    2003-01-01

    Calpain 3 (Capn3) is known as the skeletal muscle-specific member of the calpains, a family of intracellular nonlysosomal cysteine proteases. This enigmatic protease has many unique features among the calpain family and, importantly, mutations in Capn3 have been shown to be responsible for limb girdle muscular dystrophy type 2A. Here we demonstrate that the Capn3 activation mechanism is similar to the universal activation of caspases and corresponds to an autolysis within the active site of the protease. We undertook a search for substrates in immature muscle cells, as several lines of evidence suggest that Capn3 is mostly in an inactive state in muscle and needs a signal to be activated. In this model, Capn3 proteolytic activity leads to disruption of the actin cytoskeleton and disorganization of focal adhesions through cleavage of several endogenous proteins. In addition, we show that titin, a previously identified Capn3 partner, and filamin C are further substrates of Capn3. Finally, we report that Capn3 colocalizes in vivo with its substrates at various sites along cytoskeletal structures. We propose that Capn3-mediated cleavage produces an adaptive response of muscle cells to external and/or internal stimuli, establishing Capn3 as a muscle cytoskeleton regulator. PMID:14645524

  5. Evolutional and functional analysis of a serine protease in Spodoptera litura.

    Science.gov (United States)

    Yang, Li; Tang, Zhuo; Liu, Wanfei; Xiao, Jingfa; Hu, Songnian; Yang, Li; Liu, Wanfei; Deng, Huimin; Feng, Qili

    2012-11-01

    Spodoptera litura is a threatening agricultural insect in tropical and subtropical areas and accounts for tremendous annual crop losses. As seen in virtually all insect species, serine proteases (SPs) are crucial to S. litura. The expression pattern of SPs from the midgut of S. litura was studied through expressed sequence tags (ESTs) analysis. One of SP (SlSP1) was chosen for detailed study, because the expression of the gene was midgut and larvae specific. SlSP1 was conducted as a model of its evolution, structure, and potential binding activity with corresponding substrates. SlSP1 is composed of 255 amino acids including a signal peptide at N-terminal followed by a putative activation peptide and the mature protein along with five putative phosphorylation sites, three disulphide bridges, and two N-glycosylation positions. At least nine conserved motifs were obtained in multiple sequence alignments. Some conserved residues, such as the catalytic triad His84, Asp127, and Ser229 as well as six cysteines at position 66, 82, 194, 211, 223, and 247, were examined. After homology modeling and molecular dynamics simulation, the resultant three-dimensional (3D) structure of SlSP1 was docked with the substrates 2PTC-Arg and 2PTC-Lys, respectively. Molecular Mechanic/Poisson-Boltzmann surface area analysis was applied to anticipate optimal binding mode and crucial active sites of this enzyme. The residues Trp28, Gly187, Aso188, Arg249, Ile250, Lys246, and Lys278 are crucial for the substrate binding and molecule process. This information can be used in logical design of SPs inhibitors. New inhibitors may be a basis for development of a new pest control technology. © 2012 Wiley Periodicals, Inc.

  6. Human population and activities in Forsmark. Site description

    International Nuclear Information System (INIS)

    Miliander, Sofia; Punakivi, Mari; Kylaekorpi, Lasse; Rydgren, Bernt

    2004-12-01

    The Swedish Nuclear Fuel and Waste Management Co (SKB) is in the process of selecting a safe and environmentally acceptable location for a deep repository of radioactive waste. Two alternative locations are under investigation. These are Forsmark, Oesthammars kommun (kommun = municipality) and Simpevarp/Laxemar, Oskarshamns kommun. SKB has expressed the importance of describing the humans and their activities in these areas and therefore has this synthesis concerning the human population in Forsmark been produced.The description is a statistical synthesis, mainly based upon statistical data from SCB (Statistics Sweden) that has been collected, processed and analysed. The statistical data has not been verified through site inspections and interviews. When using statistical data, it is advisable to note that the data becomes more unreliable if the areas are small, with small populations.The data in this description is essential for future evaluations of the impact on the environment and its human population (Environmental Impact Assessments). The data is also important when modelling the potential flows of radio nuclides and calculating the risk of exposure in future safety assessments.The actual area for the study is in this report called 'the Forsmark area', an area of 19.5 km 2 near Forsmark nuclear power plant. The land use in the Forsmark area differs notably from the land use in Uppsala laen (laen = county). Only 0.04% of the total area is developed (built-up) compared to 4.9% in Uppsala laen and only 4% is agricultural land compared to 25% in the county. Furthermore, there are far more forest, wetlands and water areas in the Forsmark area. The forest area represents as much as 72.5% of the total area.The Forsmark area is uninhabited, and its surroundings are very sparsely populated. In 2002, the population density in Forsmark was 1.8 inhabitants per square kilometre, which was 24 times lower than in Uppsala laen. The population density in the parish has been

  7. Human population and activities in Forsmark. Site description

    Energy Technology Data Exchange (ETDEWEB)

    Miliander, Sofia; Punakivi, Mari; Kylaekorpi, Lasse; Rydgren, Bernt [SwedPower AB, Stockholm (Sweden)

    2004-12-01

    The Swedish Nuclear Fuel and Waste Management Co (SKB) is in the process of selecting a safe and environmentally acceptable location for a deep repository of radioactive waste. Two alternative locations are under investigation. These are Forsmark, Oesthammars kommun (kommun = municipality) and Simpevarp/Laxemar, Oskarshamns kommun. SKB has expressed the importance of describing the humans and their activities in these areas and therefore has this synthesis concerning the human population in Forsmark been produced.The description is a statistical synthesis, mainly based upon statistical data from SCB (Statistics Sweden) that has been collected, processed and analysed. The statistical data has not been verified through site inspections and interviews. When using statistical data, it is advisable to note that the data becomes more unreliable if the areas are small, with small populations.The data in this description is essential for future evaluations of the impact on the environment and its human population (Environmental Impact Assessments). The data is also important when modelling the potential flows of radio nuclides and calculating the risk of exposure in future safety assessments.The actual area for the study is in this report called 'the Forsmark area', an area of 19.5 km{sup 2} near Forsmark nuclear power plant. The land use in the Forsmark area differs notably from the land use in Uppsala laen (laen = county). Only 0.04% of the total area is developed (built-up) compared to 4.9% in Uppsala laen and only 4% is agricultural land compared to 25% in the county. Furthermore, there are far more forest, wetlands and water areas in the Forsmark area. The forest area represents as much as 72.5% of the total area.The Forsmark area is uninhabited, and its surroundings are very sparsely populated. In 2002, the population density in Forsmark was 1.8 inhabitants per square kilometre, which was 24 times lower than in Uppsala laen. The population density in the

  8. From phosphatases to vanadium peroxidases: a similar architecture of the active site

    NARCIS (Netherlands)

    Hemrika, W.; Renirie, R.; Dekker, H. L.; Barnett, P.; Wever, R.

    1997-01-01

    We show here that the amino acid residues contributing to the active sites of the vanadate containing haloperoxidases are conserved within three families of acid phosphatases; this suggests that the active sites of these enzymes are very similar. This is confirmed by activity measurements showing

  9. Identification of a novel phosphorylation site on TBC1D4 regulated by AMP-activated protein kinase in skeletal muscle

    DEFF Research Database (Denmark)

    Treebak, Jonas Thue; Taylor, Eric B.; Witczak, Carol A.

    2010-01-01

    TBC1D4 (also known as AS160) regulates GLUT4 translocation and glucose uptake in adipocytes and skeletal muscle. Its mode of action involves phosphorylation of Serine (S)/Threonine (T) residues by upstream kinases resulting in inactivation of Rab-GAP activity leading to GLUT4 mobilization. The ma...

  10. Use of V agents and V-analogue compounds to probe the active site of atypical butyrylcholinesterase from Oryzias latipes.

    Science.gov (United States)

    Kirkpatrick, Melanie G; diTargiani, Robert C; Sweeney, Richard E; Otto, Tamara C

    2016-11-25

    The atypical butyrylcholinesterase (aBuChE) from Oryzias latipes shares approximately 65% sequence similarity to both acetylcholinesterase and butyrylcholinesterase and was studied for its capacity to spontaneously reactivate following inhibition by organophosphorus nerve agents. Like other cholinesterases, aBuChE was inhibited by all G- and V-type nerve agents. Interestingly, aBuChE was able to undergo spontaneous reactivation after inhibition with VR (t 1/2  = 5.5 ± 0.2 h). Mass spectrometry of aBuChE after VR inhibition confirmed the presence of a covalently bound adduct of the size expected for non-aged VR on the peptide containing the active site serine. To understand the effect of substrate volume on rates of reactivation, the capacity of aBuChE to bind and spontaneously reactivate after inhibition with five V-agent analogues was examined. No appreciable reactivation was detected for enzyme inhibited by V2 (VX with O-isopropyl on retained group), V4 (VX with N-diethyl leaving group termination), or V5 (VX with N-dimethyl leaving group termination). Minimal reactivation was detected with V1 (VX with O-propyl on retained group). Conversely, spontaneous reactivation was observed when aBuChE was inhibited by V3 (VX with O-isobutyl on retained group; t 1/2  = 6.3 ± 0.4 h). The data suggest that the ability of aBuChE to spontaneously reactivate after inhibition by V-agent analogues is related to the structure of the retained group. These results provide structural information that may shed light on the design of improved small molecule reactivators of nerve agent-inhibited acetylcholinesterase or butyrylcholinesterase, and further suggest that re-engineering the active site of a cholinesterase could result in enzymes with clinically relevant rates of nerve agent hydrolysis. Published by Elsevier Ireland Ltd.

  11. Enhancement of transglutaminase activity by NMR identification of its flexible residues affecting the active site.

    Science.gov (United States)

    Shimba, Nobuhisa; Shinohara, Mina; Yokoyama, Kei ichi; Kashiwagi, Tatsuki; Ishikawa, Kohki; Ejima, Daisuke; Suzuki, Ei ichiro

    2002-04-24

    Incorporation of inter- or intramolecular covalent cross-links into food proteins with microbial transglutaminase (MTG) improves the physical and textural properties of many food proteins, such as tofu, boiled fish paste, and sausage. By using nuclear magnetic resonance, we have shown that the residues exhibiting relatively high flexibility in MTG are localized in the N-terminal region; however, the N-terminal region influences the microenvironment of the active site. These results suggest that the N-terminal region is not of primary importance for the global fold, but influences the substrate binding. Therefore, in order to increase the transglutaminase activity, the N-terminal residues were chosen as candidates for site-directed replacement and deletion. We obtained several mutants with higher activity, del1-2, del1-3, and S2R. We propose a strategy for enzyme engineering targeted toward flexible regions involved in the enzymatic activity. In addition, we also briefly describe how the number of glutamine residues in a substrate protein can be increased by mixing more than two kinds of TGases with different substrate specificities.

  12. Aromatic boronic acids as probes of the catalytic site of human plasma lecithin-cholesterol acyltransferase.

    Science.gov (United States)

    Jauhiainen, M; Ridgway, N D; Dolphin, P J

    1987-04-03

    We have recently proposed a catalytic mechanism for human plasma lecithin-cholesterol acyltransferase (EC 2.3.1.43) (J. Biol. Chem. (1986) 261, 7032-7043), implicating single serine and histidine residues in phosphatidylcholine cleavage and two cysteine residues in cholesterol esterification. We now confirm the involvement of serine and histidine in catalysing the phospholipase A2 action of lecithin-cholesterol acyltransferase by demonstrating the inhibition of this activity by phenylboronic acid (Ki = 1.23 mM) and m-aminophenylboronic acid (Ki = 2.32 mM), inhibitors of known serine/histidine hydrolases. The specificity of the interaction of aromatic boronic acids with catalytic serine and histidine residues and the putative formation of a tetrahedral adduct between boron and the lecithin-cholesterol acyltransferase serine hydroxyl group which is similar to the transition-state intermediate formed between phosphatidylcholine and the catalytic serine residue was suggested by: substrate protection against inhibition by phenylboronic acids; a much reduced incorporation of phenylmethane[35S]sulphonyl fluoride into the enzyme in the presence of phenylboronic acid; the lack of interaction of histidine- or serine-modified enzyme with immobilized phenylboronic acid in the presence of glycerol (Ve/Vo = 2.7 and 2.3 respectively) when compared to the native enzyme (Ve/Vo = 5.25). Fatty acyl-lecithin-cholesterol acyltransferase, produced by incubation of the enzyme with a lecithin-apolipoprotein A-I proteoliposome substrate, was not retarded upon the sorbent column (Ve/Vo = 1.5). Modification of the enzyme's two free cysteine residues with 5,5'-dithiobis(2-nitrobenzoic acid) or potassium ferricyanide reduced (Ve/Vo = 3.5) but did not abolish retardation on the sorbent column, indicating that these modifications resulted in steric hinderance of the interaction of the boron atom with the lecithin-cholesterol acyltransferase serine hydroxyl group. These data suggest that the

  13. NMR studies of internal dynamics of serine proteinase protein inhibitors: Binding region mobilities of intact and reactive-site hydrolyzed Cucurbita maxima trypsin inhibitor (CMTI)-III of the squash family and comparison with those of counterparts of CMTI-V of the potato I family.

    Science.gov (United States)

    Liu, J; Gong, Y; Prakash, O; Wen, L; Lee, I; Huang, J K; Krishnamoorthi, R

    1998-01-01

    Serine proteinase protein inhibitors follow the standard mechanism of inhibition (Laskowski M Jr, Kato I, 1980, Annu Rev Biochem 49:593-626), whereby an enzyme-catalyzed equilibrium between intact (I) and reactive-site hydrolyzed inhibitor (I*) is reached. The hydrolysis constant, Khyd, is defined as [I*]/[I]. Here, we explore the role of internal dynamics in the resynthesis of the scissile bond by comparing the internal mobility data of intact and cleaved inhibitors belonging to two different families. The inhibitors studied are recombinant Cucurbita maxima trypsin inhibitor III (rCMTI-III; Mr 3 kDa) of the squash family and rCMTI-V (Mr approximately 7 kDa) of the potato I family. These two inhibitors have different binding loop-scaffold interactions and different Khyd values--2.4 (CMTI-III) and 9 (CMTI-V)--at 25 degrees C. The reactive-site peptide bond (P1-P1') is that between Arg5 and Ile6 in CMTI-III, and that between Lys44 and Asp45 in CMTI-V. The order parameters (S2) of backbone NHs of uniformly 15N-labeled rCMTI-III and rCMTI-III* were determined from measurements of 15N spin-lattice and spin-spin relaxation rates, and [1H]-15N steady-state heteronuclear Overhauser effects, using the model-free formalism, and compared with the data reported previously for rCMTI-V and rCMTI-V*. The backbones of rCMTI-III [(S2) = 0.71] and rCMTI-III* [(S2) = 0.63] are more flexible than those of rCMTI-V [(S2) = 0.83] and rCMTI-V* [(S2) = 0.85]. The binding loop residues, P4-P1, in the two proteins show the following average order parameters: 0.57 (rCMTI-III) and 0.44 (rCMTI-III*); 0.70 (rCMTI-V) and 0.40 (rCMTI-V*). The P1'-P4' residues, on the other hand, are associated with (S2) values of 0.56 (rCMTI-III) and 0.47 (rCMTI-III*); and 0.73 (rCMTI-V) and 0.83 (rCMTI-V*). The newly formed C-terminal (Pn residues) gains a smaller magnitude of flexibility in rCMTI-III* due to the Cys3-Cys20 crosslink. In contrast, the newly formed N-terminal (Pn' residues) becomes more flexible

  14. Models for the active site in galactose oxidase: Structure, spectra ...

    Indian Academy of Sciences (India)

    The crystal structure of the enzyme reveals that the coordination geometry of mononuclear copper(II) ion is square pyramidal, with two histidine imidazoles, a tyrosinate, and either H2O (H 7.0) or acetate (from buffer, H 4.5) in the equatorial sites and a tyrosinate ligand weakly bound in the axial position. This paper ...

  15. Informations on the active site of palmito polyphenol oxidase.

    Science.gov (United States)

    Robert, C; Cadet, F; Rouch, C; Pabion, M

    1995-01-01

    pH studies of palmito polyphenol oxidase are carried out with either 4-methylcatechol or pyrogallol as substrates. The pH profile is independent of the nature of the substrates tested. The symmetrical behaviour and the very slight differences between the values obtained suggest the existence of only one site on the molecule for o-diphenol substrates.

  16. A Highly Conserved Bacterial D-Serine Uptake System Links Host Metabolism and Virulence.

    Directory of Open Access Journals (Sweden)

    James P R Connolly

    2016-01-01

    Full Text Available The ability of any organism to sense and respond to challenges presented in the environment is critically important for promoting or restricting colonization of specific sites. Recent work has demonstrated that the host metabolite D-serine has the ability to markedly influence the outcome of infection by repressing the type III secretion system of enterohaemorrhagic Escherichia coli (EHEC in a concentration-dependent manner. However, exactly how EHEC monitors environmental D-serine is not understood. In this work, we have identified two highly conserved members of the E. coli core genome, encoding an inner membrane transporter and a transcriptional regulator, which collectively help to "sense" levels of D-serine by regulating its uptake from the environment and in turn influencing global gene expression. Both proteins are required for full expression of the type III secretion system and diversely regulated prophage-encoded effector proteins demonstrating an important infection-relevant adaptation of the core genome. We propose that this system acts as a key safety net, sampling the environment for this metabolite, thereby promoting colonization of EHEC to favorable sites within the host.

  17. Preliminary siting activities for new waste handling facilities at the Idaho National Engineering Laboratory

    Energy Technology Data Exchange (ETDEWEB)

    Taylor, D.D.; Hoskinson, R.L.; Kingsford, C.O.; Ball, L.W.

    1994-09-01

    The Idaho Waste Processing Facility, the Mixed and Low-Level Waste Treatment Facility, and the Mixed and Low-Level Waste Disposal Facility are new waste treatment, storage, and disposal facilities that have been proposed at the Idaho National Engineering Laboratory (INEL). A prime consideration in planning for such facilities is the selection of a site. Since spring of 1992, waste management personnel at the INEL have been involved in activities directed to this end. These activities have resulted in the (a) identification of generic siting criteria, considered applicable to either treatment or disposal facilities for the purpose of preliminary site evaluations and comparisons, (b) selection of six candidate locations for siting,and (c) site-specific characterization of candidate sites relative to selected siting criteria. This report describes the information gathered in the above three categories for the six candidate sites. However, a single, preferred site has not yet been identified. Such a determination requires an overall, composite ranking of the candidate sites, which accounts for the fact that the sites under consideration have different advantages and disadvantages, that no single site is superior to all the others in all the siting criteria, and that the criteria should be assigned different weighing factors depending on whether a site is to host a treatment or a disposal facility. Stakeholder input should now be solicited to help guide the final selection. This input will include (a) siting issues not already identified in the siting, work to date, and (b) relative importances of the individual siting criteria. Final site selection will not be completed until stakeholder input (from the State of Idaho, regulatory agencies, the public, etc.) in the above areas has been obtained and a strategy has been developed to make a composite ranking of all candidate sites that accounts for all the siting criteria.

  18. Preliminary siting activities for new waste handling facilities at the Idaho National Engineering Laboratory

    International Nuclear Information System (INIS)

    Taylor, D.D.; Hoskinson, R.L.; Kingsford, C.O.; Ball, L.W.

    1994-09-01

    The Idaho Waste Processing Facility, the Mixed and Low-Level Waste Treatment Facility, and the Mixed and Low-Level Waste Disposal Facility are new waste treatment, storage, and disposal facilities that have been proposed at the Idaho National Engineering Laboratory (INEL). A prime consideration in planning for such facilities is the selection of a site. Since spring of 1992, waste management personnel at the INEL have been involved in activities directed to this end. These activities have resulted in the (a) identification of generic siting criteria, considered applicable to either treatment or disposal facilities for the purpose of preliminary site evaluations and comparisons, (b) selection of six candidate locations for siting,and (c) site-specific characterization of candidate sites relative to selected siting criteria. This report describes the information gathered in the above three categories for the six candidate sites. However, a single, preferred site has not yet been identified. Such a determination requires an overall, composite ranking of the candidate sites, which accounts for the fact that the sites under consideration have different advantages and disadvantages, that no single site is superior to all the others in all the siting criteria, and that the criteria should be assigned different weighing factors depending on whether a site is to host a treatment or a disposal facility. Stakeholder input should now be solicited to help guide the final selection. This input will include (a) siting issues not already identified in the siting, work to date, and (b) relative importances of the individual siting criteria. Final site selection will not be completed until stakeholder input (from the State of Idaho, regulatory agencies, the public, etc.) in the above areas has been obtained and a strategy has been developed to make a composite ranking of all candidate sites that accounts for all the siting criteria

  19. Identification of promiscuous ene-reductase activity by mining structural databases using active site constellations.

    Science.gov (United States)

    Steinkellner, Georg; Gruber, Christian C; Pavkov-Keller, Tea; Binter, Alexandra; Steiner, Kerstin; Winkler, Christoph; Lyskowski, Andrzej; Schwamberger, Orsolya; Oberer, Monika; Schwab, Helmut; Faber, Kurt; Macheroux, Peter; Gruber, Karl

    2014-06-23

    The exploitation of catalytic promiscuity and the application of de novo design have recently opened the access to novel, non-natural enzymatic activities. Here we describe a structural bioinformatic method for predicting catalytic activities of enzymes based on three-dimensional constellations of functional groups in active sites ('catalophores'). As a proof-of-concept we identify two enzymes with predicted promiscuous ene-reductase activity (reduction of activated C-C double bonds) and compare them with known ene-reductases, that is, members of the Old Yellow Enzyme family. Despite completely different amino acid sequences, overall structures and protein folds, high-resolution crystal structures reveal equivalent binding modes of typical Old Yellow Enzyme substrates and ligands. Biochemical and biocatalytic data show that the two enzymes indeed possess ene-reductase activity and reveal an inverted stereopreference compared with Old Yellow Enzymes for some substrates. This method could thus be a tool for the identification of viable starting points for the development and engineering of novel biocatalysts.

  20. Optimization of the Conditions for Extraction of Serine Protease from Kesinai Plant (Streblus asper Leaves Using Response Surface Methodology

    Directory of Open Access Journals (Sweden)

    Md. Zaidul Islam Sarker

    2011-11-01

    Full Text Available Response surface methodology (RSM using a central composite design (CCD was employed to optimize the conditions for extraction of serine protease from kesinai (Streblus asper leaves. The effect of independent variables, namely temperature (42.5,47.5, X1, mixing time (2–6 min, X2, buffer content (0–80 mL, X3 and buffer pH (4.5–10.5, X4 on specific activity, storage stability, temperature and oxidizing agent stability of serine protease from kesinai leaves was investigated. The study demonstrated that use of the optimum temperature, mixing time, buffer content and buffer pH conditions protected serine protease during extraction, as demonstrated by low activity loss. It was found that the interaction effect of mixing time and buffer content improved the serine protease stability, and the buffer pH had the most significant effect on the specific activity of the enzyme. The most desirable conditions of 2.5 °C temperature, 4 min mixing time, 40 mL buffer at pH 7.5 was established for serine protease extraction from kesinai leaves.

  1. Inhibition of protein phosphatase 2A induces serine/threonine phosphorylation, subcellular redistribution, and functional inhibition of STAT3

    DEFF Research Database (Denmark)

    Woetmann, A; Nielsen, M; Christensen, S T

    1999-01-01

    STAT3. We show that an inhibitor of protein phosphatases (PPs) PP1/PP2A, calyculin A, induces (i) phosphorylation of STAT3 on serine and threonine residues, (ii) inhibition of STAT3 tyrosine phosphorylation and DNA binding activity, and (iii) relocation of STAT3 from the nucleus to the cytoplasm......, whereas inhibitors of serine/threonine kinases, such as mitogen-activated protein kinase-1 extracellular-regulated kinase-kinase, mitogen-activated protein p38 kinase, and phosphatidylinositol 3-kinase, did not. In conclusion, we provide evidence that PP2A plays a crucial role in the regulation of STAT3....... Similar results were obtained with other PP2A inhibitors (okadaic acid, endothall thioanhydride) but not with inhibitors of PP1 (tautomycin) or PP2B (cyclosporine A). Pretreatment with the broad serine/threonine kinase inhibitor staurosporine partly blocked the calyculin A-induced STAT3 phosphorylation...

  2. Human population and activities at Simpevarp. Site description

    International Nuclear Information System (INIS)

    Miliander, Sofia; Punakivi, Mari; Kylaekorpi, Lasse; Rydgren, Bernt

    2004-12-01

    The Swedish Nuclear Fuel and Waste Management Co (SKB) is in the process of selecting a safe and environmentally acceptable location for a deep repository of radioactive waste. Two alternative locations are under investigation. These are Forsmark, Oesthammars kommun (kommun = municipality) and Simpevarp/Laxemar, Oskarshamns kommun. SKB has expressed the importance of describing the humans and their activities in these areas and therefore has this synthesis concerning the human population in Forsmark been produced. The description is a statistical synthesis, mainly based upon statistical data from SCB (Statistics Sweden) that has been collected, processed and analysed. The statistical data has not been verified through site inspections and interviews. When using statistical data, it is advisable to note that the data becomes more unreliable if the areas are small, with small populations. The data in this description is essential for future evaluations of the impact on the environment and its human population (environmental impacts assessments). The data is also important when modelling the potential flows of radio nuclides and calculating the risk of exposure in future safety assessments. The actual area for the study is in this report called 'the Simpevarp area', an area of 127.0 km 2 near Oskarshamn nuclear power plant. The land use in Simpevarp area differs notably from the land use in Kalmar laen. The forest area is far more dominating in Simpevarp area than in Kalmar laen and it represents as much as 89% compared to 63% of the total area. Only 4.4% of the area is arable land compared to 11.6% in Kalmar laen and only 0.3% is of other type (wetlands, bare rock, quarries, pites etc) compared to 15.6% in the county. The main observation is that Simpevarp area is a sparsely populated area located in a relatively lightly populated county. In 2002, the population density was 7.4 inhabitants/km 2 , three times lower than in Kalmar laen. The demography statistics show

  3. Human population and activities at Simpevarp. Site description

    Energy Technology Data Exchange (ETDEWEB)

    Miliander, Sofia; Punakivi, Mari; Kylaekorpi, Lasse; Rydgren, Bernt [SwedPower AB, Stockholm (Sweden)

    2004-12-01

    The Swedish Nuclear Fuel and Waste Management Co (SKB) is in the process of selecting a safe and environmentally acceptable location for a deep repository of radioactive waste. Two alternative locations are under investigation. These are Forsmark, Oesthammars kommun (kommun = municipality) and Simpevarp/Laxemar, Oskarshamns kommun. SKB has expressed the importance of describing the humans and their activities in these areas and therefore has this synthesis concerning the human population in Forsmark been produced. The description is a statistical synthesis, mainly based upon statistical data from SCB (Statistics Sweden) that has been collected, processed and analysed. The statistical data has not been verified through site inspections and interviews. When using statistical data, it is advisable to note that the data becomes more unreliable if the areas are small, with small populations. The data in this description is essential for future evaluations of the impact on the environment and its human population (environmental impacts assessments). The data is also important when modelling the potential flows of radio nuclides and calculating the risk of exposure in future safety assessments. The actual area for the study is in this report called 'the Simpevarp area', an area of 127.0 km{sup 2} near Oskarshamn nuclear power plant. The land use in Simpevarp area differs notably from the land use in Kalmar laen. The forest area is far more dominating in Simpevarp area than in Kalmar laen and it represents as much as 89% compared to 63% of the total area. Only 4.4% of the area is arable land compared to 11.6% in Kalmar laen and only 0.3% is of other type (wetlands, bare rock, quarries, pites etc) compared to 15.6% in the county. The main observation is that Simpevarp area is a sparsely populated area located in a relatively lightly populated county. In 2002, the population density was 7.4 inhabitants/km{sup 2}, three times lower than in Kalmar laen. The

  4. Early Site Permit Demonstration Program: Recommendations for communication activities and public participation in the Early Site Permit Demonstration Program

    International Nuclear Information System (INIS)

    1993-01-01

    On October 24, 1992, President Bush signed into law the National Energy Policy Act of 1992. The bill is a sweeping, comprehensive overhaul of the Nation's energy laws, the first in more than a decade. Among other provisions, the National Energy Policy Act reforms the licensing process for new nuclear power plants by adopting a new approach developed by the US Nuclear Regulatory Commission (NRC) in 1989, and upheld in court in 1992. The NRC 10 CFR Part 52 rule is a three-step process that guarantees public participation at each step. The steps are: early site permit approval; standard design certifications; and, combined construction/operating licenses for nuclear power reactors. Licensing reform increases an organization's ability to respond to future baseload electricity generation needs with less financial risk for ratepayers and the organization. Costly delays can be avoided because design, safety and siting issues will be resolved before a company starts to build a plant. Specifically, early site permit approval allows for site suitability and acceptability issues to be addressed prior to an organization's commitment to build a plant. Responsibility for site-specific activities, including communications and public participation, rests with those organizations selected to try out early site approval. This plan has been prepared to assist those companies (referred to as sponsoring organizations) in planning their communications and public involvement programs. It provides research findings, information and recommendations to be used by organizations as a resource and starting point in developing their own plans

  5. Early Site Permit Demonstration Program: Recommendations for communication activities and public participation in the Early Site Permit Demonstration Program

    Energy Technology Data Exchange (ETDEWEB)

    1993-01-27

    On October 24, 1992, President Bush signed into law the National Energy Policy Act of 1992. The bill is a sweeping, comprehensive overhaul of the Nation`s energy laws, the first in more than a decade. Among other provisions, the National Energy Policy Act reforms the licensing process for new nuclear power plants by adopting a new approach developed by the US Nuclear Regulatory Commission (NRC) in 1989, and upheld in court in 1992. The NRC 10 CFR Part 52 rule is a three-step process that guarantees public participation at each step. The steps are: early site permit approval; standard design certifications; and, combined construction/operating licenses for nuclear power reactors. Licensing reform increases an organization`s ability to respond to future baseload electricity generation needs with less financial risk for ratepayers and the organization. Costly delays can be avoided because design, safety and siting issues will be resolved before a company starts to build a plant. Specifically, early site permit approval allows for site suitability and acceptability issues to be addressed prior to an organization`s commitment to build a plant. Responsibility for site-specific activities, including communications and public participation, rests with those organizations selected to try out early site approval. This plan has been prepared to assist those companies (referred to as sponsoring organizations) in planning their communications and public involvement programs. It provides research findings, information and recommendations to be used by organizations as a resource and starting point in developing their own plans.

  6. Active-Site Hydration and Water Diffusion in Cytochrome P450cam: A Highly Dynamic Process

    Energy Technology Data Exchange (ETDEWEB)

    Miao, Yinglong [ORNL; Baudry, Jerome Y [ORNL

    2011-01-01

    Long-timescale molecular dynamics simulations (300 ns) are performed on both the apo- (i.e., camphor-free) and camphor-bound cytochrome P450cam (CYP101). Water diffusion into and out of the protein active site is observed without biased sampling methods. During the course of the molecular dynamics simulation, an average of 6.4 water molecules is observed in the camphor-binding site of the apo form, compared to zero water molecules in the binding site of the substrate-bound form, in agreement with the number of water molecules observed in crystal structures of the same species. However, as many as 12 water molecules can be present at a given time in the camphor-binding region of the active site in the case of apo-P450cam, revealing a highly dynamic process for hydration of the protein active site, with water molecules exchanging rapidly with the bulk solvent. Water molecules are also found to exchange locations frequently inside the active site, preferentially clustering in regions surrounding the water molecules observed in the crystal structure. Potential-of-mean-force calculations identify thermodynamically favored trans-protein pathways for the diffusion of water molecules between the protein active site and the bulk solvent. Binding of camphor in the active site modifies the free-energy landscape of P450cam channels toward favoring the diffusion of water molecules out of the protein active site.

  7. Recovery of serine protease inhibitor from fish roes by polyethylene glycol precipitation

    Directory of Open Access Journals (Sweden)

    Hyun Ji Lee

    2016-07-01

    Full Text Available Abstract The fractionation of serine protease inhibitor (SPI from fish roe extracts was carried out using polyethylene glycol-4000 (PEG4000 precipitation. The protease inhibitory activity of extracts and PEG fractions from Alaska pollock (AP, bastard halibut (BH, skipjack tuna (ST, and yellowfin tuna (YT roes were determined against target proteases. All of the roe extracts showed inhibitory activity toward bromelain (BR, chymotrypsin (CH, trypsin (TR, papain-EDTA (PED, and alcalase (AL as target proteases. PEG fractions, which have positive inhibitory activity and high recovery (%, were the PEG1 fraction (0–5 %, w/v against cysteine proteases (BR and PA and the PEG4 fraction (20–40 %, w/v against serine proteases (CH and TR. The strongest specific inhibitory activity toward CH and TR of PEG4 fractions was AP (9278 and 1170 U/mg followed by ST (6687 and 2064 U/mg, YT (3951 and 1536 U/mg, and BH (538 and 98 U/mg. The inhibitory activity of serine protease in extracts and PEG fractions from fish roe was stronger than that of cysteine protease toward common casein substrate. Therefore, SPI is mainly distributed in fish roe and PEG fractionation effectively isolated the SPI from fish roes.

  8. Sugarcane Serine Peptidase Inhibitors, Serine Peptidases, and Clp Protease System Subunits Associated with Sugarcane Borer (Diatraea saccharalis) Herbivory and Wounding.

    Science.gov (United States)

    Medeiros, Ane H; Mingossi, Fabiana B; Dias, Renata O; Franco, Flávia P; Vicentini, Renato; Mello, Marcia O; Moura, Daniel S; Silva-Filho, Marcio C

    2016-09-01

    Sugarcane's (Saccharum spp.) response to Diatraea saccharalis (F.) (Lepidoptera: (Crambidae) herbivory was investigated using a macroarray spotted with 248 sugarcane Expressed Sequence Tags (ESTs) encoding serine peptidase inhibitors, serine peptidases. and Clp protease system subunits. Our results showed that after nine hours of herbivory, 13 sugarcane genes were upregulated and nine were downregulated. Among the upregulated genes, nine were similar to serine peptidase inhibitors and four were similar to Bowman-Birk Inhibitors (BBIs). Phylogenetic analysis revealed that these sequences belong to a phylogenetic group of sugarcane BBIs that are potentially involved in plant defense against insect predation. The remaining four upregulated genes included serine peptidases and one homolog to the Arabidopsis AAA+ chaperone subunit ClpD, which is a member of the Clp protease system. Among the downregulated genes, five were homologous to serine peptidases and four were homologous to Arabidopsis Clp subunits (three homologous to Clp AAA+ chaperones and one to a ClpP-related ClpR subunit). Although the roles of serine peptidase inhibitors in plant defenses against herbivory have been extensively investigated, the roles of plant serine peptidases and the Clp protease system represent a new and underexplored field of study. The up- and downregulated D. saccharalis genes presented in this study may be candidate genes for the further investigation of the sugarcane response to herbivory.

  9. Sugarcane Serine Peptidase Inhibitors, Serine Peptidases, and Clp Protease System Subunits Associated with Sugarcane Borer (Diatraea saccharalis) Herbivory and Wounding

    Science.gov (United States)

    Medeiros, Ane H.; Mingossi, Fabiana B.; Dias, Renata O.; Franco, Flávia P.; Vicentini, Renato; Mello, Marcia O.; Moura, Daniel S.; Silva-Filho, Marcio C.

    2016-01-01

    Sugarcane’s (Saccharum spp.) response to Diatraea saccharalis (F.) (Lepidoptera: (Crambidae) herbivory was investigated using a macroarray spotted with 248 sugarcane Expressed Sequence Tags (ESTs) encoding serine peptidase inhibitors, serine peptidases. and Clp protease system subunits. Our results showed that after nine hours of herbivory, 13 sugarcane genes were upregulated and nine were downregulated. Among the upregulated genes, nine were similar to serine peptidase inhibitors and four were similar to Bowman-Birk Inhibitors (BBIs). Phylogenetic analysis revealed that these sequences belong to a phylogenetic group of sugarcane BBIs that are potentially involved in plant defense against insect predation. The remaining four upregulated genes included serine peptidases and one homolog to the Arabidopsis AAA+ chaperone subunit ClpD, which is a member of the Clp protease system. Among the downregulated genes, five were homologous to serine peptidases and four were homologous to Arabidopsis Clp subunits (three homologous to Clp AAA+ chaperones and one to a ClpP-related ClpR subunit). Although the roles of serine peptidase inhibitors in plant defenses against herbivory have been extensively investigated, the roles of plant serine peptidases and the Clp protease system represent a new and underexplored field of study. The up- and downregulated D. saccharalis genes presented in this study may be candidate genes for the further investigation of the sugarcane response to herbivory. PMID:27598134

  10. Identification of the catalytic residues of alpha-amino acid ester hydrolase from Acetobacter turbidans by labeling and site-directed mutagenesis

    NARCIS (Netherlands)

    Polderman - Tijmes, Jolanda j.; Jekel, Peter A.; Jeronimus-Stratingh, CM; Bruins, Andries P.; van der Laan, Jan-Metske; Sonke, Theo; Janssen, Dick B.

    2002-01-01

    The alpha-amino acid ester hydrolase from Acetobacter turbidans ATCC 9325 is capable of hydrolyzing and synthesizing the side chain peptide bond in beta-lactam antibiotics. Data base searches revealed that the enzyme contains an active site serine consensus sequence Gly-X-Ser-Tyr-X-Gly that is also

  11. Diffusion-controlled reaction rates for two active sites on a sphere.

    Science.gov (United States)

    Shoup, David E

    2014-01-01

    The diffusion-limited reaction rate of a uniform spherical reactant is generalized to anisotropic reactivity. Previous work has shown that the protein model of a uniform sphere is unsatisfactory in many cases. Competition of ligands binding to two active sites, on a spherical enzyme or cell is studied analytically. The reaction rate constant is given for two sites at opposite ends of the species of interest. This is compared with twice the reaction rate for a single site. It is found that the competition between sites lowers the reaction rate over what is expected for two sites individually. Competition between sites does not show up, until the site half angle is greater than 30 degrees. Competition between sites is negligible until the site size becomes large. The competitive effect grows as theta becomes large. The maximum effect is given for theta = pi/2.

  12. D-Serine in Neuropsychiatric Disorders: New Advances

    Directory of Open Access Journals (Sweden)

    Andrea R. Durrant

    2014-01-01

    Full Text Available D-Serine (DSR is an endogenous amino acid involved in glia-synapse interactions that has unique neurotransmitter characteristics. DSR acts as obligatory coagonist at the glycine site associated with the N-methyl-D-aspartate subtype of glutamate receptors (NMDAR and has a cardinal modulatory role in major NMDAR-dependent processes including NMDAR-mediated neurotransmission, neurotoxicity, synaptic plasticity, and cell migration. Since either over- or underfunction of NMDARs may be involved in the pathophysiology of neuropsychiatric disorders; the pharmacological manipulation of DSR signaling represents a major drug development target. A first generation of proof-of-concept animal and clinical studies suggest beneficial DSR effects in treatment-refractory schizophrenia, movement, depression, and anxiety disorders and for the improvement of cognitive performance. A related developing pharmacological strategy is the indirect modification of DSR synaptic levels by use of compounds that alter the function of main enzymes responsible for DSR production and degradation. Accumulating data indicate that, during the next decade, we will witness important advances in the understanding of DSR role that will further contribute to elucidating the causes of neuropsychiatric disorders and will be instrumental in the development of innovative treatments.

  13. Spatial patterns of microbial diversity and activity in an aged creosote-contaminated site

    OpenAIRE

    Mukherjee, Shinjini; Juottonen, Heli; Siivonen, Pauli; Lloret Quesada, Cosme; Tuomi, Pirjo; Pulkkinen, Pertti; Yrjälä, Kim

    2014-01-01

    Restoration of polluted sites via in situ bioremediation relies heavily on the indigenous microbes and their activities. Spatial heterogeneity of microbial populations, contaminants and soil chemical parameters on such sites is a major hurdle in optimizing and implementing an appropriate bioremediation regime. We performed a grid-based sampling of an aged creosote-contaminated site followed by geostatistical modelling to illustrate the spatial patterns of microbial diversity and activity and ...

  14. Probing the putative active site of YjdL

    DEFF Research Database (Denmark)

    Jensen, Johanne Mørch; Ismat, Fouzia; Szakonyi, Gerda

    2012-01-01

    pocket that opens towards the extracellular space. The C-terminal side chain faces in the opposite direction into a sub pocket that faces the cytoplasm. These data indicated a stabilizing effect on a bulky N-terminal residue by an Ala281Phe variant and on the dipeptide backbone by Trp278...... with Glu388, a preliminary orientation model of a dipeptide in the YjdL cavity is presented. Single site mutations of particularly Ala281 and Trp278 support the presented orientation. A dipeptide bound in the cavity of YjdL appears to be oriented such that the N-terminal side chain protrudes into a sub...

  15. Assessment of former uranium sites and their ongoing remediation activities

    International Nuclear Information System (INIS)

    Khakimov, N.; Adkhamov, A.; Nazarov, Kh.M.; Mahmadov, T.; Mirsaidov, I.U.

    2012-01-01

    Carried out analysis on tailing's buildings operation shows that period for engineer barrier service, taking into account any catastrophic natural impacts, is too little in comparison with life-time of long-live radionuclides. Priorities should be defined by danger degree and isolation costs (protection optimization), therefore uncommon, non-traditional methods, developed taking into account natural factors for long-live waste (radionuclides) isolation are necessary. That's why, it is necessary to carry out specialized research and development, design and exploratory and other works on monitoring of social-ecological condition of these sites, as well as on demographic public diseases, living in these regions.

  16. A proposed definition of the 'activity' of surface sites on lactose carriers for dry powder inhalation

    NARCIS (Netherlands)

    Grasmeijer, Floris; Frijlink, Henderik W.; de Boer, Anne

    2014-01-01

    A new definition of the activity of surface sites on lactose carriers for dry powder inhalation is proposed which relates to drug detachment during dispersion. The new definition is expected to improve the understanding of 'carrier surface site activity', which stimulates the unambiguous

  17. 78 FR 8190 - Commercial Wind Leasing and Site Assessment Activities on the Atlantic Outer Continental Shelf...

    Science.gov (United States)

    2013-02-05

    ... DEPARTMENT OF THE INTERIOR Bureau of Ocean Energy Management [Docket No. BOEM-2012-0090] Commercial Wind Leasing and Site Assessment Activities on the Atlantic Outer Continental Shelf (OCS) Offshore... Assessment (EA) for Commercial Wind Leasing and Site Assessment Activities on the OCS Offshore North Carolina...

  18. Facebook, Twitter Activities Sites, Location and Students' Interest in Learning

    Science.gov (United States)

    Igbo, J. N.; Ezenwaji, Ifeyinwa; Ajuziogu, Christiana U.

    2018-01-01

    This study was carried out to ascertain the influence of social networking sites activities (twitter and Facebook) on secondary school students' interest in learning It also considered the impact of these social networking sites activities on location of the students. Two research questions and two null hypotheses guided the study. Mean and…

  19. Prediction of Active Site and Distal Residues in E. coli DNA Polymerase III alpha Polymerase Activity.

    Science.gov (United States)

    Parasuram, Ramya; Coulther, Timothy A; Hollander, Judith M; Keston-Smith, Elise; Ondrechen, Mary Jo; Beuning, Penny J

    2018-02-20

    The process of DNA replication is carried out with high efficiency and accuracy by DNA polymerases. The replicative polymerase in E. coli is DNA Pol III, which is a complex of 10 different subunits that coordinates simultaneous replication on the leading and lagging strands. The 1160-residue Pol III alpha subunit is responsible for the polymerase activity and copies DNA accurately, making one error per 10 5 nucleotide incorporations. The goal of this research is to determine the residues that contribute to the activity of the polymerase subunit. Homology modeling and the computational methods of THEMATICS and POOL were used to predict functionally important amino acid residues through their computed chemical properties. Site-directed mutagenesis and biochemical assays were used to validate these predictions. Primer extension, steady-state single-nucleotide incorporation kinetics, and thermal denaturation assays were performed to understand the contribution of these residues to the function of the polymerase. This work shows that the top 15 residues predicted by POOL, a set that includes the three previously known catalytic aspartate residues, seven remote residues, plus five previously unexplored first-layer residues, are important for function. Six previously unidentified residues, R362, D405, K553, Y686, E688, and H760, are each essential to Pol III activity; three additional residues, Y340, R390, and K758, play important roles in activity.

  20. Mannan-binding lectin and mannan-binding lectin-associated serine protease 2 in acute pancreatitis

    DEFF Research Database (Denmark)

    Novovic, Srdan; Andersen, Anders Møller; Ersbøll, Annette Kjær

    2011-01-01

    Complement activation may play a prominent role in acute pancreatitis (AP). Mannan-binding lectin (MBL) and MBL-associated serine protease 2 (MASP-2) participate in complement activation. The objective of the present study was to evaluate the role of MBL and MASP-2 as markers in AP with regard...

  1. The Serine Protease Inhibitor Neuroserpin Is Required for Normal Synaptic Plasticity and Regulates Learning and Social Behavior

    Science.gov (United States)

    Reumann, Rebecca; Vierk, Ricardo; Zhou, Lepu; Gries, Frederice; Kraus, Vanessa; Mienert, Julia; Romswinkel, Eva; Morellini, Fabio; Ferrer, Isidre; Nicolini, Chiara; Fahnestock, Margaret; Rune, Gabriele; Glatzel, Markus; Galliciotti, Giovanna

    2017-01-01

    The serine protease inhibitor neuroserpin regulates the activity of tissue-type plasminogen activator (tPA) in the nervous system. Neuroserpin expression is particularly prominent at late stages of neuronal development in most regions of the central nervous system (CNS), whereas it is restricted to regions related to learning and memory in the…

  2. Gene cloning and biochemical characterization of eryngase, a serine aminopeptidase of Pleurotus eryngii belonging to the family S9 peptidases.

    Science.gov (United States)

    Arima, Jiro; Tokai, Shota; Chiba, Masanori; Ichiyanagi, Tsuyoshi; Yabuta, Yukinori; Mori, Nobuhiro; Aimi, Tadanori

    2014-01-01

    Pleurotus eryngii serine aminopeptidase that has peptide bond formation activity, redesignated as eryngase, was cloned and expressed. Eryngase has a family S9 peptidase unit in the C-terminal region having a catalytic triad of Ser, Asp, and His. In the phylogenetic relations among the subfamilies of family S9 peptidase (S9A, prolyl oligopeptidase; S9B, dipeptidyl peptidase; S9C, acylaminoacyl peptidase; S9D, glutamyl endopeptidase), eryngase existed alone in the neighbor of S9C subfamily. Mutation of the active site Ser524 of the eryngase with Ala eliminated its catalytic activity. In contrast, S524C mutant maintained low catalytic activity. Investigation of aminolysis activity using l-Phe-NH2 as a substrate showed that S524C mutant exhibited no hydrolysis reaction but synthesized a small amount of l-Phe-l-Phe-NH2 by the catalysis of aminolysis. In contrast, wild-type eryngase hydrolyzed the product of aminolysis l-Phe-l-Phe-NH2. Results show that the S524C mutant preferentially catalyzed aminolysis when on an l-Phe-NH2 substrate.

  3. Assessment of activation products in the Savannah River Site environment

    International Nuclear Information System (INIS)

    Carlton, W.H.; Denham, M.

    1996-07-01

    This document assesses the impact of radioactive activation products released from SRS facilities since the first reactor became operational late in 1953. The isotopes reported here are those whose release resulted in the highest dose to people living near SRS: 32 P, 51 Cr, 60 C, and 65 Zn. Release pathways, emission control features, and annual releases to the aqueous and atmospheric environments are discussed. No single incident has resulted in a major acute release of activation products to the environment. The releases were the result of normal operations of the reactors and separations facilities. Releases declined over the years as better controls were established and production was reduced. The overall radiological impact of SRS activation product atmospheric releases from 1954 through 1994 on the offsite maximally exposed individual can be characterized by a total dose of 0.76 mrem. During the same period, such an individual received a total dose of 14,400 mrem from non-SRS sources of ionizing radiation present in the environment. SRS activation product aqueous releases between 1954 and 1994 resulted in a total dose of 54 mrem to the offsite maximally exposed individual. The impact of SRS activation product releases on offsite populations also has been evaluated

  4. A cyclic peptidic serine protease inhibitor: increasing affinity by increasing peptide flexibility.

    Directory of Open Access Journals (Sweden)

    Baoyu Zhao

    Full Text Available Peptides are attracting increasing interest as protease inhibitors. Here, we demonstrate a new inhibitory mechanism and a new type of exosite interactions for a phage-displayed peptide library-derived competitive inhibitor, mupain-1 (CPAYSRYLDC, of the serine protease murine urokinase-type plasminogen activator (uPA. We used X-ray crystal structure analysis, site-directed mutagenesis, liquid state NMR, surface plasmon resonance analysis, and isothermal titration calorimetry and wild type and engineered variants of murine and human uPA. We demonstrate that Arg6 inserts into the S1 specificity pocket, its carbonyl group aligning improperly relative to Ser195 and the oxyanion hole, explaining why the peptide is an inhibitor rather than a substrate. Substitution of the P1 Arg with novel unnatural Arg analogues with aliphatic or aromatic ring structures led to an increased affinity, depending on changes in both P1 - S1 and exosite interactions. Site-directed mutagenesis showed that exosite interactions, while still supporting high affinity binding, differed substantially between different uPA variants. Surprisingly, high affinity binding was facilitated by Ala-substitution of Asp9 of the peptide, in spite of a less favorable binding entropy and loss of a polar interaction. We conclude that increased flexibility of the peptide allows more favorable exosite interactions, which, in combination with the use of novel Arg analogues as P1 residues, can be used to manipulate the affinity and specificity of this peptidic inhibitor, a concept different from conventional attempts at improving inhibitor affinity by reducing the entropic burden.

  5. HDAC Inhibitors without an Active Site Zn2+-Binding Group

    DEFF Research Database (Denmark)

    Vickers, Chris J.; Olsen, Christian Adam; Leman, Luke J.

    2012-01-01

    -target interactions with other metalloenzymes. As a step toward mitigating this issue, here, we describe the design, synthesis, and structure−activity characterizations of cyclic α3β-tetrapeptide HDAC inhibitors that lack the presumed indispensable Zn2+-binding group. The lead compounds (e.g., 15 and 26) display good...... potency against class 1 HDACs and are active in tissue culture against various human cancer cell lines. Importantly, enzymological analysis of 26 indicates that the cyclic α3β-tetrapeptide is a fast-on/ off competitive inhibitor of HDACs 1−3 with Ki values of 49, 33, and 37 nM, respectively. Our proof...

  6. Effects of a marine serine protease inhibitor on viability and morphology of Trypanosoma cruzi, the agent of Chagas disease.

    Science.gov (United States)

    de Almeida Nogueira, Natália Pereira; Morgado-Díaz, José Andrés; Menna-Barreto, Rubem Figueiredo Sadok; Paes, Marcia Cristina; da Silva-López, Raquel Elisa

    2013-10-01

    It has been reported that serine peptidase activities of Trypanosoma cruzi play crucial roles in parasite dissemination and host cell invasion and therefore their inhibition could affect the progress of Chagas disease. The present study investigates the interference of the Stichodactyla helianthus Kunitz-type serine protease inhibitor (ShPI-I), a 55-amino acid peptide, in T. cruzi serine peptidase activities, parasite viability, and parasite morphology. The effect of this peptide was also studied in Leishmania amazonensis promastigotes and it was proved to be a powerful inhibitor of serine proteases activities and the parasite viability. The ultrastructural alterations caused by ShPI-I included vesiculation of the flagellar pocket membrane and the appearance of a cytoplasmic vesicle that resembles an autophagic vacuole. ShPI-I, which showed itself to be an important T. cruzi serine peptidase inhibitor, reduced the parasite viability, in a dose and time dependent manner. The maximum effect of peptide on T. cruzi viability was observed when ShPI-I at 1×10(-5)M was incubated for 24 and 48h which killed completely both metacyclic trypomastigote and epimastigote forms. At 1×10(-6)M ShPI-I, in the same periods of time, reduced parasite viability about 91-95% respectively. Ultrastructural analysis demonstrated the formation of concentric membranar structures especially in the cytosol, involving organelles and small vesicles. Profiles of endoplasmic reticulum were also detected, surrounding cytosolic vesicles that resembled autophagic vacuoles. These results suggest that serine peptidases are important in T. cruzi physiology since the inhibition of their activity killed parasites in vitro as well as inducing important morphological alterations. Protease inhibitors thus appear to have a potential role as anti-trypanosomatidal agents. Copyright © 2013 Elsevier B.V. All rights reserved.

  7. Negative Role of RIG-I Serine 8 Phosphorylation in the Regulatin of Interferon-beta Production

    Energy Technology Data Exchange (ETDEWEB)

    E Nistal-Villan; M Gack; G Martinez-Delgado; N Maharaj; K Inn; H Yang; R Wang; A Aggarwal; J Jung; A Garcia-Sastre

    2011-12-31

    RIG-I (retinoic acid-inducible gene I) and TRIM25 (tripartite motif protein 25) have emerged as key regulatory factors to induce interferon (IFN)-mediated innate immune responses to limit viral replication. Upon recognition of viral RNA, TRIM25 E3 ligase binds the first caspase recruitment domain (CARD) of RIG-I and subsequently induces lysine 172 ubiquitination of the second CARD of RIG-I, which is essential for the interaction with downstream MAVS/IPS-1/CARDIF/VISA and, thereby, IFN-beta mRNA production. Although ubiquitination has emerged as a major factor involved in RIG-I activation, the potential contribution of other post-translational modifications, such as phosphorylation, to the regulation of RIG-I activity has not been addressed. Here, we report the identification of serine 8 phosphorylation at the first CARD of RIG-I as a negative regulatory mechanism of RIG-I-mediated IFN-beta production. Immunoblot analysis with a phosphospecific antibody showed that RIG-I serine 8 phosphorylation steady-state levels were decreased upon stimulation of cells with IFN-beta or virus infection. Substitution of serine 8 in the CARD RIG-I functional domain with phosphomimetic aspartate or glutamate results in decreased TRIM25 binding, RIG-I ubiquitination, MAVS binding, and downstream signaling. Finally, sequence comparison reveals that only primate species carry serine 8, whereas other animal species carry an asparagine, indicating that serine 8 phosphorylation may represent a primate-specific regulation of RIG-I activation. Collectively, these data suggest that the phosphorylation of RIG-I serine 8 operates as a negative switch of RIG-I activation by suppressing TRIM25 interaction, further underscoring the importance of RIG-I and TRIM25 connection in type I IFN signal transduction.

  8. Understanding the role of aluminum-based activators in single site iron catalysts for ethylene oligomerization.

    OpenAIRE

    Boudene , Zoubeyr; Boudier , Adrien; Breuil , Pierre-Alain; Olivier-Bourbigou , Hélène; Raybaud , Pascal; Toulhoat , Hervé; De Bruin , Theodorus

    2014-01-01

    International audience; In a combined experimental and theoretical study, the activation process of a single site ethylene oligomerization catalyst with aluminum-based activators has been studied. The results put forward a plausible deactivation reaction path of the catalyst for trimethylaluminum, while for methylaluminoxane and a novel phenoxyaluminum-based activator, the experimental catalyst's activity correlates with the energy barrier for the ethylene insertion.

  9. Site directed mutagenesis of amino acid residues at the active site of mouse aldehyde oxidase AOX1.

    Directory of Open Access Journals (Sweden)

    Silvia Schumann

    Full Text Available Mouse aldehyde oxidase (mAOX1 forms a homodimer and belongs to the xanthine oxidase family of molybdoenzymes which are characterized by an essential equatorial sulfur ligand coordinated to the molybdenum atom. In general, mammalian AOs are characterized by broad substrate specificity and an yet obscure physiological function. To define the physiological substrates and the enzymatic characteristics of mAOX1, we established a system for the heterologous expression of the enzyme in Escherichia coli. The recombinant protein showed spectral features and a range of substrate specificity similar to the native protein purified from mouse liver. The EPR data of recombinant mAOX1 were similar to those of AO from rabbit liver, but differed from the homologous xanthine oxidoreductase enzymes. Site-directed mutagenesis of amino acids Val806, Met884 and Glu1265 at the active site resulted in a drastic decrease in the oxidation of aldehydes with no increase in the oxidation of purine substrates. The double mutant V806E/M884R and the single mutant E1265Q were catalytically inactive enzymes regardless of the aldehyde or purine substrates tested. Our results show that only Glu1265 is essential for the catalytic activity by initiating the base-catalyzed mechanism of substrate oxidation. In addition, it is concluded that the substrate specificity of molybdo-flavoenzymes is more complex and not only defined by the three characterized amino acids in the active site.

  10. Location and activity specific site-management for military locations

    NARCIS (Netherlands)

    Maring, L.; Hulst, M. van; Meuken, D.

    2009-01-01

    pace is limited in the Netherlands and military activities, that may cause nuisance or environmental hazards, should therefore be considered and evaluated during the use of military locations. The last few years TNO and Deltares have worked on a research program on environmental effects due to

  11. The landscape degradation in the mining sites with suspended activity

    Directory of Open Access Journals (Sweden)

    Anca IONCE

    2009-08-01

    Full Text Available The extracting industry, through its extraction activities, of shipping the ores, of breaking the ores, of preparing the practical substances, of stowing the useless rock, of transporting the practical substances, etc. might modify the area’s relief and the quality of ground, of thesurface waters and of the air. Suceava County has an old tradition of mining, where the results of this activity are visible, especially the visual point of view, and where not taking certain measures of ecological remediation will emphasize the disappointing image of the landscape within the areas of mining activity performing.The predominant mountainous landscape, in which mining activities have been held, is being affected also by the abandoned industrial and administrative buildings, in an advanced degradation state.The hydrographic system, very rich in mining areas, has its water quality affected by the acid rock drainage- phenomenon which appeared in many mining waste deposits.

  12. Regulation of Adrenal Aldosterone Production by Serine Protease Prostasin

    Directory of Open Access Journals (Sweden)

    Takehiro Ko

    2010-01-01

    Full Text Available A serine protease prostasin has been demonstrated to have a pivotal role in the activation of the epithelial sodium channel. Systemic administration of adenovirus carrying human prostasin gene in rats resulted in an increase in plasma prostasin and aldosterone levels. However, the mechanism by which the elevation of prostasin levels in the systemic circulation stimulated the plasma aldosterone levels remains unknown. Therefore, we examined if prostasin increases the aldosterone synthesis in a human adrenocortical cell line (H295R cells. Luciferase assay using CYP11B2 promoter revealed that prostasin significantly increased the transcriptional activity of CYP11B2. Prostasin significantly increased both CYP11B2 mRNA expression and aldosterone production in a dose-dependent manner. Surprisingly, treatment with camostat mesilate, a potent prostasin inhibitor, had no effect on the aldosterone synthesis by prostasin and also a protease-dead mutant of prostasin significantly stimulated the aldosterone production. A T-type/L-type calcium channel blocker and a protein kinase C (PKC inhibitor significantly reduced the aldosterone synthesis by prostasin. Our findings suggest a stimulatory effect of prostasin on the aldosterone synthesis by adrenal gland through the nonproteolytic action and indicate a new role of prostasin in the systemic circulation.

  13. Quantifying the density and utilization of active sites in non-precious metal oxygen electroreduction catalysts

    Science.gov (United States)

    Sahraie, Nastaran Ranjbar; Kramm, Ulrike I.; Steinberg, Julian; Zhang, Yuanjian; Thomas, Arne; Reier, Tobias; Paraknowitsch, Jens-Peter; Strasser, Peter

    2015-01-01

    Carbon materials doped with transition metal and nitrogen are highly active, non-precious metal catalysts for the electrochemical conversion of molecular oxygen in fuel cells, metal air batteries, and electrolytic processes. However, accurate measurement of their intrinsic turn-over frequency and active-site density based on metal centres in bulk and surface has remained difficult to date, which has hampered a more rational catalyst design. Here we report a successful quantification of bulk and surface-based active-site density and associated turn-over frequency values of mono- and bimetallic Fe/N-doped carbons using a combination of chemisorption, desorption and 57Fe Mössbauer spectroscopy techniques. Our general approach yields an experimental descriptor for the intrinsic activity and the active-site utilization, aiding in the catalyst development process and enabling a previously unachieved level of understanding of reactivity trends owing to a deconvolution of site density and intrinsic activity. PMID:26486465

  14. Encroachment of Human Activity on Sea Turtle Nesting Sites

    Science.gov (United States)

    Ziskin, D.; Aubrecht, C.; Elvidge, C.; Tuttle, B.; Baugh, K.; Ghosh, T.

    2008-12-01

    The encroachment of anthropogenic lighting on sea turtle nesting sites poses a serious threat to the survival of these animals [Nicholas, 2001]. This danger is quantified by combining two established data sets. The first is the Nighttime Lights data produced by the NOAA National Geophysical Data Center [Elvidge et al., 1997]. The second is the Marine Turtle Database produced by the World Conservation Monitoring Centre (WCMC). The technique used to quantify the threat of encroachment is an adaptation of the method described in Aubrecht et al. [2008], which analyzes the stress on coral reef systems by proximity to nighttime lights near the shore. Nighttime lights near beaches have both a direct impact on turtle reproductive success since they disorient hatchlings when they mistake land-based lights for the sky-lit surf [Lorne and Salmon, 2007] and the lights are also a proxy for other anthropogenic threats. The identification of turtle nesting sites with high rates of encroachment will hopefully steer conservation efforts to mitigate their effects [Witherington, 1999]. Aubrecht, C, CD Elvidge, T Longcore, C Rich, J Safran, A Strong, M Eakin, KE Baugh, BT Tuttle, AT Howard, EH Erwin, 2008, A global inventory of coral reef stressors based on satellite observed nighttime lights, Geocarto International, London, England: Taylor and Francis. In press. Elvidge, CD, KE Baugh, EA Kihn, HW Kroehl, ER Davis, 1997, Mapping City Lights with Nighttime Data from the DMSP Operational Linescan System, Photogrammatic Engineering and Remote Sensing, 63:6, pp. 727-734. Lorne, JK, M Salmon, 2007, Effects of exposure to artificial lighting on orientation of hatchling sea turtles on the beach and in the ocean, Endangered Species Research, Vol. 3: 23-30. Nicholas, M, 2001, Light Pollution and Marine Turtle Hatchlings: The Straw that Breaks the Camel's Back?, George Wright Forum, 18:4, p77-82. Witherington, BE, 1999, Reducing Threats To Nesting Habitat, Research and Management Techniques for

  15. Rac1 GTPase activates the WAVE regulatory complex through two distinct binding sites

    Science.gov (United States)

    Brautigam, Chad A; Xing, Wenmin; Yang, Sheng; Henry, Lisa; Doolittle, Lynda K; Walz, Thomas

    2017-01-01

    The Rho GTPase Rac1 activates the WAVE regulatory complex (WRC) to drive Arp2/3 complex-mediated actin polymerization, which underpins diverse cellular processes. Here we report the structure of a WRC-Rac1 complex determined by cryo-electron microscopy. Surprisingly, Rac1 is not located at the binding site on the Sra1 subunit of the WRC previously identified by mutagenesis and biochemical data. Rather, it binds to a distinct, conserved site on the opposite end of Sra1. Biophysical and biochemical data on WRC mutants confirm that Rac1 binds to both sites, with the newly identified site having higher affinity and both sites required for WRC activation. Our data reveal that the WRC is activated by simultaneous engagement of two Rac1 molecules, suggesting a mechanism by which cells may sense the density of active Rac1 at membranes to precisely control actin assembly. PMID:28949297

  16. A New Bacillus licheniformis Mutant Strain Producing Serine Protease Efficient for Hvdrolvqis of Sov Meal Proteins.

    Science.gov (United States)

    Kostyleva, E V; Sereda, A S; Velikoretskaya, I A; Nefedova, L I; Sharikov, A Yu; Tsurikova, N V; Lobanov, N S; Semenova, M V; Sinitsyn, A P

    2016-07-01

    Induced mutagenesis with y-irradiation of the industrial strain Bacillus licheniformis-60 VKM B-2366,D was used to obtain a new highly active producer of an extracellular serine protease, Bacillus licheni- formis7 145. Samples of dry.concentrated preparations of serine protease produced by the original and mutant strains were obtained, and identity of their protein composition was'established. Alkaline serine protease sub- tilisin DY was the main component of the preparations. The biochemical and physicochemical properties of the Protolkheterm-145 enzyme preparation obtained from the mutant strain were studied. It exhibited pro- teolytic activity (1.5 times higher than the preparation from the initial strain) within broad ranges of pH (5- 11) and temperature (30-70'C).-Efficient hydrolysis of extruded soy meal protein at high concentrations (2 to 50%) in-the reaction mixture was.the main advantage of the Protolikheterm 145 preparation. Compared to,. the preparation obtained using the initial strain, the new preparation with increased proteolytic-activity pro- vided for more complete hydrolysis of the main non-nutritious soy,proteins.(glycinin and 0-conglycinin) with the yield of soluble protein increased by 19-28%, which decreased the cost of bioconversion of the protein- aceous material and indicated promise of the new preparation in resource-saving technologies for processing soy meals and cakes.

  17. Connecting Active-Site Loop Conformations and Catalysis in Triosephosphate Isomerase: Insights from a Rare Variation at Residue 96 in the Plasmodial Enzyme.

    Science.gov (United States)

    Pareek, Vidhi; Samanta, Moumita; Joshi, Niranjan V; Balaram, Hemalatha; Murthy, Mathur R N; Balaram, Padmanabhan

    2016-04-01

    Despite extensive research into triosephosphate isomerases (TIMs), there exists a gap in understanding of the remarkable conjunction between catalytic loop-6 (residues 166-176) movement and the conformational flip of Glu165 (catalytic base) upon substrate binding that primes the active site for efficient catalysis. The overwhelming occurrence of serine at position 96 (98% of the 6277 unique TIM sequences), spatially proximal to E165 and the loop-6 residues, raises questions about its role in catalysis. Notably, Plasmodium falciparum TIM has an extremely rare residue--phenylalanine--at this position whereas, curiously, the mutant F96S was catalytically defective. We have obtained insights into the influence of residue 96 on the loop-6 conformational flip and E165 positioning by combining kinetic and structural studies on the PfTIM F96 mutants F96Y, F96A, F96S/S73A, and F96S/L167V with sequence conservation analysis and comparative analysis of the available apo and holo structures of the enzyme from diverse organisms. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  18. Lipolytic activity from bacteria prospected in polluted portuary sites

    Directory of Open Access Journals (Sweden)

    Kaori Levy Fonseca

    2014-06-01

    This study demonstrates that these TBT resistant isolates have, at the same time, the capacity to produce enzymes with a large biotechnological potential but, nevertheless, their relationship is not well understood, representing a novel approach. It is expected for these organisms to produce highly biotechnological relevant biocatalysts, due to their severe adaptations (Suehiro et al., 2007. The fully characterization of these lipases, mostly for F3 with elevated lipolytic activity exhibited, presents also a future challenge.

  19. Molecular Basis for Enzymatic Sulfite Oxidation -- HOW THREE CONSERVED ACTIVE SITE RESIDUES SHAPE ENZYME ACTIVITY

    Energy Technology Data Exchange (ETDEWEB)

    Bailey, Susan; Rapson, Trevor; Johnson-Winters, Kayunta; Astashkin, Andrei; Enemark, John; Kappler, Ulrike

    2008-11-10

    Sulfite dehydrogenases (SDHs) catalyze the oxidation and detoxification of sulfite to sulfate, a reaction critical to all forms of life. Sulfite-oxidizing enzymes contain three conserved active site amino acids (Arg-55, His-57, and Tyr-236) that are crucial for catalytic competency. Here we have studied the kinetic and structural effects of two novel and one previously reported substitution (R55M, H57A, Y236F) in these residues on SDH catalysis. Both Arg-55 and His-57 were found to have key roles in substrate binding. An R55M substitution increased Km(sulfite)(app) by 2-3 orders of magnitude, whereas His-57 was required for maintaining a high substrate affinity at low pH when the imidazole ring is fully protonated. This effect may be mediated by interactions of His-57 with Arg-55 that stabilize the position of the Arg-55 side chain or, alternatively, may reflect changes in the protonation state of sulfite. Unlike what is seen for SDHWT and SDHY236F, the catalytic turnover rates of SDHR55M and SDHH57A are relatively insensitive to pH (~;;60 and 200 s-1, respectively). On the structural level, striking kinetic effects appeared to correlate with disorder (in SDHH57A and SDHY236F) or absence of Arg-55 (SDHR55M), suggesting that Arg-55 and the hydrogen bonding interactions it engages in are crucial for substrate binding and catalysis. The structure of SDHR55M has sulfate bound at the active site, a fact that coincides with a significant increase in the inhibitory effect of sulfate in SDHR55M. Thus, Arg-55 also appears to be involved in enabling discrimination between the substrate and product in SDH.

  20. Sequence conservation, phylogenetic relationships, and expression profiles of nondigestive serine proteases and serine protease homologs in Manduca sexta

    OpenAIRE

    Cao, Xiaolong; He, Yan; Hu, Yingxia; Zhang, Xiufeng; Wang, Yang; Zou, Zhen; Chen, Yunru; Blissard, Gary W.; Kanost, Michael R.; Jiang, Haobo

    2014-01-01

    Serine protease (SP) and serine protease homolog (SPH) genes in insects encode a large family of proteins involved in digestion, development, immunity, and other processes. While 68 digestive SPs and their close homologs are reported in a companion paper (Kuwar et al., 2015), we have identified 125 other SPs/SPHs in Manduca sexta and studied their structure, evolution, and expression. Fifty-two of them contain cystine-stabilized structures for molecular recognition, including clip, LDLa, Sush...

  1. Time Multiplexed Active Neural Probe with 1356 Parallel Recording Sites

    Directory of Open Access Journals (Sweden)

    Bogdan C. Raducanu

    2017-10-01

    Full Text Available We present a high electrode density and high channel count CMOS (complementary metal-oxide-semiconductor active neural probe containing 1344 neuron sized recording pixels (20 µm × 20 µm and 12 reference pixels (20 µm × 80 µm, densely packed on a 50 µm thick, 100 µm wide, and 8 mm long shank. The active electrodes or pixels consist of dedicated in-situ circuits for signal source amplification, which are directly located under each electrode. The probe supports the simultaneous recording of all 1356 electrodes with sufficient signal to noise ratio for typical neuroscience applications. For enhanced performance, further noise reduction can be achieved while using half of the electrodes (678. Both of these numbers considerably surpass the state-of-the art active neural probes in both electrode count and number of recording channels. The measured input referred noise in the action potential band is 12.4 µVrms, while using 678 electrodes, with just 3 µW power dissipation per pixel and 45 µW per read-out channel (including data transmission.

  2. Novel role of serine racemase in anti-apoptosis and metabolism.

    Science.gov (United States)

    Talukdar, Gourango; Inoue, Ran; Yoshida, Tomoyuki; Ishimoto, Tetsuya; Yaku, Keisuke; Nakagawa, Takashi; Mori, Hisashi

    2017-01-01

    Serine racemase (SR) catalyzes the production of d-serine, a co-agonist of the N-methyl-d-aspartate receptor (NMDAR). A previous report shows the contribution of SR in the NMDAR-mediated neuronal cell death process. To analyze the intrinsic role of SR in the cell death process, we established the epithelial human embryonic kidney 293T (HEK293T) cell lines expressing wild-type SR (SR-WT), catalytically inactive mutant SR (SR-K56G), and catalytically hyperactive mutant SR (SR-Q155D). To these cell lines, staurosporine (STS), which induces apoptosis, was introduced. The cells expressing SR-WT and SR-Q155D showed resistance to STS-induced apoptosis, compared with nontransfected HEK293T cells and cells expressing SR-K56G. The SR-WT cells also showed a significant higher viability than the SR-QD cells. Furthermore, we detected elevated phosphorylation levels of Bcl-2 at serine-70 and Akt at serine-473 and threonine-308, which are related to cell survival, in the cells expressing SR-WT and SR-Q155D. From the results of metabolite analysis, we found elevated levels of acetyl CoA and ATP in cells expressing SR-WT. Because SR has two enzymatic activities, namely, racemization and α, β-elimination, and SR-Q155D shows enhanced racemization and reduced α, β-elimination activities, we concluded that the racemization reaction catalyzed by SR may have a more protective role against apoptosis than the α, β-elimination reaction. Moreover, both of these activities are important for maximal survival and elevated levels of acetyl CoA and ATP. Our findings reveal the NMDAR-independent roles of SR in metabolism and cell survival. Copyright © 2016 Elsevier B.V. All rights reserved.

  3. Recent progress in volcanism studies: Site characterization activities for the Yucca Mountain site characterization project

    International Nuclear Information System (INIS)

    Crowe, B.M.; Valentine, G.; Morley, R.; Perry, F.V.

    1992-01-01

    Significant progress has been made on volcanism studies over the past calendar year. There are a number of major highlights from this work. Geochronology data have been obtained for the Lathrop Wells center using a range of isotopic, radiogenic, and age-calibrated methods. Initial work is encouraging but still insufficient to resolve the age of the center with confidence. Geologic mapping of the Sleeping Butte volcanic centers was completed and a report issued on the geology and chronology data. Twenty shallow trenches have been constructed in volcanic units of the Lathrop Wells volcanic center. Results of detailed studies of the trenches support a polycyclic eruptive history. New soil data from the trenches continue to support a late Pleistocene or Holocene age for many of the volcanic units at the center. Geochemical data (trace element and isotopic analysis) show that the volcanic units of the Lathrop Wells center cannot be related to one another by fractional crystallization of a single magma batch, supporting a polycyclic model of volcanism. Structural models using existing data are used to evaluate the probability of magmatic disruption of a potential repository. Several permissive models have been developed but none lead to significant differences in calculating the disruption ratio. Work was initiated on the eruptive and subsurface effects of magmatic activity on a repository. (author)

  4. Functional analysis of a missense mutation in the serine protease inhibitor SPINT2 associated with congenital sodium diarrhea.

    Directory of Open Access Journals (Sweden)

    Nicolas Faller

    Full Text Available Membrane-bound serine proteases play important roles in different biological processes. Their regulation by endogenous inhibitors is poorly understood. A Y163C mutation in the SPINT2 gene encoding the serine protease inhibitor Hepatocyte Growth Factor Inhibitor HAI-2 is associated with a congenital sodium diarrhea. The functional consequences of this mutation on HAI-2 activity and its physiological targets are unknown. We established a cellular assay in Xenopus laevis oocytes to study functional interactions between HAI-2 and candidate membrane-bound serine proteases expressed in the gastro-intestinal tract. We found that the wild-type form of HAI-2 is a potent inhibitor of nine gastro-intestinal serine proteases. The Y163C mutation in the second Kunitz domain of HAI-2 resulted in a complete loss of inhibitory activity on two intestinal proteases, prostasin and tmprss13. The effect of the mutation of the homologous Y68C in the first Kunitz domain of HAI-2 is consistent with a differential contribution of the two Kunitz domains of HAI-2 in the inhibition of serine proteases. By contrast to the Tyr to Cys, the Tyr to Ser substitution did not change the inhibitory potency of HAI-2, indicating that the thiol-group of the cysteine rather than the Tyr deletion is responsible for the HAI-2 loss of function. Our functional assay allowed us to identify membrane-bound serine proteases as cellular target for inhibition by HAI-2 wild type and mutants, and to better define the role of the Tyr in the second Kunitz domain in the inhibitory activity of HAI-2.

  5. Cooperative activation of cardiac transcription through myocardin bridging of paired MEF2 sites

    Energy Technology Data Exchange (ETDEWEB)

    Anderson, Courtney M. [Univ. of California, San Francisco, CA (United States). Cardiovascular Research Inst.; Hu, Jianxin [Univ. of California, San Francisco, CA (United States). Cardiovascular Research Inst.; Thomas, Reuben [Univ. of California, San Francisco, CA (United States). Gladstone Inst.; Gainous, T. Blair [Univ. of California, San Francisco, CA (United States). Cardiovascular Research Inst.; Celona, Barbara [Univ. of California, San Francisco, CA (United States). Cardiovascular Research Inst.; Sinha, Tanvi [Univ. of California, San Francisco, CA (United States). Cardiovascular Research Inst.; Dickel, Diane E. [Lawrence Berkeley National Lab. (LBNL), Berkeley, CA (United States). Genomics Division; Heidt, Analeah B. [Univ. of California, San Francisco, CA (United States). Cardiovascular Research Inst.; Xu, Shan-Mei [Univ. of California, San Francisco, CA (United States). Cardiovascular Research Inst.; Bruneau, Benoit G. [Univ. of California, San Francisco, CA (United States). Cardiovascular Research Inst.; Univ. of California, San Francisco, CA (United States). Gladstone Inst.; Pollard, Katherine S. [Univ. of California, San Francisco, CA (United States). Gladstone Inst.; Pennacchio, Len A. [Lawrence Berkeley National Lab. (LBNL), Berkeley, CA (United States). Genomics Division; Black, Brian L. [Univ. of California, San Francisco, CA (United States). Cardiovascular Research Inst.; Univ. of California, San Francisco, CA (United States). Dept. of

    2017-03-28

    Enhancers frequently contain multiple binding sites for the same transcription factor. These homotypic binding sites often exhibit synergy, whereby the transcriptional output from two or more binding sites is greater than the sum of the contributions of the individual binding sites alone. Although this phenomenon is frequently observed, the mechanistic basis for homotypic binding site synergy is poorly understood. Here in this paper, we identify a bona fide cardiac-specific Prkaa2 enhancer that is synergistically activated by homotypic MEF2 binding sites. We show that two MEF2 sites in the enhancer function cooperatively due to bridging of the MEF2C-bound sites by the SAP domain-containing co-activator protein myocardin, and we show that paired sites buffer the enhancer from integration site-dependent effects on transcription in vivo. Paired MEF2 sites are prevalent in cardiac enhancers, suggesting that this might be a common mechanism underlying synergy in the control of cardiac gene expression in vivo.

  6. Clozapine, but not haloperidol, enhances glial D-serine and L-glutamate release in rat frontal cortex and primary cultured astrocytes.

    Science.gov (United States)

    Tanahashi, Shunske; Yamamura, Satoshi; Nakagawa, Masanori; Motomura, Eishi; Okada, Motohiro

    2012-03-01

    Deficient transmission at the glutamate NMDA receptor is considered a key component of the pathophysiology of schizophrenia. However, the effects of antipsychotic drugs on the release of the endogenous NMDA receptor partial agonist, D-serine, remain to be clarified. We determined the interaction between antipsychotic drugs (clozapine and haloperidol) and transmission-modulating toxins (tetanus toxin, fluorocitrate, tetrodotoxin) on the release of L-glutamate and D-serine in the medial prefrontal cortex (mPFC) of freely moving rats, using microdialysis, and primary cultures of astrocytes using extreme high-pressure liquid chromatography. Release of L-glutamate and D-serine in the mPFC and in cultured astrocytes was inhibited by tetanus toxin (a synaptobrevin inhibitor) and fluorocitrate (a glial toxin), whereas tetrodotoxin (a voltage-sensitive Na(+) blocker) inhibited depolarization-induced L-glutamate release in the mPFC without affecting that of D-serine. Clozapine (1 and 5 mg·kg(-1)), but not haloperidol (0.5 and 1 mg·kg(-1)), dose-dependently increased L-glutamate and D-serine release from both astrocytes and mPFC. Clozapine-induced release of L-glutamate and D-serine was also reduced by tetanus toxin and fluorocitrate. Tetrodotoxin reduced clozapine-induced mPFC L-glutamate release but not that of D-serine. Clozapine-induced L-glutamate release preceded clozapine-induced D-serine release. MK-801 (a NMDA receptor antagonist) inhibited the delayed clozapine-induced L-glutamate release without affecting that of D-serine. Clozapine predominantly activated glial exocytosis of D-serine, and this clozapine-induced D-serine release subsequently enhances neuronal L-glutamate release via NMDA receptor activation. The enhanced D-serine associated glial transmission seems a novel mechanism of action of clozapine but not haloperidol. © 2011 The Authors. British Journal of Pharmacology © 2011 The British Pharmacological Society.

  7. Active site proton delivery and the lyase activity of human CYP17A1

    Energy Technology Data Exchange (ETDEWEB)

    Khatri, Yogan; Gregory, Michael C.; Grinkova, Yelena V.; Denisov, Ilia G.; Sligar, Stephen G., E-mail: s-sligar@illinois.edu

    2014-01-03

    equivalents and protons are funneled into non-productive pathways. This is similar to previous work with other P450 catalyzed hydroxylation. However, catalysis of carbon–carbon bond scission by the T306A mutant was largely unimpeded by disruption of the CYP17A1 acid-alcohol pair. The unique response of CYP17A1 lyase activity to mutation of Thr306 is consistent with a reactive intermediate formed independently of proton delivery in the active site, and supports involvement of a nucleophilic peroxo-anion rather than the traditional Compound I in catalysis.

  8. School Pharmacist/School Environmental Hygienic Activities at School Site.

    Science.gov (United States)

    Muramatsu, Akiyoshi

    2016-01-01

    The "School Health and Safety Act" was enforced in April 2009 in Japan, and "school environmental health standards" were established by the Minister of Education, Culture, Sports, Science and Technology. In Article 24 of the Enforcement Regulations, the duties of the school pharmacist have been clarified; school pharmacists have charged with promoting health activities in schools and carrying out complete and regular checks based on the "school environmental health standards" in order to protect the health of students and staff. In supported of this, the school pharmacist group of Japan Pharmaceutical Association has created and distributed digital video discs (DVDs) on "check methods of school environmental health standards" as support material. We use the DVD to ensure the basic issues that school pharmacists deal with, such as objectives, criteria, and methods for each item to be checked, advice, and post-measures. We conduct various workshops and classes, and set up Q&A committees so that inquiries from members are answered with the help of such activities. In addition, school pharmacists try to improve the knowledge of the school staff on environmental hygiene during their in-service training. They also conduct "drug abuse prevention classes" at school and seek to improve knowledge and recognition of drugs, including "dangerous drugs".

  9. Effects of cobalt-substitution of the active zinc ion in thermolysin on its activity and active-site microenvironment.

    Science.gov (United States)

    Kuzuya, K; Inouye, K

    2001-12-01

    Thermolysin is remarkably activated in the presence of high concentrations (1-5 M) of neutral salts [Inouye, K. (1992) J. Biochem. 112, 335-340]. The activity is enhanced 13-15 times with 4 M NaCl at pH 7.0 and 25 degrees C. Substitution of the active site zinc with other transition metals alters the activity of thermolysin [Holmquist, B. and Vallee, B.L. (1974) J. Biol. Chem. 249, 4601-4607]. Cobalt is the most effective among the transition metals and doubles the activity toward N-[3-(2-furyl)acryloyl]-glycyl-L-leucine amide. In this study, the effect of NaCl on the activity of cobalt-substituted thermolysin was examined. Cobalt-substituted thermolysin, with 2.8-fold increased activity compared with the native enzyme, is further activated by the addition of NaCl in an exponential fashion, and the activity is enhanced 13-15 times at 4 M NaCl. The effects of cobalt-substitution and the addition of salt are independent of each other. The activity of cobalt-substituted thermolysin, expressed as k(cat)/K(m), is pH-dependent and controlled by at least two ionizing residues with pK(a) values of 6.0 and 7.8, the acidic pK(a) being slightly higher compared to 5.6 of the native enzyme. These pK(a) values remain constant in the presence of 4 M NaCl, indicating that the electrostatic environment of cobalt-substituted thermolysin is more stable than that of the native enzyme, the acidic pK(a) of which shifts remarkably from 5.6 to 6.7 at 4 M NaCl. Zincov, a competitive inhibitor, binds more tightly to the cobalt-substituted than to native thermolysin at pH 4.9-9.0, probably because of its preference for cobalt in the fivefold coordination. The cobalt substitution has been shown to be a favorable tool with which to explore the active-site microenvironment of thermolysin.

  10. Chloride binding site of neurotransmitter sodium symporters

    DEFF Research Database (Denmark)

    Kantcheva, Adriana Krassimirova; Quick, Matthias; Shi, Lei

    2013-01-01

    Neurotransmitter:sodium symporters (NSSs) play a critical role in signaling by reuptake of neurotransmitters. Eukaryotic NSSs are chloride-dependent, whereas prokaryotic NSS homologs like LeuT are chloride-independent but contain an acidic residue (Glu290 in LeuT) at a site where eukaryotic NSSs...... have a serine. The LeuT-E290S mutant displays chloride-dependent activity. We show that, in LeuT-E290S cocrystallized with bromide or chloride, the anion is coordinated by side chain hydroxyls from Tyr47, Ser290, and Thr254 and the side chain amide of Gln250. The bound anion and the nearby sodium ion...

  11. Effects of serine palmitoyltransferase inhibitor ISP-I on the stratum corneum of intact mouse skin.

    Science.gov (United States)

    Mizukoshi, Koji; Matsumoto, Katsuo; Hirose, Ryouji; Fujita, Tetsuro; Ishida-Yamamoto, Akemi; Iizuka, Hajime

    2011-01-01

    Serine palmitoyltransferase (SPT) is involved in the ceramide synthesis pathway. We investigated the effects of ISP-I, a potent inhibitor of SPT, on the stratum corneum (SC) of hairless mouse skin. Application of ISP-I for one week resulted in a significant decrease in the amount of ceramide, which was associated with a decrease in SC hydration. However, there was an increase in the number of SC layers and less transepidermal water loss than control. Transmission Electron Microscopy observation revealed that the number of desmosome-like structures in the layers immediately above the stratum granulosum (SG) was significantly increased in ISP-I-treated skin compared to vehicle-treated skin. The activity of serine protease-an enzyme associated with the process of desquamation-was lower in the SC of ISP-I-treated skin than control. Furthermore, immunoelectronmicroscopy revealed that glucosylceramide and corneodesmosin tended to remain in corneocytes and were not secreted into the intercellular spaces of the SC in the ISP-I-treated skin. These results indicate that the application of ISP-I decreases ceramide and skin hydration, while at the same time increases the number of SC layers. The accumulation of corneocyte layers may originate from an aberrant desquamation process related to the decrease in the serine protease activity as well as an alteration in the transport of desquamation-related proteases by lamellar bodies.

  12. Assessment and partial purification of serine protease inhibitors from Rhipicephalus (Boophilus annulatuslarvae

    Directory of Open Access Journals (Sweden)

    Sedigheh Nabian

    Full Text Available Ticks are rich sources of serine protease inhibitors, particularly those that prevent blood clotting and inflammatory responses during blood feeding. The tick Rhipicephalus (Boophlus annulatusis an important ectoparasite of cattle. The aims of this study were to characterize and purify the serine protease inhibitors present in R. (B. annulatus larval extract. The inhibitors were characterized by means of one and two-dimensional reverse zymography, and purified using affinity chromatography on a trypsin-Sepharose column. The analysis on one and two-dimensional reverse zymography of the larval extract showed trypsin inhibitory activity at between 13 and 40 kDa. Through non-reducing SDS-PAGE and reverse zymography for proteins purified by trypsin-Sepharose affinity chromatography, some protein bands with molecular weights between 13 and 34 kDa were detected. Western blotting showed that five protein bands at 48, 70, 110, 130 and 250 kDa reacted positively with immune serum, whereas there was no positive reaction in the range of 13-40 kDa. Serine protease inhibitors from R. (B. annulatus have anti-trypsin activity similar to inhibitors belonging to several other hard tick species, thus suggesting that these proteins may be useful as targets in anti-tick vaccines.

  13. Cross genome comparisons of serine proteases in Arabidopsis and rice

    Directory of Open Access Journals (Sweden)

    Sowdhamini R

    2006-08-01

    Full Text Available Abstract Background Serine proteases are one of the largest groups of proteolytic enzymes found across all kingdoms of life and are associated with several essential physiological pathways. The availability of Arabidopsis thaliana and rice (Oryza sativa genome sequences has permitted the identification and comparison of the repertoire of serine protease-like proteins in the two plant species. Results Despite the differences in genome sizes between Arabidopsis and rice, we identified a very similar number of serine protease-like proteins in the two plant species (206 and 222, respectively. Nearly 40% of the above sequences were identified as potential orthologues. Atypical members could be identified in the plant genomes for Deg, Clp, Lon, rhomboid proteases and species-specific members were observed for the highly populated subtilisin and serine carboxypeptidase families suggesting multiple lateral gene transfers. DegP proteases, prolyl oligopeptidases, Clp proteases and rhomboids share a significantly higher percentage orthology between the two genomes indicating substantial evolutionary divergence was set prior to speciation. Single domain architectures and paralogues for several putative subtilisins, serine carboxypeptidases and rhomboids suggest they may have been recruited for additional roles in secondary metabolism with spatial and temporal regulation. The analysis reveals some domain architectures unique to either or both of the plant species and some inactive proteases, like in rhomboids and Clp proteases, which could be involved in chaperone function. Conclusion The systematic analysis of the serine protease-like proteins in the two plant species has provided some insight into the possible functional associations of previously uncharacterised serine protease-like proteins. Further investigation of these aspects may prove beneficial in our understanding of similar processes in commercially significant crop plant species.

  14. An Accessory Agonist Binding Site Promotes Activation of α4β2* Nicotinic Acetylcholine Receptors.

    Science.gov (United States)

    Wang, Jingyi; Kuryatov, Alexander; Sriram, Aarati; Jin, Zhuang; Kamenecka, Theodore M; Kenny, Paul J; Lindstrom, Jon

    2015-05-29

    Neuronal nicotinic acetylcholine receptors containing α4, β2, and sometimes other subunits (α4β2* nAChRs) regulate addictive and other behavioral effects of nicotine. These nAChRs exist in several stoichiometries, typically with two high affinity acetylcholine (ACh) binding sites at the interface of α4 and β2 subunits and a fifth accessory subunit. A third low affinity ACh binding site is formed when this accessory subunit is α4 but not if it is β2. Agonists selective for the accessory ACh site, such as 3-[3-(3-pyridyl)-1,2,4-oxadiazol-5-yl]benzonitrile (NS9283), cannot alone activate a nAChR but can facilitate more efficient activation in combination with agonists at the canonical α4β2 sites. We therefore suggest categorizing agonists according to their site selectivity. NS9283 binds to the accessory ACh binding site; thus it is termed an accessory site-selective agonist. We expressed (α4β2)2 concatamers in Xenopus oocytes with free accessory subunits to obtain defined nAChR stoichiometries and α4/accessory subunit interfaces. We show that α2, α3, α4, and α6 accessory subunits can form binding sites for ACh and NS9283 at interfaces with α4 subunits, but β2 and β4 accessory subunits cannot. To permit selective blockage of the accessory site, α4 threonine 126 located on the minus side of α4 that contributes to the accessory site, but not the α4β2 sites, was mutated to cysteine. Alkylation of this cysteine with a thioreactive reagent blocked activity of ACh and NS9283 at the accessory site. Accessory agonist binding sites are promising drug targets. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

  15. Spinal cord NR1 serine phosphorylation and NR2B subunit suppression following peripheral inflammation

    Directory of Open Access Journals (Sweden)

    Del Valle-Pinero Arseima Y

    2005-09-01

    Full Text Available Abstract Background Spinal cord N-methyl-D-aspartate (NMDA receptors are intimately involved in the development and maintenance of central sensitization. However, the mechanisms mediating the altered function of the NMDA receptors are not well understood. In this study the role of phosphorylation of NR1 splice variants and NR2 subunits was examined following hind paw inflammation in rats. We further examined the level of expression of these proteins following the injury. Results Lumbar spinal cord NR1 subunits were found to be phosphorylated on serine residues within two hours of the induction of hind paw inflammation with carrageenan. The enhanced NR1 serine phosphorylation reversed within six hours. No phosphorylation on NR1 threonine or tyrosine residues was observed. Likewise, no NR2 subunit phosphorylation was observed on serine, threonine or tyrosine residues. An analysis of NR1 and NR2 protein expression demonstrated no change in the levels of NR1 splice variants or NR2A following the inflammation. However, spinal cord NR2B expression was depressed by the hind paw inflammation. The expression of NR2B remained depressed for more than one week following initiation of the inflammation. Conclusion These data suggest that NR1 serine phosphorylation leads to an initial increase in NMDA receptor activity in the spinal cord following peripheral injury. The suppression of NR2B expression suggests compensation for the enhanced nociceptive activity. These data indicate that spinal cord NMDA receptors are highly dynamic in the development, maintenance and recovery from central sensitization following an injury. Thus, chronic pain therapies targeted to NMDA receptors should be designed for the exact configuration of NMDA receptor subunits and post-translational modifications present during specific stages of the disease.

  16. Neutron activation analysis to the profile surface sediments from several sites on the Havana Bay

    International Nuclear Information System (INIS)

    Diaz Riso, O.; Gelen, A.; Lopez, N.; Gonzalez, H.; Manso, M.V.; Graciano, A.M.; Nogueira, C.A.; Beltran, J.; Soto, J.

    2003-01-01

    Instrumental neutron activation analysis (INAA) technique was employed to analyze the surface sediments from several sites on the Havana Bay, Cuba. Measurements of heavy and trace elements in the sediments are reported. The results show that the concentration of the elements is site dependent. The data suggest that an anthropogenic input into the bay from domestic sewage and industries occurred

  17. 10 CFR 60.18 - Review of site characterization activities. 2

    Science.gov (United States)

    2010-01-01

    ... 10 Energy 2 2010-01-01 2010-01-01 false Review of site characterization activities. 2 60.18 Section 60.18 Energy NUCLEAR REGULATORY COMMISSION (CONTINUED) DISPOSAL OF HIGH-LEVEL RADIOACTIVE WASTES... Atomic Energy Act of 1954, as amended. Accordingly, neither the issuance of a site characterization...

  18. 10 CFR 63.16 - Review of site characterization activities. 2

    Science.gov (United States)

    2010-01-01

    ... 10 Energy 2 2010-01-01 2010-01-01 false Review of site characterization activities. 2 63.16 Section 63.16 Energy NUCLEAR REGULATORY COMMISSION (CONTINUED) DISPOSAL OF HIGH-LEVEL RADIOACTIVE WASTES... recommendations or stating objections to DOE's site characterization program. The Director shall invite public...

  19. Microstructure and nanomechanical properties of enamel remineralized with asparagine-serine-serine peptide

    Energy Technology Data Exchange (ETDEWEB)

    Chung, Hsiu-Ying, E-mail: hychung@mail.fcu.edu.tw; Li, Cheng Che

    2013-03-01

    A highly biocompatible peptide, triplet repeats of asparagine-serine-serine (3NSS) was designed to regulate mineral deposition from aqueous ions in saliva for the reconstruction of enamel lesions. Healthy human enamel was sectioned and acid demineralized to create lesions, then exposed to the 3NSS peptide solution, and finally immersed in artificial saliva for 24 h. The surface morphology and roughness were examined using scanning electron microscopy (SEM) and atomic force microscopy (AFM), respectively. X-ray diffraction (XRD) was used to identify the phases and crystallinity of the deposited minerals observed on the enamel surface. Attenuated total reflection Fourier transform infrared spectroscopy (ATR-FTIR) was used to quantitatively analyze the mineral variation by calculating the relative integrated-area of characteristic bands. Nanohardness and elastic modulus measured by nanoindentation at various treatment stages were utilized to evaluate the degree of recovery. Biomimetic effects were accessed according to the degree of nanohardness recovery and the amount of hydroxyapatite deposition. The charged segments in the 3NSS peptide greatly attracted aqueous ions from artificial saliva to form hydroxyapatite crystals to fill enamel caries, in particular the interrod areas, resulting in a slight reduction in overall surface roughness. Additionally, the deposited hydroxyapatites were of a small crystalline size in the presence of the 3NSS peptide, which effectively restrained the plastic deformations and thus resulted in greater improvements in nanohardness and elastic modulus. The degree of nanohardness recovery was 5 times greater for remineralized enamel samples treated with the 3NSS peptide compared to samples without peptide treatment. - Highlights: Black-Right-Pointing-Pointer The degree of nanohardness recovery of enamel was 4 times greater with the aid of 3NSS peptide. Black-Right-Pointing-Pointer 3NSS peptide promoted the formation of hydroxyapatites with

  20. Regulation of xanthine oxidase activity by substrates at active sites via cooperative interactions between catalytic subunits: implication to drug pharmacokinetics.

    Science.gov (United States)

    Tai, L A; Hwang, K C

    2011-01-01

    Three xanthine oxidase substrates (i.e., xanthine, adenine, and 2-amino-4-hydroxypterin) show a "substrate inhibition" pattern (i.e., slower turnover rates at higher substrate concentrations), whereas another two substrates (i.e., xanthopterin and lumazine) show a "substrate activation" pattern (i.e., higher turnover rates at higher substrate concentrations). Binding of a 6-formylpterin at one of the two xanthine oxidase active sites slows down the turnover rate of xanthine at the adjacent active site from 17.0 s(-1) to 10.5 s(-1), and converts the V-[S] plot from "substrate inhibition" pattern to a classical Michaelis-Menten hyperbolic saturation pattern. In contrast, binding of xanthine at an active site accelerates the turnover rate of 6-formylpterin at the neighboring active site. The experimental results demonstrate that a substrate can regulate the activity of xanthine oxidase via binding at the active sites; or a xanthine oxidase catalytic subunit can simultaneously serve as a regulatory unit. Theoretical simulation based on the velocity equation derived from the extended Michaelis-Menten model shows that the substrate inhibition and the substrate activation behavior in the V-[S] plots could be obtained by introducing cooperative interactions between two catalytic subunits in homodimeric enzymes. The current work confirms that there exist very strong cooperative interactions between the two catalytic subunits of xanthine oxidase.

  1. The balance of flexibility and rigidity in the active site residues of hen egg white lysozyme

    International Nuclear Information System (INIS)

    Qi Jian-Xun; Jiang Fan

    2011-01-01

    The crystallographic temperature factors (B factor) of individual atoms contain important information about the thermal motion of the atoms in a macromolecule. Previously the theory of flexibility of active site has been established based on the observation that the enzyme activity is sensitive to low concentration denaturing agents. It has been found that the loss of enzyme activity occurs well before the disruption of the three-dimensional structural scaffold of the enzyme. To test the theory of conformational flexibility of enzyme active site, crystal structures were perturbed by soaking in low concentration guanidine hydrochloride solutions. It was found that many lysozyme crystals tested could still diffract until the concentration of guanidine hydrochloride reached 3 M. It was also found that the B factors averaged over individually collected data sets were more accurate. Thus it suggested that accurate measurement of crystal temperature factors could be achieved for medium-high or even medium resolution crystals by averaging over multiple data sets. Furthermore, we found that the correctly predicted active sites included not only the more flexible residues, but also some more rigid residues. Both the flexible and the rigid residues in the active site played an important role in forming the active site residue network, covering the majority of the substrate binding residues. Therefore, this experimental prediction method may be useful for characterizing the binding site and the function of a protein, such as drug targeting. (interdisciplinary physics and related areas of science and technology)

  2. Cytokine-induced inside-out activation of FcaR (CD89) is mediated by a single serine residue (S263) in the intracellular domain of the receptor

    NARCIS (Netherlands)

    Bracke, M.S.G.M.; Lammers, J.W.J.; Coffer, P.J.; Koenderman, L.

    2001-01-01

    Fc receptors play an important role in leukocyte activation and the modulation of ligand binding ("activation") is a criti-cal point of regulation. Previous studies demonstrated that the Fc receptor for IgA (FcaRI/CD89) is regulated by cytokine stimulation, switching it to a

  3. Protein Kinase C-Mediated Phosphorylation of BCL11B at Serine 2 Negatively Regulates Its Interaction with NuRD Complexes during CD4+ T-Cell Activation.

    Science.gov (United States)

    Dubuissez, Marion; Loison, Ingrid; Paget, Sonia; Vorng, Han; Ait-Yahia, Saliha; Rohr, Olivier; Tsicopoulos, Anne; Leprince, Dominique

    2016-07-01

    The transcription factor BCL11B/CTIP2 is a major regulatory protein implicated in various aspects of development, function and survival of T cells. Mitogen-activated protein kinase (MAPK)-mediated phosphorylation and SUMOylation modulate BCL11B transcriptional activity, switching it from a repressor in naive murine thymocytes to a transcriptional activator in activated thymocytes. Here, we show that BCL11B interacts via its conserved N-terminal MSRRKQ motif with endogenous MTA1 and MTA3 proteins to recruit various NuRD complexes. Furthermore, we demonstrate that protein kinase C (PKC)-mediated phosphorylation of BCL11B Ser2 does not significantly impact BCL11B SUMOylation but negatively regulates NuRD recruitment by dampening the interaction with MTA1 or MTA3 (MTA1/3) and RbAp46 proteins. We detected increased phosphorylation of BCL11B Ser2 upon in vivo activation of transformed and primary human CD4(+) T cells. We show that following activation of CD4(+) T cells, BCL11B still binds to IL-2 and Id2 promoters but activates their transcription by recruiting P300 instead of MTA1. Prolonged stimulation results in the direct transcriptional repression of BCL11B by KLF4. Our results unveil Ser2 phosphorylation as a new BCL11B posttranslational modification linking PKC signaling pathway to T-cell receptor (TCR) activation and define a simple model for the functional switch of BCL11B from a transcriptional repressor to an activator during TCR activation of human CD4(+) T cells. Copyright © 2016, American Society for Microbiology. All Rights Reserved.

  4. 1993 annual report of hazardous waste activities for the Oak Ridge K-25 site

    Energy Technology Data Exchange (ETDEWEB)

    1994-02-01

    This report is a detailed listing of all of the Hazardous Waste activities occurring at Martin Marietta`s K-25 site. Contained herein are hazardous waste notification forms, waste stream reports, generator fee forms and various TSDR reports.

  5. 1993 annual report of hazardous waste activities for the Oak Ridge K-25 site

    International Nuclear Information System (INIS)

    1994-02-01

    This report is a detailed listing of all of the Hazardous Waste activities occurring at Martin Marietta's K-25 site. Contained herein are hazardous waste notification forms, waste stream reports, generator fee forms and various TSDR reports

  6. Understanding the catalytic benefits of multi=metallic active sites in microporous aluminophosphates

    OpenAIRE

    Potter, Matthew

    2015-01-01

    Structure-property correlations, which direct the formation of specific active-acid sites, in nanoporous materials have been explored with the aim to further the notion of rational catalyst design. It is demonstrated that the influence of the specific zeotype framework extends beyond modulating Brønsted acidity, with the framework topology playing a fundamental role in reaction kinetics. The structural integrity and nature of the active site has been probed using a combined catalysis and mult...

  7. XAFS Study of the Photo-Active Site of Mo/MCM-41

    International Nuclear Information System (INIS)

    Miyamoto, Daisuke; Ichikuni, Nobuyuki; Shimazu, Shogo

    2007-01-01

    An Mo/MCM-41 catalyst was prepared and used for study of propene and 1-butene photo-metathesis reactions. XAFS analysis revealed that hydrogen reduction leads to a decreased role for the Mo=O site. The Mo-O site plays an important role for the olefin photo-metathesis reaction on the H2 reduced Mo/MCM-41. From EXAFS analysis, the active site of photo-metathesis reaction is the Mo=O part for oxidized Mo/MCM-41, whereas it is the Mo-O site for reduced Mo/MCM-41

  8. Surface structural-chemical characterization of a single-site d0 heterogeneous arene hydrogenation catalyst having 100% active sites

    Science.gov (United States)

    Williams, Linda A.; Guo, Neng; Motta, Alessandro; Delferro, Massimiliano; Fragalà, Ignazio L.; Miller, Jeffrey T.; Marks, Tobin J.

    2013-01-01

    Structural characterization of the catalytically significant sites on solid catalyst surfaces is frequently tenuous because their fraction, among all sites, typically is quite low. Here we report the combined application of solid-state 13C-cross-polarization magic angle spinning nuclear magnetic resonance (13C-CPMAS-NMR) spectroscopy, density functional theory (DFT), and Zr X-ray absorption spectroscopy (XAS) to characterize the adsorption products and surface chemistry of the precatalysts (η5-C5H5)2ZrR2 (R = H, CH3) and [η5-C5(CH3)5]Zr(CH3)3 adsorbed on Brønsted superacidic sulfated alumina (AlS). The latter complex is exceptionally active for benzene hydrogenation, with ∼100% of the Zr sites catalytically significant as determined by kinetic poisoning experiments. The 13C-CPMAS-NMR, DFT, and XAS data indicate formation of organozirconium cations having a largely electrostatic [η5-C5(CH3)5]Zr(CH3)2+···AlS− interaction with greatly elongated Zr···OAlS distances of ∼2.35(2) Å. The catalytic benzene hydrogenation cycle is stepwise understandable by DFT, and proceeds via turnover-limiting H2 delivery to surface [η5-C5(CH3)5]ZrH2(benzene)+···AlS− species, observable by solid-state NMR and XAS. PMID:23269836

  9. Poisoning Experiments Aimed at Discriminating Active and Less-Active Sites of Silica-Supported Tantalum Hydride for Alkane Metathesis

    KAUST Repository

    Saggio, Guillaume

    2010-10-04

    Only 50% of the silica-supported tantalum hydride sites are active in the metathesis of propane. Indeed, more than 45% of the tantalum hydride can be eliminated by a selective oxygen poisoning of inactive sites with no significant decrease in the global turnover. Conversely, cyclopentane induces no such selective poisoning. Hence, the active tantalum hydride sites that show greater resistance to oxygen poisoning correspond to the νTa-H bands of higher wavenumbers, particularly that at 1860cm-1. These active tantalum hydride sites should correspond to tris- or monohydride species relatively far from silica surface oxygen atoms. © 2010 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  10. From phosphatases to vanadium peroxidases: a similar architecture of the active site.

    Science.gov (United States)

    Hemrika, W; Renirie, R; Dekker, H L; Barnett, P; Wever, R

    1997-03-18

    We show here that the amino acid residues contributing to the active sites of the vanadate containing haloperoxidases are conserved within three families of acid phosphatases; this suggests that the active sites of these enzymes are very similar. This is confirmed by activity measurements showing that apochloroperoxidase exhibits phosphatase activity. These observations not only reveal interesting evolutionary relationships between these groups of enzymes but may also have important implications for the research on acid phosphatases, especially glucose-6-phosphatase-the enzyme affected in von Gierke disease-of which the predicted membrane topology may have to be reconsidered.

  11. Concept for calculating dose rates from activated groundwater at accelerator sites

    CERN Document Server

    Prolingheuer, N; Vanderborght, J; Schlögl, B; Nabbi, R; Moormann, R

    Licensing of particle accelerators requires the proof that the groundwater outside of the site will not be significantly contaminated by activation products formed below accelerator and target. In order to reduce the effort for this proof, a site independent simplified but conservative method is under development. The conventional approach for calculation of activation of soil and groundwater is shortly described on example of a site close to Forschungszentrum Juelich, Germany. Additionally an updated overview of a data library for partition coefficients for relevant nuclides transported in the aquifer at the site is presented. The approximate model for transport of nuclides with ground water including exemplary results on nuclide concentrations outside of the site boundary and of resulting effective doses is described. Further applications and developments are finally outlined.

  12. Cross-phosphorylation of bacterial serine/threonine and tyrosine protein kinases on key regulatory residues

    Directory of Open Access Journals (Sweden)

    Lei eShi

    2014-09-01

    Full Text Available Bacteria possess protein serine/threonine and tyrosine kinases which resemble eukaryal kinases in their capacity to phosphorylate multiple substrates. We hypothesized that the analogy might extend further, and bacterial kinases may also undergo mutual phosphorylation and activation, which is currently considered as a hallmark of eukaryal kinase networks. In order to test this hypothesis, we explored the capacity of all members of four different classes of serine/threonine and tyrosine kinases present in the firmicute model organism Bacillus subtilis to phosphorylate each other in vitro and interact with each other in vivo. The interactomics data suggested a high degree of connectivity among all types of kinases, while phosphorylation assays revealed equally wide-spread cross-phosphorylation events. Our findings suggest that the Hanks-type kinases PrkC, PrkD and YabT exhibit the highest capacity to phosphorylate other B. subtilis kinases, while the BY-kinase PtkA and the two-component-like kinases RsbW and SpoIIAB show the highest propensity to be phosphorylated by other kinases. Analysis of phosphorylated residues on several selected recipient kinases suggests that most cross-phosphorylation events concern key regulatory residues. Therefore, cross-phosphorylation events are very likely to influence the capacity of recipient kinases to phosphorylate substrates downstream in the signal transduction cascade. We therefore conclude that bacterial serine/threonine and tyrosine kinases probably engage in a network-type behavior previously described only in eukaryal cells.

  13. Subcellular localization of an intracellular serine protease of 68 kDa in Leishmania (Leishmania amazonensis promastigotes

    Directory of Open Access Journals (Sweden)

    José Andrés Morgado-Díaz

    2005-07-01

    Full Text Available Here we report the subcellular localization of an intracellular serine protease of 68 kDa in axenic promastigotes of Leishmania (Leishmania amazonensis, using subcellular fractionation, enzymatic assays, immunoblotting, and immunocytochemistry. All fractions were evaluated by transmission electron microscopy and the serine protease activity was measured during the cell fractionation procedure using a-N-r-tosyl-L-arginine methyl ester (L-TAME as substrate, phenylmethylsulphone fluoride (PMSF and L-1-tosylamino-2-phenylethylchloromethylketone (TPCK as specific inhibitors. The enzymatic activity was detected mainly in a membranous vesicular fraction (6.5-fold enrichment relative to the whole homogenate, but also in a crude plasma membrane fraction (2.0-fold. Analysis by SDS-PAGE gelatin under reducing conditions demonstrated that the major proteolytic activity was found in a 68 kDa protein in all fractions studied. A protein with identical molecular weight was also recognized in immunoblots by a polyclonal antibody against serine protease (anti-SP, with higher immunoreactivity in the vesicular fraction. Electron microscopic immunolocalization using the same polyclonal antibody showed the enzyme present at the cell surface, as well as in cytoplasmic membranous compartments of the parasite. Our findings indicate that the internal location of this serine protease in L. amazonensis is mainly restricted to the membranes of intracellular compartments resembling endocytic/exocytic elements.

  14. The status of siting activities for a low level waste repository in the Philippines

    International Nuclear Information System (INIS)

    Valdezco, E.M.; Visitacion, M.; Palattao, B.; Marcelo, E.A.; Venida, L.L.

    2001-01-01

    The process of site selection for a low level waste repository was initiated in 1976 when the Philippine Government decided to go nuclear and constructed the first Philippine Nuclear Power Plant in the Bataan Peninsula. However, all siting activities were suspended when the nuclear power plant was mothballed and the final decision was made to convert the plant into a combined cycle power plant. In 1995, an inter-agency committee was created under the Nuclear Power Steering Committee and mandated to conduct studies on siting of radioactive waste disposal facilities, and at the same time, perform R and D activities in support of the project. This paper describes the various siting activities carried out to date. (author)

  15. Structural and Kinetic Analyses of Macrophage Migration Inhibitory Factor Active Site Interactions

    Energy Technology Data Exchange (ETDEWEB)

    Crichlow, G.; Lubetsky, J; Leng, L; Bucala, R; Lolis, E

    2009-01-01

    Macrophage migration inhibitory factor (MIF) is a secreted protein expressed in numerous cell types that counters the antiinflammatory effects of glucocorticoids and has been implicated in sepsis, cancer, and certain autoimmune diseases. Interestingly, the structure of MIF contains a catalytic site resembling the tautomerase/isomerase sites of microbial enzymes. While bona fide physiological substrates remain unknown, model substrates have been identified. Selected compounds that bind in the tautomerase active site also inhibit biological functions of MIF. It had previously been shown that the acetaminophen metabolite, N-acetyl-p-benzoquinone imine (NAPQI), covalently binds to the active site of MIF. In this study, kinetic data indicate that NAPQI inhibits MIF both covalently and noncovalently. The structure of MIF cocrystallized with NAPQI reveals that the NAPQI has undergone a chemical alteration forming an acetaminophen dimer (bi-APAP) and binds noncovalently to MIF at the mouth of the active site. We also find that the commonly used protease inhibitor, phenylmethylsulfonyl fluoride (PMSF), forms a covalent complex with MIF and inhibits the tautomerase activity. Crystallographic analysis reveals the formation of a stable, novel covalent bond for PMSF between the catalytic nitrogen of the N-terminal proline and the sulfur of PMSF with complete, well-defined electron density in all three active sites of the MIF homotrimer. Conclusions are drawn from the structures of these two MIF-inhibitor complexes regarding the design of novel compounds that may provide more potent reversible and irreversible inhibition of MIF.

  16. Discrepancy between low levels of mTOR activity and high levels of p-S6 in primary central nervous system lymphoma may be explained by PAS domain-containing serine/threonine-protein kinase-mediated phosphorylation

    DEFF Research Database (Denmark)

    Marosvari, Dora; Nagy, Noemi; Kriston, Csilla

    2018-01-01

    The primary aim of this study was to determine mTOR-pathway activity in primary central nervous system lymphoma (PCNSL), which could be a potential target for therapy. After demonstrating that p-S6 positivity largely exceeded mTOR activity, we aimed to identify other pathways that may lead to S6...... phosphorylation. We measured mTOR activity with immunohistochemistry for p-mTOR and its downstream effectors p(T389)-p70S6K1, p-S6, and p-4EBP1 in 31 cases of PCNSL and 51 cases of systemic diffuse large B-cell lymphoma (DLBCL) and evaluated alternative S6 phosphorylation pathways with p-RSK, p(T229)-p70S6K1......, and PASK antibodies. Finally, we examined the impact of PASK inhibition on S6 phosphorylation on BHD1 cell line. mTOR-pathway activity was significantly less frequent in PCNSL compared with DLBCL. p-S6 positivity was related to mTOR-pathway in DLBCL, but not in PCNSL. Among the other kinases potentially...

  17. Heterogeneity of the serine synthetic pathway in Entamoeba species.

    Science.gov (United States)

    Chiba, Yoko; Makiuchi, Takashi; Jeelani, Ghulam; Nozaki, Tomoyoshi

    2016-06-01

    Phosphoserine phosphatase (PSP) catalyzes the third step of the phosphorylated serine biosynthetic pathway, and occurred multiple times in evolution, while enzymes catalyzing the first and second steps in the pathway have single respective origins. In the present study, we examined the existence of PSP among genus Entamoeba including a human enteric parasite, Entamoeba histolytica. E. histolytica as well as majority of Entamoeba species have the first and second enzymes, but lacks PSP. In contrast, a reptilian enteric parasite, Entamoeba invadens possesses canonical PSP. Thus, there are variations in the existence of the serine biosynthetic ability among Entamoeba species. Copyright © 2016 Elsevier B.V. All rights reserved.

  18. Convergent synthesis of a deuterium-labeled serine dipeptide lipid for analysis of biological samples.

    Science.gov (United States)

    Dietz, Christopher; Clark, Robert B; Nichols, Frank C; Smith, Michael B

    2017-05-30

    Bacterial serine dipeptide lipids are known to promote inflammatory processes and are detected in human tissues associated with periodontal disease or atherosclerosis. Accurate quantification of bacterial serine lipid, specifically lipid 654 [((S)-15-methyl-3-((13-methyltetradecanoyl)oxy)hexadecanoyl)glycyl-l-serine, (3S)-l-serine] isolated from Porphyromonas gingivalis, in biological samples requires the preparation of a stable isotope internal standard for sample supplementation and subsequent mass spectrometric analysis. This report describes the convergent synthesis of a deuterium-substituted serine dipeptide lipid, which is an isotopically labeled homologue that represents a dominant form of serine dipeptide lipid recovered in bacteria. Copyright © 2017 John Wiley & Sons, Ltd.

  19. Feasibility, Safety, and Efficacy of the Combination of -Serine and Computerized Cognitive Retraining in Schizophrenia: An International Collaborative Pilot Study

    OpenAIRE

    D'Souza, Deepak C; Radhakrishnan, Rajiv; Perry, Edward; Bhakta, Savita; Singh, Nagendra M; Yadav, Richa; Abi-Saab, Danielle; Pittman, Brian; Chaturvedi, Santosh K; Sharma, Mahendra P; Bell, Morris; Andrade, Chittaranjan

    2012-01-01

    The combination of pharmacotherapy and cognitive retraining (CRT) for the cognitive deficits of schizophrenia may be more efficacious than either approach alone, but this has not yet been tested. This study evaluated the feasibility, safety, tolerability, and efficacy of 12 weeks of -serine, combined with CRT in the treatment of cognitive deficits in schizophrenia at two academic sites in parallel, in India and the United States. In a randomized, partial double-blind, placebo-controlled, par...

  20. Effects of resource activities upon repository siting and waste containment with reference to bedded salt

    Energy Technology Data Exchange (ETDEWEB)

    Ashby, J.; Rowe, J.

    1980-02-01

    The primary consideration for the suitability of a nuclear waste repository site is the overall ability of the repository to safely contain radioactive waste. This report is a discussion of the past, present, and future effects of resource activities on waste containment. Past and present resource activities which provide release pathways (i.e., leaky boreholes, adjacent mines) will receive initial evaluation during the early stages of any repository site study. However, other resource activities which may have subtle effects on containment (e.g., long-term pumping causing increased groundwater gradients, invasion of saline water causing lower retardation) and all potential future resource activities must also be considered during the site evaluation process. Resource activities will affect both the siting and the designing of repositories. Ideally, sites should be located in areas of low resource activity and low potential for future activity, and repository design should seek to eliminate or minimize the adverse effects of any resource activity. Buffer zones should be created to provide areas in which resource activities that might adversely affect containment can be restricted or curtailed. This could mean removing large areas of land from resource development. The impact of these frozen assets should be assessed in terms of their economic value and of their effect upon resource reserves. This step could require a major effort in data acquisition and analysis followed by extensive numerical modeling of regional fluid flow and mass transport. Numerical models should be used to assess the effects of resource activity upon containment and should include the cumulative effects of different resource activities. Analysis by other methods is probably not possible except for relatively simple cases.

  1. Effects of resource activities upon repository siting and waste containment with reference to bedded salt

    International Nuclear Information System (INIS)

    Ashby, J.; Rowe, J.

    1980-02-01

    The primary consideration for the suitability of a nuclear waste repository site is the overall ability of the repository to safely contain radioactive waste. This report is a discussion of the past, present, and future effects of resource activities on waste containment. Past and present resource activities which provide release pathways (i.e., leaky boreholes, adjacent mines) will receive initial evaluation during the early stages of any repository site study. However, other resource activities which may have subtle effects on containment (e.g., long-term pumping causing increased groundwater gradients, invasion of saline water causing lower retardation) and all potential future resource activities must also be considered during the site evaluation process. Resource activities will affect both the siting and the designing of repositories. Ideally, sites should be located in areas of low resource activity and low potential for future activity, and repository design should seek to eliminate or minimize the adverse effects of any resource activity. Buffer zones should be created to provide areas in which resource activities that might adversely affect containment can be restricted or curtailed. This could mean removing large areas of land from resource development. The impact of these frozen assets should be assessed in terms of their economic value and of their effect upon resource reserves. This step could require a major effort in data acquisition and analysis followed by extensive numerical modeling of regional fluid flow and mass transport. Numerical models should be used to assess the effects of resource activity upon containment and should include the cumulative effects of different resource activities. Analysis by other methods is probably not possible except for relatively simple cases

  2. Unveiling Active Sites for the Hydrogen Evolution Reaction on Monolayer MoS2.

    Science.gov (United States)

    Zhang, Jing; Wu, Jingjie; Guo, Hua; Chen, Weibing; Yuan, Jiangtan; Martinez, Ulises; Gupta, Gautam; Mohite, Aditya; Ajayan, Pulickel M; Lou, Jun

    2017-11-01

    Here, the hydrogen evolution reaction (HER) activities at the edge and basal-plane sites of monolayer molybdenum disulfide (MoS 2 ) synthesized by chemical vapor deposition (CVD) are studied using a local probe method enabled by selected-area lithography. Reaction windows are opened by e-beam lithography at sites of interest on poly(methyl methacrylate) (PMMA)-covered monolayer MoS 2 triangles. The HER properties of MoS 2 edge sites are obtained by subtraction of the activity of the basal-plane sites from results containing both basal-plane and edge sites. The catalytic performances in terms of turnover frequencies (TOFs) are calculated based on the estimated number of active sites on the selected areas. The TOFs follow a descending order of 3.8 ± 1.6, 1.6 ± 1.2, 0.008 ± 0.002, and 1.9 ± 0.8 × 10 -4 s -1 , found for 1T'-, 2H-MoS 2 edges, and 1T'-, 2H-MoS 2 basal planes, respectively. Edge sites of both 2H- and 1T'-MoS 2 are proved to have comparable activities to platinum (≈1-10 s -1 ). When fitted into the HER volcano plot, the MoS 2 active sites follow a trend distinct from conventional metals, implying a possible difference in the reaction mechanism between transition-metal dichalcogenides (TMDs) and metal catalysts. © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  3. Cyanide does more to inhibit heme enzymes, than merely serving as an active-site ligand

    Energy Technology Data Exchange (ETDEWEB)

    Parashar, Abhinav [Center for Biomedical Research, VIT University, Vellore, Tamil Nadu, 632014 India (India); Venkatachalam, Avanthika [REDOx Lab, PSG Institute of Advanced Studies, Avinashi Road, Peelamedu, Coimbatore, Tamil Nadu, 641004 (India); Gideon, Daniel Andrew [Center for Biomedical Research, VIT University, Vellore, Tamil Nadu, 632014 India (India); Manoj, Kelath Murali, E-mail: satyamjayatu@yahoo.com [REDOx Lab, PSG Institute of Advanced Studies, Avinashi Road, Peelamedu, Coimbatore, Tamil Nadu, 641004 (India)

    2014-12-12

    Highlights: • Cyanide (CN) is a well-studied toxic principle, known to inhibit heme-enzymes. • Inhibition is supposed to result from CN binding at the active site as a ligand. • Diverse heme enzymes’ CN inhibition profiles challenge prevailing mechanism. • Poor binding efficiency of CN at low enzyme concentrations and ligand pressures. • CN-based diffusible radicals cause ‘non-productive electron transfers’ (inhibition). - Abstract: The toxicity of cyanide is hitherto attributed to its ability to bind to heme proteins’ active site and thereby inhibit their activity. It is shown herein that the long-held interpretation is inadequate to explain several observations in heme-enzyme reaction systems. Generation of cyanide-based diffusible radicals in heme-enzyme reaction milieu could shunt electron transfers (by non-active site processes), and thus be detrimental to the efficiency of oxidative outcomes.

  4. Water molecule network and active site flexibility of apo protein tyrosine phosphatase 1B

    DEFF Research Database (Denmark)

    Pedersen, A.K.; Peters, Günther H.J.; Møller, K.B.

    2004-01-01

    the conformation and flexibility of active-site residues as well as the water-molecule network, is a key issue in understanding ligand binding and enzyme kinetics and in structure-based drug design. A 1.95 Angstrom apo PTP1B structure has been obtained, showing four highly coordinated water molecules in the active......Protein tyrosine phosphatase 1B (PTP1B) plays a key role as a negative regulator of insulin and leptin signalling and is therefore considered to be an important molecular target for the treatment of type 2 diabetes and obesity. Detailed structural information about the structure of PTP1B, including......-site pocket of the enzyme; hence, the active site is highly solvated in the apo state. Three of the water molecules are located at positions that approximately correspond to the positions of the phosphate O atoms of the natural substrate phosphotyrosine and form a similar network of hydrogen bonds. The active...

  5. Purification and biochemical characterization of the haloalkaliphilic archaeon Natronococcus occultus extracellular serine protease

    DEFF Research Database (Denmark)

    Studdert, C A; Herrera Seitz, M K; Plasencia, I

    2001-01-01

    other halobacteria nor with commercial proteases except subtilisin. The amino acid sequences of three tryptic peptides obtained from Natronococcus occultus protease did not show significant similarity to other known proteolytic enzymes. This fact, in addition to its high molecular mass suggests......A serine protease was purified from Natronococcus occultus stationary phase culture medium (328-fold, yield 19%) and characterized at the biochemical level. The enzyme has a native molecular mass of 130 kDa, has chymotrypsin-like activity, is stable and active in a broad pH range (5...... that Natronococcus occultus extracellular protease may be a novel enzyme. Udgivelsesdato: 2001-null...

  6. Enhancement in catalytic activity of Aspergillus niger XynB by selective site-directed mutagenesis of active site amino acids.

    Science.gov (United States)

    Wu, Xiuyun; Tian, Zhennan; Jiang, Xukai; Zhang, Qun; Wang, Lushan

    2018-01-01

    XynB from Aspergillus niger ATCC1015 (AnXynB) is a mesophilic glycoside hydrolase (GH) family 11 xylanase which holds great potentials in a wide variety of industrial applications. In the present study, the catalytic activity and stability of AnXynB were improved by a combination of computational and experimental approaches. Virtual mutation and molecular dynamics simulations indicated that the introduction of Glu and Asn altered the interaction network at the - 3 subsite. Interestingly, the double mutant S41N/T43E displayed 72% increase in catalytic activity when compared to the wild type (WT). In addition, it also showed a better thermostability than the WT enzyme. Kinetic determination of the T43E and S41N/T43E mutants suggested that the higher xylanase activity is probably due to the increasing binding affinity of enzyme and substrate. Consequently, the enzyme activity and thermostability of AnXynB was both increased by selective site-directed mutagenesis at the - 3 subsite of its active site architecture which provides a good example for a successfully engineered enzyme for potential industrial application. Moreover, the molecular evolution approach adopted in this study led to the design of a library of sequences that captures a meaningful functional diversity in a limited number of protein variants.

  7. Optimization of serine protease purification from mango (Mangifera indica cv. Chokanan) peel in polyethylene glycol/dextran aqueous two phase system.

    Science.gov (United States)

    Mehrnoush, Amid; Mustafa, Shuhaimi; Sarker, Md Zaidul Islam; Yazid, Abdul Manap Mohd

    2012-01-01

    Mango peel is a good source of protease but remains an industrial waste. This study focuses on the optimization of polyethylene glycol (PEG)/dextran-based aqueous two-phase system (ATPS) to purify serine protease from mango peel. The activity of serine protease in different phase systems was studied and then the possible relationship between the purification variables, namely polyethylene glycol molecular weight (PEG, 4000-12,000 g·mol(-1)), tie line length (-3.42-35.27%), NaCl (-2.5-11.5%) and pH (4.5-10.5) on the enzymatic properties of purified enzyme was investigated. The most significant effect of PEG was on the efficiency of serine protease purification. Also, there was a significant increase in the partition coefficient with the addition of 4.5% of NaCl to the system. This could be due to the high hydrophobicity of serine protease compared to protein contaminates. The optimum conditions to achieve high partition coefficient (84.2) purification factor (14.37) and yield (97.3%) of serine protease were obtained in the presence of 8000 g·mol(-1) of PEG, 17.2% of tie line length and 4.5% of NaCl at pH 7.5. The enzymatic properties of purified serine protease using PEG/dextran ATPS showed that the enzyme could be purified at a high purification factor and yield with easy scale-up and fast processing.

  8. Large zinc cation occupancy of octahedral sites in mechanically activated zinc ferrite powders

    International Nuclear Information System (INIS)

    Oliver, S. A.; Harris, V. G.; Hamdeh, H. H.; Ho, J. C.

    2000-01-01

    The cation site occupancy of a mechanically activated nanocrystalline zinc ferrite powder was determined as (Zn 0.55 2+ Fe 0.18 3+ ) tet [Zr 0.45 2+ Fe 1.82 3+ ] oct O 4 through analysis of extended x-ray absorption fine structure measurements, showing a large redistribution of cations between sites compared to normal zinc ferrite samples. The overpopulation of cations in the octahedral sites was attributed to the ascendance in importance of the ionic radii over the crystal energy and bonding coordination in determining which interstitial sites are occupied in this structurally disordered powder. Slight changes are observed in the local atomic environment about the zinc cations, but not the iron cations, with respect to the spinel structure. The presence of Fe 3+ on both sites is consistent with the measured room temperature magnetic properties. (c) 2000 American Institute of Physics

  9. Evaluation of physical activity web sites for use of behavior change theories.

    Science.gov (United States)

    Doshi, Amol; Patrick, Kevin; Sallis, James F; Calfas, Karen

    2003-01-01

    Physical activity (PA) Web sites were assessed for their use of behavior change theories, including constructs of the health belief model, Transtheoretical Model, social cognitive theory, and the theory of reasoned action and planned behavior. An evaluation template for assessing PA Web sites was developed, and content validity and interrater reliability were demonstrated. Two independent raters evaluated 24 PA Web sites. Web sites varied widely in application of theory-based constructs, ranging from 5 to 48 on a 100-point scale. The most common intervention strategies were general information, social support, and realistic goal areas. Coverage of theory-based strategies was low, varying from 26% for social cognitive theory to 39% for health belief model. Overall, PA Web sites provided little assessment, feedback, or individually tailored assistance for users. They were unable to substantially tailor the on-line experience for users at different stages of change or different demographic characteristics.

  10. Exploring the molecular origins of protein dynamics in the active site of human carbonic anhydrase II.

    Science.gov (United States)

    Hill, Sarah E; Bandaria, Jigar N; Fox, Michelle; Vanderah, Elizabeth; Kohen, Amnon; Cheatum, Christopher M

    2009-08-20

    We present three-pulse vibrational echo measurements of azide ion bound to the active site Zn of human carbonic anhydrase II (HCA II) and of two separate active-site mutants Thr199 --> Ala (T199A) and Leu198 --> Phe (L198F). Because structural motions of the protein active site influence the frequency of bound ligands, the differences in the time scales of the frequency-frequency correlation functions (FFCFs) obtained from global fits to each set of data allow us to make inferences about the time scales of the active site dynamics of HCA II. Surprisingly, the deletion of a potential electrostatic interaction in results in very little change in the FFCF, but the insertion of the bulky phenylalanine ring in causes much faster dynamics. We conclude that the fast, sub-picosecond time scale in the correlation function is attributable to hydrogen bond dynamics, and the slow, apparently static contribution is due to the conformational flexibility of Zn-bound azide in the active site.

  11. A simplified method for active-site titration of lipases immobilised on hydrophobic supports.

    Science.gov (United States)

    Nalder, Tim D; Kurtovic, Ivan; Barrow, Colin J; Marshall, Susan N

    2018-06-01

    The aim of this work was to develop a simple and accurate protocol to measure the functional active site concentration of lipases immobilised on highly hydrophobic supports. We used the potent lipase inhibitor methyl 4-methylumbelliferyl hexylphosphonate to titrate the active sites of Candida rugosa lipase (CrL) bound to three highly hydrophobic supports: octadecyl methacrylate (C18), divinylbenzene crosslinked methacrylate (DVB) and styrene. The method uses correction curves to take into account the binding of the fluorophore (4-methylumbelliferone, 4-MU) by the support materials. We showed that the uptake of the detection agent by the three supports is not linear relative to the weight of the resin, and that the uptake occurs in an equilibrium that is independent of the total fluorophore concentration. Furthermore, the percentage of bound fluorophore varied among the supports, with 50 mg of C18 and styrene resins binding approximately 64 and 94%, respectively. When the uptake of 4-MU was calculated and corrected for, the total 4-MU released via inhibition (i.e. the concentration of functional lipase active sites) could be determined via a linear relationship between immobilised lipase weight and total inhibition. It was found that the functional active site concentration of immobilised CrL varied greatly among different hydrophobic supports, with 56% for C18, compared with 14% for DVB. The described method is a simple and robust approach to measuring functional active site concentration in immobilised lipase samples. Copyright © 2018 Elsevier Inc. All rights reserved.

  12. Substrate Shuttling Between Active Sites of Uroporphyrinogen Decarboxylase in Not Required to Generate Coproporphyrinogen

    Energy Technology Data Exchange (ETDEWEB)

    Phillips, J.; Warby, C; Whitby, F; Kushner, J; Hill, C

    2009-01-01

    Uroporphyrinogen decarboxylase (URO-D; EC 4.1.1.37), the fifth enzyme of the heme biosynthetic pathway, is required for the production of heme, vitamin B12, siroheme, and chlorophyll precursors. URO-D catalyzes the sequential decarboxylation of four acetate side chains in the pyrrole groups of uroporphyrinogen to produce coproporphyrinogen. URO-D is a stable homodimer, with the active-site clefts of the two subunits adjacent to each other. It has been hypothesized that the two catalytic centers interact functionally, perhaps by shuttling of reaction intermediates between subunits. We tested this hypothesis by construction of a single-chain protein (single-chain URO-D) in which the two subunits were connected by a flexible linker. The crystal structure of this protein was shown to be superimposable with wild-type activity and to have comparable catalytic activity. Mutations that impaired one or the other of the two active sites of single-chain URO-D resulted in approximately half of wild-type activity. The distributions of reaction intermediates were the same for mutant and wild-type sequences and were unaltered in a competition experiment using I and III isomer substrates. These observations indicate that communication between active sites is not required for enzyme function and suggest that the dimeric structure of URO-D is required to achieve conformational stability and to create a large active-site cleft.

  13. Risks due to industrial activities and to transports around nuclear installations sites

    International Nuclear Information System (INIS)

    Doumenc, A.; Faure, J.

    1988-01-01

    In order to verify that the human activities around a site under consideration are not incompatible with the installation conception, they should be analyzed before the definitive site selection, then watched over and if necessary limited during the installation construction and operation. Taking account of the aggression sources diversity, there is to consider different distances according to the risks. 6 tabs., 5 refs. (F.M.)

  14. Expression and characterization of Coprothermobacter proteolyticus alkaline serine protease

    Science.gov (United States)

    TECHNICAL ABSTRACT A putative protease gene (aprE) from the thermophilic bacterium Coprothermobacter proteolyticus was cloned and expressed in Bacillus subtilis. The enzyme was determined to be a serine protease based on inhibition by PMSF. Biochemical characterization demonstrated the enzyme had...

  15. Fatal cerebral edema associated with serine deficiency in CSF

    NARCIS (Netherlands)

    Keularts, Irene M. L. W.; Leroy, Piet L. J. M.; Rubio-Gozalbo, Estela M.; Spaapen, Leo J. M.; Weber, Biene; Dorland, Bert; de Koning, Tom J.; Verhoeven-Duif, Nanda M.

    2010-01-01

    Two young girls without a notable medical history except for asthma presented with an acute toxic encephalopathy with very low serine concentrations both in plasma and cerebrospinal fluid (CSF) comparable to patients with 3-phosphoglycerate dehydrogenase (3-PGDH) deficiency. Clinical symptoms and

  16. Serine protease from midgut of Bombus terrestris males

    Czech Academy of Sciences Publication Activity Database

    Brabcová, Jana; Kindl, Jiří; Valterová, Irena; Pichová, Iva; Zarevúcka, Marie; Brabcová, J.; Jágr, Michal; Mikšík, Ivan

    2013-01-01

    Roč. 82, č. 3 (2013), s. 117-128 ISSN 0739-4462 R&D Projects: GA ČR GA203/09/1446; GA TA ČR TA01020969 Institutional support: RVO:61388963 ; RVO:67985823 Keywords : Bombus terrestris * midgut * serine protease * bumblebee Subject RIV: CE - Biochemistry; CE - Biochemistry (FGU-C) Impact factor: 1.160, year: 2013

  17. Distribution pattern of histone H3 phosphorylation at serine 10

    Indian Academy of Sciences (India)

    Histones are the major eukaryotic DNA-binding proteins. Posttranslational modifications on N-terminal tails of histones that form nucleosomes are often associated with distinct biological functions. Some theories suggest that one of these modifications, the phosphorylation of histone H3 at serine 10 (H3S10ph) plays a role ...

  18. Metabolism of serine and ethanolamine plasmalogens in Megasphaera elsdenii

    NARCIS (Netherlands)

    Prins, R.A.; Akkermans-Kruyswijk, J.; Franklin-Klein, W.; Lankhorst, A.; Golde, L.M.G. van

    1974-01-01

    1. 1. Megasphaera elsdenii appears to be a very suitable organism for studies on the metabolism of plasmalogens in anaerobic bacteria due to its extremely high content of both serine and ethanolamine plasmalogen. 2. 2. Growth of this organism in the presence of either 32Pi or [2-3H]glycerol

  19. Production, purification and characterization of a thermotolerant alkaline serine protease from a novel species Bacillus caseinilyticus.

    Science.gov (United States)

    Mothe, Thirumala; Sultanpuram, Vishnuvardhan Reddy

    2016-06-01

    Alkaline proteases are important enzymes in many industrial applications, especially as additives in laundry detergent industry. Though there are a number of Bacillus species which are reported to be producing proteases, the efficiency of a protease produced by a novel strain has to be studied in comparison to the others. Hence, in this study, an alkaline serine protease produced by a novel species Bacillus caseinilyticus was purified and characterized for its possible usage in detergent industry. Ammonium sulphate, dialysis and DEAE column chromatographic methods were used for purification of the isolated alkaline protease. The molecular weight of the protease was determined by SDS-PAGE and it was found to be 66 kDa. Peptide mass fingerprinting (PMF) was carried out using MALDI-TOF-TOF mass spectrometry and the peptides were found to be similar to that of subtilisin protease. Specific activity of purified protein was found to be 89.2 U/mg. Optimum pH and temperature for enzyme activity were at pH 8 and 60 °C, respectively, showing stability with 10 mM CaCl 2 . Phenyl methyl sulphonyl fluoride (PMSF) at both 5 and 10 mM concentrations completely inhibited the enzyme activity suggesting its serine nature. EDTA, metal ions Mg 2+ and Ca 2+ increased the enzyme activity. The one factor at a time optimisation of the protease production was carried to identify the important factors that affect its production. After optimisation, the protease was produced at lab scale, purified and characterised. This alkali, thermotolerant serine protease was found to be significantly stable in the presence of various surfactants and H 2 O 2. Also, it was successfully able to remove blood stain when used as an additive along with commercial detergent suggesting its potential application in the laundry detergent industry.

  20. Vpu serine 52 dependent counteraction of tetherin is required for HIV-1 replication in macrophages, but not in ex vivo human lymphoid tissue

    Directory of Open Access Journals (Sweden)

    Specht Anke

    2010-01-01

    Full Text Available Abstract Background The human immunodeficiency virus type 1 (HIV-1 Vpu protein degrades CD4 and counteracts a restriction factor termed tetherin (CD317; Bst-2 to enhance virion release. It has been suggested that both functions can be genetically separated by mutation of a serine residue at position 52. However, recent data suggest that the S52 phosphorylation site is also important for the ability of Vpu to counteract tetherin. To clarify this issue, we performed a comprehensive analysis of HIV-1 with a mutated casein kinase-II phosphorylation site in Vpu in various cell lines, primary blood lymphocytes (PBL, monocyte-derived macrophages (MDM and ex vivo human lymphoid tissue (HLT. Results We show that mutation of serine 52 to alanine (S52A entirely disrupts Vpu-mediated degradation of CD4 and strongly impairs its ability to antagonize tetherin. Furthermore, casein-kinase II inhibitors blocked the ability of Vpu to degrade tetherin. Overall, Vpu S52A could only overcome low levels of tetherin, and its activity decreased in a manner dependent on the amount of transiently or endogenously expressed tetherin. As a consequence, the S52A Vpu mutant virus was unable to replicate in macrophages, which express high levels of this restriction factor. In contrast, HIV-1 Vpu S52A caused CD4+ T-cell depletion and spread efficiently in ex vivo human lymphoid tissue and PBL, most likely because these cells express comparably low levels of tetherin. Conclusion Our data explain why the effect of the S52A mutation in Vpu on virus release is cell-type dependent and suggest that a reduced ability of Vpu to counteract tetherin impairs HIV-1 replication in macrophages, but not in tissue CD4+ T cells.

  1. Structural and functional characterization of cleavage and inactivation of human serine protease inhibitors by the bacterial SPATE protease EspPα from enterohemorrhagic E. coli.

    Directory of Open Access Journals (Sweden)

    André Weiss

    Full Text Available EspPα and EspI are serine protease autotransporters found in enterohemorrhagic Escherichia coli. They both belong to the SPATE autotransporter family and are believed to contribute to pathogenicity via proteolytic cleavage and inactivation of different key host proteins during infection. Here, we describe the specific cleavage and functional inactivation of serine protease inhibitors (serpins by EspPα and compare this activity with the related SPATE EspI. Serpins are structurally related proteins that regulate vital protease cascades, such as blood coagulation and inflammatory host response. For the rapid determination of serpin cleavage sites, we applied direct MALDI-TOF-MS or ESI-FTMS analysis of coincubations of serpins and SPATE proteases and confirmed observed cleavage positions using in-gel-digest of SDS-PAGE-separated degradation products. Activities of both serpin and SPATE protease were assessed in a newly developed photometrical assay using chromogenic peptide substrates. EspPα cleaved the serpins α1-protease inhibitor (α1-PI, α1-antichymotrypsin, angiotensinogen, and α2-antiplasmin. Serpin cleavage led to loss of inhibitory function as demonstrated for α1-PI while EspPα activity was not affected. Notably, EspPα showed pronounced specificity and cleaved procoagulatory serpins such as α2-antiplasmin while the anticoagulatory antithrombin III was not affected. Together with recently published research, this underlines the interference of EspPα with hemostasis or inflammatory responses during infection, while the observed interaction of EspI with serpins is likely to be not physiologically relevant. EspPα-mediated serpin cleavage occurred always in flexible loops, indicating that this structural motif might be required for substrate recognition.

  2. Sphingoid bases and the serine catabolic enzyme CHA1 define a novel feedforward/feedback mechanism in the response to serine availability.

    Science.gov (United States)

    Montefusco, David J; Newcomb, Benjamin; Gandy, Jason L; Brice, Sarah E; Matmati, Nabil; Cowart, L Ashley; Hannun, Yusuf A

    2012-03-16

    Targets of bioactive sphingolipids in Saccharomyces cerevisiae were previously identified using microarray experiments focused on sphingolipid-dependent responses to heat stress. One of these heat-induced genes is the serine deamidase/dehydratase Cha1 known to be regulated by increased serine availability. This study investigated the hypothesis that sphingolipids may mediate the induction of Cha1 in response to serine availability. The results showed that inhibition of de novo synthesis of sphingolipids, pharmacologically or genetically, prevented the induction of Cha1 in response to increased serine availability. Additional studies implicated the sphingoid bases phytosphingosine and dihydrosphingosine as the likely mediators of Cha1 up-regulation. The yeast protein kinases Pkh1 and Pkh2, known sphingoid base effectors, were found to mediate CHA1 up-regulation via the transcription factor Cha4. Because the results disclosed a role for sphingolipids in negative feedback regulation of serine metabolism, we investigated the effects of disrupting this mechanism on sphingolipid levels and on cell growth. Intriguingly, exposure of the cha1Δ strain to high serine resulted in hyperaccumulation of endogenous serine and in turn a significant accumulation of sphingoid bases and ceramides. Under these conditions, the cha1Δ strain displayed a significant growth defect that was sphingolipid-dependent. Together, this work reveals a feedforward/feedback loop whereby the sphingoid bases serve as sensors of serine availability and mediate up-regulation of Cha1 in response to serine availability, which in turn regulates sphingolipid levels by limiting serine accumulation.

  3. Fragment-based identification of determinants of conformational and spectroscopic change at the ricin active site

    Directory of Open Access Journals (Sweden)

    Soares Alexei S

    2007-11-01

    Full Text Available Abstract Background Ricin is a potent toxin and known bioterrorism threat with no available antidote. The ricin A-chain (RTA acts enzymatically to cleave a specific adenine base from ribosomal RNA, thereby blocking translation. To understand better the relationship between ligand binding and RTA active site conformational change, we used a fragment-based approach to find a minimal set of bonding interactions able to induce rearrangements in critical side-chain positions. Results We found that the smallest ligand stabilizing an open conformer of the RTA active site pocket was an amide group, bound weakly by only a few hydrogen bonds to the protein. Complexes with small amide-containing molecules also revealed a switch in geometry from a parallel towards a splayed arrangement of an arginine-tryptophan cation-pi interaction that was associated with an increase and red-shift in tryptophan fluorescence upon ligand binding. Using the observed fluorescence signal, we determined the thermodynamic changes of adenine binding to the RTA active site, as well as the site-specific binding of urea. Urea binding had a favorable enthalpy change and unfavorable entropy change, with a ΔH of -13 ± 2 kJ/mol and a ΔS of -0.04 ± 0.01 kJ/(K*mol. The side-chain position of residue Tyr80 in a complex with adenine was found not to involve as large an overlap of rings with the purine as previously considered, suggesting a smaller role for aromatic stacking at the RTA active site. Conclusion We found that amide ligands can bind weakly but specifically to the ricin active site, producing significant shifts in positions of the critical active site residues Arg180 and Tyr80. These results indicate that fragment-based drug discovery methods are capable of identifying minimal bonding determinants of active-site side-chain rearrangements and the mechanistic origins of spectroscopic shifts. Our results suggest that tryptophan fluorescence provides a sensitive probe for the

  4. Threatened and endangered wildlife species of the Hanford Site related to CERCLA characterization activities

    Energy Technology Data Exchange (ETDEWEB)

    Fitzner, R.E. [Pacific Northwest Lab., Richland, WA (United States); Weiss, S.G.; Stegen, J.A. [Westinghouse Hanford Co., Richland, WA (United States)

    1994-06-01

    The US Department of Energy`s (DOE) Hanford Site has been placed on the National Priorities List, which requires that it be remediated under the Comprehensive Environmental Response, Compensation, and Liability Act (CERCLA) or Superfund. Potentially contaminated areas of the Hanford Site were grouped into operable units, and detailed characterization and investigation plans were formulated. The DOE Richland Operations Office requested Westinghouse Hanford Company (WHC) to conduct a biological assessment of the potential impact of these characterization activities on the threatened, endangered, and sensitive wildlife species of the Hanford Site. Additional direction for WHC compliances with wildlife protection can be found in the Environmental Compliance Manual. This document is intended to meet these requirements, in part, for the CERCLA characterization activities, as well as for other work comparable in scope. This report documents the biological assessment and describes the pertinent components of the Hanford Site as well as the planned characterization activities. Also provided are accounts of endangered, threatened, and federal candidate wildlife species on the Hanford Site and information as to how human disturbances can affect these species. Potential effects of the characterization activities are described with recommendations for mitigation measures.

  5. Testing the applicability of rapid on-site enzymatic activity detection for surface water monitoring

    Science.gov (United States)

    Stadler, Philipp; Vogl, Wolfgang; Juri, Koschelnik; Markus, Epp; Maximilian, Lackner; Markus, Oismüller; Monika, Kumpan; Peter, Strauss; Regina, Sommer; Gabriela, Ryzinska-Paier; Farnleitner Andreas, H.; Matthias, Zessner

    2015-04-01

    On-site detection of enzymatic activities has been suggested as a rapid surrogate for microbiological pollution monitoring of water resources (e.g. using glucuronidases, galactosidases, esterases). Due to the possible short measuring intervals enzymatic methods have high potential as near-real time water quality monitoring tools. This presentation describes results from a long termed field test. For twelve months, two ColiMinder devices (Vienna Water Monitoring, Austria) for on-site determination of enzymatic activity were tested for stream water monitoring at the experimental catchment HOAL (Hydrological Open Air Laboratory, Center for Water Resource Systems, Vienna University of Technology). The devices were overall able to follow and reflect the diverse hydrological and microbiological conditions of the monitored stream during the test period. Continuous data in high temporal resolution captured the course of enzymatic activity in stream water during diverse rainfall events. The method also proofed sensitive enough to determine diurnal fluctuations of enzymatic activity in stream water during dry periods. The method was able to capture a seasonal trend of enzymatic activity in stream water that matches the results gained from Colilert18 analysis for E. coli and coliform bacteria of monthly grab samples. Furthermore the comparison of ColiMinder data with measurements gained at the same test site with devices using the same method but having different construction design (BACTcontrol, microLAN) showed consistent measuring results. Comparative analysis showed significant differences between measured enzymatic activity (modified fishman units and pmol/min/100ml) and cultivation based analyses (most probable number, colony forming unit). Methods of enzymatic activity measures are capable to detect ideally the enzymatic activity caused by all active target bacteria members, including VBNC (viable but nonculturable) while cultivation based methods cannot detect VBNC

  6. Insights into the glycyl radical enzyme active site of benzylsuccinate synthase: a computational study.

    Science.gov (United States)

    Bharadwaj, Vivek S; Dean, Anthony M; Maupin, C Mark

    2013-08-21

    The fumarate addition reaction, catalyzed by the enzyme benzylsuccinate synthase (BSS), is considered to be one of the most intriguing and energetically challenging reactions in biology. BSS belongs to the glycyl radical enzyme family and catalyzes the fumarate addition reaction, which enables microorganisms to utilize hydrocarbons as an energy source under anaerobic conditions. Unfortunately, the extreme sensitivity of the glycyl radical to oxygen has hampered the structural and kinetic characterization of BSS, thereby limiting our knowledge on this enzyme. To enhance our molecular-level understanding of BSS, a computational approach involving homology modeling, docking studies, and molecular dynamics (MD) simulations has been used to deduce the structure of BSS's catalytic subunit (BSSα) and illuminate the molecular basis for the fumarate addition reaction. We have identified two conserved and distinct binding pockets at the BSSα active site: a hydrophobic pocket for toluene binding and a polar pocket for fumaric acid binding. Subsequent dynamical and energetic evaluations have identified Glu509, Ser827, Leu390, and Phe384 as active site residues critical for substrate binding. The orientation of substrates at the active site observed in MD simulations is consistent with experimental observations of the syn addition of toluene to fumaric acid. It is also found that substrate binding tightens the active site and restricts the conformational flexibility of the thiyl radical, leading to hydrogen transfer distances conducive to the proposed reaction mechanism. The stability of substrates at the active site and the occurrence of feasible radical transfer distances between the thiyl radical, substrates, and the active site glycine indicate a substrate-assisted radical transfer pathway governing fumarate addition.

  7. Fluorescence studies on the active sites of porcine pepsin and Rhizopus-pepsin.

    Science.gov (United States)

    Sachdev, G P; Brownstein, A D; Fruton, J S

    1975-01-25

    Fluorescence studies on the interaction, with porcine pepsin, of oligopeptides bearing a mansyl (Mns, 6-(N-methylanilino)-2-naphthalenesulfonyl) or dansyl (Dns, 5-dimethylaminonaphthalene-1-sulfonyl) group at the NH2 or COOH terminus have provided further evidence showing that the probe group is drawn into the extended active site largely as a consequence of the specific binding of the peptide portion of the substrate. The active site does not appear to have appreciable intrinsic affinity for the mansyl or dansyl group, and the principal contribution to the specific peptide-protein interaction is provided by the sensitive L-phenylalanyl-L-phenylalanyl (Phe-Phe) unit of the substrates tested. The pepsin inhibitor pepstatin can displace substrates such as Mns-(Gly)n-Phe-Phe-OR or Gly-Gly-Phe-Phe-NHNH-Mns from the active site of porcine pepsin; in these circumstances the mansyl group is bound weakly at a separate, nonspecific locus, distinct from the active site, which can accept the mansyl group of Mns-Gly-Gly-OR or mansylamide. In the interaction with substrates such as Mns-(Gly)n-Phe-Phe-OR or Dns-(Gly)n-Phe-Phe-OR, the above conclusions for porcine pepsin also apply to Rhizopus-pepsin. With substrates such as Gly-Gly-Phe-Phe-NHNH-Mns, however, the active site of Rhizopus-pepsin shows less affinity for the fluorescent probe group than does that of porcine pepsin, suggesting structural differences between the two acid proteinases in the region of their extended active sites which bind the COOH-terminal portion of small oligopeptide substrates.

  8. A Tale of Two Isomerases: Compact versus Extended Active Sites in Ketosteroid Isomerase and Phosphoglucose Isomerase

    Energy Technology Data Exchange (ETDEWEB)

    Somarowthu, Srinivas; Brodkin, Heather R.; D’Aquino, J. Alejandro; Ringe, Dagmar; Ondrechen, Mary Jo; Beuning, Penny J. (Brandeis); (NEU)

    2012-07-11

    Understanding the catalytic efficiency and specificity of enzymes is a fundamental question of major practical and conceptual importance in biochemistry. Although progress in biochemical and structural studies has enriched our knowledge of enzymes, the role in enzyme catalysis of residues that are not nearest neighbors of the reacting substrate molecule is largely unexplored experimentally. Here computational active site predictors, THEMATICS and POOL, were employed to identify functionally important residues that are not in direct contact with the reacting substrate molecule. These predictions then guided experiments to explore the active sites of two isomerases, Pseudomonas putida ketosteroid isomerase (KSI) and human phosphoglucose isomerase (PGI), as prototypes for very different types of predicted active sites. Both KSI and PGI are members of EC 5.3 and catalyze similar reactions, but they represent significantly different degrees of remote residue participation, as predicted by THEMATICS and POOL. For KSI, a compact active site of mostly first-shell residues is predicted, but for PGI, an extended active site in which residues in the first, second, and third layers around the reacting substrate are predicted. Predicted residues that have not been previously tested experimentally were investigated by site-directed mutagenesis and kinetic analysis. In human PGI, single-point mutations of the predicted second- and third-shell residues K362, H100, E495, D511, H396, and Q388 show significant decreases in catalytic activity relative to that of the wild type. The results of these experiments demonstrate that, as predicted, remote residues are very important in PGI catalysis but make only small contributions to catalysis in KSI.

  9. Secretory Phospholipase A2 Hydrolysis Phospholipid Analogs is Dependent on Water Accessibility to the Active Site

    DEFF Research Database (Denmark)

    Peters, Günther H.J.; Møller, Martin S.; Jørgensen, Kent

    2007-01-01

    A new and unnatural type of phospholipids with the head group attached to the 2-position of the glycerol backbone has been synthesized and shown to be a good substrate for secretory phospholipase A2 (sPLA2). To investigate the unexpected sPLA2 activity, we have compared three different phospholip...... the correct position such that hydrolysis can occur is reduced for the unnatural lipids. The relative water count follows 1R > 2R > 3S. This is in good agreement with experimental data that indicate the same trend for sPLA2 activity:  1R > 2R > 3S....... observed experimentally in sPLA2 activity might be caused by reduced access of water molecules to the active site. We have monitored the number of water molecules that enter the active site region for the different sPLA2−phospholipid complexes and found that the probability of a water molecule reaching...

  10. A novel anti-plant viral protein from coelomic fluid of the earthworm Eisenia foetida: purification, characterization and its identification as a serine protease.

    Science.gov (United States)

    Ueda, Mitsuhiro; Noda, Kanako; Nakazawa, Masami; Miyatake, Kazutaka; Ohki, Satoshi; Sakaguchi, Minoru; Inouye, Kuniyo

    2008-12-01

    A novel protein showing strong antiviral activities against cucumber mosaic virus (CMV) and tomato mosaic virus (TMV) was purified from the coelomic fluid of the earthworm Eisenia foetida. The protein was characterized as a cold-adapted serine protease. Its molecular weight was estimated to be 27,000 by SDS-PAGE. The enzyme was most active at pH 9.5 and 40-50 degrees C. The protease activity at 4 degrees C was 60% of that obtained at the optimal temperature. The activity was suppressed by various serine protease inhibitors. Partial N-terminal amino acid sequence of the enzyme showed homology with serine proteases of earthworms, E. foetida and Lumbricus rubellus previously studied. Our results suggest that the enzyme can be applicable as a potential antiviral factor against CMV, TMV, and other plant viruses.

  11. Preliminary examination of the impacts of repository site characterization activities and facility construction and operation activities on Hanford air quality

    Energy Technology Data Exchange (ETDEWEB)

    Glantz, C.S.; Ramsdell, J.V.

    1986-04-01

    Air quality impacts that would result from site characterization activities and from the construction and operation of a high-level nuclear wste repository at Hanford are estimated using two simple atmospheric dispersion models, HANCHI and CHISHORT. Model results indicate that pollutant concentrations would not exceed ambient air quality standards at any point outside the Hanford fenceline or at any publicly accessible location within the Hanford Site. The increase in pollutant concentrations in nearby communities due to site activities would be minimal. HANCHI and CHISHORT are documented in the appendices of this document. Further study of the repository's impact on air quality will be conducted when more detailed project plans and work schedules are available.

  12. Mutations in SERPINB7, Encoding a Member of the Serine Protease Inhibitor Superfamily, Cause Nagashima-type Palmoplantar Keratosis

    Science.gov (United States)

    Kubo, Akiharu; Shiohama, Aiko; Sasaki, Takashi; Nakabayashi, Kazuhiko; Kawasaki, Hiroshi; Atsugi, Toru; Sato, Showbu; Shimizu, Atsushi; Mikami, Shuji; Tanizaki, Hideaki; Uchiyama, Masaki; Maeda, Tatsuo; Ito, Taisuke; Sakabe, Jun-ichi; Heike, Toshio; Okuyama, Torayuki; Kosaki, Rika; Kosaki, Kenjiro; Kudoh, Jun; Hata, Kenichiro; Umezawa, Akihiro; Tokura, Yoshiki; Ishiko, Akira; Niizeki, Hironori; Kabashima, Kenji; Mitsuhashi, Yoshihiko; Amagai, Masayuki

    2013-01-01

    “Nagashima-type” palmoplantar keratosis (NPPK) is an autosomal recessive nonsyndromic diffuse palmoplantar keratosis characterized by well-demarcated diffuse hyperkeratosis with redness, expanding on to the dorsal surfaces of the palms and feet and the Achilles tendon area. Hyperkeratosis in NPPK is mild and nonprogressive, differentiating NPPK clinically from Mal de Meleda. We performed whole-exome and/or Sanger sequencing analyses of 13 unrelated NPPK individuals and identified biallelic putative loss-of-function mutations in SERPINB7, which encodes a cytoplasmic member of the serine protease inhibitor superfamily. We identified a major causative mutation of c.796C>T (p.Arg266∗) as a founder mutation in Japanese and Chinese populations. SERPINB7 was specifically present in the cytoplasm of the stratum granulosum and the stratum corneum (SC) of the epidermis. All of the identified mutants are predicted to cause premature termination upstream of the reactive site, which inhibits the proteases, suggesting a complete loss of the protease inhibitory activity of SERPINB7 in NPPK skin. On exposure of NPPK lesional skin to water, we observed a whitish spongy change in the SC, suggesting enhanced water permeation into the SC due to overactivation of proteases and a resultant loss of integrity of the SC structure. These findings provide an important framework for developing pathogenesis-based therapies for NPPK. PMID:24207119

  13. Interaction of activator of G-protein signaling 3 (AGS3) with LKB1, a serine/threonine kinase involved in cell polarity and cell cycle progression: phosphorylation of the G-protein regulatory (GPR) motif as a regulatory mechanism for the interaction of GPR motifs with Gi alpha.

    Science.gov (United States)

    Blumer, Joe B; Bernard, Michael L; Peterson, Yuri K; Nezu, Jun-ichi; Chung, Peter; Dunican, Dara J; Knoblich, Juergen A; Lanier, Stephen M

    2003-06-27

    Activator of G-protein signaling 3 (AGS3) has a modular domain structure consisting of seven tetratricopeptide repeats (TPRs) and four G-protein regulatory (GPR) motifs. Each GPR motif binds to the alpha subunit of Gi/Go (Gialpha > Goalpha) stabilizing the GDP-bound conformation of Galpha and apparently competing with Gbetagamma for GalphaGDP binding. As an initial approach to identify regulatory mechanisms for AGS3-G-protein interactions, a yeast two-hybrid screen was initiated using the TPR and linker region of AGS3 as bait. This screen identified the serine/threonine kinase LKB1, which is involved in the regulation of cell cycle progression and polarity. Protein interaction assays in mammalian systems using transfected cells or brain lysate indicated the regulated formation of a protein complex consisting of LKB1, AGS3, and G-proteins. The interaction between AGS3 and LKB1 was also observed with orthologous proteins in Drosophila where both proteins are involved in cell polarity. LKB1 immunoprecipitates from COS7 cells transfected with LKB1 phosphorylated the GPR domains of AGS3 and the related protein LGN but not the AGS3-TPR domain. GPR domain phosphorylation was completely blocked by a consensus GPR motif peptide, and placement of a phosphate moiety within a consensus GPR motif reduced the ability of the peptide to interact with G-proteins. These data suggest that phosphorylation of GPR domains may be a general mechanism regulating the interaction of GPR-containing proteins with G-proteins. Such a mechanism may be of particular note in regard to localized signal processing in the plasma membrane involving G-protein subunits and/or intracellular functions regulated by heterotrimeric G-proteins that occur independently of a typical G-protein-coupled receptor.

  14. Human activities in Natura 2000 sites: a highly diversified conservation network.

    Science.gov (United States)

    Tsiafouli, Maria A; Apostolopoulou, Evangelia; Mazaris, Antonios D; Kallimanis, Athanasios S; Drakou, Evangelia G; Pantis, John D

    2013-05-01

    The Natura 2000 network was established across the European Union's (EU) Member States with the aim to conserve biodiversity, while ensuring the sustainability of human activities. However, to what kind and to what extent Natura 2000 sites are subject to human activities and how this varies across Member States remains unspecified. Here, we analyzed 111,269 human activity records from 14,727 protected sites in 20 Member States. The frequency of occurrence of activities differs among countries, with more than 86 % of all sites being subjected to agriculture or forestry. Activities like hunting, fishing, urbanization, transportation, and tourism are more frequently recorded in south European sites than in northern or eastern ones. The observed variations indicate that Natura 2000 networks are highly heterogeneous among EU Member States. Our analysis highlights the importance of agriculture in European landscapes and indicates possible targets for policy interventions at national, European, or "sub-European" level. The strong human presence in the Natura 2000 network throughout Member States, shows that conservation initiatives could succeed only by combining social and ecological sustainability and by ensuring the integration of policies affecting biodiversity.

  15. Orthogonal use of a human tRNA synthetase active site to achieve multi-functionality

    Science.gov (United States)

    Zhou, Quansheng; Kapoor, Mili; Guo, Min; Belani, Rajesh; Xu, Xiaoling; Kiosses, William B.; Hanan, Melanie; Park, Chulho; Armour, Eva; Do, Minh-Ha; Nangle, Leslie A.; Schimmel, Paul; Yang, Xiang-Lei

    2011-01-01

    Protein multi-functionality is an emerging explanation for the complexity of higher organisms. In this regard, while aminoacyl tRNA synthetases catalyze amino acid activation for protein synthesis, some also act in pathways for inflammation, angiogenesis, and apoptosis. How multiple functions evolved and their relationship to the active site is not clear. Here structural modeling analysis, mutagenesis, and cell-based functional studies show that the potent angiostatic, natural fragment of human TrpRS associates via Trp side chains that protrude from the cognate cellular receptor VE-cadherin. Modeling indicates that (I prefer the way it was because the conclusion was reached not only by modeling, but more so by experimental studies.)VE-cadherin Trp side chains fit into the Trp-specific active site of the synthetase. Thus, specific side chains of the receptor mimic (?) amino acid substrates and expand the functionality of the active site of the synthetase. We propose that orthogonal use of the same active site may be a general way to develop multi-functionality of human tRNA synthetases and other proteins. PMID:20010843

  16. Endonuclease active site plasticity allows DNA cleavage with diverse alkaline Earth and transition metal ions.

    Science.gov (United States)

    Vasu, Kommireddy; Saravanan, Matheshwaran; Nagaraja, Valakunja

    2011-09-16

    A majority of enzymes show a high degree of specificity toward a particular metal ion in their catalytic reaction. However, Type II restriction endonuclease (REase) R.KpnI, which is the first member of the HNH superfamily of REases, exhibits extraordinary diversity in metal ion dependent DNA cleavage. Several alkaline earth and transition group metal ions induce high fidelity and promiscuous cleavage or inhibition depending upon their concentration. The metal ions having different ionic radii and co-ordination geometries readily replace each other from the enzyme's active site, revealing its plasticity. Ability of R.KpnI to cleave DNA with both alkaline earth and transition group metal ions having varied ionic radii could imply utilization of different catalytic site(s). However, mutation of the invariant His residue of the HNH motif caused abolition of the enzyme activity with all of the cofactors, indicating that the enzyme follows a single metal ion catalytic mechanism for DNA cleavage. Indispensability of His in nucleophile activation together with broad cofactor tolerance of the enzyme indicates electrostatic stabilization function of metal ions during catalysis. Nevertheless, a second metal ion is recruited at higher concentrations to either induce promiscuity or inhibit the DNA cleavage. Regulation of the endonuclease activity and fidelity by a second metal ion binding is a unique feature of R.KpnI among REases and HNH nucleases. The active site plasticity of R.KpnI opens up avenues for redesigning cofactor specificities and generation of mutants specific to a particular metal ion.

  17. Serine protease inhibitor Serp-1 strongly impairs atherosclerotic lesion formation and induces a stable plaque phenotype in ApoE-/-mice

    NARCIS (Netherlands)

    Bot, Ilze; von der Thüsen, Jan H.; Donners, Marjo M. P. C.; Lucas, Alexandra; Fekkes, Madelon L.; de Jager, Saskia C. A.; Kuiper, Johan; Daemen, Mat J. A. P.; van Berkel, Theo J. C.; Heeneman, Sylvia; Biessen, Erik A. L.

    2003-01-01

    The myxoma virus protein Serp-1 is a member of the serine protease inhibitor superfamily. Serp-1 potently inhibits human serum proteases including plasmin, urokinase-type plasminogen activator (uPA), and tissue-type plasminogen activator (tPA). Serp-1 also displays a high antiinflammatory activity,

  18. The active site of a carbohydrate esterase displays divergent catalytic and noncatalytic binding functions.

    Directory of Open Access Journals (Sweden)

    Cedric Montanier

    2009-03-01

    Full Text Available Multifunctional proteins, which play a critical role in many biological processes, have typically evolved through the recruitment of different domains that have the required functional diversity. Thus the different activities displayed by these proteins are mediated by spatially distinct domains, consistent with the specific chemical requirements of each activity. Indeed, current evolutionary theory argues that the colocalization of diverse activities within an enzyme is likely to be a rare event, because it would compromise the existing activity of the protein. In contrast to this view, a potential example of multifunctional recruitment into a single protein domain is provided by CtCel5C-CE2, which contains an N-terminal module that displays cellulase activity and a C-terminal module, CtCE2, which exhibits a noncatalytic cellulose-binding function but also shares sequence identity with the CE2 family of esterases. Here we show that, unlike other CE2 members, the CtCE2 domain displays divergent catalytic esterase and noncatalytic carbohydrate binding functions. Intriguingly, these diverse activities are housed within the same site on the protein. Thus, a critical component of the active site of CtCE2, the catalytic Ser-His dyad, in harness with inserted aromatic residues, confers noncatalytic binding to cellulose whilst the active site of the domain retains its esterase activity. CtCE2 catalyses deacetylation of noncellulosic plant structural polysaccharides to deprotect these substrates for attack by other enzymes. Yet it also acts as a cellulose-binding domain, which promotes the activity of the appended cellulase on recalcitrant substrates. The CE2 family encapsulates the requirement for multiple activities by biocatalysts that attack challenging macromolecular substrates, including the grafting of a second, powerful and discrete noncatalytic binding functionality into the active site of an enzyme. This article provides a rare example of

  19. Crystal structures of E. coli native MenH and two active site mutants.

    Directory of Open Access Journals (Sweden)

    Jodie M Johnston

    Full Text Available Recent revision of the biosynthetic pathway for menaquinone has led to the discovery of a previously unrecognized enzyme 2-succinyl-6-hydroxy-2,4-cyclohexadiene-1-carboxylate synthase, also known as MenH. This enzyme has an α/β hydrolase fold with a catalytic triad comprising Ser86, His232, and Asp210. Mutational studies identified a number of conserved residues of importance to activity, and modeling further implicated the side chains of Tyr85 and Trp147 in formation of a non-standard oxyanion hole. We have solved the structure of E. coli MenH (EcMenH at 2.75 Å resolution, together with the structures of the active site mutant proteins Tyr85Phe and Arg124Ala, both at 2.5 Å resolution. EcMenH has the predicted α/β hydrolase fold with its core α/β domain capped by a helical lid. The active site, a long groove beneath the cap, contains a number of conserved basic residues and is found to bind exogeneous anions, modeled as sulfate and chloride, in all three crystal structures. Docking studies with the MenH substrate and a transition state model indicate that the bound anions mark the binding sites for anionic groups on the substrate. The docking studies, and careful consideration of the active site geometry, further suggest that the oxyanion hole is of a conventional nature, involving peptide NH groups, rather than the proposed site involving Tyr85 and Trp147. This is in accord with conclusions from the structure of S. aureus MenH. Comparisons with the latter do, however, indicate differences in the periphery of the active site that could be of relevance to selective inhibition of MenH enzymes.

  20. Bacillus subtilis Two-Component System Sensory Kinase DegS Is Regulated by Serine Phosphorylation in Its Input Domain

    DEFF Research Database (Denmark)

    Jers, Carsten; Kobir, Ahasanul; Søndergaard, Elsebeth Oline

    2011-01-01

    Bacillus subtilis two-component system DegS/U is well known for the complexity of its regulation. The cytosolic sensory kinase DegS does not receive a single predominant input signal like most two-component kinases, instead it integrates a wide array of metabolic inputs that modulate its activity......S phosphorylation at serine 76 and the level of DegU phosphorylation, establishing this post-translational modification as an additional trigger for this two-component system....

  1. Ixodes scapularis tick serine proteinase inhibitor (serpin gene family; annotation and transcriptional analysis

    Directory of Open Access Journals (Sweden)

    Chalaire Katelyn C

    2009-05-01

    Full Text Available Abstract Background Serine proteinase inhibitors (Serpins are a large superfamily of structurally related, but functionally diverse proteins that control essential proteolytic pathways in most branches of life. Given their importance in the biology of many organisms, the concept that ticks might utilize serpins to evade host defenses and immunizing against or disrupting their functions as targets for tick control is an appealing option. Results A sequence homology search strategy has allowed us to identify at least 45 tick serpin genes in the Ixodes scapularis genome that are structurally segregated into 32 intronless and 13 intron-containing genes. Nine of the intron-containing serpins occur in a cluster of 11 genes that span 170 kb of DNA sequence. Based on consensus amino acid residues in the reactive center loop (RCL and signal peptide scanning, 93% are putatively inhibitory while 82% are putatively extracellular. Among the 11 different amino acid residues that are predicted at the P1 sites, 16 sequences possess basic amino acid (R/K residues. Temporal and spatial expression analyses revealed that 40 of the 45 serpins are differentially expressed in salivary glands (SG and/or midguts (MG of unfed and partially fed ticks. Ten of the 38 serpin genes were expressed from six to 24 hrs of feeding while six and fives genes each are predominantly or exclusively expressed in either MG and SG respectively. Conclusion Given the diversity among tick species, sizes of tick serpin families are likely to be variable. However this study provides insight on the potential sizes of serpin protein families in ticks. Ticks must overcome inflammation, complement activation and blood coagulation to complete feeding. Since these pathways are regulated by serpins that have basic residues at their P1 sites, we speculate that I. scapularis may utilize some of the serpins reported in this study to manipulate host defense. We have discussed our data in the context of

  2. Increased production of L-serine in Escherichia coli through Adaptive Laboratory Evolution

    DEFF Research Database (Denmark)

    Mundhada, Hemanshu; Seoane, Jose Miguel; Schneider, Konstantin

    2017-01-01

    L-serine is a promising building block biochemical with a high theoretical production yield from glucose. Toxicity of L-serine is however prohibitive for high-titer production in E. coli. Here, E. coli lacking L-serine degradation pathways was evolved for improved tolerance by gradually increasin...

  3. Serine/threonine/tyrosine phosphorylation regulates DNA binding of bacterial transcriptional regulators

    DEFF Research Database (Denmark)

    Kalantari, Aida; Derouiche, Abderahmane; Shi, Lei

    2015-01-01

    of residue, i.e. serine, threonine, tyrosine and cysteine, is also quite common. The phosphorylation of the ester type (phospho-serine/threonine/tyrosine) is more stable than the aspartate phosphorylation of TCSs. The kinases which catalyse these phosphorylation events (Hanks-type serine/threonine protein...

  4. Engineering of High Yield Production of L-serine in Escherichia coli

    DEFF Research Database (Denmark)

    Mundhada, Hemanshu; Schneider, Konstantin; Christensen, Hanne Bjerre

    2016-01-01

    high serine production yield (0.45 g/g glucose) during small-scale batch fermentation in minimal medium. Serine, however, was found to be highly toxic even at low concentrations to this strain, which lead to slow growth and production during fed batch fermentation, resulting in a serine production of 8...

  5. Structural insight into the active site of mushroom tyrosinase using phenylbenzoic acid derivatives.

    Science.gov (United States)

    Oyama, Takahiro; Yoshimori, Atsushi; Takahashi, Satoshi; Yamamoto, Tetsuya; Sato, Akira; Kamiya, Takanori; Abe, Hideaki; Abe, Takehiko; Tanuma, Sei-Ichi

    2017-07-01

    So far, many inhibitors of tyrosinase have been discovered for cosmetic and clinical agents. However, the molecular mechanisms underlying the inhibition in the active site of tyrosinase have not been well understood. To explore this problem, we examined here the inhibitory effects of 4'-hydroxylation and methoxylation of phenylbenzoic acid (PBA) isomers, which have a unique scaffold to inhibit mushroom tyrosinase. The inhibitory effect of 3-PBA, which has the most potent inhibitory activity among the isomers, was slightly decreased by 4'-hydroxylation and further decreased by 4'-methoxylation against mushroom tyrosinase. Surprisingly, 4'-hydroxylation but not methoxylation of 2-PBA appeared inhibitory activity. On the other hand, both 4'-hydroxylation and methoxylation of 4-PBA increased the inhibitory activity against mushroom tyrosinase. In silico docking analyses using the crystallographic structure of mushroom tyrosinase indicated that the carboxylic acid or 4'-hydroxyl group of PBA derivatives could chelate with cupric ions in the active site of mushroom tyrosinase, and that the interactions of Asn260 and Phe264 in the active site with the adequate-angled biphenyl group are involved in the inhibitory activities of the modified PBAs, by parallel and T-shaped π-π interactions, respectively. Furthermore, Arg268 could fix the angle of the aromatic ring of Phe264, and Val248 is supposed to interact with the inhibitors as a hydrophobic manner. These results may enhance the structural insight into mushroom tyrosinase for the creation of novel tyrosinase inhibitors. Copyright © 2017 Elsevier Ltd. All rights reserved.

  6. Campylobacter jejuni gene cj0511 encodes a serine peptidase essential for colonisation

    Directory of Open Access Journals (Sweden)

    A.V. Karlyshev

    2014-01-01

    Full Text Available According to MEROPS peptidase database, Campylobacter species encode 64 predicted peptidases. However, proteolytic properties of only a few of these proteins have been confirmed experimentally. In this study we identified and characterised a Campylobacter jejuni gene cj0511 encoding a novel peptidase. The proteolytic activity associated with this enzyme was demonstrated in cell lysates. Moreover, enzymatic studies conducted with a purified protein confirmed a prediction of it being a serine peptidase. Furthermore, cj0511 mutant was found to be severely attenuated in chicken colonisation model, suggesting a role of the Cj0511 protein in infection.

  7. Macrophage migration inhibitory factor (MIF) modulates trophic signaling through interaction with serine protease HTRA1

    DEFF Research Database (Denmark)

    Fex Svenningsen, Åsa; Loering, Svenja; Sørensen, Anna Lahn

    2017-01-01

    its function is far from clear. Here, we report the finding of a new binding partner to MIF, the ser-ine protease HTRA1. This enzyme cleaves several growth factors, extracellular matrix molecules and is implicated in some of the same diseases as MIF. We show that the func-tion of the binding between...... MIF and HTRA1 is to inhibit the proteolytic activity of HTRA1, modulating the availability of molecules that can change cell growth and differentiation. MIF is therefore the first endogenous inhibitor ever found for HTRA1. It was found that both molecules were present in astrocytes...

  8. Active catalytic sites in the ammoxidation of propane and propene over V-Sb-O catalysts

    Energy Technology Data Exchange (ETDEWEB)

    Buchholz, S.A.; Zanthoff, H.W. [Bochum Univ. (Germany). Lehrstuhl fuer Technische Chemie

    1998-12-31

    The ammoxidation of propane over VSb{sub y}O{sub x} catalysts (y=1, 2, 5) was investigated with respect to the role of different oxygen species in the selective and non selective reaction steps using transient experiments in the Temporal Analysis of Products (TAP) reactor. Only lattice oxygen is involved in the oxidation reactions. Using isotopic labelled oxygen it is shown that two different active sites exist on the surface. On site A, which can be reoxidized faster by gas phase oxygen compared to site B, mainly CO is formed. On site B CO{sub 2} and acrolein as well as NO and N{sub 2}O in the presence of ammonia in the feed gas are formed and reoxidation mainly occurs with bulk lattice oxygen. (orig.)

  9. Deepest and hottest hydrothermal activity in the Okinawa Trough: the Yokosuka site at Yaeyama Knoll

    Science.gov (United States)

    Miyazaki, Junichi; Kawagucci, Shinsuke; Makabe, Akiko; Takahashi, Ayu; Kitada, Kazuya; Torimoto, Junji; Matsui, Yohei; Tasumi, Eiji; Shibuya, Takazo; Nakamura, Kentaro; Horai, Shunsuke; Sato, Shun; Ishibashi, Jun-ichiro; Kanzaki, Hayato; Nakagawa, Satoshi; Hirai, Miho; Takaki, Yoshihiro; Okino, Kyoko; Watanabe, Hiromi Kayama; Kumagai, Hidenori; Chen, Chong

    2017-12-01

    Since the initial discovery of hydrothermal vents in 1977, these `extreme' chemosynthetic systems have been a focus of interdisciplinary research. The Okinawa Trough (OT), located in the semi-enclosed East China Sea between the Eurasian continent and the Ryukyu arc, hosts more than 20 known vent sites but all within a relatively narrow depth range (600-1880 m). Depth is a significant factor in determining fluid temperature and chemistry, as well as biological composition. However, due to the narrow depth range of known sites, the actual influence of depth here has been poorly resolved. Here, the Yokosuka site (2190 m), the first OT vent exceeding 2000 m depth is reported. A highly active hydrothermal vent site centred around four active vent chimneys reaching 364°C in temperature, it is the hottest in the OT. Notable Cl depletion (130 mM) and both high H2 and CH4 concentrations (approx. 10 mM) probably result from subcritical phase separation and thermal decomposition of sedimentary organic matter. Microbiota and fauna were generally similar to other sites in the OT, although with some different characteristics. In terms of microbiota, the H2-rich vent fluids in Neuschwanstein chimney resulted in the dominance of hydrogenotrophic chemolithoautotrophs such as Thioreductor and Desulfobacterium. For fauna, the dominance of the deep-sea mussel Bathymodiolus aduloides is surprising given other nearby vent sites are usually dominated by B. platifrons and/or B. japonicus, and a sponge field in the periphery dominated by Poecilosclerida is unusual for OT vents. Our insights from the Yokosuka site implies that although the distribution of animal species may be linked to depth, the constraint is perhaps not water pressure and resulting chemical properties of the vent fluid but instead physical properties of the surrounding seawater. The potential significance of these preliminary results and prospect for future research on this unique site are discussed.

  10. Deepest and hottest hydrothermal activity in the Okinawa Trough: the Yokosuka site at Yaeyama Knoll.

    Science.gov (United States)

    Miyazaki, Junichi; Kawagucci, Shinsuke; Makabe, Akiko; Takahashi, Ayu; Kitada, Kazuya; Torimoto, Junji; Matsui, Yohei; Tasumi, Eiji; Shibuya, Takazo; Nakamura, Kentaro; Horai, Shunsuke; Sato, Shun; Ishibashi, Jun-Ichiro; Kanzaki, Hayato; Nakagawa, Satoshi; Hirai, Miho; Takaki, Yoshihiro; Okino, Kyoko; Watanabe, Hiromi Kayama; Kumagai, Hidenori; Chen, Chong

    2017-12-01

    Since the initial discovery of hydrothermal vents in 1977, these 'extreme' chemosynthetic systems have been a focus of interdisciplinary research. The Okinawa Trough (OT), located in the semi-enclosed East China Sea between the Eurasian continent and the Ryukyu arc, hosts more than 20 known vent sites but all within a relatively narrow depth range (600-1880 m). Depth is a significant factor in determining fluid temperature and chemistry, as well as biological composition. However, due to the narrow depth range of known sites, the actual influence of depth here has been poorly resolved. Here, the Yokosuka site (2190 m), the first OT vent exceeding 2000 m depth is reported. A highly active hydrothermal vent site centred around four active vent chimneys reaching 364°C in temperature, it is the hottest in the OT. Notable Cl depletion (130 mM) and both high H 2 and CH 4 concentrations (approx. 10 mM) probably result from subcritical phase separation and thermal decomposition of sedimentary organic matter. Microbiota and fauna were generally similar to other sites in the OT, although with some different characteristics. In terms of microbiota, the H 2 -rich vent fluids in Neuschwanstein chimney resulted in the dominance of hydrogenotrophic chemolithoautotrophs such as Thioreductor and Desulfobacterium . For fauna, the dominance of the deep-sea mussel Bathymodiolus aduloides is surprising given other nearby vent sites are usually dominated by B. platifrons and/or B. japonicus , and a sponge field in the periphery dominated by Poecilosclerida is unusual for OT vents. Our insights from the Yokosuka site implies that although the distribution of animal species may be linked to depth, the constraint is perhaps not water pressure and resulting chemical properties of the vent fluid but instead physical properties of the surrounding seawater. The potential significance of these preliminary results and prospect for future research on this unique site are discussed.

  11. Structural characterization of a non-heme iron active site in zeolites that hydroxylates methane.

    Science.gov (United States)

    Snyder, Benjamin E R; Böttger, Lars H; Bols, Max L; Yan, James J; Rhoda, Hannah M; Jacobs, Ariel B; Hu, Michael Y; Zhao, Jiyong; Alp, E Ercan; Hedman, Britt; Hodgson, Keith O; Schoonheydt, Robert A; Sels, Bert F; Solomon, Edward I

    2018-04-02

    Iron-containing zeolites exhibit unprecedented reactivity in the low-temperature hydroxylation of methane to form methanol. Reactivity occurs at a mononuclear ferrous active site, α-Fe(II), that is activated by N 2 O to form the reactive intermediate α-O. This has been defined as an Fe(IV)=O species. Using nuclear resonance vibrational spectroscopy coupled to X-ray absorption spectroscopy, we probe the bonding interaction between the iron center, its zeolite lattice-derived ligands, and the reactive oxygen. α-O is found to contain an unusually strong Fe(IV)=O bond resulting from a constrained coordination geometry enforced by the zeolite lattice. Density functional theory calculations clarify how the experimentally determined geometric structure of the active site leads to an electronic structure that is highly activated to perform H-atom abstraction.

  12. A three-dimensional model of mammalian tyrosinase active site accounting for loss of function mutations.

    Science.gov (United States)

    Schweikardt, Thorsten; Olivares, Concepción; Solano, Francisco; Jaenicke, Elmar; García-Borrón, José Carlos; Decker, Heinz

    2007-10-01

    Tyrosinases are the first and rate-limiting enzymes in the synthesis of melanin pigments responsible for colouring hair, skin and eyes. Mutation of tyrosinases often decreases melanin production resulting in albinism, but the effects are not always understood at the molecular level. Homology modelling of mouse tyrosinase based on recently published crystal structures of non-mammalian tyrosinases provides an active site model accounting for loss-of-function mutations. According to the model, the copper-binding histidines are located in a helix bundle comprising four densely packed helices. A loop containing residues M374, S375 and V377 connects the CuA and CuB centres, with the peptide oxygens of M374 and V377 serving as hydrogen acceptors for the NH-groups of the imidazole rings of the copper-binding His367 and His180. Therefore, this loop is essential for the stability of the active site architecture. A double substitution (374)MS(375) --> (374)GG(375) or a single M374G mutation lead to a local perturbation of the protein matrix at the active site affecting the orientation of the H367 side chain, that may be unable to bind CuB reliably, resulting in loss of activity. The model also accounts for loss of function in two naturally occurring albino mutations, S380P and V393F. The hydroxyl group in S380 contributes to the correct orientation of M374, and the substitution of V393 for a bulkier phenylalanine sterically impedes correct side chain packing at the active site. Therefore, our model explains the mechanistic necessity for conservation of not only active site histidines but also adjacent amino acids in tyrosinase.

  13. Identification and characterization of radioactively contaminated sites in Ukraine and planning for environmental restoration activities

    International Nuclear Information System (INIS)

    Soroka, Y.

    2000-01-01

    In the Pridniprovsk-Krivoy Rog region uranium, titanium, iron and manganese ores were mined and milled beginning in the 1950s. These activities have caused radioactive contamination of the environment at some sites. In recent times intensive works concerning the surveying of contaminated areas and substantiating the need for remediation have been initiated. The research methodologies applied and the results from radiation surveys are presented for the site of the first uranium mine in the Ukraine, for tailings originating from the Pridniprovsk Chemical Plant (PChP), for the recultivated dump-site of the former 'O'-mine, as well as for the wastes, raw materials and production of the Nicopol Ferro-Alloy Plant. The planning procedure for the remediation activities at the town of Zhovty Vody is described. (author)

  14. Remediation of uranium contaminated sites: clean-up activities in Serbia

    International Nuclear Information System (INIS)

    Raicevic, S.; Raicevic, J. . E-mail address of corresponding author: raich@beotel.yu; Raicevic, S.)

    2005-01-01

    One of the serious environmental problems in Serbia represent sites contaminated with depleted uranium (DU) during past war activities. According to UNEP reports and our findings there are two types of contamination: (i) localized points of high, concentrated contamination where DU penetrators enter the soil, and (ii) low level of widespread DU contamination, which indicates that during the conflict DU dust was dispersed into the environment. Remediation of these sites is an urgent need because they represent a permanent threat to the population living in this area. Here we give a brief description of approaches commonly used in remediation of DU contaminated sites, and an overview of current clean-up activities performed in Serbia. (author)

  15. Screening Approach to the Activation of Soil and Contamination of Groundwater at Linear Proton Accelerator Sites

    CERN Document Server

    Otto, Thomas

    The activation of soil and the contamination of groundwater at proton accelerator sites with the radionuclides 3H and 22Na are estimated with a Monte-Carlo calculation and a conservative soil- and ground water model. The obtained radionuclide concentrations show that the underground environment of future accelerators must be adequately protected against a migration of activation products. This study is of particular importance for the proton driver accelerator in the planned EURISOL facility.

  16. Thiolactomycin inhibits D-aspartate oxidase: a novel approach to probing the active site environment.

    Science.gov (United States)

    Katane, Masumi; Saitoh, Yasuaki; Hanai, Toshihiko; Sekine, Masae; Furuchi, Takemitsu; Koyama, Nobuhiro; Nakagome, Izumi; Tomoda, Hiroshi; Hirono, Shuichi; Homma, Hiroshi

    2010-10-01

    D-Aspartate oxidase (DDO) and D-amino acid oxidase (DAO) are flavin adenine dinucleotide (FAD)-containing flavoproteins that catalyze the oxidative deamination of D-amino acids. While several functionally and structurally important amino acid residues have been identified in the DAO protein, little is known about the structure-function relationships of DDO. In the search for a potent DDO inhibitor as a novel tool for investigating its structure-function relationships, a large number of biologically active compounds of microbial origin were screened for their ability to inhibit the enzymatic activity of mouse DDO. We discovered several compounds that inhibited the activity of mouse DDO, and one of the compounds identified, thiolactomycin (TLM), was then characterized and evaluated as a novel DDO inhibitor. TLM reversibly inhibited the activity of mouse DDO with a mixed type of inhibition more efficiently than meso-tartrate and malonate, known competitive inhibitors of mammalian DDOs. The selectivity of TLM was investigated using various DDOs and DAOs, and it was found that TLM inhibits not only DDO, but also DAO. Further experiments with apoenzymes of DDO and DAO revealed that TLM is most likely to inhibit the activities of DDO and DAO by competition with both the substrate and the coenzyme, FAD. Structural models of mouse DDO/TLM complexes supported this finding. The binding mode of TLM to DDO was validated further by site-directed mutagenesis of an active site residue, Arg-237. Collectively, our findings show that TLM is a novel, active site-directed DDO inhibitor that will be useful for elucidating the molecular details of the active site environment of DDO. Copyright © 2010 Elsevier Masson SAS. All rights reserved.

  17. Active layer dynamics in three sites with contrasted topography in the Byers Peninsula (Livingston Island, Antarctica)

    Science.gov (United States)

    Oliva, Marc; Ruiz-Fernández, Jesús; Vieira, Gonçalo

    2015-04-01

    Topography exerts a key role on permafrost distribution in areas where mean annual temperatures are slightly negative. This is the case of low-altitude environments in Maritime Antarctica, namely in the South Shetland Islands, where permafrost is marginal to discontinuous until elevations of 20-40 m asl turning to continuous at higher areas. Consequently, the active layer dynamics is also strongly conditioned by the geomorphological setting. In January 2014 we installed three sites for monitoring the active layer dynamics across the Byers Peninsula (Livingston Island, South Shetland Islands) in different geomorphological environments at elevations between 60 and 100 m. The purpose was to examine the role of the topography and microclimatic conditions on the active layer dynamics. At each site a set of loggers was set up to monitor: air temperatures, snow thickness, ground temperatures until 80 cm together with the coupling atmosphere-ground temperatures. During the first year of monitoring the mean annual air temperatures show similar values in the three sites, in all cases slightly below freezing. The snowy conditions during this year in this archipelago have resulted in a late melting of snow, which has also conditioned the duration of frozen conditions in the uppermost soil layers. Topography has a strong influence on snow cover duration, which in turn affects frozen ground conditions. The Domo site is located in a higher position with respect to the central plateau of Byers; here, the wind is stronger and snow cover thinner, which has conditioned a longer thawing season than in the other two sites (Cerro Negro, Escondido). These two sites are located in topographically protected areas favouring snow accumulation. The longer persistence of snow conditions a longer duration of negative temperatures in the active layer of the permafrost. This research was financially supported by the HOLOANTAR project (Portuguese Science Foundation) and the AXA Research Fund.

  18. Effect of quarrying activity on biodiversity: Case study of Ogbere site ...

    African Journals Online (AJOL)

    USER

    Full Length Research Paper. Effect of quarrying activity on biodiversity: Case study of Ogbere site, Ogun State Nigeria. G. A. Lameed1* and A. E. Ayodele2. 1Department of Wildlife and Fisheries Management, Faculty of Agriculture and Forestry University of Ibadan, Ibadan,. Nigeria. 2Department of Botany and Microbiology, ...

  19. 113Cd NMR as a Probe of the Active Sites of Metalloenzymes

    NARCIS (Netherlands)

    Armitage, Ian M.; Schoot Uiterkamp, Antonius J.M.; Chlebowski, Jan F.; Coleman, Joseph E.

    1978-01-01

    113Cd NMR has been used to study the active site metal ion(s) of the 113Cd(II) derivatives of four Zn(II) metalloenzymes, carboxypeptidase A, carbonic anhydrases, alkaline phosphatase, and superoxide dismutase. The resonances of the enzyme-bound 113Cd(II) ions are extremely sensitive to ligand

  20. Active-site residues of procarboxypeptidase Y are accessible to chemical modification

    DEFF Research Database (Denmark)

    Sørensen, S O; Winther, Jakob R.

    1994-01-01

    The accessibility of the active-site cleft of procarboxypeptidase Y from Saccharomyces cerevisiae has been studied by chemical modifications of two specific amino-acid residues. Previous studies have shown that these residues, Cys-341 and Met-398 in the mature enzyme, are located in the S1 and S'1...

  1. Examination of a social-networking site activities scale (SNSAS) using Rasch analysis

    NARCIS (Netherlands)

    Alhaythami, Hassan; Karpinski, Aryn; Kirschner, Paul A.; Bolden, Edward

    2018-01-01

    This study examined the psychometric properties of a socialnetworking site (SNS) activities scale using Rasch Analysis. Items were also examined with Rasch Principal Components Analysis (PCA) and Differential Item Functioning (DIF) across groups of university students (i.e., males and females from

  2. Dynamics of water molecules in the active-site cavity of human cytochromes P450

    DEFF Research Database (Denmark)

    Rydberg, Patrik; Rod, Thomas Holm; Olsen, Lars

    2007-01-01

    We have studied the dynamics of water molecules in six crystal structures of four human cytochromes P450, 2A6, 2C8, 2C9, and 3A4, with molecular dynamics simulations. In the crystal structures, only a few water molecules are seen and the reported sizes of the active-site cavity vary a lot. In the...

  3. Comment on "Active sites for CO2 hydrogenation to methanol on Cu/ZnO catalysts"

    DEFF Research Database (Denmark)

    Nakamura, Junji; Fujitani, Tadahiro; Kuld, Sebastian

    2017-01-01

    Kattel et al (Reports, 24 March 2017, p. 1296) report that a zinc on copper (Zn/Cu) surface undergoes oxidation to zinc oxide/copper (ZnO/Cu) during carbon dioxide (CO2) hydrogenation to methanol and conclude that the Cu-ZnO interface is the active site for methanol synthesis. Similar experiments...

  4. Application of QA grading to Yucca Mountain Site Characterization Project items and activities

    International Nuclear Information System (INIS)

    Murthy, R.B.; Smith, S.C.

    1991-01-01

    Grading is the act of selecting the quality assurance (QA) measures necessary to develop and maintain confidence in the quality of an item or activity. The list of QA measures from which this selection is made are the 20 criteria of the Yucca Mountain Site Characterization Project Quality Assurance Requirements Document

  5. Structural Basis of Eukaryotic Nitrate Reduction: Crystal Structures of the Nitrate Reductase Active Site

    Science.gov (United States)

    Fischer, Katrin; Barbier, Guillaume G.; Hecht, Hans-Juergen; Mendel, Ralf R.; Campbell, Wilbur H.; Schwarz, Guenter

    2005-01-01

    Nitrate assimilation in autotrophs provides most of the reduced nitrogen on earth. In eukaryotes, reduction of nitrate to nitrite is catalyzed by the molybdenum-containing NAD(P)H:nitrate reductase (NR; EC 1.7.1.1-3). In addition to the molybdenum center, NR contains iron-heme and flavin adenine dinucleotide as redox cofactors involved in an internal electron transport chain from NAD(P)H to nitrate. Recombinant, catalytically active Pichia angusta nitrate-reducing, molybdenum-containing fragment (NR-Mo) was expressed in P. pastoris and purified. Crystal structures for NR-Mo were determined at 1.7 and 2.6 Å. These structures revealed a unique slot for binding nitrate in the active site and identified key Arg and Trp residues potentially involved in nitrate binding. Dimeric NR-Mo is similar in overall structure to sulfite oxidases, with significant differences in the active site. Sulfate bound in the active site caused conformational changes, as compared with the unbound enzyme. Four ordered water molecules located in close proximity to Mo define a nitrate binding site, a penta-coordinated reaction intermediate, and product release. Because yeast NAD(P)H:NR is representative of the family of eukaryotic NR, we propose a general mechanism for nitrate reduction catalysis. PMID:15772287

  6. Active site electrostatics protect genome integrity by blocking abortive hydrolysis during DNA recombination

    Science.gov (United States)

    Ma, Chien-Hui; Rowley, Paul A; Macieszak, Anna; Guga, Piotr; Jayaram, Makkuni

    2009-01-01

    Water, acting as a rogue nucleophile, can disrupt transesterification steps of important phosphoryl transfer reactions in DNA and RNA. We have unveiled this risk, and identified safeguards instituted against it, during strand cleavage and joining by the tyrosine site-specific recombinase Flp. Strand joining is threatened by a latent Flp endonuclease activity (type I) towards the 3′-phosphotyrosyl intermediate resulting from strand cleavage. This risk is not alleviated by phosphate electrostatics; neutralizing the negative charge on the scissile phosphate through methylphosphonate (MeP) substitution does not stimulate type I endonuclease. Rather, protection derives from the architecture of the recombination synapse and conformational dynamics within it. Strand cleavage is protected against water by active site electrostatics. Replacement of the catalytic Arg-308 of Flp by alanine, along with MeP substitution, elicits a second Flp endonuclease activity (type II) that directly targets the scissile phosphodiester bond in DNA. MeP substitution, combined with appropriate active site mutations, will be useful in revealing anti-hydrolytic mechanisms engendered by systems that mediate DNA relaxation, DNA transposition, site-specific recombination, telomere resolution, RNA splicing and retrohoming of mobile introns. PMID:19440204

  7. Substrate binding in the active site of cytochrome P450cam

    NARCIS (Netherlands)

    Swart, M.; Groenhof, A.R.; Ehlers, A.W.; Lammertsma, K.

    2005-01-01

    We have studied the binding of camphor in the active site of cytochrome P450cam with density functional theory (DFT) calculations. A strong hydrogen bond (>6 kcal/mol) to a tyrosine residue (Tyr96) is observed, that may account for the high specificity of the reaction taking place. The DFT

  8. 77 FR 74218 - Commercial Wind Leasing and Site Assessment Activities on the Atlantic Outer Continental Shelf...

    Science.gov (United States)

    2012-12-13

    ...] Commercial Wind Leasing and Site Assessment Activities on the Atlantic Outer Continental Shelf (OCS) Offshore... initiative is designed to identify areas that appear to be suitable for wind energy leasing on the OCS, known... responsible renewable energy leasing process. More information on the ``Smart from the Start'' initiative can...

  9. Aberration-corrected imaging of active sites on industrial catalyst nanoparticles

    DEFF Research Database (Denmark)

    Gontard, Lionel Cervera; Chang, L-Y; Hetherington, CJD

    2007-01-01

    Picture perfect: Information about the local topologies of active sites on commercial nanoparticles can be gained with atomic resolution through spherical-aberration-corrected transmission electron microscopy (TEM). A powder of Pt nanoparticles on carbon black was examined with two advanced TEM t...

  10. The Thumbs Up Ecology Curriculum: A Fun Group of School Site Activities for Sixth Graders.

    Science.gov (United States)

    Smith, John; And Others

    This guide is a collection of "fun" school site activities for sixth graders. Some of the topics covered are: animals, trees, energy and lifestyle, land use and you, energy conservation, and car-pooling. Each section offers both introductory information about the topic as well as questions to ponder such as what, so what, now what, and another way…

  11. Gasification under CO2–Steam Mixture: Kinetic Model Study Based on Shared Active Sites

    Directory of Open Access Journals (Sweden)

    Xia Liu

    2017-11-01

    Full Text Available In this work, char gasification of two coals (i.e., Shenfu bituminous coal and Zunyi anthracite and a petroleum coke under a steam and CO2 mixture (steam/CO2 partial pressures, 0.025–0.075 MPa; total pressures, 0.100 MPa and CO2/steam chemisorption of char samples were conducted in a Thermogravimetric Analyzer (TGA. Two conventional kinetic models exhibited difficulties in exactly fitting the experimental data of char–steam–CO2 gasification. Hence, a modified model based on Langmuir–Hinshelwood model and assuming that char–CO2 and char–steam reactions partially shared active sites was proposed and had indicated high accuracy for estimating the interactions in char–steam–CO2 reaction. Moreover, it was found that two new model parameters (respectively characterized as the amount ratio of shared active sites to total active sites in char–CO2 and char–steam reactions in the modified model hardly varied with gasification conditions, and the results of chemisorption indicate that these two new model parameters mainly depended on the carbon active sites in char samples.

  12. United States and European students’ social-networking site activities and academic performance

    NARCIS (Netherlands)

    Karpinski, Aryn; Kirschner, Paul A.; Shreffler, Anthony; Albert, Patricia; Tomko, Carrie

    2018-01-01

    Different cultures communicate differently. Research is beginning to examine the differences in culture related to social-networking site (SNS) use. Differences in specific SNS activities related to academic performance among United States (US; n = 446) and European (n = 394) university students

  13. Modeling the active site of [FeFe]-hydrogenase: Electro-catalytic ...

    Indian Academy of Sciences (India)

    dithiolate-bridged di-iron com- plexes have been investigated in modelling the active sites of [FeFe]-H2ases. However, the reports of elec- trochemical data on ene-1,2-dithiolate-bridged-{Fe2S2} complexes are scarce. In this study, compound [Fe2{µ-.

  14. Metabolic impact of overexpression of liver glycogen synthase with serine-to-alanine substitutions in rat primary hepatocytes.

    Science.gov (United States)

    Kadotani, Akito; Fujimura, Maho; Nakamura, Takao; Ohyama, Sumika; Harada, Naomoto; Maruki, Hiroko; Tamai, Yoshitaka; Kanatani, Akio; Eiki, Jun-Ichi; Nagata, Yasufumi

    2007-10-15

    To investigate the effect of elevation of liver glycogen synthase (GYS2) activity on glucose and glycogen metabolism, we performed adenoviral overexpression of the mutant GYS2 with six serine-to-alanine substitutions in rat primary hepatocytes. Cell-free assays demonstrated that the serine-to-alanine substitutions caused constitutive activity and electrophoretic mobility shift. In rat primary hepatocytes, overexpression of the mutant GYS2 significantly reduced glucose production by 40% and dramatically induced glycogen synthesis via the indirect pathway rather than the direct pathway. Thus, we conclude that elevation of glycogen synthase activity has an inhibitory effect on glucose production in hepatocytes by shunting gluconeogenic precursors into glycogen. In addition, although intracellular compartmentation of glucose-6-phosphate (G6P) remains unclear in hepatocytes, our results imply that there are at least two G6P pools via gluconeogenesis and due to glucose phosphorylation, and that G6P via gluconeogenesis is preferentially used for glycogen synthesis in hepatocytes.

  15. Realizing Serine/Threonine Ligation: Scope and Limitations and Mechanistic Implication Thereof

    Directory of Open Access Journals (Sweden)

    Clarence T. T. Wong

    2014-05-01

    Full Text Available Serine/Threonine ligation (STL has emerged as an alternative tool for protein chemical synthesis, bioconjugations as well as macrocyclization of peptides of various sizes. Owning to the high abundance of Ser/Thr residues in natural peptides and proteins, STL is expected to find a wide range of applications in chemical biology research. Herein, we have fully investigated the compatibility of the serine/threonine ligation strategy for X-Ser/Thr ligation sites, where X is any of the 20 naturally occurring amino acids. Our studies have shown that 17 amino acids are suitable for ligation, while Asp, Glu, and Lys are not compatible. Among the working 17 C-terminal amino acids, the retarded reaction resulted from the bulky β-branched amino acid (Thr, Val and Ile is not seen under the current ligation condition. We have also investigated the chemoselectivity involving the amino group of the internal lysine which may compete with the N-terminal Ser/Thr for reaction with the C-terminal salicylaldehyde (SAL ester aldehyde group. The result suggested that the free internal amino group does not adversely slow down the ligation rate.

  16. Agonist activation of α7 nicotinic acetylcholine receptors via an allosteric transmembrane site

    Science.gov (United States)

    Gill, JasKiran K.; Savolainen, Mari; Young, Gareth T.; Zwart, Ruud; Sher, Emanuele; Millar, Neil S.

    2011-01-01

    Conventional nicotinic acetylcholine receptor (nAChR) agonists, such as acetylcholine, act at an extracellular “orthosteric” binding site located at the interface between two adjacent subunits. Here, we present evidence of potent activation of α7 nAChRs via an allosteric transmembrane site. Previous studies have identified a series of nAChR-positive allosteric modulators (PAMs) that lack agonist activity but are able to potentiate responses to orthosteric agonists, such as acetylcholine. It has been shown, for example, that TQS acts as a conventional α7 nAChR PAM. In contrast, we have found that a compound with close chemical similarity to TQS (4BP-TQS) is a potent allosteric agonist of α7 nAChRs. Whereas the α7 nAChR antagonist metyllycaconitine acts competitively with conventional nicotinic agonists, metyllycaconitine is a noncompetitive antagonist of 4BP-TQS. Mutation of an amino acid (M253L), located in a transmembrane cavity that has been proposed as being the binding site for PAMs, completely blocks agonist activation by 4BP-TQS. In contrast, this mutation had no significant effect on agonist activation by acetylcholine. Conversely, mutation of an amino acid located within the known orthosteric binding site (W148F) has a profound effect on agonist potency of acetylcholine (resulting in a shift of ∼200-fold in the acetylcholine dose-response curve), but had little effect on the agonist dose-response curve for 4BP-TQS. Computer docking studies with an α7 homology model provides evidence that both TQS and 4BP-TQS bind within an intrasubunit transmembrane cavity. Taken together, these findings provide evidence that agonist activation of nAChRs can occur via an allosteric transmembrane site. PMID:21436053

  17. Management of Ground and Groundwater Contamination on a Compact Site Constrained by Ongoing Activities

    International Nuclear Information System (INIS)

    Eilbeck, K.E.; Reeve, P.

    2009-01-01

    Sellafield Site is a compact and complex site which since the 1940's has been home to a range of facilities associated with the production and reprocessing of fissile material. The site contains the UK equivalent of the Chicago Pile-1 reactor, Hanford B Reactor, Rocky Flats Buildings 771 and 774, West Valley Main Process Plant Building, Savannah River Vitrification Plant, Savannah River MOX Plant, Savannah River F Canyon, Hanford 222 Analytical Laboratory, Savannah River K-, L-, and P-Basins, and the Fort St. Vrain Reactor all in an area of approximately 1000 acres. Spent fuel reprocessing is still undertaken on site; however waste management and decommissioning activities are of increasing importance. These include the emptying and removal of fragile ponds and silos containing significant radioactive inventories, the decommissioning of reactors (including the world's first commercial reactor for power generation and the Windscale Piles, the site of a reactor fire in the late 1950's) and the construction of a new generation of vitrification and encapsulation plants. Leaks, spills and on-site disposals during the site's industrial lifetime have resulted in a legacy of fission products and other radionuclides in the ground and groundwater. Volumes of contaminated ground have been estimated as being as much as 18 million m 3 and an estimated below ground inventory of approximately 1.8 E16 Bq. These have all occurred within close proximity to a range of receptors including farm land and the sea. The cramped nature of the facilities on site, overlapping source terms and ongoing decommissioning, waste management and operating activities all raise significant challenges in the management and remediation of contaminated land and groundwater. The strategy to address these challenges includes: 1. Data collection, management and interpretation. The congested nature of the site and the age of some of the monitoring facilities has resulted in particular difficulties. For

  18. Interaction of protein C inhibitor with the type II transmembrane serine protease enteropeptidase.

    Directory of Open Access Journals (Sweden)

    Thomas A Prohaska

    Full Text Available The serine protease inhibitor protein C inhibitor (PCI is expressed in many human tissues and exhibits broad protease reactivity. PCI binds glycosaminoglycans and certain phospholipids, which modulate its inhibitory activity. Enteropeptidase (EP is a type II transmembrane serine protease mainly found on the brush border membrane of epithelial cells in the duodenum, where it activates trypsinogen to initiate the digestion of food proteins. Some active EP is also present in duodenal fluid and has been made responsible for causing pancreatitis in case of duodeno-pancreatic reflux. Together with its substrate trypsinogen, EP is furthermore present in the epidermis and in some cancer cells. In this report, we show that PCI inhibited EP with an apparent 2nd order rate constant of 4.48 × 10(4 M(-1 s(-1. Low molecular weight (LMWH and unfractionated heparin (UFH slightly reduced the inhibitory effect of PCI. The SI (stoichiometry of inhibition value for the inhibition of EP by PCI was 10.8 in the absence and 17.9 in the presence of UFH (10 U/ml. By inhibiting trypsin, chymotrypsin, and additionally EP, PCI might play a role in the protection of the pancreas from autodigestion. Furthermore the interaction of PCI with EP may influence the regulation of epithelial differentiation.

  19. Number of active transcription factor binding sites is essential for the Hes7 oscillator

    Directory of Open Access Journals (Sweden)

    de Angelis Martin

    2006-02-01

    Full Text Available Abstract Background It is commonly accepted that embryonic segmentation of vertebrates is regulated by a segmentation clock, which is induced by the cycling genes Hes1 and Hes7. Their products form dimers that bind to the regulatory regions and thereby repress the transcription of their own encoding genes. An increase of the half-life of Hes7 protein causes irregular somite formation. This was shown in recent experiments by Hirata et al. In the same work, numerical simulations from a delay differential equations model, originally invented by Lewis, gave additional support. For a longer half-life of the Hes7 protein, these simulations exhibited strongly damped oscillations with, after few periods, severely attenuated the amplitudes. In these simulations, the Hill coefficient, a crucial model parameter, was set to 2 indicating that Hes7 has only one binding site in its promoter. On the other hand, Bessho et al. established three regulatory elements in the promoter region. Results We show that – with the same half life – the delay system is highly sensitive to changes in the Hill coefficient. A small increase changes the qualitative behaviour of the solutions drastically. There is sustained oscillation and hence the model can no longer explain the disruption of the segmentation clock. On the other hand, the Hill coefficient is correlated with the number of active binding sites, and with the way in which dimers bind to them. In this paper, we adopt response functions in order to estimate Hill coefficients for a variable number of active binding sites. It turns out that three active transcription factor binding sites increase the Hill coefficient by at least 20% as compared to one single active site. Conclusion Our findings lead to the following crucial dichotomy: either Hirata's model is correct for the Hes7 oscillator, in which case at most two binding sites are active in its promoter region; or at least three binding sites are active, in which

  20. Fibromodulin Interacts with Collagen Cross-linking Sites and Activates Lysyl Oxidase*

    Science.gov (United States)

    Bihan, Dominique; Bonna, Arkadiusz; Rubin, Kristofer; Farndale, Richard W.

    2016-01-01

    The hallmark of fibrotic disorders is a highly cross-linked and dense collagen matrix, a property driven by the oxidative action of lysyl oxidase. Other fibrosis-associated proteins also contribute to the final collagen matrix properties, one of which is fibromodulin. Its interactions with collagen affect collagen cross-linking, packing, and fibril diameter. We investigated the possibility that a specific relationship exists between fibromodulin and lysyl oxidase, potentially imparting a specific collagen matrix phenotype. We mapped the fibromodulin-collagen interaction sites using the collagen II and III Toolkit peptide libraries. Fibromodulin interacted with the peptides containing the known collagen cross-linking sites and the MMP-1 cleavage site in collagens I and II. Interestingly, the interaction sites are closely aligned within the quarter-staggered collagen fibril, suggesting a multivalent interaction between fibromodulin and several collagen helices. Furthermore, we detected an interaction between fibromodulin and lysyl oxidase (a major collagen cross-linking enzyme) and mapped the interaction site to 12 N-terminal amino acids on fibromodulin. This interaction also increases the activity of lysyl oxidase. Together, the data suggest a fibromodulin-modulated collagen cross-linking mechanism where fibromodulin binds to a specific part of the collagen domain and also forms a complex with lysyl oxidase, targeting the enzyme toward specific cross-linking sites. PMID:26893379

  1. Fibromodulin Interacts with Collagen Cross-linking Sites and Activates Lysyl Oxidase.

    Science.gov (United States)

    Kalamajski, Sebastian; Bihan, Dominique; Bonna, Arkadiusz; Rubin, Kristofer; Farndale, Richard W

    2016-04-08

    The hallmark of fibrotic disorders is a highly cross-linked and dense collagen matrix, a property driven by the oxidative action of lysyl oxidase. Other fibrosis-associated proteins also contribute to the final collagen matrix properties, one of which is fibromodulin. Its interactions with collagen affect collagen cross-linking, packing, and fibril diameter. We investigated the possibility that a specific relationship exists between fibromodulin and lysyl oxidase, potentially imparting a specific collagen matrix phenotype. We mapped the fibromodulin-collagen interaction sites using the collagen II and III Toolkit peptide libraries. Fibromodulin interacted with the peptides containing the known collagen cross-linking sites and the MMP-1 cleavage site in collagens I and II. Interestingly, the interaction sites are closely aligned within the quarter-staggered collagen fibril, suggesting a multivalent interaction between fibromodulin and several collagen helices. Furthermore, we detected an interaction between fibromodulin and lysyl oxidase (a major collagen cross-linking enzyme) and mapped the interaction site to 12 N-terminal amino acids on fibromodulin. This interaction also increases the activity of lysyl oxidase. Together, the data suggest a fibromodulin-modulated collagen cross-linking mechanism where fibromodulin binds to a specific part of the collagen domain and also forms a complex with lysyl oxidase, targeting the enzyme toward specific cross-linking sites. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

  2. Locomotor activity influences muscle architecture and bone growth but not muscle attachment site morphology

    Science.gov (United States)

    Rabey, Karyne N.; Green, David J.; Taylor, Andrea B.; Begun, David R.; Richmond, Brian G.; McFarlin, Shannon C.

    2014-01-01

    The ability to make behavioural inferences from skeletal remains is critical to understanding the lifestyles and activities of past human populations and extinct animals. Muscle attachment site (enthesis) morphology has long been assumed to reflect muscle strength and activity during life, but little experimental evidence exists to directly link activity patterns with muscle development and the morphology of their attachments to the skeleton. We used a mouse model to experimentally test how the level and type of activity influences forelimb muscle architecture of spinodeltoideus, acromiodeltoideus, and superficial pectoralis, bone growth rate and gross morphology of their insertion sites. Over an 11-week period, we collected data on activity levels in one control group and two experimental activity groups (running, climbing) of female wild-type mice. Our results show that both activity type and level increased bone growth rates influenced muscle architecture, including differences in potential muscular excursion (fibre length) and potential force production (physiological cross-sectional area). However, despite significant influences on muscle architecture and bone development, activity had no observable effect on enthesis morphology. These results suggest that the gross morphology of entheses is less reliable than internal bone structure for making inferences about an individual’s past behaviour. PMID:25467113

  3. Integral Public Activities as a Support to the Site Selection Process for LILW Repository

    International Nuclear Information System (INIS)

    Zeleznik, N.; Kralj, M.

    2008-01-01

    The first site selection process for low and intermediate level radioactive waste (LILW) repository took place between 1990 and 1993 in Slovenia was stopped unsuccessfully with very strong public opposition at local level, followed by political withdrawal on national level. As one of the consequences ARAO started to develop new approach to the site selection based also on the findings from sociology, psychology and other human sciences. The recommendations on public involvement and transparency were so strong that ARAO started with first limited public relation (PR) activities which later grew to the PR process which supports all technical activities in ARAO. Presently the PR process covers communication, information and research activities and assures careful planning, prompt responds and involvement of the highest responsible persons at ARAO. Integral public relation activities are divided in several parts. Majority of activities support the on-going site selection process where activities are presently focused on functioning of local partnerships developed as a basic communication tool to involve as much citizens and public as possible on local level. Presently two local partnerships are working in Krsko and Brezice community with clear role to enhance public involvement according to Aarchus convention. Each of the partnerships is organized in a specific way adjusted to the local needs. Communication activities are organized also for different other projects and are preparing the necessary basis for the work with different groups of stake holders and in different situations. As a foundation very broad information material, such as books, leaflets, reports, magazines, video cassettes, CD and DVD on the radioactive waste management is prepared and used for different purposes. We also try to be proactive with web pages and have a well organized visitors' center. Improvement of public relation process is achieved through constant survey and feed-back information

  4. Improvement of the activity and thermostability of microbial transglutaminase by multiple-site mutagenesis.

    Science.gov (United States)

    Mu, Dongdong; Lu, Jiaojiao; Shu, Chang; Li, Haowen; Li, Xingjiang; Cai, Jing; Luo, Shuizhong; Yang, Peizhou; Jiang, Shaotong; Zheng, Zhi

    2018-01-01

    Microbial transglutaminase (MTG) is an enzyme widely used in the food industry. Mutiple-site mutagenesis of Streptomyces mobaraensis transglutaminase was performed in Escherichia coli. According to enzymatic assay and thermostability study, among three penta-site MTG mutants (DM01-03), DM01 exhibited the highest enzymatic activity of 55.7 ± 1.4 U/mg and longest half-life at 50 °C (418.2 min) and 60 °C (24.8 min).

  5. Surface binding sites in carbohydrate active enzymes: An emerging picture of structural and functional diversity

    DEFF Research Database (Denmark)

    Svensson, Birte; Cockburn, Darrell

    2013-01-01

    identified in enzymes from a wide variety of families, though almost half are found in the α-amylase family GH13. The roles attributed to SBSs are not limited to targeting the enzyme to the substrate, but also include a variety of others such as guiding the substrate into the active site, altering enzyme...... specificity and acting as an allosteric site. Although SBSs share many roles with CBMs they may not simply be an alternative to CBMs, but rather complementary as SBSs and CBMs frequently co-occur in enzymes. Despite a relatively long history, it is only in recent years that SBSs have been studied in great...

  6. Electrochemical probing into the active sites of graphitic-layer encapsulated iron oxygen reduction reaction electrocatalysts

    DEFF Research Database (Denmark)

    Zhong, Lijie; Jensen, Jens Oluf; Cleemann, Lars Nilausen

    2017-01-01

    is still unclear compared with the well-recognized surface coordinated FeNx/C structure. Using the strong complexing effect of the iron component with anions, cyanide (CN−) in alkaline and thiocyanate (SCN−) in acidic media, the metal containing active sites are electrochemically probed. Three...... significant shifts of half-wave potentials indicating that surface Fe coordinated sites in all cases. The G@Fe catalyst showed the weakest poisoning effect (the lowest shifts of half-wave potential) compared to the Fe/N/C and FePc catalysts in both electrolytes. These results could be explained...

  7. Technical basis for classification of low-activity waste fraction from Hanford site tanks

    Energy Technology Data Exchange (ETDEWEB)

    Petersen, C.A., Westinghouse Hanford

    1996-07-17

    The overall objective of this report is to provide a technical basis to support a U.S. Nuclear Regulatory Commission determination to classify the low-activity waste from the Hanford Site single-shell and double-shell tanks as `incidental` wastes after removal of additional radionuclides and immobilization.The proposed processing method, in addition to the previous radionuclide removal efforts, will remove the largest practical amount of total site radioactivity, attributable to high-level wastes, for disposal in a deep geologic repository. The remainder of the waste would be considered `incidental` waste and could be disposed onsite.

  8. Technical basis for classification of low-activity waste fraction from Hanford site tanks

    Energy Technology Data Exchange (ETDEWEB)

    Petersen, C.A.

    1996-09-20

    The overall objective of this report is to provide a technical basis to support a U.S. Nuclear Regulatory Commission determination to classify the low-activity waste from the Hanford Site single-shell and double-shell tanks as `incidental` wastes after removal of additional radionuclides and immobilization.The proposed processing method, in addition to the previous radionuclide removal efforts, will remove the largest practical amount of total site radioactivity, attributable to high-level waste, for disposal is a deep geologic repository. The remainder of the waste would be considered `incidental` waste and could be disposed onsite.

  9. Final Report - Independent Verification Survey Activities at the Seperations Process Research Unit Sites, Niskayuna, New York

    Energy Technology Data Exchange (ETDEWEB)

    Evan Harpenau

    2011-03-15

    The Separations Process Research Unit (SPRU) complex located on the Knolls Atomic Power Laboratory (KAPL) site in Niskayuna, New York, was constructed in the late 1940s to research the chemical separation of plutonium and uranium (Figure A-1). SPRU operated as a laboratory scale research facility between February 1950 and October 1953. The research activities ceased following the successful development of the reduction oxidation and plutonium/uranium extraction processes. The oxidation and extraction processes were subsequently developed for large scale use by the Hanford and Savannah River sites (aRc 2008a). Decommissioning of the SPRU facilities began in October 1953 and continued through the 1990s.

  10. Regulatory inspection activities on nuclear power plant sites during construction in the United Kingdom

    International Nuclear Information System (INIS)

    Jeffery, J.V.

    1977-01-01

    The work of regulatory inspection of the construction of the plant on the site is performed not only by the inspector who has been allocated to inspection duties for that site but also by the specialist staff who are involved with the safety assessment of the plant. The co-ordination of this work is described in the paper and examples are given of inspection activities associated with the enforcement requirements of licence conditions as well as those related to the inspection of the plant itself. (author)

  11. Radiological audit of remedial action activities at the processing site, transfer site, and Cheney disposal site Grand Junction, Colorado: Audit date, August 9--11, 1993

    International Nuclear Information System (INIS)

    1993-08-01

    The Uranium Mill Tailing Remedial Action (UMTRA) Project's Technical Assistance Contractor (TAC) performed a radiological audit of the Remedial Action Contractor (RAC), MK-Ferguson and CWM Federal Environmental Services, Inc., at the processing site, transfer site, and Cheney disposal site in Grand Junction, Colorado. Jim Hylko and Bill James of the TAC conducted this audit August 9 through 11, 1993. Bob Cornish and Frank Bosiljevec represented the US Department of Energy (DOE). This report presents one programmatic finding, eleven site-specific observations, one good practice, and four programmatic observations

  12. Assessment of national systems for obtaining local acceptance of nuclear waste management siting activities

    International Nuclear Information System (INIS)

    Paige, H.W.; Owens, J.E.

    1984-01-01

    On behalf of the United States Department of Energy (DOE), International Energy Associates Limited (IEAL) of Washington, D.C. has conducted surveys and analyses of fourteen countries' plans and approaches for dealing with the problems of obtaining local siting acceptance for nuclear waste management facilities. It was determined that the following elements of the formal systems generally facilitate and/or expedite waste management siting decisions: (1) a clear-cut pro-nuclear power position on the part of the government; (2) a willingness on the part of the central government to exert (with prudence and restraint) its pre-emptive rights in nuclear matters; (3) political structures in which the heads of regional or provincial governments are appointed by the central government; (4) national laws that link reactor licensing with a detailed plan for waste management; (5) an established and stable policy with regard to reprocessing. In contrast, it was determined that the following elements of the formal system generally hinder waste management siting activities: (1) historically strong local land used veto laws; (2) the use of national referenda for making nuclear decisions; (3) requirements for public hearings. The informal approaches fall into the following five categories: (1) political: e.g. assertion of will by political leaders, activities to enlist support of local politicians, activities to broaden involvement in decision-making; (2) economic: e.g. emphasis on normal benefits, provision for additional economic benefits; (3) siting: e.g. at or near existing nuclear facilities, on government or utility property, at multiple locations to spread the political burden; (4) timing: e.g. decoupling drilling activities from ultimate repository site decision, deliberate deferral to (long-range) future; (5) education: e.g. creation of special government programmes, enlisting of media support

  13. Controlling activation site density by low-energy far-field stimulation in cardiac tissue

    Science.gov (United States)

    Hörning, Marcel; Takagi, Seiji; Yoshikawa, Kenichi

    2012-06-01

    Tachycardia and fibrillation are potentially fatal arrhythmias associated with the formation of rotating spiral waves in the heart. Presently, the termination of these types of arrhythmia is achieved by use of antitachycardia pacing or cardioversion. However, these techniques have serious drawbacks, in that they either have limited application or produce undesirable side effects. Low-energy far-field stimulation has recently been proposed as a superior therapy. This proposed therapeutic method would exploit the phenomenon in which the application of low-energy far-field shocks induces a large number of activation sites (“virtual electrodes”) in tissue. It has been found that the formation of such sites can lead to the termination of undesired states in the heart and the restoration of normal beating. In this study we investigate a particular aspect of this method. Here we seek to determine how the activation site density depends on the applied electric field through in vitro experiments carried out on neonatal rat cardiac tissue cultures. The results indicate that the activation site density increases exponentially as a function of the intracellular conductivity and the level of cell isotropy. Additionally, we report numerical results obtained from bidomain simulations of the Beeler-Reuter model that are quantitatively consistent with our experimental results. Also, we derive an intuitive analytical framework that describes the activation site density and provides useful information for determining the ratio of longitudinal to transverse conductivity in a cardiac tissue culture. The results obtained here should be useful in the development of an actual therapeutic method based on low-energy far-field pacing. In addition, they provide a deeper understanding of the intrinsic properties of cardiac cells.

  14. Synthesis and Activity of A Single Active Site N-doped Electro-catalyst for Oxygen Reduction

    International Nuclear Information System (INIS)

    Bayati, Maryam; Scott, Keith

    2016-01-01

    Nitrogen doped carbon materials are promising oxygen reduction reaction (ORR) catalysts which could potentially replace platinum. However, despite extensive studies, their active sites are still controversial and their impact on overall ORR remains obscure. Herein, we present a method for preparation of a single active site catalyst based on cycling an iron-inserted N-doped carbon catalyst in a wide potential window firstly in sulfuric acid and later in alkaline solution to study the contribution of the remaining one active group in overall activity. Following preparation of the metal-inserted N-doped carbon catalyst (MINC), its morphology was characterized using X-ray photoelectron spectroscopy (XPS), high resolution transmission electron microscopy (HRTEM), X-ray diffraction (XRD), and the electro-catalytic behavior was investigated by employing linear sweep voltammetry (LSV) using a rotating ring disk electrode (RRDE). XPS revealed that graphitic nitrogen was the only remaining active nitrogen-containing group after elimination the pyridinic and pyrrolic groups and also iron nano-materials removal confirmed from auger peak of Fe LMMa and poisoning reaction with cyanide. The C1 s XPS region data showed an increase in the oxygen reduction intermediate C-OH peak, after the reaction, which indicates electrocatalytic activity of the graphitic carbon. Electrochemical studies revealed no significant changes in limiting current, a small increase in H 2 O 2 production and 47 mV shift in half wave potential for degraded catalyst which is in line with previous theoretical calculations.

  15. Differential regulation of the transcriptional activity of the glucocorticoid receptor through site-specific phosphorylation

    Directory of Open Access Journals (Sweden)

    Raj Kumar

    2008-08-01

    Full Text Available Raj Kumar1, William J Calhoun21Division of Gastroenterology; 2Division of Allergy, Pulmonary, Immunology, Critical Care, and Sleep (APICS, Department of Internal Medicine, University of Texas Medical Branch, Galveston, TX, USAAbstract: Post-translational modifications such as phosphorylation are known to play an important role in the gene regulation by the transcription factors including the nuclear hormone receptor superfamily of which the glucocorticoid receptor (GR is a member. Protein phosphorylation often switches cellular activity from one state to another. Like many other transcription factors, the GR is a phosphoprotein, and phosphorylation plays an important role in the regulation of GR activity. Cell signaling pathways that regulate phosphorylation of the GR and its associated proteins are important determinants of GR function under various physiological conditions. While the role of many phosphorylation sites in the GR is still not fully understood, the role of others is clearer. Several aspects of transcription factor function, including DNA binding affinity, interaction of transactivation domains with the transcription initiation complex, and shuttling between the cytoplasmic compartments, have all been linked to site-specific phosphorylation. All major phosphorylation sites in the human GR are located in the N-terminal domain including the major transactivation domain, AF1. Available literature clearly indicates that many of these potential phosphorylation sites are substrates for multiple kinases, suggesting the potential for a very complex regulatory network. Phosphorylated GR interacts favorably with critical coregulatory proteins and subsequently enhances transcriptional activity. In addition, the activities and specificities of coregulators may be subject to similar regulation by phosphorylation. Regulation of the GR activity due to phosphorylation appears to be site-specific and dependent upon specific cell signaling cascade

  16. Progress report on decommissioning activities at the Fernald Environmental Management Project (FEMP) site

    International Nuclear Information System (INIS)

    1998-01-01

    The Fernald Environmental Management Project (FEMP), is located about 18 miles northwest of Cincinnati, Ohio. Between 1953 and 1989, the facility, then called the Feed Material Production Center or FMPC, produced uranium metal products used in the eventual production of weapons grade material for use by other US Department of Energy (DOE) sites. In 1989, FMPC's production was suspended by the federal government in order to focus resources on environmental restoration versus defense production. In 1992, Fluor Daniel Fernald assumed responsibility for managing all cleanup activities at the FEMP under contract to the DOE. In 1990, as part of the remediation effort, the site was divided into five operable units based on physical proximity of contaminated areas, similar amounts of types of contamination, or the potential for a similar technology to be used in cleanup activities. This report continues the outline of the decontamination and decommissioning (D and D) activities at the FEMP site Operable Unit 3 (OU3) and provides an update on the status of the decommissioning activities. OU3, the Facilities Closure and Demolition Project, involves the remediation of more than 200 uranium processing facilities. The mission of the project is to remove nuclear materials stored in these buildings, then perform the clean out of the buildings and equipment, and decontaminate and dismantle the facilities

  17. Active site dynamics in NADH oxidase from Thermus thermophilus studied by NMR spin relaxation

    International Nuclear Information System (INIS)

    Miletti, Teresa; Farber, Patrick J.; Mittermaier, Anthony

    2011-01-01

    We have characterized the backbone dynamics of NADH oxidase from Thermus thermophilus (NOX) using a recently-developed suite of NMR experiments designed to isolate exchange broadening, together with 15 N R 1 , R 1ρ , and { 1 H}- 15 N steady-state NOE relaxation measurements performed at 11.7 and 18.8 T. NOX is a 54 kDa homodimeric enzyme that belongs to a family of structurally homologous flavin reductases and nitroreductases with many potential biotechnology applications. Prior studies have suggested that flexibility is involved in the catalytic mechanism of the enzyme. The active site residue W47 was previously identified as being particularly important, as its level of solvent exposure correlates with enzyme activity, and it was observed to undergo “gating” motions in computer simulations. The NMR data are consistent with these findings. Signals from W47 are dynamically broadened beyond detection and several other residues in the active site have significant R ex contributions to transverse relaxation rates. In addition, the backbone of S193, whose side chain hydroxyl proton hydrogen bonds directly with the FMN cofactor, exhibits extensive mobility on the ns–ps timescale. We hypothesize that these motions may facilitate structural rearrangements of the active site that allow NOX to accept both FMN and FAD as cofactors.

  18. Analysis of a two-domain binding site for the urokinase-type plasminogen activator-plasminogen activator inhibitor-1 complex in low-density-lipoprotein-receptor-related protein.

    Science.gov (United States)

    Andersen, O M; Petersen, H H; Jacobsen, C; Moestrup, S K; Etzerodt, M; Andreasen, P A; Thøgersen, H C

    2001-07-01

    The low-density-lipoprotein-receptor (LDLR)-related protein (LRP) is composed of several classes of domains, including complement-type repeats (CR), which occur in clusters that contain binding sites for a multitude of different ligands. Each approximately 40-residue CR domain contains three conserved disulphide linkages and an octahedral Ca(2+) cage. LRP is a scavenging receptor for ligands from extracellular fluids, e.g. alpha(2)-macroglobulin (alpha(2)M)-proteinase complexes, lipoprotein-containing particles and serine proteinase-inhibitor complexes, like the complex between urokinase-type plasminogen activator (uPA) and the plasminogen activator inhibitor-1 (PAI-1). In the present study we analysed the interaction of the uPA-PAI-1 complex with an ensemble of fragments representing a complete overlapping set of two-domain fragments accounting for the ligand-binding cluster II (CR3-CR10) of LRP. By ligand blotting, solid-state competition analysis and surface-plasmon-resonance analysis, we demonstrate binding to multiple CR domains, but show a preferential interaction between the uPA-PAI-1 complex and a two-domain fragment comprising CR domains 5 and 6 of LRP. We demonstrate that surface-exposed aspartic acid and tryptophan residues at identical positions in the two homologous domains, CR5 and CR6 (Asp(958,CR5), Asp(999,CR6), Trp(953,CR5) and Trp(994,CR6)), are critical for the binding of the complex as well as for the binding of the receptor-associated protein (RAP) - the folding chaperone/escort protein required for transport of LRP to the cell surface. Accordingly, the present work provides (1) an identification of a preferred binding site within LRP CR cluster II; (2) evidence that the uPA-PAI-1 binding site involves residues from two adjacent protein domains; and (3) direct evidence identifying specific residues as important for the binding of uPA-PAI-1 as well as for the binding of RAP.

  19. Active Site Flexibility as a Hallmark for Efficient PET Degradation by I. sakaiensis PETase.

    Science.gov (United States)

    Fecker, Tobias; Galaz-Davison, Pablo; Engelberger, Felipe; Narui, Yoshie; Sotomayor, Marcos; Parra, Loreto P; Ramírez-Sarmiento, César A

    2018-03-27

    Polyethylene terephthalate (PET) is one of the most-consumed synthetic polymers, with an annual production of 50 million tons. Unfortunately, PET accumulates as waste and is highly resistant to biodegradation. Recently, fungal and bacterial thermophilic hydrolases were found to catalyze PET hydrolysis with optimal activities at high temperatures. Strikingly, an enzyme from Ideonella sakaiensis, termed PETase, was described to efficiently degrade PET at room temperature, but the molecular basis of its activity is not currently understood. Here, a crystal structure of PETase was determined at 2.02 Å resolution and employed in molecular dynamics simulations showing that the active site of PETase has higher flexibility at room temperature than its thermophilic counterparts. This flexibility is controlled by a novel disulfide bond in its active site, with its removal leading to destabilization of the catalytic triad and reduction of the hydrolase activity. Molecular docking of a model substrate predicts that PET binds to PETase in a unique and energetically favorable conformation facilitated by several residue substitutions within its active site when compared to other enzymes. These computational predictions are in excellent agreement with recent mutagenesis and PET film degradation analyses. Finally, we rationalize the increased catalytic activity of PETase at room temperature through molecular dynamics simulations of enzyme-ligand complexes for PETase and other thermophilic PET-degrading enzymes at 298, 323, and 353 K. Our results reveal that both the binding pose and residue substitutions within PETase favor proximity between the catalytic residues and the labile carbonyl of the substrate at room temperature, suggesting a more favorable hydrolytic reaction. These results are valuable for enabling detailed evolutionary analysis of PET-degrading enzymes and for rational design endeavors aiming at increasing the efficiency of PETase and similar enzymes toward plastic

  20. Preliminary characterisation of extracellular serine proteases of Dermatophilus congolensis isolates from cattle, sheep and horses.

    Science.gov (United States)

    Ambrose, N C; Mijinyawa, M S; Hermoso de Mendoza, J

    1998-08-15

    Dermatophilus congolensis is a filamentous branching actinomycete that causes dermatophilosis, an exudative dermatitis in ruminants. The pathogenesis of this disease is poorly understood and virulence factors of D. congolensis have not been characterised. Culture filtrate (CF) of 14 D. congolensis isolates from cattle, 15 from sheep and four from horses were examined for proteolytic activity using azocasein as a non-specific substrate. The isolates were from a variety of geographical locations. All the isolates examined produced extracellular proteolytic activity. CF from 24 and 48 h cultures and from first and third passages contained proteases. Proteolytic activity was greatest in neutral to alkaline pH (pH 7-10). CF of bovine isolates contained more proteolytic activity than that of ovine isolates. Furthermore, in substrate SDS-PAGE gels containing azocasein the number of proteolytic bands and their molecular weights in CF of bovine, ovine and equine isolates were different, giving distinctive band patterns for isolates from each host species. Three out of four bovine isolates from Antigua gave a fourth band pattern. Bands of equivalent molecular weights to the proteases could not be identified in silver stained SDS-PAGE gels of CF. Serine protease inhibitors had a concentration-dependent inhibitory effect on proteolytic activity in CF and inhibited activity of all proteolytic bands in substrate gels. With the exception of EDTA which had a variable-enhancing effect on activity, inhibitors of other classes of protease had no effect on activity. We conclude that D. congolensis produces a number of extracellular alkaline serine proteases, our results suggest the presence of host-specific variation between isolates and to a lesser extent between isolates from the same host species.

  1. Large zinc cation occupancy of octahedral sites in mechanically activated zinc ferrite powders

    Energy Technology Data Exchange (ETDEWEB)

    Oliver, S. A. [Center for Electromagnetic Research, Northeastern University, Boston, Massachusetts 02115 (United States); Harris, V. G. [Complex Materials Section, Code 6342, Naval Research Laboratory, Washington, DC 20375 (United States); Hamdeh, H. H. [Department of Physics, Wichita State University, Wichita, Kansas 67260 (United States); Ho, J. C. [Department of Physics, Wichita State University, Wichita, Kansas 67260 (United States)

    2000-05-08

    The cation site occupancy of a mechanically activated nanocrystalline zinc ferrite powder was determined as (Zn{sub 0.55}{sup 2+}Fe{sub 0.18}{sup 3+}){sub tet}[Zr{sub 0.45}{sup 2+}Fe{sub 1.82}{sup 3+}]{sub oct}O{sub 4} through analysis of extended x-ray absorption fine structure measurements, showing a large redistribution of cations between sites compared to normal zinc ferrite samples. The overpopulation of cations in the octahedral sites was attributed to the ascendance in importance of the ionic radii over the crystal energy and bonding coordination in determining which interstitial sites are occupied in this structurally disordered powder. Slight changes are observed in the local atomic environment about the zinc cations, but not the iron cations, with respect to the spinel structure. The presence of Fe{sup 3+} on both sites is consistent with the measured room temperature magnetic properties. (c) 2000 American Institute of Physics.

  2. Anti-nuclear activities and critics concerning nuclear power plant sites

    International Nuclear Information System (INIS)

    Rhee, We-Beg

    2000-01-01

    Korea has dynamic nuclear power expansion programs, operating 16 nuclear units producing 13710 MW in total located on 4 different sites. Last year, nuclear power supplied over 40 % of national total electricity demands. In 1998, Korean government initiated re-designation work investigating circumstance changes to rule out the unnecessary sites in consideration of a long-term power supply. Korean government has determined to expand the Ulchin site and to designate one point of Woolju county as a new candidate site, and ruled out the rest candidate sites at the end of 1998. About such a governmental measure, the two areas show different reactions. Ulchin where nuclear power plant has been operated safely for about 10 years was likely to accept the governmental determination in spite of some opposition and called for several financial supports for local development. WooIju county, however, showed a strong opposition among local environmental groups and autonomous politicians, and they presented a variety of anti-nuclear activities including demonstrations mainly at the neighbouring metropolis, Ulsan city

  3. Site I Inactivation Impacts Calmodulin Calcium Binding and Activation of Bordetella pertussis Adenylate Cyclase Toxin.

    Science.gov (United States)

    Johns, Christian W; Finley, Natosha L

    2017-11-30

    Site I inactivation of calmodulin (CaM) was used to examine the importance of aspartic acid 22 at position 3 in CaM calcium binding, protein folding, and activation of the Bordetella pertussis adenylate cyclase toxin domain (CyaA-ACD). NMR calcium titration experiments showed that site I in the CaM mutant (D22A) remained largely unperturbed, while sites II, III, and IV exhibited calcium-induced conformational changes similar to wild-type CaM (CaMWt). Circular dichroism analyses revealed that D22A had comparable α -helical content to CaMWt, and only modest differences in α -helical composition were detected between CaMWt-CyaA-ACD and D22A-CyaA-ACD complexes. However, the thermal stability of the D22A-CyaA-ACD complex was reduced, as compared to the CaMWt-CyaA-ACD complex. Moreover, CaM-dependent activity of CyaA-ACD decreased 87% in the presence of D22A. Taken together, our findings provide evidence that D22A engages CyaA-ACD, likely through C -terminal mediated binding, and that site I inactivation exerts functional effects through the modification of stabilizing interactions that occur between N -terminal CaM and CyaA-ACD.

  4. Den site activity patterns of adult male and female swift foxes, Vulpes velox, in Northwestern Texas

    Science.gov (United States)

    Lemons, P.R.; Ballard, W.B.; Sullivan, R.M.; Sovada, M.A.

    2003-01-01

    Activity of Swift Foxes (Vulpes velox) at den sites was studied in northwestern Texas during pup rearing seasons in 2000 and 2001 to determine role of males in parental care. Twenty-four percent of radio-collared females with a potential to breed successfully raised pups to eight weeks of age. We intensively monitored presence and absence of male and female Swift Foxes at two den sites each year. Females were present >2.6 times more at den sites than males during the pup rearing season. Female and male Swift Foxes largely stayed at dens during diurnal hours and were active away from dens during nocturnal and crepuscular hours. Females and males spent 12.4% and 3.0% more time at dens before pups emerged, than after pups emerged, respectively. Following depredation of one male parent, the female spent 29% less time at the den site. Decrease in time spent at the den by the female following loss of her mate suggested that loss of one parent might severely impact recruitment of Swift Foxes. Our observations indicated that intense Coyote (Canis latrans) depredation may severely impact pup-rearing success as well as the parental care within Swift Fox family groups.

  5. Assessment of Tritium Activity in Groundwater at the Nuclear Objects Sites in Lithuania

    Directory of Open Access Journals (Sweden)

    Vigilija Cidzikienė

    2014-01-01

    Full Text Available The assessment of nuclear objects sites in Lithuania, including groundwater characterization, took place in the last few years. Tritium activity in groundwater is a very useful tool for determining how groundwater systems function. Natural and artificial tritium was measured in 8 wells in different layers (from 1.5 to 15 meters depth. The results were compared with other regions of Lithuania also. The evaluated tritium activities varied from 1.8 to 6.4 Bq/L at nuclear objects sites in Lithuania and they are coming to background level (1.83 Bq/L and other places in Lithuania. The data show, that groundwater at the nuclear power objects sites is not contaminated with artificial tritium. In this work, the vertical tritium transfer from soil water to the groundwater well at nuclear objects site was estimated. The data show that the main factor for vertical tritium transfer to the well depends on the depth of wells.

  6. Active-site titration analysis of surface influence on immobilized Candida antarctica Lipase B activity

    Science.gov (United States)

    Matrix morphology and surface polarity effects were investigated for Candida antarctica lipase B immobilization. Measurements of the amount of lipase immobilized (bicinchoninic acid method) and the catalyst’s tributyrin hydrolysis activity, coupled with a determination of the lipase’s functional fr...

  7. Discovery of Active Hydrothermal Sites Along the Mariana Volcanic Arc, Western Pacific Ocean

    Science.gov (United States)

    Baker, E. T.; Embley, R. W.; Resing, J. A.; Lupton, J. E.; Massoth, G. J.; de Ronde, C. E.; Nakamura, K.; Walker, S. L.

    2003-12-01

    Some 20,000 km of volcanic arcs, roughly one-third the total length of the global midocean ridge (MOR) system, rim the western Pacific Ocean. But compared to 25 years of hydrothermal investigations along MORs, exploration of similar activity on the estimated 600 submarine arc volcanoes is only beginning. In February 2003, as part of the Submarine Ring of Fire project funded by NOAA's Ocean Exploration Program, we made the first systematic survey of hydrothermal activity along the 1270-km-long Mariana intraoceanic volcanic arc, which lies almost entirely within the US EEZ. Prior fieldwork had documented active (but low-temperature) hydrothermal discharge on only three volcanoes: Kasuga 2, Kasuga 3, and Esmeralda Bank. During the cruise, we conducted 70 CTD operations over more than 50 individual volcanoes from 13° N to 23° N, plus a continuous CTD survey along 75 km of the back-arc spreading center (13° 15'N to 13° 41'N) adjacent to the southern end of the arc. We found evidence for active hydrothermal venting at 11 submarine volcanoes with summit (or caldera floor) depths ranging from 50 to 1550 m. Two additional sites were identified on the back-arc spreading center. Ongoing analyses of collected water samples could increase these totals. Our results confirmed continuing hydrothermal activity at Kasuga 2 (but not Kasuga 3) and Esmeralda Bank, in addition to newly discovered sites on nine other volcanoes. Many of these sites produce intense and widely dispersed plumes indicative of vigorous, high-temperature discharge. The volcanoes with active hydrothermal systems are about equally divided between those with and without summit calderas. The addition of the Marianas data greatly improves our view of hydrothermal sources along arcs. The 20,000 km of Pacific arcs can be divided between 6380 km of intraoceanic (i.e., mostly submarine) arcs and 13,880 km of island (i.e., mostly subaerial) arcs. At present, ˜15% of the total length of Pacific arcs has been surveyed

  8. Metal ion site engineering indicates a global toggle switch model for seven-transmembrane receptor activation

    DEFF Research Database (Denmark)

    Elling, Christian E; Frimurer, Thomas M; Gerlach, Lars-Ole

    2006-01-01

    Much evidence indicates that, during activation of seven-transmembrane (7TM) receptors, the intracellular segments of the transmembrane helices (TMs) move apart with large amplitude, rigid body movements of especially TM-VI and TM-VII. In this study, AspIII:08 (Asp113), the anchor point...... in sites constructed between positions III:08 (Asp or His), VI:16 (preferentially Cys), and/or VII:06 (preferentially Cys). In molecular models built over the backbone conformation of the inactive rhodopsin structure, the heavy atoms that coordinate the metal ion were located too far away from each other...... ion sites, we propose a global toggle switch mechanism for 7TM receptor activation in which inward movement of the extracellular segments of especially TM-VI and, to some extent, TM-VII is coupled to the well established outward movement of the intracellular segments of these helices. We suggest...

  9. Use of the Multi-Agency Radiological Laboratory Analytical Protocols Manual (MARLAP) for site cleanup activities

    International Nuclear Information System (INIS)

    Griggs, J.

    1999-01-01

    MARLAP is being developed as a multi-agency guidance manual for project managers and radioanalytical laboratories. The document uses a performance based approach and will provide guidance and a framework to assure that laboratory radioanalytical data meets the specific project or program needs and requirements. MARLAP supports a wide range of data collection activities including site characterization and compliance demonstration activities. Current participants include: US Environmental Protection Agency (EPA), US Department of Energy (DOE), US Nuclear Regulatory Commission (NRC), US Department of Defense (DoD), US National Institutes of Standards and Technology (NIST), US Geologic Survey (USGS), US Food and Drug Administration (FDA), Commonwealth of Kentucky, and the State of California. MARLAP is the radioanalytical laboratory counterpart to the Multi-Agency Radiological Survey and Site Investigation Manual (MARSSIM). MARLAP is currently in a preliminary draft stage. (author)

  10. Isolation and Biochemical Characterization of a New Thrombin-Like Serine Protease from Bothrops pirajai Snake Venom

    Directory of Open Access Journals (Sweden)

    Kayena D. Zaqueo

    2014-01-01

    Full Text Available This paper presents a novel serine protease (SP isolated from Bothrops pirajai, a venomous snake found solely in Brazil that belongs to the Viperidae family. The identified SP, named BpirSP-39, was isolated by three chromatographic steps (size exclusion, bioaffinity, and reverse phase chromatographies. The molecular mass of BpirSP-39 was estimated by SDS-PAGE and confirmed by mass spectrometry (39,408.32 Da. The protein was able to form fibrin networks, which was not observed in the presence of serine protease inhibitors, such as phenylmethylsulfonyl fluoride (PMSF. Furthermore, BpirSP-39 presented considerable thermal stability and was apparently able to activate factor XIII of the blood coagulation cascade, unlike most serine proteases. BpirSP-39 was capable of hydrolyzing different chromogenic substrates tested (S-2222, S-2302, and S-2238 while Cu2+ significantly diminished BspirSP-39 activity on the three tested substrates. The enzyme promoted platelet aggregation and also exhibited fibrinogenolytic, fibrinolytic, gelatinolytic, and amidolytic activities. The multiple alignment showed high sequence similarity to other thrombin-like enzymes from snake venoms. These results allow us to conclude that a new SP was isolated from Bothrops pirajai snake venom.

  11. Effects of Army Training Activities on Bird Communities at the Pinon Canyon Maneuver Site, Colorado

    Science.gov (United States)

    1991-09-01

    the course of the study. They are brood parasites that lay their eggs in the nests of other species, sometimes substantially reducing the nesting...of Biological Sciences, Northern Arizona University, 1979). 42. May, R.M., and SK. Robinson, "Population Dynamics of Brood Parasitism ," American...Effects of Army Training Activities on Bird Communities at the Piflon Canyon Maneuver Site, Colorado by David J. Tazik This report describes a study

  12. Disposal Activities and the Unique Waste Streams at the Nevada National Security Site (NNSS)

    Energy Technology Data Exchange (ETDEWEB)

    Arnold, P.

    2012-10-31

    This slide show documents waste disposal at the Nevada National Security Site. Topics covered include: radionuclide requirements for waste disposal; approved performance assessment (PA) for depleted uranium disposal; requirements; program approval; the Waste Acceptance Review Panel (WARP); description of the Radioactive Waste Acceptance Program (RWAP); facility evaluation; recent program accomplishments, nuclear facility safety changes; higher-activity waste stream disposal; and, large volume bulk waste streams.

  13. Identification of the provenience of Majolica from sites in the Caribbean using neutron activation analysis

    Energy Technology Data Exchange (ETDEWEB)

    Olin, J.S.; Sayre, E.V.

    1975-01-01

    Tin-enamelled earthenware pottery from five early Spanish Colonial sites in the Dominican Republic and Venezuela were sampled and analyzed by neutron activation analysis in an attempt to determine whether these sherds had a common source. The tentative conclusion was that although several sources were indicated for the specimens analyzed the overall similarity in composition indicated that these sources were probably closely related. (JSR)

  14. Interaction of mining activities and aquatic environment: A review from Greek mine sites.

    Science.gov (United States)

    Vasileiou, Eleni; Kallioras, Andreas

    2016-04-01

    In Greece a significant amount of mineral and ore deposits have been recorded accompanied by large industrial interest and a long mining history. Today many active and/or abandoned mine sites are scattered within the country; while mining activities take place in different sites for exploiting various deposits (clay, limestone, slate, gypsum, kaolin, mixed sulphide ores (lead, zinc, olivine, pozzolan, quartz lignite, nickel, magnesite, aluminum, bauxite, gold, marbles etc). The most prominent recent ones are: (i) the lignite exploitation that is extended in the area of Ptolemais (Western Macedonia) and Megalopolis (Central Peloponnese); and (ii) the major bauxite deposits located in central Greece within the Parnassos-Ghiona geotectonic zone and on Euboea Island. In the latter area, significant ores of magnesite were exploited and mixed sulphide ores. Centuries of intensive mining exploitation and metallurgical treatment of lead-silver deposits in Greece, have also resulted in significant abandoned sites, such as the one in Lavrion. Mining activities in Lavrio, were initiated in ancient times and continued until the 1980s, resulting in the production of significant waste stockpiles deposited in the area, crucial for the local water resources. Ιn many mining sites, environmental pressures are also recorded after the mine closure to the aquatic environment, as the surface waters flow through waste dump areas and contaminated soils. This paper aims to the geospatial visualization of the mining activities in Greece, in connection to their negative (surface- and/or ground-water pollution; overpumping due to extensive dewatering practices) or positive (enhanced groundwater recharge; pit lakes, improvement of water budget in the catchment scale) impacts on local water resources.

  15. Atomistic characterization of the active-site solvation dynamics of a model photocatalyst

    DEFF Research Database (Denmark)

    Brandt van Driel, Tim; Kjær, Kasper Skov; Hartsock, Robert W.

    2016-01-01

    and simulated with Born-Oppenheimer Molecular Dynamics. Both methods provide direct access to the solute-solvent pair distribution function, enabling the solvation dynamics around the catalytically active iridium sites to be robustly characterized. Our results provide evidence for the coordination...... of the iridium atoms by the acetonitrile solvent and demonstrate the viability of using diffuse X-ray scattering at free-electron laser sources for studying the dynamics of photocatalysis....

  16. Crystal Structure of Albaflavenone Monooxygenase Containing a Moonlighting Terpene Synthase Active Site

    Energy Technology Data Exchange (ETDEWEB)

    Zhao, Bin; Lei, Li; Vassylyev, Dmitry G.; Lin, Xin; Cane, David E.; Kelly, Steven L.; Yuan, Hang; Lamb, David C.; Waterman, Michael R.; (Vanderbilt); (UAB); (Brown); (Swansea)

    2010-01-08

    Albaflavenone synthase (CYP170A1) is a monooxygenase catalyzing the final two steps in the biosynthesis of this antibiotic in the soil bacterium, Streptomyces coelicolor A3(2). Interestingly, CYP170A1 shows no stereo selection forming equal amounts of two albaflavenol epimers, each of which is oxidized in turn to albaflavenone. To explore the structural basis of the reaction mechanism, we have studied the crystal structures of both ligand-free CYP170A1 (2.6 {angstrom}) and complex of endogenous substrate (epi-isozizaene) with CYP170A1 (3.3 {angstrom}). The structure of the complex suggests that the proximal epi-isozizaene molecules may bind to the heme iron in two orientations. In addition, much to our surprise, we have found that albaflavenone synthase also has a second, completely distinct catalytic activity corresponding to the synthesis of farnesene isomers from farnesyl diphosphate. Within the cytochrome P450 {alpha}-helical domain both the primary sequence and x-ray structure indicate the presence of a novel terpene synthase active site that is moonlighting on the P450 structure. This includes signature sequences for divalent cation binding and an {alpha}-helical barrel. This barrel is unusual because it consists of only four helices rather than six found in all other terpene synthases. Mutagenesis establishes that this barrel is essential for the terpene synthase activity of CYP170A1 but not for the monooxygenase activity. This is the first bifunctional P450 discovered to have another active site moonlighting on it and the first time a terpene synthase active site is found moonlighting on another protein.

  17. Discovery and Characterization of Non-ATP Site Inhibitors of the Mitogen Activated Protein (MAP) Kinases

    Energy Technology Data Exchange (ETDEWEB)

    Comess, Kenneth M.; Sun, Chaohong; Abad-Zapatero, Cele; Goedken, Eric R.; Gum, Rebecca J.; Borhani, David W.; Argiriadi, Maria; Groebe, Duncan R.; Jia, Yong; Clampit, Jill E.; Haasch, Deanna L.; Smith, Harriet T.; Wang, Sanyi; Song, Danying; Coen, Michael L.; Cloutier, Timothy E.; Tang, Hua; Cheng, Xueheng; Quinn, Christopher; Liu, Bo; Xin, Zhili; Liu, Gang; Fry, Elizabeth H.; Stoll, Vincent; Ng, Teresa I.; Banach, David; Marcotte, Doug; Burns, David J.; Calderwood, David J.; Hajduk, Philip J. (Abbott)

    2012-03-02

    Inhibition of protein kinases has validated therapeutic utility for cancer, with at least seven kinase inhibitor drugs on the market. Protein kinase inhibition also has significant potential for a variety of other diseases, including diabetes, pain, cognition, and chronic inflammatory and immunologic diseases. However, as the vast majority of current approaches to kinase inhibition target the highly conserved ATP-binding site, the use of kinase inhibitors in treating nononcology diseases may require great selectivity for the target kinase. As protein kinases are signal transducers that are involved in binding to a variety of other proteins, targeting alternative, less conserved sites on the protein may provide an avenue for greater selectivity. Here we report an affinity-based, high-throughput screening technique that allows nonbiased interrogation of small molecule libraries for binding to all exposed sites on a protein surface. This approach was used to screen both the c-Jun N-terminal protein kinase Jnk-1 (involved in insulin signaling) and p38{alpha} (involved in the formation of TNF{alpha} and other cytokines). In addition to canonical ATP-site ligands, compounds were identified that bind to novel allosteric sites. The nature, biological relevance, and mode of binding of these ligands were extensively characterized using two-dimensional {sup 1}H/{sup 13}C NMR spectroscopy, protein X-ray crystallography, surface plasmon resonance, and direct enzymatic activity and activation cascade assays. Jnk-1 and p38{alpha} both belong to the MAP kinase family, and the allosteric ligands for both targets bind similarly on a ledge of the protein surface exposed by the MAP insertion present in the CMGC family of protein kinases and distant from the active site. Medicinal chemistry studies resulted in an improved Jnk-1 ligand able to increase adiponectin secretion in human adipocytes and increase insulin-induced protein kinase PKB phosphorylation in human hepatocytes, in

  18. Active site structure and catalytic mechanism of phosphodiesterase for degradation of intracellular second messengers

    Science.gov (United States)

    Zhan, Chang-Guo

    2002-03-01

    Phosphodiesterases are clinical targets for a variety of biological disorders, because this superfamily of enzymes regulate intracellular concentration of cyclic nucleotides that serve as the second messengers playing a critical role in a variety of physiological processes. Understanding structure and mechanism of a phosphodiesterase will provide a solid basis for rational design of the more efficient therapeutics. Although a three-dimensional X-ray crystal structure of the catalytic domain of human phosphodiesterase 4B2B was recently reported, it was uncertain whether a critical bridging ligand in the active site is a water molecule or a hydroxide ion. The identity of this bridging ligand has been determined by performing first-principles quantum chemical calculations on models of the active site. All the results obtained indicate that this critical bridging ligand in the active site of the reported X-ray crystal structure is a hydroxide ion, rather than a water molecule, expected to serve as the nucleophile to initialize the catalytic degradation of the intracellular second messengers.

  19. Days of dismantling activities of installations and rehabilitation of contaminated sites in France

    International Nuclear Information System (INIS)

    2008-01-01

    The objective of these days, organized by the section environment of the French society of radiation protection, is to present a panorama of the activities of nuclear installations dismantling and contaminated sites rehabilitation in France, by leaning in the same time on practical cases and by stating the French rule and the national and international recommendations on the subject. These days have also for object to approach the stakes associated with the sectors of waste management and the materials generated by these activities and in a more general way, the stakes to come for the different actors of the dismantling and the rehabilitation. (N.C.)

  20. Regulating retrotransposon activity through the use of alternative transcription start sites

    DEFF Research Database (Denmark)

    Persson, Jenna; Steglich, Babett; Smialowska, Agata

    2016-01-01

    . This enforces the use of a downstream TSS and the production of a truncated RNA incapable of reverse transcription and retrotransposition. However, in stressed cells, nucleosome occupancy at LTR elements is reduced, and the TSS shifts to allow for productive transcription. We propose that controlled...... a new mechanism of retrotransposon regulation through transcription start site (TSS) selection by altered nucleosome occupancy. We show that Fun30 chromatin remodelers cooperate to maintain a high level of nucleosome occupancy at retrotransposon-flanking long terminal repeat (LTR) elements...... retrotransposon transcription from a nonproductive TSS allows for rapid stress-induced activation, while preventing uncontrolled transposon activity in the genome....

  1. Altered binding of thioflavin t to the peripheral anionic site of acetylcholinesterase after phosphorylation of the active site by chlorpyrifos oxon or dichlorvos

    International Nuclear Information System (INIS)

    Sultatos, L.G.; Kaushik, R.

    2008-01-01

    The peripheral anionic site of acetylcholinesterase, when occupied by a ligand, is known to modulate reaction rates at the active site of this important enzyme. The current report utilized the peripheral anionic site specific fluorogenic probe thioflavin t to determine if the organophosphates chlorpyrifos oxon and dichlorvos bind to the peripheral anionic site of human recombinant acetylcholinesterase, since certain organophosphates display concentration-dependent kinetics when inhibiting this enzyme. Incubation of 3 nM acetylcholinesterase active sites with 50 nM or 2000 nM inhibitor altered both the B max and K d for thioflavin t binding to the peripheral anionic site. However, these changes resulted from phosphorylation of Ser203 since increasing either inhibitor from 50 nM to 2000 nM did not alter further thioflavin t binding kinetics. Moreover, the organophosphate-induced decrease in B max did not represent an actual reduction in binding sites, but instead likely resulted from conformational interactions between the acylation and peripheral anionic sites that led to a decrease in the rigidity of bound thioflavin t. A drop in fluorescence quantum yield, leading to an apparent decrease in B max , would accompany the decreased rigidity of bound thioflavin t molecules. The organophosphate-induced alterations in K d represented changes in binding affinity of thioflavin t, with diethylphosphorylation of Ser203 increasing K d , and dimethylphosphorylation of Ser203 decreasing K d . These results indicate that chlorpyrifos oxon and dichlorvos do not bind directly to the peripheral anionic site of acetylcholinesterase, but can affect binding to that site through phosphorylation of Ser203

  2. Serine protease inhibitor attenuates ovalbumin induced inflammation in mouse model of allergic airway disease.

    Directory of Open Access Journals (Sweden)

    Sanjay Saw

    Full Text Available BACKGROUND: Serine proteases promote inflammation and tissue remodeling by activating proteinase-activated receptors, urokinase, metalloproteinases and angiotensin. In the present study, 4-(2-Aminoethyl benzenesulfonyl fluoride (AEBSF a serine protease inhibitor was evaluated for prophylactic and therapeutic treatment in mouse model of airway allergy. METHODS: BALB/c mice were sensitized by i.p route and challenged with ovalbumin. They were treated i.n. with 2, 10 and 50 µg of AEBSF, one hour before or after challenge and euthanized to collect BALF (bronchoalveolar lavage fluid, blood and lungs. Proteolytic activity, total cell/eosinophil/neutrophil count eosinophil peroxidase activity (EPO, IL-4, IL-5, IL-10, IL-13, cysteinyl leukotrienes and 8-isoprostane were determined in BALF and immunoglobulins were measured in serum. H&E and PAS stained lung sections were examined for cellular infiltration and airway inflammation. RESULTS: Mice exposed to ovalbumin and treated with PBS showed increased cellular infiltration in lungs and higher serum IgE, IgG1 and IgG2a levels as compared to sham mice. Treatment with AEBSF reduced total cells/eosinophil/neutrophil infiltration. Both prophylactic and therapeutic AEBSF treatment of 10 or 50 µg reduced serum IgE and IgG1 significantly (p<0.05 than control. AEBSF treatment reduced the proteolytic activity in BALF. IL-4 IL-5 and IL-13 levels decreased significantly (p<0.05 after AEBSF treatment while IL-10 levels increased significantly (p<0.05 in BALF. Airway inflammation and goblet cell hyperplasia reduced as demonstrated by lung histopathology, EPO activity and cysteinyl leukotrienes in BALF after treatment. AEBSF treatment also suppressed oxidative stress in terms of 8-isoprostane in BALF. Among the treatment doses, 10 or 50 µg of AEBSF were most effective in reducing the inflammatory parameters. CONCLUSIONS: Prophylactic and therapeutic treatment with serine protease inhibitor attenuates the airway

  3. The growth hormone dependent serine protease inhibitor, Spi 2.1 inhibits the des (1-3) insulin-like growth factor-I generating protease.

    Science.gov (United States)

    Maake, C; Yamamoto, H; Murphy, L J

    1997-12-01

    The conversion of insulin-like growth factor-I (IGF-I) to the biologically more active des (1-3) IGF-I variant is catalyzed by a ubiquitous protease. This proteolytic activity is inhibited by human alpha1-antitrypsin and soy-bean trypsin inhibitor and is up-regulated in serum and tissue extracts of hypophysectomized rats. These observations lead us to investigate whether the growth hormone regulated, serine protease inhibitor, Spi 2.1 was able to inhibit the des (1-3) IGF-I generating protease. Dihydrofolate reductase deficient Chinese hamster ovary (CHO(dhfr-ve)) cells were transfected with a rat Spi 2.1 expression vector containing the dhfr and neomycin resistance gene. Stable transfectants were selected using G418 and amplified using methotrexate. Conditioned medium from Spi 2.1 transfected CHO cells potently inhibited proteolytic activity directed against a synthetic hexa-peptide with a sequence identical to the N-terminal of IGF-I. In contrast conditioned medium from wild-type CHO cells had little effect. Based upon these observations we suggest that our previous finding of enhanced des (1-3) IGF-I generating protease activity in growth hormone deficient rats may be, at least partly explained by reduced levels of Spi 2.1. Furthermore, we propose that the regulation of the generation of des (1-3) IGF-I may be an additional potential site of growth hormone regulation of IGF-I action.

  4. Orthogonal electrode catheter array for mapping of endocardial focal site of ventricular activation

    Energy Technology Data Exchange (ETDEWEB)

    Desai, J.M.; Nyo, H.; Vera, Z.; Seibert, J.A.; Vogelsang, P.J. (Division of Cardiovascular Medicine, University of California, School of Medicine, Davis (USA))

    1991-04-01

    Precise location of the endocardial site of origin of ventricular tachycardia may facilitate surgical and catheter ablation of this arrhythmia. The endocardial catheter mapping technique can locate the site of ventricular tachycardia within 4-8 cm2 of the earliest site recorded by the catheter. This report describes an orthogonal electrode catheter array (OECA) for mapping and radiofrequency ablation (RFA) of endocardial focal site of origin of a plunge electrode paced model of ventricular activation in dogs. The OECA is an 8 F five pole catheter with four peripheral electrodes and one central electrode (total surface area 0.8 cm{sup 2}). In eight mongrel dogs, mapping was performed by arbitrarily dividing the left ventricle (LV) into four segments. Each segment was mapped with OECA to find the earliest segment. Bipolar and unipolar electrograms were obtained. The plunge electrode (not visible on fluoroscopy) site was identified by the earliest wave front arrival times of -30 msec or earlier at two or more electrodes (unipolar electrograms) with reference to the earliest recorded surface ECG (I, AVF, and V1). Validation of the proximity of the five electrodes of the OECA to the plunge electrode was performed by digital radiography and RFA. Pathological examination was performed to document the proximity of the OECA to the plunge electrode and also for the width, depth, and microscopic changes of the ablation. To find the segment with the earliest LV activation a total of 10 {plus minus} 3 (mean {plus minus} SD) positions were mapped. Mean arrival times at the two earlier electrodes were -39 {plus minus} 4 msec and -35 {plus minus} 3 msec. Digital radiography showed the plunge electrode to be within the area covered by all five electrodes in all eight dogs. The plunge electrode was within 1 cm2 area of the region of RFA in all eight dogs.

  5. Spatial patterns of microbial diversity and activity in an aged creosote-contaminated site

    Science.gov (United States)

    Mukherjee, Shinjini; Juottonen, Heli; Siivonen, Pauli; Lloret Quesada, Cosme; Tuomi, Pirjo; Pulkkinen, Pertti; Yrjälä, Kim

    2014-01-01

    Restoration of polluted sites via in situ bioremediation relies heavily on the indigenous microbes and their activities. Spatial heterogeneity of microbial populations, contaminants and soil chemical parameters on such sites is a major hurdle in optimizing and implementing an appropriate bioremediation regime. We perf