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Sample records for acidothermus cellulolyticus e1

  1. Crystal structure of thermostable family 5 endocellulase E1 from Acidothermus cellulolyticus in complex with cellotetraose.

    PubMed

    Sakon, J; Adney, W S; Himmel, M E; Thomas, S R; Karplus, P A

    1996-08-20

    The crystal structure of the catalytic domain of the thermostable endocellulase E1 from Acidothermus cellulolyticus in complex with cellotetraose has been solved by multiple isomorphous replacement and refined at 2.4 A resolution to an R-factor of 0.18 (Rfree = 0.24). E1cd is a member of the 4/7 superfamily of hydrolases, and as expected, its structure is an (alpha/beta)8 barrel, which constitutes a prototype for family 5-subfamily 1 cellulases. The cellotetraose molecule binds in a manner consistent with the expected Michaelis complex for the glycosylation half-reaction and reveals that all eight residues conserved in family 5 enzymes are involved in recognition of the glycosyl group attacked during cleavage. Whereas only three residues are conserved in the whole 4/7 superfamily (the Asn/Glu duo and the Glu from which the name is derived), structural comparisons show that all eight residues conserved in family 5 have functional equivalents in the other 4/7 superfamily members, strengthening the case that mechanistic details are conserved throughout the superfamily. On the basis of the structure, a detailed sequence of physical steps of the cleavage mechanism is proposed. A close approach of two key glutamate residues provides an elegant mechanism for the shift in the pKa of the acid/base for the glycosylation and deglycosylation half-reactions. Finally, purely structural based comparisons are used to show that significant differences exist in structural similarity scores resulting from different methods and suggest that caution should be exercised in interpreting such results in terms of implied evolutional relationships. PMID:8718854

  2. Quantitation of Acidothermus cellulolyticus E1 endoglucanase and Thermomonospora fusca E{sub 3} exoglucanase using enzyme-linked immunosorbent assay (ELISA)

    SciTech Connect

    Nieves, R.A.; Chou, Y.C.; Himmel, M.E.

    1995-12-31

    Two distinct quantitative indirect ELISAs were developed to determine the concentration of recombinant cellulose enzymes in culture filtrates. A monoclonal antibody (E1P7) was used as the primary antibody in developing an ELISA specific for Acidothermus cellulolyticus E1 endoglucanase. Likewise, a polyclonal rabbit serum (Ab684) was used to develop an ELISA specific for Thermomonospora fusca E{sub 3} exoglucanase. Dose-response curves indicated a dynamic range for both assays between 0.01 and 0.08 {mu}g/mL (1-8 ng/assay) when purified enzymes were used as standards. These assays have been used to estimate concentrations of secreted recombinant E1 and/or E{sub 3} in culture supernatants of Streptomyces lividans strain TK24 in which the corresponding genes have been cloned and expressed.

  3. Thermal tolerant avicelase from Acidothermus cellulolyticus

    DOEpatents

    Ding, Shi-You; Adney, William S.; Vinzant, Todd B.; Himmel, Michael E.

    2009-05-26

    The invention provides a thermal tolerant (thermostable) cellulase, AviIII, that is a member of the glycoside hydrolase (GH) family. AviIII was isolated and characterized from Acidothermus cellulolyticus and, like many cellulases, the disclosed polypeptide and/or its derivatives may be useful for the conversion of biomass into biofuels and chemicals.

  4. Thermal tolerant avicelase from Acidothermus cellulolyticus

    DOEpatents

    Ding, Shi-You; Adney, William S.; Vinzant, Todd B.; Himmel, Michael E.

    2008-04-29

    The invention provides a thermal tolerant (thermostable) cellulase, AviIII, that is a member of the glycoside hydrolase (GH) family. AviIII was isolated and characterized from Acidothermus cellulolyticus and, like many cellulases, the disclosed polypeptide and/or its derivatives may be useful for the conversion of biomass into biofuels and chemicals.

  5. Thermal tolerant exoglucanase from Acidothermus cellulolyticus

    DOEpatents

    Adney, William S.; Ding, Shi-You; Vinzant, Todd B.; Himmel, Michael E.; Decker, Stephen R.; McCarter, Suzanne Lantz

    2008-07-01

    The invention provides a thermal tolerant cellulase that is a member of the glycoside hydrolase family. The invention further discloses this cellulase as Gux1. Gux1 has been isolated and characterized from Acidothermus cellulolyticus. The invention further provides recombinant forms of the identified Gux1. Methods of making and using Gux1 polypeptides, including fusions, variants, and derivatives, are also disclosed.

  6. Thermal tolerant cellulase from Acidothermus cellulolyticus

    DOEpatents

    Ding, Shi-You; Adney, William S.; Vinzant, Todd B.; Himmel, Michael E.; Decker, Stephen R.

    2006-06-13

    The invention provides a thermal tolerant cellulase that is a member of the glycoside hydrolase family. The invention further discloses this cellulase as GuxA. GuxA has been isolated and characterized from Acidothermus cellulolyticus. The invention further provides recombinant forms of the identified GuxA. Methods of making and using GuxA polypeptides, including fusions, variants, and derivatives, are also disclosed.

  7. Thermal Tolerant Cellulase from Acidothermus Cellulolyticus

    DOEpatents

    Ding, S. Y.; Adney, W. S.; Vinzant, T. B.; Himmel, M. E.; Decker, S. R.

    2006-06-13

    The invention provides a thermal tolerant cellulase that is a member of the glycoside hydrolase family. The invention further discloses this cellulase as GuxA. GuxA has been isolated and characterized from Acidothermus cellulolyticus. The invention further provides recombinant forms of the identified GuxA. Methods of making and using GuxA polypeptides, including fusions, variants, and derivatives, are also disclosed.

  8. Thermal tolerant mannanase from acidothermus cellulolyticus

    DOEpatents

    Ding, Shi-You; Adney, William S.; Vinzant, Todd B.; Himmel, Michael E.

    2006-09-26

    The invention provides a thermal tolerant mannanase that is a member of the glycoside hydrolase family. The invention further discloses this mannanase as ManA. ManA has been isolated and characterized from Acidothermus cellulolyticus. The invention further provides recombinant forms of the identified ManA. Methods of making ManA polypeptides, including fusions, variants, and derivatives, are also disclosed. Methods of using mannanase A, including for the processing of food and for use in food stuffs as bulking agents and the like, are also disclosed.

  9. Optimization of Acidothermus cellulolyticus endoglucanase (E1) production in transgenic tobacco plants by transcriptional, post-transcription and post-translational modification.

    PubMed

    Dai, Ziyu; Hooker, Brian S; Quesenberry, Ryan D; Thomas, Steven R

    2005-10-01

    An attempt was made to obtain a high-level production of intact Acidothermus cellulolyticus endoglucanase (E1) in transgenic tobacco plants. The E1 expression was examined under the control of the constitutive and strong Mac promoter or light-inducible tomato Rubisco small sub-unit (RbcS-3C) promoter with its original or Alfalfa Mosaic Virus (AMV) RNA4 5'-untranslated leader (UTL) and targeted to different sub-cellular compartments via transit peptides. The transit peptides included native E1, endoplasmic reticulum, vacuole, apoplast, and chloroplast. E1 expression and its stability in transgenic plants were determined via E1 activity, protein immunoblotting, and RNA gel-blotting analyses. Effects of sub-cellular compartments on E1 production and its stability were determined in transgenic tobacco plants carrying one of six transgene expression vectors, where the E1 was under the control of Mac promoter, mannopine synthase transcription terminator, and one of the five transit peptides. Transgenic tobacco plants with an apoplastic transit peptide had the highest average E1 activity and protein accumulation, which was about 0.25% of total leaf soluble proteins estimated via E1 specific activity and protein gel blots. Intercellular fluid analyses confirmed that E1 signal peptide functioned properly in tobacco cells to secret E1 protein into the apoplast. By replacing RbcS-3C UTL with AMV RNA4 UTL E1 production was enhanced more than twofold, while it was less effective than the mannopine synthase UTL. It was observed that RbcS-3C promoter was more favorable for E1 expression in transgenic plants than the Mac promoter. E1 activity in dried tobacco seeds stored one year at room temperature was 45% higher than that observed immediately after harvesting, suggesting that E1 protein can be stored at room temperature for a long period. E1 stability in different sub-cellular compartments and the optimal combination of promoter, 5'-UTL, and sub-cellular compartmentation for

  10. Methods of using thermal tolerant avicelase from Acidothermus cellulolyticus

    DOEpatents

    Adney, William S.; Vinzant, Todd B.; Ding, Shih-You; Himmel, Michael E.

    2011-04-26

    The invention provides a thermal tolerant (thermostable) cellulase, AviIII, that is a member of the glycoside hydrolase (GH) family. AviIII was isolated and characterized from Acidothermus cellulolyticus, and, like many cellulases, the disclosed polypeptide and/or its derivatives may be useful for the conversion of biomass into biofuels and chemicals.

  11. Expression of the Acidothermus cellulolyticus E1 endoglucanase in Caldicellulosiruptor bescii enhances its ability to deconstruct crystalline cellulose

    DOE PAGESBeta

    Chung, Daehwan; Young, Jenna; Cha, Minseok; Brunecky, Roman; Bomble, Yannick J.; Himmel, Michael E.; Westpheling, Janet

    2015-08-13

    The Caldicellulosiruptor bescii genome encodes a potent set of carbohydrate-active enzymes (CAZymes), found primarily as multi-domain enzymes that exhibit high cellulolytic and hemicellulolytic activity on and allow utilization of a broad range of substrates, including plant biomass without conventional pretreatment. CelA, the most abundant cellulase in the C. bescii secretome, uniquely combines a GH9 endoglucanase and a GH48 exoglucanase in one protein. The most effective commercial enzyme cocktails used in vitro to pretreat biomass are derived from fungal cellulases (cellobiohydrolases, endoglucanases and a β-d-glucosidases) that act synergistically to release sugars for microbial conversion. The C. bescii genome contains six GH5more » domains in five different open reading frames. Four exist in multi-domain proteins and two as single catalytic domains. E1 is a GH5 endoglucanase reported to have high specific activity and simple architecture and is active at the growth temperature of C. bescii. E1 is an endo-1,4-β-glucanase linked to a family 2 carbohydrate-binding module shown to bind primarily to cellulosic substrates. As a result, we tested if the addition of this protein to the C. bescii secretome would improve its cellulolytic activity.« less

  12. Thermostable purified endoglucanase from thermophilic bacterium acidothermus cellulolyticus

    DOEpatents

    Tucker, Melvin P.; Grohmann, Karel; Himmel, Michael E.; Mohagheghi, Ali

    1992-01-01

    A substantially purified high molecular weight cellulase enzyme having a molecular weight of between about 156,000 to about 203,400 daltons isolated from the bacterium Acidothermus cellulolyticus (ATCC 43068) and a method of producing it are disclosed. The enzyme is water soluble, possesses both C.sub.1 and C.sub.x types of enzymatic activity, has a high degree of stability toward heat and exhibits both a high optimum temperature activity and high inactivation characteristics.

  13. Thermostable purified endoglucanase II from Acidothermus cellulolyticus ATCC

    DOEpatents

    Adney, William S.; Thomas, Steven R.; Nieves, Rafael A.; Himmel, Michael E.

    1994-01-01

    A purified low molecular weight endoglucanase II from Acidothermus cellulolyticus (ATCC 43068) is disclosed. The endoglucanase is water soluble, possesses both C.sub.1, and C.sub.x types of enzyme activity, a high degree of stability toward heat, and exhibits optimum temperature activity at about 81.degree. C. at pH's from about 2 to about 9, and at a inactivation temperature of about 100.degree. C. at pH's from about 2 to about 9.

  14. Thermostable purified endoglucanase II from Acidothermus cellulolyticus ATCC

    DOEpatents

    Adney, W.S.; Thomas, S.R.; Nieves, R.A.; Himmel, M.E.

    1994-11-22

    A purified low molecular weight endoglucanase II from Acidothermus cellulolyticus (ATCC 43068) is disclosed. The endoglucanase is water soluble, possesses both C[sub 1], and C[sub x] types of enzyme activity, a high degree of stability toward heat, and exhibits optimum temperature activity at about 81 C at pH's from about 2 to about 9, and at a inactivation temperature of about 100 C at pH's from about 2 to about 9. 9 figs.

  15. Cloning of cellulase genes from Acidothermus cellulolyticus

    DOEpatents

    Lastick, S.M.; Tucker, M.P.; Grohmann, K.

    1996-05-07

    A process is described for moving fragments that code for cellulase activity from the genome of A. cellulolyticus to several plasmid vectors and the subsequent expression of active cellulase activity in E. coli. 5 figs.

  16. Cloning of cellulase genes from acidothermus cellulolyticus

    DOEpatents

    Lastick, deceased, Stanley M.; Tucker, Melvin P.; Grohmann, Karel

    1996-01-01

    A process is described for moving fragments that code for cellulase activity from the genome of A. cellulolyticus to several plasmid vectors and the subsequent expression of active cellulase acitivty in E. coli.

  17. Thermostable purified endoglucanas from acidothermus cellulolyticus ATCC 43068

    DOEpatents

    Himmel, Michael E.; Adney, William S.; Tucker, Melvin P.; Grohmann, Karel

    1994-01-01

    A purified low molecular weight cellulase endoglucanase I having a molecular weight of between about 57,420 to about 74,580 daltons from Acidothermus cellulolyticus (ATCC 43068). The cellulase is water soluble, possesses both C.sub.1 and C.sub.x types of enzyme activity, a high degree of stability toward heat, and exhibits optimum temperature activity at about 83.degree. C. at pH's from about 2 to about 9, and in inactivation temperature of about 110.degree. C. at pH's from about 2 to about 9.

  18. Thermostable purified endoglucanase from Acidothermus cellulolyticus ATCC 43068

    DOEpatents

    Himmel, M.E.; Adney, W.S.; Tucker, M.P.; Grohmann, K.

    1994-01-04

    A purified low molecular weight cellulase endoglucanase I having a molecular weight of between about 57,420 to about 74,580 daltons from Acidothermus cellulolyticus (ATCC 43068) is presented. The cellulase is water soluble, possesses both C[sub 1] and C[sub x] types of enzyme activity, a high degree of stability toward heat, and exhibits optimum temperature activity at about 83 C at pH's from about 2 to about 9, and in inactivation temperature of about 110 C at pH's from about 2 to about 9. 7 figures.

  19. Expression of the Acidothermus cellulolyticus E1 endoglucanase in Caldicellulosiruptor bescii enhances its ability to deconstruct crystalline cellulose

    SciTech Connect

    Chung, Daehwan; Young, Jenna; Cha, Minseok; Brunecky, Roman; Bomble, Yannick J.; Himmel, Michael E.; Westpheling, Janet

    2015-08-13

    The Caldicellulosiruptor bescii genome encodes a potent set of carbohydrate-active enzymes (CAZymes), found primarily as multi-domain enzymes that exhibit high cellulolytic and hemicellulolytic activity on and allow utilization of a broad range of substrates, including plant biomass without conventional pretreatment. CelA, the most abundant cellulase in the C. bescii secretome, uniquely combines a GH9 endoglucanase and a GH48 exoglucanase in one protein. The most effective commercial enzyme cocktails used in vitro to pretreat biomass are derived from fungal cellulases (cellobiohydrolases, endoglucanases and a β-d-glucosidases) that act synergistically to release sugars for microbial conversion. The C. bescii genome contains six GH5 domains in five different open reading frames. Four exist in multi-domain proteins and two as single catalytic domains. E1 is a GH5 endoglucanase reported to have high specific activity and simple architecture and is active at the growth temperature of C. bescii. E1 is an endo-1,4-β-glucanase linked to a family 2 carbohydrate-binding module shown to bind primarily to cellulosic substrates. As a result, we tested if the addition of this protein to the C. bescii secretome would improve its cellulolytic activity.

  20. Structure of Acidothermus cellulolyticus family 74 glycoside hydrolase at 1.82 Å resolution

    PubMed Central

    Alahuhta, Markus; Adney, William S.; Himmel, Michael E.; Lunin, Vladimir V.

    2013-01-01

    Here, a 1.82 Å resolution X-ray structure of a glycoside hydrolase family 74 (GH74) enzyme from Acidothermus cellulolyticus is reported. The resulting structure was refined to an R factor of 0.150 and an R free of 0.196. Structural analysis shows that five related structures have been reported with a secondary-structure similarity of between 75 and 89%. The five similar structures were all either Clostridium thermocellum or Geotrichum sp. M128 GH74 xyloglucanases. Structural analysis indicates that the A. cellulolyticus GH74 enzyme is an endoxyloglucanase, as it lacks a characteristic loop that blocks one end of the active site in exoxyloglucanases. Superimposition with the C. thermocellum GH74 shows that Asp451 and Asp38 are the catalytic residues. PMID:24316824

  1. Low molecular weight thermostable .beta.-D-glucosidase from acidothermus cellulolyticus

    DOEpatents

    Himmel, Michael E.; Tucker, Melvin P.; Adney, William S.; Nieves, Rafael A.

    1995-01-01

    A purified low molecular weight .beta.-D-glucosidase is produced from Acidothermus cellulolyticus ATCC 43068. The enzyme is water soluble, possesses activity against pNP-.beta.-D-glucopyranoside, has a high of degree of stability toward heat, exhibits optimal temperature activity at about 65.degree. C. at a pH range of from about 2 to about 7, has an inactivation temperature of about 80.degree. C. at a pH range of from about 2 to about 7 and has a molecular weight of about 50.5-54.5 kD as determineded by SDS-PAGE.

  2. Low molecular weight thermostable {beta}-D-glucosidase from Acidothermus cellulolyticus

    DOEpatents

    Himmel, M.E.; Tucker, M.P.; Adney, W.S.; Nieves, R.A.

    1995-07-11

    A purified low molecular weight {beta}-D-glucosidase is produced from Acidothermus cellulolyticus ATCC 43068. The enzyme is water soluble, possesses activity against pNP-{beta}-D-glucopyranoside, has a high of degree of stability toward heat, exhibits optimal temperature activity at about 65 C at a pH range of from about 2 to about 7, has an inactivation temperature of about 80 C at a pH range of from about 2 to about 7 and has a molecular weight of about 50.5--54.5 kD as determined by SDS-PAGE. 6 figs.

  3. Expression and Characterization of Acidothermus celluloyticus E1 Endoglucanase in Transgenic Duckweed Lemna minor 8627

    SciTech Connect

    Sun, Y.; Cheng, J. J.; Himmel, M. E.; Skory, C. D.; Adney, W. S.; Thomas, S. R.; Tisserat, B.; Nishimura, Y.; Yamamoto, Y. T.

    2007-01-01

    Endoglucanase E1 from Acidothermus cellulolyticus was expressed cytosolically under control of the cauliflower mosaic virus 35S promoter in transgenic duckweed, Lemna minor 8627 without any obvious observable phenotypic effects on morphology or rate of growth. The recombinant enzyme co-migrated with the purified catalytic domain fraction of the native E1 protein on western blot analysis, revealing that the cellulose-binding domain was cleaved near or in the linker region. The duckweed-expressed enzyme was biologically active and the expression level was up to 0.24% of total soluble protein. The endoglucanase activity with carboxymethylcellulose averaged 0.2 units mg protein{sup -1} extracted from fresh duckweed. The optimal temperature and pH for E1 enzyme activity were about 80 C and pH 5, respectively. While extraction with HEPES (N-[2-hydroxyethyl]piperazine-N{prime}-[2-ethanesulfonic acid]) buffer (pH 8) resulted in the highest recovery of total soluble proteins and E1 enzyme, extraction with citrate buffer (pH 4.8) at 65 C enriched relative amounts of E1 enzyme in the extract. This study demonstrates that duckweed may offer new options for the expression of cellulolytic enzymes in transgenic plants.

  4. Complete genome of the cellyloytic thermophile Acidothermus cellulolyticus 11B provides insights into its ecophysiological and evloutionary adaptations

    SciTech Connect

    Barabote, Ravi D.; Xie, Gary; Leu, David H.; Normand, Philippe; Necsulea, Anamaria; Daubin, Vincent; Medigue, Claudine; Adney, William S.; Xu,Xin Clare; Lapidus, Alla; Detter, Chris; Pujic, Petar; Bruce, David; Lavire, Celine; Challacombe, Jean F.; Brettin, Thomas S.; Berry, Alison M.

    2009-01-01

    We present here the complete 2.4 Mb genome of the cellulolytic actinobacterial thermophile, Acidothermus cellulolyticus 11B. New secreted glycoside hydrolases and carbohydrate esterases were identified in the genome, revealing a diverse biomass-degrading enzyme repertoire far greater than previously characterized, and significantly elevating the industrial value of this organism. A sizable fraction of these hydrolytic enzymes break down plant cell walls and the remaining either degrade components in fungal cell walls or metabolize storage carbohydrates such as glycogen and trehalose, implicating the relative importance of these different carbon sources. A novel feature of the A. cellulolyticus secreted cellulolytic and xylanolytic enzymes is that they are fused to multiple tandemly arranged carbohydrate binding modules (CBM), from families 2 and 3. Interestingly, CBM3 was found to be always N-terminal to CBM2, suggesting a functional constraint driving this organization. While the catalytic domains of these modular enzymes are either diverse or unrelated, the CBMs were found to be highly conserved in sequence and may suggest selective substrate-binding interactions. For the most part, thermophilic patterns in the genome and proteome of A. cellulolyticus were weak, which may be reflective of the recent evolutionary history of A. cellulolyticus since its divergence from its closest phylogenetic neighbor Frankia, a mesophilic plant endosymbiont and soil dweller. However, ribosomal proteins and non-coding RNAs (rRNA and tRNAs) in A. cellulolyticus showed thermophilic traits suggesting the importance of adaptation of cellular translational machinery to environmental temperature. Elevated occurrence of IVYWREL amino acids in A. cellulolyticus orthologs compared to mesophiles, and inverse preferences for G and A at the first and third codon positions also point to its ongoing thermoadaptation. Additional interesting features in the genome of this cellulolytic, hot

  5. Complete genome of the cellulolytic thermophile Acidothermus cellulolyticus 11B provides insights into its ecophysiological and evolutionary adaptations

    SciTech Connect

    Xie, Gary; Detter, John C; Bruce, David C; Challacombe, Jean F; Brettin, Thomas S; Necsulea, Anamaria; Daubin, Vincent; Medigue, Claudine; Adney, William S; Xu, Xin C; Lapidus, Alla; Pujic, Pierre; Berry, Alison M; Barabote, Ravi D; Leu, David; Normand, Phillipe

    2009-01-01

    We present here the complete 2.4 MB genome of the actinobacterial thermophile, Acidothermus cellulolyticus 11B, that surprisingly reveals thermophilic amino acid usage in only the cytosolic subproteome rather than its whole proteome. Thermophilic amino acid usage in the partial proteome implies a recent, ongoing evolution of the A. cellulolyticus genome since its divergence about 200-250 million years ago from its closest phylogenetic neighbor Frankia, a mesophilic plant symbiont. Differential amino acid usage in the predicted subproteomes of A. cellulolyticus likely reflects a stepwise evolutionary process of modern thermophiles in general. An unusual occurrence of higher G+C in the non-coding DNA than in the transcribed genome reinforces a late evolution from a higher G+C common ancestor. Comparative analyses of the A. cellulolyticus genome with those of Frankia and other closely-related actinobacteria revealed that A. cellulolyticus genes exhibit reciprocal purine preferences at the first and third codon positions, perhaps reflecting a subtle preference for the dinucleotide AG in its mRNAs, a possible adaptation to a thermophilic environment. Other interesting features in the genome of this cellulolytic, hot-springs dwelling prokaryote reveal streamlining for adaptation to its specialized ecological niche. These include a low occurrence of pseudo genes or mobile genetic elements, a flagellar gene complement previously unknown in this organism, and presence of laterally-acquired genomic islands of likely ecophysiological value. New glycoside hydrolases relevant for lignocellulosic biomass deconstruction were identified in the genome, indicating a diverse biomass-degrading enzyme repertoire several-fold greater than previously characterized, and significantly elevating the industrial value of this organism.

  6. Complete genome of the cellulolytic thermophile Acidothermus cellulolyticus 11B provides insights into its ecophysiological and evolutionary adaptations

    SciTech Connect

    Xie, Gary; Detter, Chris; Bruce, David; Challacome, Jean F; Brettin, Thomas S; Barabote, Ravi D; Leu, David; Normand, Philippe; Necsula, Anamaria; Daubin, Vincent; Medigue, Claudine; Xu, Xin C; Lapidus, Alla; Pujic, Pierre; Richardson, Paul; Berry, Alison M

    2008-01-01

    We present here the complete 2.4 MB genome of the actinobacterial thermophile, Acidothermus cellulolyticus lIB, that surprisingly reveals thermophilic amino acid usage in only the cytosolic subproteome rather than its whole proteome. Thermophilic amino acid usage in the partial proteome implies a recent, ongoing evolution of the A. cellulolyticus genome since its divergence about 200-250 million years ago from its closest phylogenetic neighbor Frankia, a mesophilic plant symbiont. Differential amino acid usage in the predicted subproteomes of A. cellulolyticus likely reflects a stepwise evolutionary process of modern thermophiles in general. An unusual occurrence of higher G+C in the non-coding DNA than in the transcribed genome reinforces a late evolution from a higher G+C common ancestor. Comparative analyses of the A. cellulolyticus genome with those of Frankia and other closely-related actinobacteria revealed that A. cellulolyticus genes exhibit reciprocal purine preferences at the first and third codon positions, perhaps reflecting a subtle preference for the dinucleotide AG in its mRNAs, a possible adaptation to a thermophilic environment. Other interesting features in the genome of this cellulolytic, hot-springs dwelling prokaryote reveal streamlining for adaptation to its specialized ecological niche. These include a low occurrence of pseudogenes or mobile genetic elements, a flagellar gene complement previously unknown in this organism, and presence of laterally-acquired genomic islands of likely ecophysiological value. New glycoside hydrolases relevant for lignocellulosic biomass deconstruction were identified in the genome, indicating a diverse biomass-degrading enzyme repertoire several-fold greater than previously characterized, and significantly elevating the industrial value of this organism.

  7. High level expression of Acidothermus cellulolyticus β-1, 4-endoglucanase in transgenic rice enhances the hydrolysis of its straw by cultured cow gastric fluid

    SciTech Connect

    Chou, Hong L.; Dai, Ziyu; Hsieh, Chia W.; Ku, Maurice S.

    2011-12-10

    Large-scale production of effective cellulose hydrolytic enzymes is the key to the bioconversion of agricultural residues to ethanol. The goal of this study was to develop a rice plant as a bioreactor for the large-scale production of cellulose hydrolytic enzymes via genetic transformation, and to simultaneously improve rice straw as an efficient biomass feedstock for conversion of cellulose to glucose. In this study, the cellulose hydrolytic enzyme {beta}-1, 4-endoglucanase (E1) from the thermophilic bacterium Acidothermus cellulolyticus was overexpressed in rice through Agrobacterium-mediated transformation. The expression of the bacterial gene in rice was driven by the constitutive Mac promoter, a hybrid promoter of Ti plasmid mannopine synthetase promoter and cauliflower mosaic virus 35S promoter enhancer with the signal peptide of tobacco pathogenesis-related protein for targeting the protein to the apoplastic compartment for storage. A total of 52 transgenic rice plants from six independent lines expressing the bacterial enzyme were obtained, which expressed the gene at high levels with a normal phenotype. The specific activities of E1 in the leaves of the highest expressing transgenic rice lines were about 20 fold higher than those of various transgenic plants obtained in previous studies and the protein amounts accounted for up to 6.1% of the total leaf soluble protein. Zymogram and temperature-dependent activity analyses demonstrated the thermostability of the enzyme and its substrate specificity against cellulose, and a simple heat treatment can be used to purify the protein. In addition, hydrolysis of transgenic rice straw with cultured cow gastric fluid yielded almost twice more reducing sugars than wild type straw. Taken together, these data suggest that transgenic rice can effectively serve as a bioreactor for large-scale production of active, thermostable cellulose hydrolytic enzymes. As a feedstock, direct expression of large amount of cellulases in

  8. High level expression of Acidothermus cellulolyticus β-1, 4-endoglucanase in transgenic rice enhances the hydrolysis of its straw by cultured cow gastric fluid

    PubMed Central

    2011-01-01

    Background Large-scale production of effective cellulose hydrolytic enzymes is the key to the bioconversion of agricultural residues to ethanol. The goal of this study was to develop a rice plant as a bioreactor for the large-scale production of cellulose hydrolytic enzymes via genetic transformation, and to simultaneously improve rice straw as an efficient biomass feedstock for conversion of cellulose to glucose. Results In this study, the cellulose hydrolytic enzyme β-1, 4-endoglucanase (E1) gene, from the thermophilic bacterium Acidothermus cellulolyticus, was overexpressed in rice through Agrobacterium-mediated transformation. The expression of the bacterial E1 gene in rice was driven by the constitutive Mac promoter, a hybrid promoter of Ti plasmid mannopine synthetase promoter and cauliflower mosaic virus 35S promoter enhancer, with the signal peptide of tobacco pathogenesis-related protein for targeting the E1 protein to the apoplastic compartment for storage. A total of 52 transgenic rice plants from six independent lines expressing the bacterial E1 enzyme were obtained that expressed the gene at high levels without severely impairing plant growth and development. However, some transgenic plants exhibited a shorter stature and flowered earlier than the wild type plants. The E1 specific activities in the leaves of the highest expressing transgenic rice lines were about 20-fold higher than those of various transgenic plants obtained in previous studies and the protein amounts accounted for up to 6.1% of the total leaf soluble protein. A zymogram and temperature-dependent activity analyses demonstrated the thermostability of the E1 enzyme and its substrate specificity against cellulose, and a simple heat treatment can be used to purify the protein. In addition, hydrolysis of transgenic rice straw with cultured cow gastric fluid for one hour at 39°C and another hour at 81°C yielded 43% more reducing sugars than wild type rice straw. Conclusion Taken together

  9. Cloning, Expression, and Characterization of a Thermophilic Endoglucanase, AcCel12B from Acidothermus cellulolyticus 11B

    PubMed Central

    Wang, Junling; Gao, Gui; Li, Yuwei; Yang, Liangzhen; Liang, Yanli; Jin, Hanyong; Han, Weiwei; Feng, Yan; Zhang, Zuoming

    2015-01-01

    The gene ABK52392 from the thermophilic bacterium Acidothermus cellulolyticus 11B was predicted to be endoglucanase and classified into glycoside hydrolase family 12. ABK52392 encodes a protein containing a catalytic domain and a carbohydrate binding module. ABK52392 was cloned and functionally expressed in Escherichia coli. After purification by Ni-NTA agarose affinity chromatography and Q-Sepharose® Fast Flow chromatography, the properties of the recombinant protein (AcCel12B) were characterized. AcCel12B exhibited optimal activity at pH 4.5 and 75 °C. The half-lives of AcCel12B at 60 and 70 °C were about 90 and 2 h, respectively, under acidic conditions. The specific hydrolytic activities of AcCel12B at 70 °C and pH 4.5 for sodium carboxymethylcellulose (CMC) and regenerated amorphous cellulose (RAC) were 118.3 and 104.0 U·mg−1, respectively. The Km and Vmax of AcCel12B for CMC were 25.47 mg·mL−1 and 131.75 U·mg−1, respectively. The time course of hydrolysis for RAC was investigated by measuring reducing ends in the soluble and insoluble phases. The total hydrolysis rate rapidly decreased after the early stage of incubation and the generation of insoluble reducing ends decreased earlier than that of soluble reducing ends. High thermostability of the cellulase indicates its potential commercial significance and it could be exploited for industrial application in the future. PMID:26506341

  10. Gene coding for the E1 endoglucanase

    DOEpatents

    Thomas, Steven R.; Laymon, Robert A.; Himmel, Michael E.

    1996-01-01

    The gene encoding Acidothermus cellulolyticus E1 endoglucanase is cloned and expressed in heterologous microorganisms. A new modified E1 endoglucanase enzyme is produced along with variants of the gene and enzyme. The E1 endoglucanase is useful for hydrolyzing cellulose to sugars for simultaneous or later fermentation into alcohol.

  11. Gene coding for the E1 endoglucanase

    DOEpatents

    Thomas, S.R.; Laymon, R.A.; Himmel, M.E.

    1996-07-16

    The gene encoding Acidothermus cellulolyticus E1 endoglucanase is cloned and expressed in heterologous microorganisms. A new modified E1 endoglucanase enzyme is produced along with variants of the gene and enzyme. The E1 endoglucanase is useful for hydrolyzing cellulose to sugars for simultaneous or later fermentation into alcohol. 6 figs.

  12. Optimization of Acidothermus Celluloyticus Endoglucanase (E1) Production in Transgenic Tobacco Plants by Transcriptional, Post-transcription and Post-Translational Modification

    SciTech Connect

    Dai, Ziyu; Hooker, Brian S.; Quesenberry, Ryan D.; Thomas, S. R.

    2005-10-01

    Biochemical characteristics of Acidothermus cellulolyticus endoglucanase (E1) and its physiological effects in transgenic tobacco (Nicotiana tabacum) has been studied previously. In an attempt to obtain a high level of production of intact E1 in transgenic plants, the E1 gene was expressed under the control of strong Mac promoter (a hybrid promoter of manopine synthase promoter and cauliflower mosaic virus 35S promoter enhancer region) or tomato Rubisco small subunit (RbcS-3C) promoter with different 5’ untranslated leader (UTL) sequence and targeted to different subcellular comartmentations with various transit peptides. The expression of E1 protein in transgenic tobacco plants was determined via E1 activity, protein immunobloting, and RNA gel-blotting analyses. Effects of different transit peptides on E1 protein production and its stability were examined in transgenic tobacco plants carrying one of six transgene expression vectors with the same (Mac) promoter and transcription terminator (Tmas). Transgenic tobacco plants with apoplast transit peptide (Mm-apo) had the highest average E1 activity and protein accumulation , while E1 protein was more stable in transgenic plants with no transit peptide (Mm) than others. The E1 expression under tomato RbcS-3C promoter was higher than that under Mac promoter based on the average E1 activity, E1 protein accumulation, and RNA gel-blotting. The E1 expression was increased more than two fold when the 5’-UTL of alfalfa mosaic virus RNA4 gene replaced the UTL of RbcS-3C promoter, while the UTL of alfalfa mosaic virus RNA4 gene was less effective than the UTL of Mac promoter. The optimal combination of promoter, 5’-UTL, and subcellular compartmentation (transit peptide) for E1 protein production in transgenic tobacco plants are discussed.

  13. In planta expression of A. cellulolyticus Cel5A endocellulase reduces cell wall recalcitrance in tobacco and maize

    PubMed Central

    2011-01-01

    The glycoside hydrolase family 5 endocellulase, E1 (Cel5A), from Acidothermus cellulolyticus was transformed into both Nicotiana tabacum and Zea mays with expression targeted to the cell wall under a constitutive promoter. Here we explore the possibility that in planta expression of endocellulases will allow these enzymes to access their substrates during cell wall construction, rendering cellulose more amenable to pretreatment and enzyme digestion. Tobacco and maize plants were healthy and developed normally compared with the wild type (WT). After thermochemical pretreatment and enzyme digestion, transformed plants were clearly more digestible than WT, requiring lower pretreatment severity to achieve comparable conversion levels. Furthermore, the decreased recalcitrance was not due to post-pretreatment residual E1 activity and could not be reproduced by the addition of exogenous E1 to the biomass prior to pretreatment, indicating that the expression of E1 during cell wall construction altered the inherent recalcitrance of the cell wall. PMID:21269444

  14. Improved Plant-based Production of E1 endoglucanase Using Potato: Expression Optimization and Tissue Targeting

    SciTech Connect

    Dai, Ziyu; Hooker, Brian S.; Anderson, Daniel B.; Thomas, Steven R.

    2000-06-01

    Optimization of Acidothermus cellulolyticus endoglucanase (E1) gene expression in transgenic potato (Solanum tuberosum L.) was examined in this study, where the E1 coding sequence was transcribed under control of a leaf specific promoter (tomato RbcS-3C) or the Mac promoter (a hybrid promoter of mannopine synthase promoter and cauliflower mosaic virus 35S promoter enhancer region). Average E1 activity in leaf extracts of potato transformants, in which E1 protein was targeted by a chloroplast signal peptide and an apoplast signal peptide were much higher than those by an E1 native signal peptide and a vacuole signal peptide. E1 protein accumulated up to 2.6% of total leaf soluble protein, where E1 gene was under control of the RbcS-3C promoter, alfalfa mosaic virus 5-untranslated leader, and RbcS-2A signal peptide. E1 protein production, based on average E1 activity and E1 protein accumulation in leaf extracts, is higher in potato than those measured previously in transgenic tobacco bearing the same transgene constructs. Comparisons of E1 activity, protein accumulation, and relative mRNA levels showed that E1 expression under control of tomato RbcS-3C promoter was specifically localized in leaf tissues, while E1 gene was expressed in both leaf and tuber tissues under control of Mac promoter. This suggests dual-crop applications in which potato vines serve as enzyme production `bioreactors' while tubers are preserved for culinary applications.

  15. Lactose enhances cellulase production by the filamentous fungus Acremonium cellulolyticus.

    PubMed

    Fang, Xu; Yano, Shinichi; Inoue, Hiroyuki; Sawayama, Shigeki

    2008-08-01

    Acremonium cellulolyticus is a fungus that produces cellulase and has been exploited by enzyme industry. To promote cellulase production by A. cellulolyticus strain C-1, we evaluated the effects of the saccharides: Solka Floc (cellulose), soluble soybean polysaccharide (SSPS), pullulan, lactose, trehalose, sophorose, cellobiose, galactose, sorbose, lactobionic acid, and mixtures as carbon sources for cellulase production. Solka Floc with SSPS enhanced cellulase production. Lactose as the sole carbon source induced cellulase synthesis in this fungus, and the synergistic effects between lactose and Solka Floc was observed. Various enzyme activities and the protein composition of crude enzyme produced by cultures with or without addition of lactose were analyzed. The results showed that lactose addition greatly improves the production of various proteins with cellulase activity by A. cellulolyticus. To our knowledge, this is the first report on production of cellulases by lactose in the A. cellulolyticus. PMID:18804052

  16. Crystal structure of Talaromyces cellulolyticus (formerly known as Acremonium cellulolyticus) GH family 11 xylanase.

    PubMed

    Kataoka, Misumi; Akita, Fusamichi; Maeno, Yuka; Inoue, Benchaporn; Inoue, Hiroyuki; Ishikawa, Kazuhiko

    2014-10-01

    Talaromyces cellulolyticus (formerly known as Acremonium cellulolyticus) is one of the mesophilic fungi that can produce high levels of cellulose-related enzymes and are expected to be used for the degradation of polysaccharide biomass. In silico analysis of the genome sequence of T. cellulolyticus detected seven open reading frames (ORFs) showing homology to xylanases from glycoside hydrolase (GH) family 11. The gene encoding the GH11 xylanase C (TcXylC) with the highest activity was used for overproduction and purification of the recombinant enzyme, permitting solving of the crystal structure to a resolution of 1.98 Å. In the asymmetric unit, two kinds of the crystal structures of the xylanase were identified. The main structure of the protein showed a β-jelly roll structure. We hypothesize that one of the two structures represents the open form and the other shows the close form. The changing of the flexible region between the two structures is presumed to induce and accelerate the enzyme reaction. The specificity of xylanase toward the branched xylan is discussed in the context of this structural data and by comparison with the other published structures of xylanases. PMID:25138599

  17. Characterization of a feruloyl esterase B from Talaromyces cellulolyticus.

    PubMed

    Watanabe, Masahiro; Yoshida, Erika; Fukada, Hiroaki; Inoue, Hiroyuki; Tokura, Mitsunori; Ishikawa, Kazuhiko

    2015-01-01

    A feruloyl esterase catalyzes the hydrolysis of the 4-hydroxy-3-methoxycinnamoyl (feruloyl) group from esterified sugars in plant cell walls. Talaromyces cellulolyticus is a high cellulolytic-enzyme producing fungus. However, there is no report for feruloyl esterase activity of T. cellulolyticus. Analysis of the genome database of T. cellulolyticus identified a gene encoding a putative feruloyl esterase B. The recombinant enzyme was prepared using a T. cellulolyticus homologous expression system and characterized. The purified enzyme exhibited hydrolytic activity toward p-nitrophenyl acetate, p-nitrophenyl trans-ferulate, methyl ferulate, rice husk, and bagasse. HPLC assays showed that the enzyme released ferulic acid and p-coumaric acid from hydrothermal-treated rice husk and bagasse. Trichoderma sp. is well-known high cellulolytic-enzyme producing fungus useful for the lignocellulosic biomass saccharification. Interestingly, no feruloyl esterase has been reported from Trichoderma sp. The results show that this enzyme is expected to be industrially useful for biomass saccharification. PMID:26110915

  18. Draft Genome Sequence of Talaromyces cellulolyticus Strain Y-94, a Source of Lignocellulosic Biomass-Degrading Enzymes

    PubMed Central

    Fujii, Tatsuya; Koike, Hideaki; Sawayama, Shigeki; Yano, Shinichi

    2015-01-01

    Talaromyces cellulolyticus (formerly Acremonium cellulolyticus) is a promising fungus for cellulase production. Here, we present the draft genome sequence of T. cellulolyticus strain Y-94. The genome is 36.4 Mbp long and contains genes for several enzymes involved in the degradation of lignocellulosic biomass, including cellulases, hemicellulases, pectinases, and amylases. PMID:25720677

  19. Identification and characterization of core cellulolytic enzymes from Talaromyces cellulolyticus (formerly Acremonium cellulolyticus) critical for hydrolysis of lignocellulosic biomass

    DOE PAGESBeta

    Inoue, Hiroyuki; Decker, Stephen R.; Taylor, Larry E.; Yano, Shinichi; Sawayama, Shigeki

    2014-10-09

    Background: Enzymatic hydrolysis of pretreated lignocellulosic biomass is an essential process for the production of fermentable sugars for industrial use. A better understanding of fungal cellulase systems will provide clues for maximizing the hydrolysis of target biomass. Talaromyces cellulolyticus is a promising fungus for cellulase production and efficient biomass hydrolysis. Several cellulolytic enzymes purified from T. cellulolyticus were characterized in earlier studies, but the core enzymes critical for hydrolysis of lignocellulosic biomass remain unknown. Results: Six cellulolytic enzymes critical for the hydrolysis of crystalline cellulose were purified from T. cellulolyticus culture supernatant using an enzyme assay based on synergistic hydrolysismore » of Avicel. The purified enzymes were identified by their substrate specificities and analyses of trypsin-digested peptide fragments and were classified into the following glycosyl hydrolase (GH) families: GH3 (β-glucosidase, Bgl3A), GH5 (endoglucanase, Cel5A), GH6 (cellobiohydrolase II, Cel6A), GH7 (cellobiohydrolase I and endoglucanase, Cel7A and Cel7B, respectively), and GH10 (xylanase, Xyl10A). Hydrolysis of dilute acid-pretreated corn stover (PCS) with mixtures of the purified enzymes showed that Cel5A, Cel7B, and Xyl10A each had synergistic effects with a mixture of Cel6A and Cel7A. Cel5A seemed to be more effective in the synergistic hydrolysis of the PCS than Cel7B. The ratio of Cel5A, Cel6A, Cel7A, and Xyl10A was statistically optimized for the hydrolysis of PCS glucan in the presence of Bgl3A. The resultant mixture achieved higher PCS glucan hydrolysis at lower enzyme loading than a culture filtrate from T. cellulolyticus or a commercial enzyme preparation, demonstrating that the five enzymes play a role as core enzymes in the hydrolysis of PCS glucan. In Conclusion: Core cellulolytic enzymes in the T. cellulolyticus cellulase system were identified to Cel5A, Cel6A, Cel7A, Xyl10A, and Bgl3A

  20. Identification and characterization of core cellulolytic enzymes from Talaromyces cellulolyticus (formerly Acremonium cellulolyticus) critical for hydrolysis of lignocellulosic biomass

    SciTech Connect

    Inoue, Hiroyuki; Decker, Stephen R.; Taylor, Larry E.; Yano, Shinichi; Sawayama, Shigeki

    2014-10-09

    Background: Enzymatic hydrolysis of pretreated lignocellulosic biomass is an essential process for the production of fermentable sugars for industrial use. A better understanding of fungal cellulase systems will provide clues for maximizing the hydrolysis of target biomass. Talaromyces cellulolyticus is a promising fungus for cellulase production and efficient biomass hydrolysis. Several cellulolytic enzymes purified from T. cellulolyticus were characterized in earlier studies, but the core enzymes critical for hydrolysis of lignocellulosic biomass remain unknown. Results: Six cellulolytic enzymes critical for the hydrolysis of crystalline cellulose were purified from T. cellulolyticus culture supernatant using an enzyme assay based on synergistic hydrolysis of Avicel. The purified enzymes were identified by their substrate specificities and analyses of trypsin-digested peptide fragments and were classified into the following glycosyl hydrolase (GH) families: GH3 (β-glucosidase, Bgl3A), GH5 (endoglucanase, Cel5A), GH6 (cellobiohydrolase II, Cel6A), GH7 (cellobiohydrolase I and endoglucanase, Cel7A and Cel7B, respectively), and GH10 (xylanase, Xyl10A). Hydrolysis of dilute acid-pretreated corn stover (PCS) with mixtures of the purified enzymes showed that Cel5A, Cel7B, and Xyl10A each had synergistic effects with a mixture of Cel6A and Cel7A. Cel5A seemed to be more effective in the synergistic hydrolysis of the PCS than Cel7B. The ratio of Cel5A, Cel6A, Cel7A, and Xyl10A was statistically optimized for the hydrolysis of PCS glucan in the presence of Bgl3A. The resultant mixture achieved higher PCS glucan hydrolysis at lower enzyme loading than a culture filtrate from T. cellulolyticus or a commercial enzyme preparation, demonstrating that the five enzymes play a role as core enzymes in the hydrolysis of PCS glucan. In Conclusion: Core cellulolytic enzymes in the T. cellulolyticus cellulase system were identified to Cel5A, Cel6A, Cel7A, Xyl10A, and Bgl3A and

  1. Cellulose-inducible xylanase Xyl10A from Acremonium cellulolyticus: Purification, cloning and homologous expression.

    PubMed

    Kishishita, Seiichiro; Yoshimi, Miho; Fujii, Tatsuya; Taylor, Larry E; Decker, Stephen R; Ishikawa, Kazuhiko; Inoue, Hiroyuki

    2014-02-01

    Cellulose-inducible endo-β-1,4-xylanase (Xyl10A) from the mesophilic fungus Acremonium cellulolyticus was purified, characterized, and expressed by a homologous expression system. A. cellulolyticus CF-2612 produces a high level of xylanase upon induction by Solka-Floc cellulose. To identify this xylanase, the major fraction showing xylanase activity was purified from the CF-2612 culture supernatant, and its gene was identified from the genome sequence. Amino acid sequence homology of Xyl10A revealed that the purified xylanase, designated Xyl10A, exhibited significant homology to family 10 of the glycoside hydrolases (GH10), possessing a cellulose-binding module 1 in the C-terminal region. The xyl10A gene was cloned and expressed in A. cellulolyticus under the control of a glucoamylase promoter. Two recombinant Xyl10As (rXyl10A-I, 53kDa, and rXyl10A-II, 51kDa) were purified that have slightly different molecular weights based on SDS-PAGE. The rXyl10As had the same physicochemical and enzymatic properties as wtXyl10A: high thermostability (Tm 80.5°C), optimum pH 5.0 and specific activity 232-251U/mg for birchwood xylan. The molecular weights of N-deglycosylated rXyl10As were consistent with that of wild-type Xyl10A (wtXyl10A, 51kDa). PMID:24211645

  2. Method for increasing thermostability in cellulase ennzymes

    DOEpatents

    Adney, William S.; Thomas, Steven R.; Baker, John O.; Himmel, Michael E.; Chou, Yat-Chen

    1998-01-01

    The gene encoding Acidothermus cellulolyticus E1 endoglucanase is cloned and expressed in Pichia pastoris. A new modified E1 endoglucanase enzyme comprising the catalytic domain of the full size E1 enzyme demonstrates enhanced thermostability and is produced by two methods. The first method of producing the new modified E1 is proteolytic cleavage to remove the cellulose binding domain and linker peptide of the full size E1. The second method of producing the new modified E1 is genetic truncation of the gene encoding the full size E1 so that the catalytic domain is expressed in the expression product.

  3. Method for increasing thermostability in cellulase ennzymes

    DOEpatents

    Adney, W.S.; Thomas, S.R.; Baker, J.O.; Himmel, M.E.; Chou, Y.C.

    1998-01-27

    The gene encoding Acidothermus cellulolyticus E1 endoglucanase is cloned and expressed in Pichia pastoris. A new modified E1 endoglucanase enzyme comprising the catalytic domain of the full size E1 enzyme demonstrates enhanced thermostability and is produced by two methods. The first method of producing the new modified E1 is proteolytic cleavage to remove the cellulose binding domain and linker peptide of the full size E1. The second method of producing the new modified E1 is genetic truncation of the gene encoding the full size E1 so that the catalytic domain is expressed in the expression product. 8 figs.

  4. The E1 proteins

    SciTech Connect

    Bergvall, Monika; Melendy, Thomas; Archambault, Jacques

    2013-10-15

    E1, an ATP-dependent DNA helicase, is the only enzyme encoded by papillomaviruses (PVs). It is essential for replication and amplification of the viral episome in the nucleus of infected cells. To do so, E1 assembles into a double-hexamer at the viral origin, unwinds DNA at the origin and ahead of the replication fork and interacts with cellular DNA replication factors. Biochemical and structural studies have revealed the assembly pathway of E1 at the origin and how the enzyme unwinds DNA using a spiral escalator mechanism. E1 is tightly regulated in vivo, in particular by post-translational modifications that restrict its accumulation in the nucleus. Here we review how different functional domains of E1 orchestrate viral DNA replication, with an emphasis on their interactions with substrate DNA, host DNA replication factors and modifying enzymes. These studies have made E1 one of the best characterized helicases and provided unique insights on how PVs usurp different host-cell machineries to replicate and amplify their genome in a tightly controlled manner. - Highlights: • The papillomavirus E1 helicase orchestrates replication of the viral DNA genome. • E1 assembles into a double-hexamer at the viral origin with the help of E2. • E1 interacts with cellular DNA replication factors. • E1 unwinds DNA using a spiral escalator mechanism. • Nuclear accumulation of E1 is regulated by post-translational modifications.

  5. THE E1 PROTEINS

    PubMed Central

    Bergvall, Monika; Melendy, Thomas; Archambault, Jacques

    2013-01-01

    E1, an ATP-dependent DNA helicase, is the only enzyme encoded by papillomaviruses (PVs). It is essential for replication and amplification of the viral episome in the nucleus of infected cells. To do so, E1 assembles into a double-hexamer at the viral origin, unwinds DNA at the origin and ahead of the replication fork and interacts with cellular DNA replication factors. Biochemical and structural studies have revealed the assembly pathway of E1 at the origin and how the enzyme unwinds DNA using a spiral escalator mechanism. E1 is tightly regulated in vivo, in particular by post-translational modifications that restrict its accumulation in the nucleus. Here we review how different functional domains of E1 orchestrate viral DNA replication, with an emphasis on their interactions with substrate DNA, host DNA replication factors and modifying enzymes. These studies have made E1 one of the best characterized helicases and provided unique insights on how PVs usurp different host-cell machineries to replicate and amplify their genome in a tightly controlled manner. PMID:24029589

  6. Enhancing cellulase production by overexpression of xylanase regulator protein gene, xlnR, in Talaromyces cellulolyticus cellulase hyperproducing mutant strain.

    PubMed

    Okuda, Naoyuki; Fujii, Tatsuya; Inoue, Hiroyuki; Ishikawa, Kazuhiko; Hoshino, Tamotsu

    2016-10-01

    We obtained strains with the xylanase regulator gene, xlnR, overexpressed (HXlnR) and disrupted (DXlnR) derived from Talaromyces cellulolyticus strain C-1, which is a cellulase hyperproducing mutant. Filter paper degrading enzyme activity and cellobiohydrolase I gene expression was the highest in HXlnR, followed by C-1 and DXlnR. These results indicate that the enhancement of cellulase productivity was succeeded by xlnR overexpression. PMID:27309759

  7. One-pot bioethanol production from cellulose by co-culture of Acremonium cellulolyticus and Saccharomyces cerevisiae

    PubMed Central

    2012-01-01

    Background While the ethanol production from biomass by consolidated bioprocess (CBP) is considered to be the most ideal process, simultaneous saccharification and fermentation (SSF) is the most appropriate strategy in practice. In this study, one-pot bioethanol production, including cellulase production, saccharification of cellulose, and ethanol production, was investigated for the conversion of biomass to biofuel by co-culture of two different microorganisms such as a hyper cellulase producer, Acremonium cellulolyticus C-1 and an ethanol producer Saccharomyces cerevisiae. Furthermore, the operational conditions of the one-pot process were evaluated for maximizing ethanol concentration from cellulose in a single reactor. Results Ethanol production from cellulose was carried out in one-pot bioethanol production process. A. cellulolyticus C-1 and S. cerevisiae were co-cultured in a single reactor. Cellulase producing-medium supplemented with 2.5 g/l of yeast extract was used for productions of both cellulase and ethanol. Cellulase production was achieved by A. cellulolyticus C-1 using Solka-Floc (SF) as a cellulase-inducing substrate. Subsequently, ethanol was produced with addition of both 10%(v/v) of S. cerevisiae inoculum and SF at the culture time of 60 h. Dissolved oxygen levels were adjusted at higher than 20% during cellulase producing phase and at lower than 10% during ethanol producing phase. Cellulase activity remained 8–12 FPU/ml throughout the one-pot process. When 50–300 g SF/l was used in 500 ml Erlenmeyer flask scale, the ethanol concentration and yield based on initial SF were as 8.7–46.3 g/l and 0.15–0.18 (g ethanol/g SF), respectively. In 3-l fermentor with 50–300 g SF/l, the ethanol concentration and yield were 9.5–35.1 g/l with their yields of 0.12–0.19 (g/g) respectively, demonstrating that the one-pot bioethanol production is a reproducible process in a scale-up bioconversion of cellulose to ethanol. Conclusion A. cellulolyticus

  8. Complete Genome Sequences of Caldicellulosiruptor sp. Strain Rt8.B8, Caldicellulosiruptor sp. Strain Wai35.B1, and "Thermoanaerobacter cellulolyticus".

    PubMed

    Lee, Laura L; Izquierdo, Javier A; Blumer-Schuette, Sara E; Zurawski, Jeffrey V; Conway, Jonathan M; Cottingham, Robert W; Huntemann, Marcel; Copeland, Alex; Chen, I-Min A; Kyrpides, Nikos; Markowitz, Victor; Palaniappan, Krishnaveni; Ivanova, Natalia; Mikhailova, Natalia; Ovchinnikova, Galina; Andersen, Evan; Pati, Amrita; Stamatis, Dimitrios; Reddy, T B K; Shapiro, Nicole; Nordberg, Henrik P; Cantor, Michael N; Hua, Susan X; Woyke, Tanja; Kelly, Robert M

    2015-01-01

    The genus Caldicellulosiruptor contains extremely thermophilic, cellulolytic bacteria capable of lignocellulose deconstruction. Currently, complete genome sequences for eleven Caldicellulosiruptor species are available. Here, we report genome sequences for three additional Caldicellulosiruptor species: Rt8.B8 DSM 8990 (New Zealand), Wai35.B1 DSM 8977 (New Zealand), and "Thermoanaerobacter cellulolyticus" strain NA10 DSM 8991 (Japan). PMID:25977428

  9. Development of A Flexible System for the Simultaneous Conversion of Biomass to Industrial Chemicals and the Production of Industrial Biocatalysts

    SciTech Connect

    Gao, Johnway; Hooker, Brian S.; Skeen, R S.; Anderson, D B.; Lankey, R. L.; Anastas, P. T.

    2002-01-01

    A flexible system was developed for the simultaneous conversion of biomass to industrial chemicals and the production of industrial biocatalysts. In particular, the expression of a bacterial enzyme, beta-glucuronidase (GUS), was investigated using a genetically modified starch-degrading Saccharomyces strain in suspension cultures in starch media. Different sources of starch including corn and waste potato starch were used for yeast biomass accumulation and GUS expression studies under controls of inducible and constitutive promoters. A thermostable bacterial cellulase, Acidothermus cellulolyticus E1 endoglucanase gene was also cloned into an episomal plasmid expression vector and expressed in the starch-degrading Saccharomyces strain.

  10. A Novel Cellulosomal Scaffoldin from Acetivibrio cellulolyticus That Contains a Family 9 Glycosyl Hydrolase

    PubMed Central

    Ding, Shi-You; Bayer, Edward A.; Steiner, David; Shoham, Yuval; Lamed, Raphael

    1999-01-01

    A novel cellulosomal scaffoldin gene, termed cipV, was identified and sequenced from the mesophilic cellulolytic anaerobe Acetivibrio cellulolyticus. Initial identification of the protein was based on a combination of properties, including its high molecular weight, cellulose-binding activity, glycoprotein nature, and immuno-cross-reactivity with the cellulosomal scaffoldin of Clostridium thermocellum. The cipV gene is 5,748 bp in length and encodes a 1,915-residue polypeptide with a calculated molecular weight of 199,496. CipV contains an N-terminal signal peptide, seven type I cohesin domains, an internal family III cellulose-binding domain (CBD), and an X2 module of unknown function in tandem with a type II dockerin domain at the C terminus. Surprisingly, CipV also possesses at its N terminus a catalytic module that belongs to the family 9 glycosyl hydrolases. Sequence analysis indicated the following. (i) The repeating cohesin domains are very similar to each other, ranging between 70 and 90% identity, and they also have about 30 to 40% homology with each of the other known type I scaffoldin cohesins. (ii) The internal CBD belongs to family III but differs from other known scaffoldin CBDs by the omission of a 9-residue stretch that constitutes a characteristic loop previously associated with the scaffoldins. (iii) The C-terminal type II dockerin domain is only the second such domain to have been discovered; its predicted “recognition codes” differ from those proposed for the other known dockerins. The putative calcium-binding loop includes an unusual insert, lacking in all the known type I and type II dockerins. (iv) The X2 module has about 60% sequence homology with that of C. thermocellum and appears at the same position in the scaffoldin. (v) Unlike the other known family 9 catalytic modules of bacterial origin, the CipV catalytic module is not accompanied by a flanking helper module, e.g., an adjacent family IIIc CBD or an immunoglobulin-like domain

  11. Bioprocessing of some agro-industrial residues for endoglucanase production by the new subsp.; Streptomyces albogriseolus subsp. cellulolyticus strain NEAE-J

    PubMed Central

    El-Naggar, Noura El-Ahmady; Abdelwahed, Nayera A.M.; Saber, Wesam I.A.; Mohamed, Asem A.

    2014-01-01

    The use of low cost agro-industrial residues for the production of industrial enzymes is one of the ways to reduce significantly production costs. Cellulase producing actinomycetes were isolated from soil and decayed agricultural wastes. Among them, a potential culture, strain NEAE-J, was selected and identified on the basis of morphological, cultural, physiological and chemotaxonomic properties, together with 16S rDNA sequence. It is proposed that strain NEAE-J should be included in the species Streptomyces albogriseolus as a representative of a novel sub-species, Streptomyces albogriseolus subsp. cellulolyticus strain NEAE-J and sequencing product was deposited in the GenBank database under accession number JN229412. This organism was tested for its ability to produce endoglucanase and release reducing sugars from agro-industrial residues as substrates. Sugarcane bagasse was the most suitable substrate for endoglucanase production. Effects of process variables, namely incubation time, temperature, initial pH and nitrogen source on production of endoglucanase by submerged fermentation using Streptomyces albogriseolus subsp. cellulolyticus have been studied. Accordingly optimum conditions have been determined. Incubation temperature of 30 °C after 6 days, pH of 6.5, 1% sugarcane bagasse as carbon source and peptone as nitrogen source were found to be the optimum for endoglucanase production. Optimization of the process parameters resulted in about 2.6 fold increase in the endoglucanase activity. Therefore, Streptomyces albogriseolus subsp. cellulolyticus coud be potential microorganism for the intended application. PMID:25242966

  12. 26 CFR 1.367(e)-1 - Distributions described in section 367(e)(1).

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    ... 26 Internal Revenue 4 2013-04-01 2013-04-01 false Distributions described in section 367(e)(1). 1.367(e)-1 Section 1.367(e)-1 Internal Revenue INTERNAL REVENUE SERVICE, DEPARTMENT OF THE TREASURY (CONTINUED) INCOME TAX (CONTINUED) INCOME TAXES (CONTINUED) Effects on Corporation § 1.367(e)-1 Distributions described in section 367(e)(1)....

  13. Overexpression, purification, crystallization and preliminary X-ray characterization of the fourth scaffoldin A cohesin from Acetivibrio cellulolyticus in complex with a dockerin from a family 5 glycoside hydrolase

    PubMed Central

    Bule, Pedro; Correia, Ana; Cameron, Kate; Alves, Victor D.; Cardoso, Vânia; Fontes, Carlos M. G. A.; Najmudin, Shabir

    2014-01-01

    Cellulosomes are cell-bound multienzyme complexes secreted by anaerobic bacteria that play a crucial role in carbon turnover by degrading plant cell walls to simple sugars. Integration of cellulosomal components occurs via highly ordered protein–protein interactions between cohesin modules located in a molecular scaffold and dockerin modules found in cellulosomal enzymes. Acetivibrio cellulolyticus possesses a complex cellulosome arrangement which is organized by a primary enzyme-binding scaffoldin (ScaA), two anchoring scaffoldins (ScaC and ScaD) and an unusual adaptor scaffoldin (ScaB). A dockerin from a family 5 glycoside hydrolase (GH5), which was engineered to inactivate one of the two putative cohesin-binding interfaces, complexed with one of the ScaA cohesins from A. cellulolyticus has been purified and crystallized, and data were processed to a resolution of 1.57 Å in the orthorhombic space group P212121. PMID:25084383

  14. Crystallization and preliminary X-ray analysis of Acetivibrio cellulolyticus cellulosomal type II cohesin module: two versions having different linker lengths

    SciTech Connect

    Noach, Ilit; Alber, Orly; Bayer, Edward A.; Lamed, Raphael; Levy-Assaraf, Maly; Shimon, Linda J. W.; Frolow, Felix

    2008-01-01

    The cloning, expression, purification, crystallization and preliminary X-ray characterization of two protein constructs of the second type II cohesin module from A. cellulolyticus ScaB are described. Both constructs contain the native N-terminal linker, but only one of them contains the full-length 45-residue C-terminal linker; the other contains a five-residue segment of this linker. The second type II cohesin module of the cellulosomal scaffoldin polypeptide ScaB from Acetivibrio cellulolyticus (CohB2) was cloned into two constructs: one containing a short (five-residue) C-terminal linker (CohB2-S) and the second incorporating the full native 45-residue linker (CohB2-L). Both constructs encode proteins that also include the full native six-residue N-terminal linker. The CohB2-S and CohB2-L proteins were expressed, purified and crystallized in the orthorhombic crystal system, but with different unit cells and symmetries: space group P2{sub 1}2{sub 1}2{sub 1} with unit-cell parameters a = 90.36, b = 68.65, c = 111.29 Å for CohB2-S and space group P2{sub 1}2{sub 1}2 with unit-cell parameters a = 68.76, b = 159.22, c = 44.21 Å for CohB2-L. The crystals diffracted to 2.0 and 2.9 Å resolution, respectively. The asymmetric unit of CohB2-S contains three cohesin molecules, while that of CohB2-L contains two molecules.

  15. 26 CFR 1.367(e)-1 - Distributions described in section 367(e)(1).

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... 26 Internal Revenue 4 2010-04-01 2010-04-01 false Distributions described in section 367(e)(1). 1.367(e)-1 Section 1.367(e)-1 Internal Revenue INTERNAL REVENUE SERVICE, DEPARTMENT OF THE TREASURY (CONTINUED) INCOME TAX (CONTINUED) INCOME TAXES Effects on Corporation § 1.367(e)-1 Distributions described in section 367(e)(1). (a) Purpose and...

  16. Adenovirus E1B 19-kilodalton protein overcomes the cytotoxicity of E1A proteins.

    PubMed Central

    White, E; Cipriani, R; Sabbatini, P; Denton, A

    1991-01-01

    Infection with adenovirus mutants carrying either point mutations or deletions in the coding region for the 19-kDa E1B gene product (19K protein) causes degradation of host cell and viral DNAs (deg phenotype) and enhanced cytopathic effect (cyt phenotype). Therefore, one function of the E1B 19K protein is to protect nuclear DNA integrity and preserve cytoplasmic architecture during productive adenovirus infection. When placed in the background of a virus incapable of expressing a functional E1A gene product, however, E1B 19K gene mutations do not result in the appearance of the cyt and deg phenotypes. This demonstrated that expression of the E1A proteins was responsible for inducing the appearance of the cyt and deg phenotypes. By constructing a panel of viruses possessing E1A mutations spanning each of the three E1A conserved regions in conjunction with E1B 19K gene mutations, we mapped the induction of the cyt and deg phenotypes to the amino-terminal region of E1A. Viruses that fail to express conserved region 3 (amino acids 140 to 185) and/or 2, (amino acids 121 to 185) or nonconserved sequences between conserved regions 2 and 1 of E1A (amino acids 86 to 120) were still capable of inducing cyt and deg. This indicated that activities associated with these regions, such as transactivation and binding to the product of the retinoblastoma susceptibility gene, were dispensable for induction of E1A-dependent cytotoxic effects. In contrast, deletion of sequences in the amino terminus of E1A (amino acids 22 to 107) resulted in extragenic suppression of the cyt and deg phenotypes. Therefore, a function affected by deletion of amino acids 22 to 86 of E1A is responsible for exerting cytotoxic effects in virally infected cells. Furthermore, transient high-level expression of the E1A region using a cytomegalovirus promoter plasmid expression vector was sufficient to induce the cyt and deg phenotypes, demonstrating that E1A expression alone is sufficient to exert these

  17. Production and characterization of cellulases and hemicellulases by Acremonium cellulolyticus using rice straw subjected to various pretreatments as the carbon source.

    PubMed

    Hideno, Akihiro; Inoue, Hiroyuki; Tsukahara, Kenichiro; Yano, Shinichi; Fang, Xu; Endo, Takashi; Sawayama, Shigeki

    2011-02-01

    Cellulases and hemicellulases are key enzymes in the production of alternative fuels and chemicals from lignocellulosic biomass-an abundant renewable resource. Carbon source selection is an important factor in the production of cellulases and hemicellulases. Rice straw--a potential ethanol source--has recently gained considerable interest in Asian countries. Here, we investigated the production of cellulases by using rice straw subjected to various pretreatments as substrates in order to produce cellulases at low costs; we also identified the enzymes' characteristics. Rice straw cutter milled to <3mm was pretreated by wet disk milling, dry ball milling, or hot-compressed water treatment (HCWT). Pretreated rice straw and commercial cellulose, Solka Floc (SF), were used as carbon sources for cellulase production by the fungus Acremonium cellulolyticus. Filter paper cellulase, β-xylanase, and β-xylosidase production from ball- and disk-milled samples were higher than those from SF. Enzymatic activity was absent in cultures where HCWT rice straw was used as carbon source. Wet disk-milled rice straw cultures were more suitable for enzymatic hydrolysis of pretreated rice straw than SF cultures. Thus, wet disk milling may be a suitable pretreatment for producing substrates for enzymatic hydrolysis and generating inexpensive carbon sources for cellulase production. PMID:22112826

  18. Prostaglandin E1 in hand angiography

    SciTech Connect

    Levy, J.M.; Joseph, R.B.; Bodell, L.S.; Nykamp, P.W.; Hessel, S.J.

    1983-11-01

    Prostaglandin E1 (PG1) is a rapid, potent vasodilator which, when infused into the arterial system in low doses by bolus injection, has no significant systemic effects and has a relatively long duration of action. Sixty-three hand angiograms were done on 55 patients, comparing PGE1 to tolazoline and to angiograms done with no vasodilation. There was no significant difference between PGE1 and tolazoline in digital artery opacification; however, venous opacification was very significantly better with PGE1. PGE1 should be a drug of choice in hand angiography.

  19. Adenovirus E1A/E1B Transformed Amniotic Fluid Cells Support Human Cytomegalovirus Replication

    PubMed Central

    Krömmelbein, Natascha; Wiebusch, Lüder; Schiedner, Gudrun; Büscher, Nicole; Sauer, Caroline; Florin, Luise; Sehn, Elisabeth; Wolfrum, Uwe; Plachter, Bodo

    2016-01-01

    The human cytomegalovirus (HCMV) replicates to high titers in primary human fibroblast cell cultures. A variety of primary human cells and some tumor-derived cell lines do also support permissive HCMV replication, yet at low levels. Cell lines established by transfection of the transforming functions of adenoviruses have been notoriously resistant to HCMV replication and progeny production. Here, we provide first-time evidence that a permanent cell line immortalized by adenovirus type 5 E1A and E1B (CAP) is supporting the full HCMV replication cycle and is releasing infectious progeny. The CAP cell line had previously been established from amniotic fluid cells which were likely derived from membranes of the developing fetus. These cells can be grown under serum-free conditions. HCMV efficiently penetrated CAP cells, expressed its immediate-early proteins and dispersed restrictive PML-bodies. Viral DNA replication was initiated and viral progeny became detectable by electron microscopy in CAP cells. Furthermore, infectious virus was released from CAP cells, yet to lower levels compared to fibroblasts. Subviral dense bodies were also secreted from CAP cells. The results show that E1A/E1B expression in transformed cells is not generally repressive to HCMV replication and that CAP cells may be a good substrate for dense body based vaccine production. PMID:26848680

  20. In Vitro and In Vivo Biology of Recombinant Adenovirus Vectors with E1, E1/E2A, or E1/E4 Deleted

    PubMed Central

    Lusky, M.; Christ, M.; Rittner, K.; Dieterle, A.; Dreyer, D.; Mourot, B.; Schultz, H.; Stoeckel, F.; Pavirani, A.; Mehtali, M.

    1998-01-01

    Isogenic, E3-deleted adenovirus vectors defective in E1, E1 and E2A, or E1 and E4 were generated in complementation cell lines expressing E1, E1 and E2A, or E1 and E4 and characterized in vitro and in vivo. In the absence of complementation, deletion of both E1 and E2A completely abolished expression of early and late viral genes, while deletion of E1 and E4 impaired expression of viral genes, although at a lower level than the E1/E2A deletion. The in vivo persistence of these three types of vectors was monitored in selected strains of mice with viral genomes devoid of transgenes to exclude any interference by immunogenic transgene-encoded products. Our studies showed no significant differences among the vectors in the short-term maintenance and long-term (4-month) persistence of viral DNA in liver and lung cells of immunocompetent and immunodeficient mice. Furthermore, all vectors induced similar antibody responses and comparable levels of adenovirus-specific cytotoxic T lymphocytes. These results suggest that in the absence of transgenes, the progressive deletion of the adenovirus genome does not extend the in vivo persistence of the transduced cells and does not reduce the antivirus immune response. In addition, our data confirm that, in the absence of transgene expression, mouse cellular immunity to viral antigens plays a minor role in the progressive elimination of the virus genome. PMID:9499056

  1. 26 CFR 1.167(e)-1 - Change in method.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... to December 30, 2003 (§ 1.167(e)-1 as contained in 26 CFR part 1 edition revised as of April 1, 2003). ... 26 Internal Revenue 2 2010-04-01 2010-04-01 false Change in method. 1.167(e)-1 Section 1.167(e)-1...) INCOME TAXES (CONTINUED) Itemized Deductions for Individuals and Corporations § 1.167(e)-1 Change...

  2. CYP2E1 and Oxidative Liver Injury by Alcohol

    PubMed Central

    Lu, Yongke; Cederbaum, Arthur I.

    2008-01-01

    Ethanol-induced oxidative stress appears to play a major role in mechanisms by which ethanol causes liver injury. Many pathways have been suggested to contribute to the ability of ethanol to induce a state of oxidative stress. One central pathway appears to be the induction of cytochrome P450 2E1 (CYP2E1) by ethanol. CYP2E1 metabolizes and activates many toxicological substrates, including ethanol, to more reactive, toxic products. Levels of CYP2E1 are elevated under a variety of physiological and pathophysiological conditions, and after acute and chronic alcohol treatment. CYP2E1 is also an effective generator of reactive oxygen species such as the superoxide anion radical and hydrogen peroxide, and in the presence of iron catalysts, produces powerful oxidants such as the hydroxyl radical. This Review Article summarizes some of the biochemical and toxicological properties of CYP2E1, and briefly describes the use of cell lines developed to constitutively express CYP2E1 in assessing the actions of CYP2E1. Possible therapeutic implications for treatment of alcoholic liver injury by inhibition of CYP2E1 or CYP2E1-dependent oxidative stress will be discussed, followed by some future directions which may help to understand the actions of CYP2E1 and its role in alcoholic liver injury. PMID:18078827

  3. Activation of adenovirus 5 E1A transcription by region E1B in transformed primary rat cells.

    PubMed Central

    Jochemsen, A G; Peltenburg, L T; te Pas, M F; de Wit, C M; Bos, J L; van der Eb, A J

    1987-01-01

    The human adenovirus 5 E1A region can immortalize primary cultures of baby rat kidney cells, but requires the presence of the E1B region for complete oncogenic transformation. One of the effects of the E1B region in the transformation process is the activation of E1A expression. We have investigated the mechanism of this stimulation of E1A expression using nuclear run-on assays with nuclei from Ad5 E1A- and Ad5 E1-transformed cells. It was found that E1B enhances E1A at the level of transcription-initiation. This activation is mainly observed when the E1A and E1B regions are integrated simultaneously into the cellular genome and only minimally when these genes are integrated separately, strongly suggesting that a close physical linkage of these regions is essential for the observed effect. Images Fig. 1. Fig. 2. Fig. 3. Fig. 4. Fig. 5. Fig. 6. PMID:2962857

  4. Activation of NF-κB by Human Papillomavirus 16 E1 Limits E1-Dependent Viral Replication through Degradation of E1

    PubMed Central

    Nakahara, Tomomi; Tanaka, Katsuyuki; Ohno, Shin-ichi; Egawa, Nagayasu; Yugawa, Takashi

    2015-01-01

    ABSTRACT NF-κB is a family of transcription factors that regulate gene expression involved in many processes, such as the inflammatory response and cancer progression. Little is known about associations of NF-κB with the human papillomavirus (HPV) life cycle. We have developed a tissue culture system to conditionally induce E1-dependent replication of the human papillomavirus 16 (HPV16) genome in human cervical keratinocytes and found that expression of HPV16 E1, a viral helicase, results in reduction of IκBα and subsequent activation of NF-κB in a manner dependent on helicase activity. Exogenous expression of a degradation-resistant mutant of IκBα, which inhibits the activation of NF-κB, enhanced E1-dependent replication of the viral genome. Wortmannin, a broad inhibitor of phosphoinositide 3-kinases (PI3Ks), and, to a lesser extent, VE-822, an ATR kinase inhibitor, but not KU55933, an ATM kinase inhibitor, suppressed the activation of NF-κB and augmented E1-dependent replication of the HPV16 genome. Interestingly, the enhancement of E1-dependent replication of the viral genome was associated with increased stability of E1 in the presence of wortmannin as well as the IκBα mutant. Collectively, we propose that expression of E1 induces NF-κB activation at least in part through the ATR-dependent DNA damage response and that NF-κB in turn limits E1-dependent replication of HPV16 through degradation of E1, so that E1 and NF-κB may constitute a negative feedback loop. IMPORTANCE A major risk factor in human papillomavirus (HPV)-associated cancers is persistent infection with high-risk HPVs. To eradicate viruses from infected tissue, it is important to understand molecular mechanisms underlying the establishment and maintenance of persistent infection. In this study, we obtained evidence that human papillomavirus 16 (HPV16) E1, a viral DNA helicase essential for amplification of the viral genomes, induces NF-κB activation and that this limits E1-dependent

  5. The nuclear receptor NR2E1/TLX controls senescence

    PubMed Central

    Krusche, Benjamin; Pemberton, Helen; Alonso, Marta M.; Chandler, Hollie; Brookes, Sharon; Parrinello, Simona; Peters, Gordon; Gil, Jesús

    2014-01-01

    The nuclear receptor NR2E1 (also known as TLX or tailless) controls the self-renewal of neural stem cells (NSCs) and has been implied as an oncogene which initiates brain tumours including glioblastomas. Despite NR2E1 regulating targets like p21CIP1 or PTEN we still lack a full explanation for its role in NSC self-renewal and tumorigenesis. We know that Polycomb repressive complexes (PRC) also control stem cell self-renewal and tumorigenesis, but so far, no formal connection has been established between NR2E1 and PRCs. In a screen for transcription factors regulating the expression of the Polycomb protein CBX7, we identified NR2E1 as one of its more prominent regulators. NR2E1 binds at the CBX7 promoter, inducing its expression. Notably CBX7 represses NR2E1 as part of a regulatory loop. Ectopic NR2E1 expression inhibits cellular senescence, extending cellular lifespan in fibroblasts via CBX7-mediated regulation of p16INK4a and direct repression of p21CIP1. In addition NR2E1 expression also counteracts oncogene-induced senescence (OIS). The importance of NR2E1 to restrain senescence is highlighted through the process of knocking down its expression, which causes premature senescence in human fibroblasts and epithelial cells. We also confirmed that NR2E1 regulates CBX7 and restrains senescence in NSCs. Finally, we observed that the expression of NR2E1 directly correlates with that of CBX7 in human glioblastoma multiforme. Overall we identified control of senescence and regulation of Polycomb action as two possible mechanisms that can join those so far invoked to explain the role of NR2E1 in control of NSC self-renewal and cancer. PMID:25328137

  6. Chemical characteristics for optimizing CYP2E1 inhibition.

    PubMed

    van de Wier, B; Balk, J M; Bast, A; Koek, G H; Haenen, G R M M

    2015-12-01

    Cytochrome P450 2E1 (CYP2E1) expression and activity in the liver is associated with the degree of liver damage in patients with alcoholic steatohepatitis (ASH) as well as non-alcoholic steatohepatitis (NASH). CYP2E1 is known to generate reactive oxygen species, which leads to oxidative stress, one of the hallmarks of both diseases. Apart from ROS, toxic metabolites can be formed by CYP2E1 metabolism, further potentiating liver injury. Therefore, CYP2E1 is implicated in the pathogenesis of ASH and NASH. The aim of this study was to determine the chemical characteristics of compounds that are important to inhibit CYP2E1. To this end, structurally related analogs that differed in their lipophilic, steric and electronic properties were tested. In addition, homologues series of aliphatic primary alcohols, secondary alcohols, aldehydes, ketones and carboxylic acids were tested. It was found that inhibition of the CYP2E1 activity is primarily governed by lipophilicity. The optimal log D7.4 (octanol/water distribution coefficient at pH 7.4) value for inhibition of CYP2E1 was approximately 2.4. In the carboxylic acids series the interaction of the carboxylate group with polar residues lining the CYP2E1 active site also has to be considered. This study sketches the basic prerequisites in the search for inhibitors of CYP2E1, which would strengthen our therapeutic armamentarium against CYP2E1 associated diseases, such as ASH and NASH. PMID:26428356

  7. 26 CFR 1.665(e)-1 - Preceding taxable year.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... 26 Internal Revenue 8 2010-04-01 2010-04-01 false Preceding taxable year. 1.665(e)-1 Section 1.665... (CONTINUED) INCOME TAXES Treatment of Excess Distributions of Trusts Applicable to Taxable Years Beginning Before January 1, 1969 § 1.665(e)-1 Preceding taxable year. (a) Definition. For purposes of subpart...

  8. 26 CFR 1.665(e)-1 - Preceding taxable year.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... 26 Internal Revenue 8 2011-04-01 2011-04-01 false Preceding taxable year. 1.665(e)-1 Section 1.665... (CONTINUED) INCOME TAXES (CONTINUED) Treatment of Excess Distributions of Trusts Applicable to Taxable Years Beginning Before January 1, 1969 § 1.665(e)-1 Preceding taxable year. (a) Definition. For purposes...

  9. 26 CFR 1.927(e)-1 - Special sourcing rule.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ...(e)-1 Internal Revenue INTERNAL REVENUE SERVICE, DEPARTMENT OF THE TREASURY (CONTINUED) INCOME TAX (CONTINUED) INCOME TAXES (CONTINUED) Earned Income of Citizens of United States § 1.927(e)-1 Special sourcing rule. (a) Source rules for related persons—(1) In general. The income of a person described in...

  10. 26 CFR 1.927(e)-1 - Special sourcing rule.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ...(e)-1 Internal Revenue INTERNAL REVENUE SERVICE, DEPARTMENT OF THE TREASURY (CONTINUED) INCOME TAX (CONTINUED) INCOME TAXES Earned Income of Citizens of United States § 1.927(e)-1 Special sourcing rule. (a) Source rules for related persons—(1) In general. The income of a person described in section 482 from...

  11. 26 CFR 1.514(e)-1 - Allocation rules.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... 26 Internal Revenue 7 2010-04-01 2010-04-01 true Allocation rules. 1.514(e)-1 Section 1.514(e)-1 Internal Revenue INTERNAL REVENUE SERVICE, DEPARTMENT OF THE TREASURY (CONTINUED) INCOME TAX (CONTINUED) INCOME TAXES (CONTINUED) Taxation of Business Income of Certain Exempt Organizations §...

  12. 26 CFR 1.642(e)-1 - Depreciation and depletion.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... 26 Internal Revenue 8 2010-04-01 2010-04-01 false Depreciation and depletion. 1.642(e)-1 Section 1... (CONTINUED) INCOME TAXES Estates, Trusts, and Beneficiaries § 1.642(e)-1 Depreciation and depletion. An estate or trust is allowed the deductions for depreciation and depletion, but only to the extent...

  13. 26 CFR 1.665(e)-1 - Preceding taxable year.

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ... 26 Internal Revenue 8 2012-04-01 2012-04-01 false Preceding taxable year. 1.665(e)-1 Section 1.665... (CONTINUED) INCOME TAXES (CONTINUED) Treatment of Excess Distributions of Trusts Applicable to Taxable Years Beginning Before January 1, 1969 § 1.665(e)-1 Preceding taxable year. (a) Definition. For purposes...

  14. 26 CFR 1.665(e)-1 - Preceding taxable year.

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ... 26 Internal Revenue 8 2014-04-01 2014-04-01 false Preceding taxable year. 1.665(e)-1 Section 1.665... (CONTINUED) INCOME TAXES (CONTINUED) Treatment of Excess Distributions of Trusts Applicable to Taxable Years Beginning Before January 1, 1969 § 1.665(e)-1 Preceding taxable year. (a) Definition. For purposes...

  15. Resolvin E1 Regulates ADP Activation of Human Platelets

    PubMed Central

    Fredman, Gabrielle; Van Dyke, Thomas E.; Serhan, Charles N.

    2010-01-01

    Objective Resolvin E1 (RvE1) is an eicosapentaenoic acid (EPA)-derived specialized pro-resolving mediator generated during resolution of acute inflammation. RvE1 exhibits potent organ-protective actions in vivo and acts on specific cell types including platelets. Here, we investigated the ability of RvE1 to regulate adenosine diphosphate (ADP) activation of platelets via specific receptors because RvE1 reduces platelet aggregation with certain agonists including ADP. Methods and Results RvE1 (0.1nM–100nM) incubated with platelets gave reduced ADP-stimulated P-selectin mobilization (IC50 ~1.6×10−12 M) and polymerized actin content compared to control platelets. RvE1 (1–100nM) did not stimulate or block intracellular calcium mobilization. Using a new P2Y12-β-arrestin-coupled cell system, ADP-activated P2Y12 with an EC50 of 5×10−6 M and RvE1 did not directly stimulate P2Y12 or block ADP-P2Y12 signals. In this system, another eicosanoid LTE4 (EC50 1.3×10−11 M) dose dependently activated P2Y12. When recombinant P2Y12-expressing cells were transiently transfected with an RvE1 receptor, human ChemR23 (present on human platelets), addition of RvE1 (0.1nM-10.0nM) blocked ADP signals (IC50 ~1.6×10−11 M) in P2Y12-ChemR23-expressing cells compared to mock transfections. Conclusions These results demonstrate that RvE1’s regulatory actions (i.e reducing ADP-stimulated P-selectin mobilization and actin polymerization) are hChemR23-dependent. Moreover, they document specific platelet actions of RvE1 selectively engaged with ADP-activated platelets that illuminate a new cellular mechanism and impact of omega-3 EPA that may contribute to both resolution of vascular inflammation and ADP-dependent platelet activation relevant in pathologic cardiovascular events. PMID:20702811

  16. Establishment of Class e1 Mass Standard of 50 kg

    NASA Astrophysics Data System (ADS)

    Yao, Hong; Wang, Jian; Ding, Jingan; Zhong, Ruilin; Ren, Xiaoping

    Because of the equipment limit, the dissemination of large mass has been realized by a large amount of higher class of 20 kg weights since 1950s in China. But with improvement of the technique and customer's requirements, it is necessary to establish the mass standard of 50 kg weight. In 1990s, mass standard laboratory has set up Class E1 weight sets from 20 kg to 1 mg. To extend the mass capacity up to 50 kg of Class E1, it is not only to produce Class E1 50 kg weight and import a mass comparator, but also need to lift the heavy weight from weight box to balance receptor safely. Up to now, the mass comparator has been installed in Hepingli campus of NIM. Two pieces of Class E1 50 kg weights are determined by combination weighing method. A lifting device has been mounted close to the mass comparator in order to move the 50 kg easily.

  17. Influence of neutron surface on E1 resonance properties

    NASA Astrophysics Data System (ADS)

    Goncharova, N. G.; Tretyakova, T. Yu.; Fedorov, N. A.

    2016-01-01

    The E1 strength distributions in even-even Si isotopes were calculated in the "particle-core coupling" version of the shell model taking into account the fragmentation of the hole configuration among the states of the daughter nuclei. The comparison of calculated strength distributions in different isotopes of the same element shows the peculiarities of a neutron surface influence on the E1 resonance fragmentation.

  18. A Two-Photon E1-M1 Optical Clock

    NASA Astrophysics Data System (ADS)

    Alden, Emily A.

    Innovations in precision frequency measurement advance popular technologies such as global positioning systems (GPS), permit the testing of fundamental physics constants, and have the potential to measure local variations in gravity. Driving optical transitions for frequency measurement using an E1-M1 excitation scheme in a hot mercury (Hg) vapor cell is viable and could be the basis of a portable optical frequency standard with comparable accuracy to the most precise atomic clocks in the world. This dissertation explores the fundamental physics of the new E1-M1 method of high-precision frequency measurement in an optical, atomic clock and describes the construction of a high-power E1-M1 clock laser. The value of this new scheme compared to existing optical frequency standards is the simplicity and portability of the experimental setup. Such an optical frequency standard would permit frequency measurement in far-flung locations on earth and in space. Analysis of both the E1-M1 optical transition and thermal properties of the candidate clock atoms are presented. These models allow a stability estimate of an E1-M1 optical clock and recommend experimental settings to optimize the standard. The experimental work that has been performed in pursuit of observing the E1-M1 clock transition in Hg is also discussed. An optical clock operates by making a precision frequency measurement of a laser that has been brought into resonance with a clock atom's oscillator: a high quality atomic level transition. Group II type atoms, such as Hg, have the 1S0-3P0 transition that is an ideal basis for a clock. The E1-M1 excitation is performed by driving the two-photon allowed transition 1S0-3P1-3P0. This is in contrast to the single-photon E1 transition used in other systems. Single-photon schemes must use ultracold atoms to reduce atomic motion to attain high levels of accuracy. Driving the clock transition with a pair of degenerate counter-propagating photons in an E1-M1 scheme

  19. Impotence evaluated by the use of prostaglandin E1

    SciTech Connect

    Hwang, T.I.; Yang, C.R.; Wang, S.J.; Chang, C.L.; Tzai, T.S.; Chang, C.H.; Wu, H.C.

    1989-06-01

    We screened 80 patients at our hospital for the differential diagnosis of impotence using intracavernous injection of prostaglandin E1 (20 micrograms). The rate of positive response was 78.8 per cent (63 patients). Neither systemic reactions nor priapism occurred. However, a considerable incidence (23.8 per cent, 19 of 80 patients) of tolerable injection pain was encountered. The 133-xenon penile washout study was conducted routinely in impotent men for hemodynamic evaluation of penile vascularity. In 80 patients a positive correlation between the response of intracavernous prostaglandin E1 injection and the result of the washout study was found (r equals 0.381, p less than 0.0002). We selected 14 subjects randomly to receive additional intravenous infusions of prostaglandin E1 (6 ampules, 120 micrograms total) for 3 days, after which another 133-xenon washout study was done. The washout studies before and after intravenous prostaglandin E1 infusion were compared, and 10 patients (71.4 per cent) appeared to obtain improvement in half-time clearance and penile blood flow. However, only 3 patients noticed improvement subjectively. We suggest that prostaglandin E1 could be a desirable alternative for the diagnosis and treatment of impotence.

  20. Oncolytic Replication of E1b-Deleted Adenoviruses

    PubMed Central

    Cheng, Pei-Hsin; Wechman, Stephen L.; McMasters, Kelly M.; Zhou, Heshan Sam

    2015-01-01

    Various viruses have been studied and developed for oncolytic virotherapies. In virotherapy, a relatively small amount of viruses used in an intratumoral injection preferentially replicate in and lyse cancer cells, leading to the release of amplified viral particles that spread the infection to the surrounding tumor cells and reduce the tumor mass. Adenoviruses (Ads) are most commonly used for oncolytic virotherapy due to their infection efficacy, high titer production, safety, easy genetic modification, and well-studied replication characteristics. Ads with deletion of E1b55K preferentially replicate in and destroy cancer cells and have been used in multiple clinical trials. H101, one of the E1b55K-deleted Ads, has been used for the treatment of late-stage cancers as the first approved virotherapy agent. However, the mechanism of selective replication of E1b-deleted Ads in cancer cells is still not well characterized. This review will focus on three potential molecular mechanisms of oncolytic replication of E1b55K-deleted Ads. These mechanisms are based upon the functions of the viral E1B55K protein that are associated with p53 inhibition, late viral mRNA export, and cell cycle disruption. PMID:26561828

  1. Oncolytic Replication of E1b-Deleted Adenoviruses.

    PubMed

    Cheng, Pei-Hsin; Wechman, Stephen L; McMasters, Kelly M; Zhou, Heshan Sam

    2015-11-01

    Various viruses have been studied and developed for oncolytic virotherapies. In virotherapy, a relatively small amount of viruses used in an intratumoral injection preferentially replicate in and lyse cancer cells, leading to the release of amplified viral particles that spread the infection to the surrounding tumor cells and reduce the tumor mass. Adenoviruses (Ads) are most commonly used for oncolytic virotherapy due to their infection efficacy, high titer production, safety, easy genetic modification, and well-studied replication characteristics. Ads with deletion of E1b55K preferentially replicate in and destroy cancer cells and have been used in multiple clinical trials. H101, one of the E1b55K-deleted Ads, has been used for the treatment of late-stage cancers as the first approved virotherapy agent. However, the mechanism of selective replication of E1b-deleted Ads in cancer cells is still not well characterized. This review will focus on three potential molecular mechanisms of oncolytic replication of E1b55K-deleted Ads. These mechanisms are based upon the functions of the viral E1B55K protein that are associated with p53 inhibition, late viralmRNAexport, and cell cycle disruption. PMID:26561828

  2. Acetaldehyde and parkinsonism: role of CYP450 2E1

    PubMed Central

    Vaglini, Francesca; Viaggi, Cristina; Piro, Valentina; Pardini, Carla; Gerace, Claudio; Scarselli, Marco; Corsini, Giovanni Umberto

    2013-01-01

    The present review update the relationship between acetaldehyde (ACE) and parkinsonism with a specific focus on the role of P450 system and CYP 2E1 isozyme particularly. We have indicated that ACE is able to enhance the parkinsonism induced in mice by 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine, a neurotoxin able to damage the nigrostriatal dopaminergic pathway. Similarly diethyldithiocarbamate, the main metabolite of disulfiram, a drug widely used to control alcoholism, diallylsulfide (DAS) and phenylisothiocyanate also markedly enhance the toxin-related parkinsonism. All these compounds are substrate/inhibitors of CYP450 2E1 isozyme. The presence of CYP 2E1 has been detected in the dopamine (DA) neurons of rodent Substantia Nigra (SN), but a precise function of the enzyme has not been elucidated yet. By treating CYP 2E1 knockout (KO) mice with the neurotoxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine, the SN induced lesion was significantly reduced when compared with the lesion observed in wild-type animals. Several in vivo and in vitro studies led to the conclusion that CYP 2E1 may enhance the 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine toxicity in mice by increasing free radical production inside the dopaminergic neurons. ACE is a good substrate for CYP 2E1 enzyme as the other substrate-inhibitors and by this way may facilitate the susceptibility of dopaminergic neurons to toxic events. The literature suggests that ethanol and/or disulfiram may be responsible for toxic parkinsonism in human and it indicates that basal ganglia are the major targets of disulfiram toxicity. A very recent study reports that there are a decreased methylation of the CYP 2E1 gene and increased expression of CYP 2E1 mRNA in Parkinson's disease (PD) patient brains. This study suggests that epigenetic variants of this cytochrome contribute to the susceptibility, thus confirming multiples lines of evidence which indicate a link between environmental toxins and PD. PMID:23801948

  3. A Conserved Regulatory Module at the C Terminus of the Papillomavirus E1 Helicase Domain Controls E1 Helicase Assembly

    PubMed Central

    Schuck, Stephen

    2014-01-01

    ABSTRACT Viruses frequently combine multiple activities into one polypeptide to conserve coding capacity. This strategy creates regulatory challenges to ascertain that the combined activities are compatible and do not interfere with each other. The papillomavirus E1 protein, as many other helicases, has the intrinsic ability to form hexamers and double hexamers (DH) that serve as the replicative DNA helicase. However, E1 also has the more unusual ability to generate local melting by forming a double trimer (DT) complex that can untwist the double-stranded origin of DNA replication (ori) DNA in preparation for DH formation. Here we describe a switching mechanism that allows the papillomavirus E1 protein to form these two different kinds of oligomers and to transition between them. We show that a conserved regulatory module attached to the E1 helicase domain blocks hexamer and DH formation and promotes DT formation. In the presence of the appropriate trigger, the inhibitory effect of the regulatory module is relieved and the transition to DH formation can occur. IMPORTANCE This study provides a mechanistic understanding into how a multifunctional viral polypeptide can provide different, seemingly incompatible activities. A conserved regulatory sequence module attached to the AAA+ helicase domain in the papillomavirus E1 protein allows the formation of different oligomers with different biochemical activities. PMID:25378487

  4. Multi-Laboratory Validation of Estrone (E1) ELISA Methods

    EPA Science Inventory

    This project is a round-robin evaluation of commercially available Enzyme-Linked Immunosorbent Assay (ELISA) technology to quantitatively or qualitatively measure the hormone estrone (E1) in combined animal feeding operation (CAFO) receiving streams. ELISA is meant to be a simpl...

  5. 26 CFR 53.4941(e)-1 - Definitions.

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ... TAXES (CONTINUED) FOUNDATION AND SIMILAR EXCISE TAXES Taxes on Self-Dealing § 53.4941(e)-1 Definitions... and the liabilities of the parties have been fixed. Thus, for example, if a private foundation gives a disqualified person a binding option on June 15, 1971, to purchase property owned by the foundation at any...

  6. 26 CFR 53.4941(e)-1 - Definitions.

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    ... TAXES (CONTINUED) FOUNDATION AND SIMILAR EXCISE TAXES Taxes on Self-Dealing § 53.4941(e)-1 Definitions... and the liabilities of the parties have been fixed. Thus, for example, if a private foundation gives a disqualified person a binding option on June 15, 1971, to purchase property owned by the foundation at any...

  7. 26 CFR 53.4941(e)-1 - Definitions.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... TAXES (CONTINUED) FOUNDATION AND SIMILAR EXCISE TAXES Taxes on Self-Dealing § 53.4941(e)-1 Definitions... and the liabilities of the parties have been fixed. Thus, for example, if a private foundation gives a disqualified person a binding option on June 15, 1971, to purchase property owned by the foundation at any...

  8. 26 CFR 53.4941(e)-1 - Definitions.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... TAXES (CONTINUED) FOUNDATION AND SIMILAR EXCISE TAXES Taxes on Self-Dealing § 53.4941(e)-1 Definitions... and the liabilities of the parties have been fixed. Thus, for example, if a private foundation gives a disqualified person a binding option on June 15, 1971, to purchase property owned by the foundation at any...

  9. 26 CFR 53.4941(e)-1 - Definitions.

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ... TAXES (CONTINUED) FOUNDATION AND SIMILAR EXCISE TAXES Taxes on Self-Dealing § 53.4941(e)-1 Definitions... and the liabilities of the parties have been fixed. Thus, for example, if a private foundation gives a disqualified person a binding option on June 15, 1971, to purchase property owned by the foundation at any...

  10. 24. SPILLWAY CHANNEL WALLS REINFORCEMENT DETAILS; MONOLITHS E1 TO ...

    Library of Congress Historic Buildings Survey, Historic Engineering Record, Historic Landscapes Survey

    24. SPILLWAY CHANNEL WALLS - REINFORCEMENT DETAILS; MONOLITHS E-1 TO F-4 INCL. & NO. 34. Sheet S-11, June, 1939. File no. SA 342/24(?). - Prado Dam, Spillway, Santa Ana River near junction of State Highways 71 & 91, Corona, Riverside County, CA

  11. RP-1 delivered to E-1 Test Stand

    NASA Technical Reports Server (NTRS)

    2010-01-01

    NASA John C. Stennis Space Center employee Dustan Ladner (left) assists tanker driver David Velasco in transferring RP-1 fuel to a 20,000-gallon underground tank at the E-1 Test Stand during a March 30 delivery. The rocket propellant will be used for testing Aerojet AJ26 rocket engines beginning this summer. Stennis is testing the engines for Orbital Sciences Corporation, which has partnered with NASA to provide eight supply missions to the International Space Station through 2015. The partnership is part of NASA's Commercial Orbital Transportation Services initiative to work closer with companies to provide commercial space transport once the space shuttle is retired later this year.

  12. E1 strength in Pb208 within the shell model

    NASA Astrophysics Data System (ADS)

    Schwengner, R.; Massarczyk, R.; Brown, B. A.; Beyer, R.; Dönau, F.; Erhard, M.; Grosse, E.; Junghans, A. R.; Kosev, K.; Nair, C.; Rusev, G.; Schilling, K. D.; Wagner, A.

    2010-05-01

    The dipole response of the doubly magic nuclide Pb208 was studied in photon-scattering experiments at the electron linear accelerator ELBE with bremsstrahlung produced at kinetic electron energies of 9.0 and 15.0 MeV. The present (γ,γ') data combined with (γ,n) data from the literature are compared with results of shell-model calculations and calculations using a quasiparticle random-phase approximation. The shell-model calculations including (2p-2h) excitations describe the experimental E1 strength well and reproduce the spreading of the giant dipole resonance by applying a small smearing width only.

  13. Octupole deformation in sup 221 Fr; E1 transition rates

    SciTech Connect

    Liang, C.F.; Peghaire, A. ); Sheline, R.K. )

    1990-07-10

    Experimental data following the alpha decay of{sup 225}Ac are interpreted in terms of a spectroscopy in {sup 221}Fr consistent with octupole deformation. However, the measured E1 transition probabilities suggest that the low lying bands in {sup 221}Fr are considerably more mixed than in nuclei with slightly higher mass number. It is suggested that this mixing of states in {sup 221}Fr is indicative of the partial collapse of Nilsson-like orbitals into more degenerate shell model orbitals.

  14. Crossover Analysis of CHANG'E-1 Laser Altimeter Data

    NASA Astrophysics Data System (ADS)

    Hu, W.; Yue, Z.; Di, K.

    2011-08-01

    This paper presents a preliminary result of crossover analysis and adjustment of Chang'E-1(CE-1) Laser Altimeter (LAM) data of the Moon for global and regional mapping applications. During the operation of Chang'E-1 from November 28, 2007 to December 4, 2008, the laser altimeter acquired 1400 orbital profiles with about 9.12 million altimetric points. In our experiment, we derived more than 1.38 million crossovers from 1395 ground tracks covering the entire lunar surface after eliminating outliers of orbits and altimetric points. A method of least-squares crossover adjustment with a series of basis functions of time (trigonometric functions and polynomials) is developed to reconcile the LAM data by minimizing the crossover residuals globally. The normal equations are very large but sparse; therefore they are stored and solved using sparse matrix technique. In a test area (0°N~60°N, 50°W~0°W), the crossover residuals are reduced from 62.1m to 32.8m, and the quality of the DEM generated from the adjusted LAM data is improved accordingly. We will optimize the method for the global adjustment to generate a high precision consistent global DEM, which can be used as absolute control for lunar mapping with orbital images.

  15. Origin of replication of colicin E1 plasmid DNA.

    PubMed Central

    Tomizawa, J I; Ohmori, H; Bird, R E

    1977-01-01

    Cleavage maps of colicin E1 plasmid DNA and its smaller derivative, pNT1 DNA, were constructed by using restriction endonucleases. The nucleotide sequence of a region that contains the orgin of replication was determined. The site of the nucleotide from which DNA replication is initiated was determined with 6S L-fragments, the DNA fragment first made on colicin E1 plasmid DNA. The fragments were labeled with [gamma-32P]ATP and polynucleotide 5'-hydroxyl-kinase (ATP:5'-dephosphopolynucleotide 5'-phosphotransferase, EC 2.7.1.78) at the 5'-OH groups which were uncovered by alkali treatment. The site is one of three consecutive nucleotides, dA, dA, and dC, located at a unique position. One or a few rA residues were found to be attached to some of the DNA molecules. The transition from the primer RNA to DNA occurs in a region consisting of a segment of five A residues. Both sides of this segment are rich in G and C. Images PMID:325558

  16. Metabolism of prostaglandin E1 in dog kidneys

    PubMed Central

    Nakano, J.

    1970-01-01

    1. The biotransformation of prostaglandin E1 (PGE1) was studied in the isolated, perfused dog kidneys. 2. An average 43% of PGE1 was converted into the less polar metabolite I by a single passage through the kidney. As the re-circulation of the perfusate continued, PGE1 was converted not only into metabolite I but also the least polar metabolite II. The velocity of the conversion of PGE1 into metabolite I was significantly greater than that into metabolite II. Usually, six passages elapsed before maximum degradation of PGE1 occurred. 3. Further separation with silicic acid column chromatography and gas-liquid chromatography showed that metabolite II consists of two individual metabolites, metabolite IIa and metabolite IIb. 4. The present study indicates that the kidney biotransforms PGE1 rather rapidly into three metabolites which are less polar than PGE1. PMID:5492900

  17. Transgenic Plants Lower the Costs of Cellulosic Biofuels (Fact Sheet)

    SciTech Connect

    Not Available

    2011-11-01

    A new transgenic maize was observed to be less recalcitrant than wild-type biomass, as manifested through lower severity requirements to achieve comparable levels of conversion. Expression of a single gene derived from bacteria in plants has resulted in transgenic plants that are easier and cheaper to convert into biofuels. Part of the high production cost of cellulosic biofuels is the relatively poor accessibility of substrates to enzymes due to the strong associations between plant cell wall components. This biomass recalcitrance makes costly thermochemical pretreatment necessary. Scientists at the National Renewable Energy Laboratory (NREL) have created transgenic maize expressing an active glycosyl hydrolase enzyme, E1 endoglucanase, originally isolated from a thermophilic bacterium, Acidothermus cellulolyticus. This engineered feedstock was observed to be less recalcitrant than wild-type biomass when subjected to reduced severity pretreatments and post-pretreatment enzymatic hydrolysis. This reduction in recalcitrance was manifested through lower severity requirements to achieve comparable levels of conversion of wild-type biomass. The improvements observed are significant enough to positively affect the economics of the conversion process through decreased capital construction costs and decreased degradation products and inhibitor formation.

  18. E1-forbidden transition rates in ions of astrophysical interest

    NASA Astrophysics Data System (ADS)

    Träbert, E.

    2014-11-01

    Transition rates in atomic systems may appear to be of little importance in steady-state plasmas that are observed at great distances from Earth. However, some of the transition rates compete with collision rates, and in these cases certain line intensity ratios are affected and can serve as remote indicators of density. In the low-density environments of stellar coronae and planetary nebulae, the transition rates of interest are mostly spin-forbidden E1 decays, higher-multipole order transitions (M1, E2, M2, M3), and hyperfine-induced transitions. On Earth, measurements of the long upper level lifetimes of these atomic systems require the use of ion traps. A fair number of test cases with lifetimes in the range from nanoseconds to many seconds have been treated successfully, and the evolution of calculations along with the experimental progress is notable. A new generation of cold ion traps is expected to extend the atomic lifetime measurements on multiply charged ions into the range of many minutes.

  19. Human adenovirus 2 E1B-19K and E1B-53K tumor antigens: antipeptide antibodies targeted to the NH2 and COOH termini.

    PubMed Central

    Green, M; Brackmann, K H; Lucher, L A; Symington, J S; Kramer, T A

    1983-01-01

    The human adenovirus 2 (Ad2) transforming region is located in the left 11.1% of the viral genome and encodes two early transcription units, E1A and E1B. Based on the amino acid sequence deduced from the Ad2 E1B DNA sequence (Gingeras et al., J. Biol. Chem. 257:13475-13491, 1982), we have prepared antibodies against synthetic peptides, 8 to 16 amino acids in length, encoded at the NH2 and COOH termini of the major E1B-19K and E1B-53K tumor antigens. The antipeptide antibodies immunoprecipitated the targeted E1B-19K or E1B-53K tumor antigens from extracts of Ad2-infected cells. The specificity of the peptide competition studies. Antipeptide antibodies directed to the NH2 and COOH termini immunoprecipitated the E1B-19K and E1B-53K tumor antigens from two Ad2-transformed rat cell lines, F17 and F4, providing evidence that identical tumor antigens are synthesized in Ad2-infected and Ad2-transformed cells. These results show that the E1B-19K and E1B-53K T antigens are not processed proteolytically at either the NH2 or COOH terminus. Our data provide strong evidence at the protein level that the E1B-19K and E1B-53K tumor antigens partially overlap in DNA sequence, with the E1B-19K initiating translation at the first ATG at nucleotide 1711 in translation reading frame 1 and the E1B-53K tumor antigen initiating translation at the second ATG at nucleotide 2016 in reading frame 3. This confirms the results of others on the N-terminal amino acid sequence of E1B-19K and theoretical deductions based on the DNA sequence. Our findings prove that the large E1B-53K T antigen initiates translation at the second ATG at nucleotide 2016 and not at equally plausible initiation codons located farther downstream at nucleotides 2202 and 2235. Thus, the E1B-53K T antigen is another example of a protein which initiates translation at an internal ATG rather than at the 5'-proximal ATG. Images PMID:6632083

  20. Lack of cytochrome P450 2E1 (CYP2E1) induction in the rat liver by starvation without coprophagy.

    PubMed

    Chung, H C; Sung, S H; Kim, J S; Kim, Y C; Kim, S G

    2001-03-01

    Starvation potentiates the hepatotoxicity of a variety of small molecules, including chlorinated hydrocarbons and nitrosamines, through the induction of CYP2E1. A change in CYP2E1 expression during starvation may also alter the pharmacokinetic profiles of xenobiotics. Northern blot and Western blot analyses revealed that hepatic CYP2E1 was not induced during starvation in rats placed in metabolic or wire-bottom cages in contrast to the induction of CYP2E1 in animals housed in solid-bottom cages. We studied the effect of coprophagy on the expression of hepatic CYP2E1 during starvation. The extent of coprophagy was 24% in fed rats. Fecal matter of starving rats was reduced to 14% of control and starving rats re-ingested ~1.6 g of feces per day. The effect of fecal matter on CYP2E1 expression (i.e., 1.6 g/kg/day for 3 days) was assessed in fed or starving rats. Starving rats gavaged with fecal matter for 3 days resulted in a 3.5-fold increase in the level of CYP2E1 mRNA, while fed rats gavaged with feces failed to show an increase in the mRNA. The increase in the CYP2E1 mRNA level accompanied the induction of CYP2E1. Starving rats gavaged with methanol extract of feces (500 mg/kg/day for 3 days) showed a 3.3-fold increase in CYP2E1 mRNA level in the liver. These results provide evidence that CYP2E1 is not induced by starvation without coprophagy, raising the contention that the mechanistic basis for CYP2E1 induction by starvation should be reevaluated. PMID:11181486

  1. Two classes of human papillomavirus type 16 E1 mutants suggest pleiotropic conformational constraints affecting E1 multimerization, E2 interaction, and interaction with cellular proteins.

    PubMed Central

    Yasugi, T; Vidal, M; Sakai, H; Howley, P M; Benson, J D

    1997-01-01

    Random mutagenesis of human papillomavirus type 16 (HPV16) E1 was used to generate E1 missense mutants defective for interaction with either hUBC9 or 16E1-BP, two cDNAs encoding proteins that have been identified by their ability to interact with HPV16 E1 in two-hybrid assays. hUBC9, the human counterpart of Saccharomyces cerevisiae UBC9, is a ubiquitin-conjugating enzyme known to be involved in cell cycle progression. 16E1-BP encodes a protein of no known function but does contain an ATPase signature motif. Eight hUBC9 or 16E1-BP interaction-defective HPV16 E1 missense mutants were identified and characterized for origin-dependent transient DNA replication, ATPase activity, and various protein-protein interaction phenotypes. Six of these mutant E1 proteins were significantly impaired for replication. Among these, two classes of replication-defective HPV16 E1 missense mutants were observed. One class, represented by the S330R replication-defective mutant (containing an S-to-R change at position 330), remained competent for all protein-protein interactions tested, with the exception of hUBC9 association. Furthermore, this mutant, unlike the other replication-defective HPV16 E1 missense mutants, had a strong dominant negative replication phenotype in transient-replication assays. The other class, represented by five of the missense mutants, was defective for multiple protein-protein interactions, usually including, but not limited to, the interaction defect for which each mutant was originally selected. In many cases, a single missense mutation in one region of HPV16 E1 had pleiotropic effects, even upon activities thought to be associated with other domains of HPV16 E1. This suggests that E1 proteins are not modular but may instead be composed of multiple structurally and/or functionally interdependent domains. PMID:9223484

  2. Crystal structure of the DNA binding domain of the replication initiation protein E1 from papillomavirus.

    PubMed

    Enemark, E J; Chen, G; Vaughn, D E; Stenlund, A; Joshua-Tor, L

    2000-07-01

    Papillomaviral infection causes both benign and malignant lesions and is a necessary cause of cervical carcinoma. Replication of this virus requires the replication initiation proteins E1 and E2, which bind cooperatively at the origin of replication (ori) as an (E1)2-(E2)2-DNA complex. This is a precursor to larger E1 complexes that distort and unwind the ori. We present the crystal structure of the E1 DNA binding domain refined to 1.9 A resolution. Residues critical for DNA binding are located on an extended loop and an alpha helix. We identify the E1 dimerization surface by selective mutations at an E1/E1 interface observed in the crystal and propose a model for the (E1)2-DNA complex. These and other observations suggest how the E1 DNA binding domain orchestrates assembly of the hexameric helicase on the ori. PMID:10949036

  3. The dual effect of adenovirus type 5 E1A 13S protein on NF-kappaB activation is antagonized by E1B 19K.

    PubMed Central

    Schmitz, M L; Indorf, A; Limbourg, F P; Städtler, H; Traenckner, E B; Baeuerle, P A

    1996-01-01

    The genomes of human adenoviruses encode several regulatory proteins, including the two differentially spliced gene products E1A and E1B. Here, we show that the 13S but not the 12S splice variant of E1A of adenovirus type 5 can activate the human transcription factor NF-kappaB in a bimodal fashion. One mode is the activation of NF-kappaB containing the p65 subunit from the cytoplasmic NF-kappaB-IkappaB complex. This activation required reactive oxygen intermediates and the phosphorylation of IkappaBalpha at serines 32 and 36, followed by IkappaBalpha degradation and the nuclear uptake of NF-kappaB. In addition, 13S E1A stimulated the transcriptional activity of the C-terminal 80 amino acids of p65 at a core promoter with either a TATA box or an initiator (INR) element. The C-terminal 80 amino acids of p65 were found to associate with E1A in vitro. The activation of NF-kappaB-dependent reporter gene transcription by E1A was potently suppressed upon coexpression of the E1B 19-kDa protein (19K). E1B 19K prevented both the activation of NF-kappaB and the E1A-mediated transcriptional enhancement of p65. These inhibitory effects were not found for the 55-kDa splice variant of the E1B protein. We suggest that the inductive effect of E1A 13S on the host factor NF-kappaB, whose activation is important for the transcription of various adenovirus genes, must be counteracted by the suppressive effect of E1B 19K so that the adenovirus-infected cell can escape the immune-stimulatory and apoptotic effects of NF-kappaB. PMID:8754803

  4. The Human Adenovirus Type 5 E4orf6/E1B55K E3 Ubiquitin Ligase Complex Enhances E1A Functional Activity

    PubMed Central

    Dallaire, Frédéric; Schreiner, Sabrina; Blair, G. Eric; Dobner, Thomas; Branton, Philip E.

    2015-01-01

    ABSTRACT Human adenovirus (Ad) E1A proteins have long been known as the central regulators of virus infection as well as the major source of adenovirus oncogenic potential. Not only do they activate expression of other early viral genes, they make viral replication possible in terminally differentiated cells, at least in part, by binding to the retinoblastoma (Rb) tumor suppressor family of proteins to activate E2F transcription factors and thus viral and cellular DNA synthesis. We demonstrate in an accompanying article (F. Dallaire et al., mSphere 1:00014-15, 2016) that the human adenovirus E3 ubiquitin ligase complex formed by the E4orf6 and E1B55K proteins is able to mimic E1A activation of E2F transactivation factors. Acting alone in the absence of E1A, the Ad5 E4orf6 protein in complex with E1B55K was shown to bind E2F, disrupt E2F/Rb complexes, and induce hyperphosphorylation of Rb, leading to induction of viral and cellular DNA synthesis, as well as stimulation of early and late viral gene expression and production of viral progeny. While these activities were significantly lower than those exhibited by E1A, we report here that this ligase complex appeared to enhance E1A activity in two ways. First, the E4orf6/E1B55K complex was shown to stabilize E1A proteins, leading to higher levels in infected cells. Second, the complex was demonstrated to enhance the activation of E2F by E1A products. These findings indicated a new role of the E4orf6/E1B55K ligase complex in promoting adenovirus replication. IMPORTANCE Following our demonstration that adenovirus E3 ubiquitin ligase formed by the viral E4orf6 and E1B55K proteins is able to mimic the activation of E2F by E1A, we conducted a series of studies to determine if this complex might also promote the ability of E1A to do so. We found that the complex both significantly stabilizes E1A proteins and also enhances their ability to activate E2F. This finding is of significance because it represents an entirely new

  5. The Human Adenovirus Type 5 E4orf6/E1B55K E3 Ubiquitin Ligase Complex Enhances E1A Functional Activity.

    PubMed

    Dallaire, Frédéric; Schreiner, Sabrina; Blair, G Eric; Dobner, Thomas; Branton, Philip E; Blanchette, Paola

    2016-01-01

    Human adenovirus (Ad) E1A proteins have long been known as the central regulators of virus infection as well as the major source of adenovirus oncogenic potential. Not only do they activate expression of other early viral genes, they make viral replication possible in terminally differentiated cells, at least in part, by binding to the retinoblastoma (Rb) tumor suppressor family of proteins to activate E2F transcription factors and thus viral and cellular DNA synthesis. We demonstrate in an accompanying article (F. Dallaire et al., mSphere 1:00014-15, 2016) that the human adenovirus E3 ubiquitin ligase complex formed by the E4orf6 and E1B55K proteins is able to mimic E1A activation of E2F transactivation factors. Acting alone in the absence of E1A, the Ad5 E4orf6 protein in complex with E1B55K was shown to bind E2F, disrupt E2F/Rb complexes, and induce hyperphosphorylation of Rb, leading to induction of viral and cellular DNA synthesis, as well as stimulation of early and late viral gene expression and production of viral progeny. While these activities were significantly lower than those exhibited by E1A, we report here that this ligase complex appeared to enhance E1A activity in two ways. First, the E4orf6/E1B55K complex was shown to stabilize E1A proteins, leading to higher levels in infected cells. Second, the complex was demonstrated to enhance the activation of E2F by E1A products. These findings indicated a new role of the E4orf6/E1B55K ligase complex in promoting adenovirus replication. IMPORTANCE Following our demonstration that adenovirus E3 ubiquitin ligase formed by the viral E4orf6 and E1B55K proteins is able to mimic the activation of E2F by E1A, we conducted a series of studies to determine if this complex might also promote the ability of E1A to do so. We found that the complex both significantly stabilizes E1A proteins and also enhances their ability to activate E2F. This finding is of significance because it represents an entirely new function for

  6. The adenovirus E1A proteins induce apoptosis, which is inhibited by the E1B 19-kDa and Bcl-2 proteins.

    PubMed Central

    Rao, L; Debbas, M; Sabbatini, P; Hockenbery, D; Korsmeyer, S; White, E

    1992-01-01

    Cooperation between the adenovirus E1A and E1B oncogenes is required for transformation of primary quiescent rodent cells. Although expression of E1A alone will stimulate cell proliferation sufficient to initiate transformed focus formation, proliferation fails to be sustained and foci degenerate. Coexpression of either the 19-kDa or 55-kDa E1B oncoproteins with E1A permits high-frequency transformation by overcoming this cytotoxic response. Without E1B 19-kDa protein expression, however, transformants remain susceptible to induction of cell death. Rapid loss of viability is coincident with nucleolytic cleavage of DNA in intranucleosomal regions and chromatin condensation, hallmarks of programmed cell death (apoptosis). Furthermore, overexpression of a known suppressor of apoptosis, the Bcl-2 protooncogene, can rescue E1A-induced focus degeneration. Thus E1A-dependent stimulation of cell proliferation is accompanied by apoptosis and thereby insufficient to singly induce transformation. High-frequency transformation requires a second function encoded by the E1B 19-kDa protein to block apoptosis. Images PMID:1457005

  7. Sequence-independent autoregulation of the adenovirus type 5 E1A transcription unit.

    PubMed Central

    Hearing, P; Shenk, T

    1985-01-01

    The adenovirus E1A gene is known to be autoregulated at the level of transcription. Autoregulation was found to be mediated by products of the E1A 13S mRNA, which induced a fivefold increase in E1A transcription rate. Deletion analysis suggested that the autoregulation did not require any specific sequence in the E1A transcriptional control region. This conclusion was reinforced by the demonstration that a cellular alpha-globin gene substituted for the E1A gene on the adenovirus chromosome was also positively regulated by E1A gene products. Images PMID:2943984

  8. Metabolic inactivation of resolvin E1 and stabilization of its anti-inflammatory actions.

    PubMed

    Arita, Makoto; Oh, Sungwhan F; Chonan, Tomomichi; Hong, Song; Elangovan, Siva; Sun, Yee-Ping; Uddin, Jasim; Petasis, Nicos A; Serhan, Charles N

    2006-08-11

    The resolvins (Rv) are lipid mediators derived from omega-3 polyunsaturated fatty acids that act within a local inflammatory milieu to stop leukocyte recruitment and promote resolution. Resolvin E1 (RvE1; (5S,12R,18R)-trihydroxy-6Z,8E,10E,14Z,16E-eicosapentaenoic acid) is an oxygenase product derived from omega-3 eicosapentaenoic acid that displays potent anti-inflammation/pro-resolution actions in vivo. Here, we determined whether oxidoreductase enzymes catalyze the conversion of RvE1 and assessed the biological activity of the RvE1 metabolite. With NAD+ as a cofactor, recombinant 15-hydroxyprostaglandin dehydrogenase acted as an 18-hydroxyl dehydrogenase to form 18-oxo-RvE1. In the murine lung, dehydrogenation of the hydroxyl group at carbon 18 position to form 18-oxo-RvE1 represented the major initial metabolic route for RvE1. At a concentration where RvE1 potently reduced polymorphonuclear leukocyte (PMN) recruitment in zymosan-induced peritonitis, 18-oxo-RvE1 was devoid of activity. In human neutrophils, carbon 20 hydroxylation of RvE1 was the main route of conversion. An RvE1 analog, i.e. 19-(p-fluorophenoxy)-RvE1, was synthesized that resisted rapid metabolic inactivation and proved to retain biological activity reducing PMN infiltration and pro-inflammatory cytokine/chemokine production in vivo. These results established the structure of a novel RvE1 initial metabolite, indicating that conversion of RvE1 to the oxo product represents a mode of RvE1 inactivation. Moreover, the designed RvE1 analog, which resisted further metabolism/inactivation, could be a useful tool to evaluate the actions of RvE1 in complex disease models. PMID:16757471

  9. Functional conservation and diversification of the soybean maturity gene E1 and its homologs in legumes

    PubMed Central

    Zhang, Xingzheng; Zhai, Hong; Wang, Yaying; Tian, Xiaojie; Zhang, Yupeng; Wu, Hongyan; Lü, Shixiang; Yang, Guang; Li, Yuqiu; Wang, Lu; Hu, Bo; Bu, Qingyun; Xia, Zhengjun

    2016-01-01

    Gene regulatory networks involved in flowering time and photoperiodic responses in legumes remain unknown. Although the major maturity gene E1 has been successfully deciphered in soybean, knowledge on the functional conservation of this gene is limited to a certain extent to E1 homologs in legumes. The ectopic expression of Phvul.009G204600 (PvE1L), an E1 homolog from common bean, delayed the onset of flowering in soybean. By contrast, the ectopic expression of Medtr2g058520 (MtE1L) from Medicago truncatula did not affect the flowering of soybean. Characterization of the late-flowering mte1l mutant indicated that MtE1L promoted flowering in Medicago truncatula. Moreover, all transgenic E1, PvE1L and MtE1L soybean lines exhibited phenotypic changes in terms of plant height. Transgenic E1 or PvE1L plants were taller than the wild-type, whereas transgenic MtE1L plants produced dwarf phenotype with few nodes and short internode. Thus, functional conservation and diversification of E1 family genes from legumes in the regulation of flowering and plant growth may be associated with lineage specification and genomic duplication. PMID:27405888

  10. Cell-based analysis of Chikungunya virus E1 protein in membrane fusion

    PubMed Central

    2012-01-01

    Background Chikungunya fever is a pandemic disease caused by the mosquito-borne Chikungunya virus (CHIKV). E1 glycoprotein mediation of viral membrane fusion during CHIKV infection is a crucial step in the release of viral genome into the host cytoplasm for replication. How the E1 structure determines membrane fusion and whether other CHIKV structural proteins participate in E1 fusion activity remain largely unexplored. Methods A bicistronic baculovirus expression system to produce recombinant baculoviruses for cell-based assay was used. Sf21 insect cells infected by recombinant baculoviruses bearing wild type or single-amino-acid substitution of CHIKV E1 and EGFP (enhanced green fluorescence protein) were employed to investigate the roles of four E1 amino acid residues (G91, V178, A226, and H230) in membrane fusion activity. Results Western blot analysis revealed that the E1 expression level and surface features in wild type and mutant substituted cells were similar. However, cell fusion assay found that those cells infected by CHIKV E1-H230A mutant baculovirus showed little fusion activity, and those bearing CHIKV E1-G91D mutant completely lost the ability to induce cell-cell fusion. Cells infected by recombinant baculoviruses of CHIKV E1-A226V and E1-V178A mutants exhibited the same membrane fusion capability as wild type. Although the E1 expression level of cells bearing monomeric-E1-based constructs (expressing E1 only) was greater than that of cells bearing 26S-based constructs (expressing all structural proteins), the sizes of syncytial cells induced by infection of baculoviruses containing 26S-based constructs were larger than those from infections having monomeric-E1 constructs, suggesting that other viral structure proteins participate or regulate E1 fusion activity. Furthermore, membrane fusion in cells infected by baculovirus bearing the A226V mutation constructs exhibited increased cholesterol-dependences and lower pH thresholds. Cells bearing the V178

  11. 26 CFR 1.501(e)-1 - Cooperative hospital service organizations.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... state they were in on completion of the exempt function (e.g., the sale of silver waste produced in the...(e)-1 Section 1.501(e)-1 Internal Revenue INTERNAL REVENUE SERVICE, DEPARTMENT OF THE TREASURY (CONTINUED) INCOME TAX (CONTINUED) INCOME TAXES (CONTINUED) Exempt Organizations § 1.501(e)-1...

  12. 26 CFR 1.501(e)-1 - Cooperative hospital service organizations.

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ... state they were in on completion of the exempt function (e.g., the sale of silver waste produced in the...(e)-1 Section 1.501(e)-1 Internal Revenue INTERNAL REVENUE SERVICE, DEPARTMENT OF THE TREASURY (CONTINUED) INCOME TAX (CONTINUED) INCOME TAXES (CONTINUED) Exempt Organizations § 1.501(e)-1...

  13. 26 CFR 1.501(e)-1 - Cooperative hospital service organizations.

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    ... state they were in on completion of the exempt function (e.g., the sale of silver waste produced in the....501(e)-1 Section 1.501(e)-1 Internal Revenue INTERNAL REVENUE SERVICE, DEPARTMENT OF THE TREASURY (CONTINUED) INCOME TAX (CONTINUED) INCOME TAXES (CONTINUED) Exempt Organizations § 1.501(e)-1...

  14. 26 CFR 1.501(e)-1 - Cooperative hospital service organizations.

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ... state they were in on completion of the exempt function (e.g., the sale of silver waste produced in the....501(e)-1 Section 1.501(e)-1 Internal Revenue INTERNAL REVENUE SERVICE, DEPARTMENT OF THE TREASURY (CONTINUED) INCOME TAX (CONTINUED) INCOME TAXES (CONTINUED) Exempt Organizations § 1.501(e)-1...

  15. 26 CFR 1.501(e)-1 - Cooperative hospital service organizations.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... state they were in on completion of the exempt function (e.g., the sale of silver waste produced in the...(e)-1 Section 1.501(e)-1 Internal Revenue INTERNAL REVENUE SERVICE, DEPARTMENT OF THE TREASURY (CONTINUED) INCOME TAX (CONTINUED) INCOME TAXES (CONTINUED) Exempt Organizations § 1.501(e)-1...

  16. 26 CFR 1.1397E-1 - Qualified zone academy bonds.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... 26 Internal Revenue 11 2010-04-01 2010-04-01 true Qualified zone academy bonds. 1.1397E-1 Section 1.1397E-1 Internal Revenue INTERNAL REVENUE SERVICE, DEPARTMENT OF THE TREASURY (CONTINUED) INCOME TAX (CONTINUED) INCOME TAXES Empowerment Zone Employment Credit § 1.1397E-1 Qualified zone...

  17. 26 CFR 1.669(e)-1A - Pro rata portion of taxes deemed distributed.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... 26 Internal Revenue 8 2010-04-01 2010-04-01 false Pro rata portion of taxes deemed distributed. 1.669(e)-1A Section 1.669(e)-1A Internal Revenue INTERNAL REVENUE SERVICE, DEPARTMENT OF THE TREASURY... Taxable Years Beginning Before January 1, 1969 § 1.669(e)-1A Pro rata portion of taxes deemed...

  18. 26 CFR 1.924(e)-1 - Activities relating to the disposition of export property.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... 26 Internal Revenue 10 2010-04-01 2010-04-01 false Activities relating to the disposition of export property. 1.924(e)-1 Section 1.924(e)-1 Internal Revenue INTERNAL REVENUE SERVICE, DEPARTMENT OF THE TREASURY (CONTINUED) INCOME TAX (CONTINUED) INCOME TAXES Earned Income of Citizens of United States § 1.924(e)-1 Activities relating to...

  19. 26 CFR 1.1402(e)-1A - Application of regulations under section 1402(e).

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... 26 Internal Revenue 12 2010-04-01 2010-04-01 false Application of regulations under section 1402(e). 1.1402(e)-1A Section 1.1402(e)-1A Internal Revenue INTERNAL REVENUE SERVICE, DEPARTMENT OF THE TREASURY (CONTINUED) INCOME TAX (CONTINUED) INCOME TAXES Tax on Self-Employment Income §...

  20. 26 CFR 1.1402(e)-1A - Application of regulations under section 1402(e).

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... 26 Internal Revenue 12 2011-04-01 2011-04-01 false Application of regulations under section 1402(e). 1.1402(e)-1A Section 1.1402(e)-1A Internal Revenue INTERNAL REVENUE SERVICE, DEPARTMENT OF THE TREASURY (CONTINUED) INCOME TAX (CONTINUED) INCOME TAXES (CONTINUED) Tax on Self-Employment Income §...

  1. 26 CFR 1.263(e)-1 - Expenditures in connection with certain railroad rolling stock.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... by the Interstate Commerce Commission (49 CFR Part 1201), but only if (i) such unit exclusively moves... rolling stock. 1.263(e)-1 Section 1.263(e)-1 Internal Revenue INTERNAL REVENUE SERVICE, DEPARTMENT OF THE TREASURY (CONTINUED) INCOME TAX (CONTINUED) INCOME TAXES Items Not Deductible § 1.263(e)-1 Expenditures...

  2. 26 CFR 1.1059(e)-1 - Non-pro rata redemptions.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... 26 Internal Revenue 11 2011-04-01 2011-04-01 false Non-pro rata redemptions. 1.1059(e)-1 Section 1... (CONTINUED) INCOME TAXES (CONTINUED) Special Rules § 1.1059(e)-1 Non-pro rata redemptions. (a) In general... under section 1059(e)(1). For example, if a redemption of stock is not pro rata as to all...

  3. 26 CFR 1.669(e)-1A - Pro rata portion of taxes deemed distributed.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... 26 Internal Revenue 8 2011-04-01 2011-04-01 false Pro rata portion of taxes deemed distributed. 1.669(e)-1A Section 1.669(e)-1A Internal Revenue INTERNAL REVENUE SERVICE, DEPARTMENT OF THE TREASURY... Applicable to Taxable Years Beginning Before January 1, 1969 § 1.669(e)-1A Pro rata portion of taxes...

  4. 26 CFR 1.509(e)-1 - Definition of gross investment income.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... 26 Internal Revenue 7 2010-04-01 2010-04-01 true Definition of gross investment income. 1.509(e)-1 Section 1.509(e)-1 Internal Revenue INTERNAL REVENUE SERVICE, DEPARTMENT OF THE TREASURY (CONTINUED) INCOME TAX (CONTINUED) INCOME TAXES (CONTINUED) Private Foundations § 1.509(e)-1 Definition of...

  5. 26 CFR 1.509(e)-1 - Definition of gross investment income.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... 26 Internal Revenue 7 2011-04-01 2009-04-01 true Definition of gross investment income. 1.509(e)-1 Section 1.509(e)-1 Internal Revenue INTERNAL REVENUE SERVICE, DEPARTMENT OF THE TREASURY (CONTINUED) INCOME TAX (CONTINUED) INCOME TAXES (CONTINUED) Private Foundations § 1.509(e)-1 Definition of...

  6. 26 CFR 1.149(e)-1 - Information reporting requirements for tax-exempt bonds.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... 26 Internal Revenue 2 2010-04-01 2010-04-01 false Information reporting requirements for tax-exempt bonds. 1.149(e)-1 Section 1.149(e)-1 Internal Revenue INTERNAL REVENUE SERVICE, DEPARTMENT OF THE TREASURY (CONTINUED) INCOME TAX (CONTINUED) INCOME TAXES (CONTINUED) Tax Exemption Requirements for State and Local Bonds § 1.149(e)-1...

  7. 26 CFR 1.6050E-1 - Reporting of State and local income tax refunds.

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    .... 1.6050E-1 Section 1.6050E-1 Internal Revenue INTERNAL REVENUE SERVICE, DEPARTMENT OF THE TREASURY (CONTINUED) INCOME TAX (CONTINUED) INCOME TAXES (CONTINUED) Information Returns § 1.6050E-1 Reporting of... individual in any calendar year. (b) Definitions. For purposes of this section— (1) The term refund...

  8. 26 CFR 1.6050E-1 - Reporting of State and local income tax refunds.

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    .... 1.6050E-1 Section 1.6050E-1 Internal Revenue INTERNAL REVENUE SERVICE, DEPARTMENT OF THE TREASURY (CONTINUED) INCOME TAX (CONTINUED) INCOME TAXES (CONTINUED) Information Returns § 1.6050E-1 Reporting of... individual in any calendar year. (b) Definitions. For purposes of this section— (1) The term refund...

  9. 26 CFR 1.6050E-1 - Reporting of State and local income tax refunds.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    .... 1.6050E-1 Section 1.6050E-1 Internal Revenue INTERNAL REVENUE SERVICE, DEPARTMENT OF THE TREASURY (CONTINUED) INCOME TAX (CONTINUED) INCOME TAXES Information Returns § 1.6050E-1 Reporting of State and local... year. (b) Definitions. For purposes of this section— (1) The term refund officer means the officer...

  10. 42 CFR 52e.1 - To what programs do these regulations apply?

    Code of Federal Regulations, 2010 CFR

    2010-10-01

    ... 42 Public Health 1 2010-10-01 2010-10-01 false To what programs do these regulations apply? 52e.1 Section 52e.1 Public Health PUBLIC HEALTH SERVICE, DEPARTMENT OF HEALTH AND HUMAN SERVICES GRANTS NATIONAL HEART, LUNG, AND BLOOD INSTITUTE GRANTS FOR PREVENTION AND CONTROL PROJECTS § 52e.1 To what programs...

  11. 42 CFR 52e.1 - To what programs do these regulations apply?

    Code of Federal Regulations, 2013 CFR

    2013-10-01

    ... 42 Public Health 1 2013-10-01 2013-10-01 false To what programs do these regulations apply? 52e.1 Section 52e.1 Public Health PUBLIC HEALTH SERVICE, DEPARTMENT OF HEALTH AND HUMAN SERVICES GRANTS NATIONAL HEART, LUNG, AND BLOOD INSTITUTE GRANTS FOR PREVENTION AND CONTROL PROJECTS § 52e.1 To what programs...

  12. 42 CFR 52e.1 - To what programs do these regulations apply?

    Code of Federal Regulations, 2012 CFR

    2012-10-01

    ... 42 Public Health 1 2012-10-01 2012-10-01 false To what programs do these regulations apply? 52e.1 Section 52e.1 Public Health PUBLIC HEALTH SERVICE, DEPARTMENT OF HEALTH AND HUMAN SERVICES GRANTS NATIONAL HEART, LUNG, AND BLOOD INSTITUTE GRANTS FOR PREVENTION AND CONTROL PROJECTS § 52e.1 To what programs...

  13. 42 CFR 52e.1 - To what programs do these regulations apply?

    Code of Federal Regulations, 2011 CFR

    2011-10-01

    ... 42 Public Health 1 2011-10-01 2011-10-01 false To what programs do these regulations apply? 52e.1 Section 52e.1 Public Health PUBLIC HEALTH SERVICE, DEPARTMENT OF HEALTH AND HUMAN SERVICES GRANTS NATIONAL HEART, LUNG, AND BLOOD INSTITUTE GRANTS FOR PREVENTION AND CONTROL PROJECTS § 52e.1 To what programs...

  14. Identification of a nuclear export signal sequence for bovine papillomavirus E1 protein

    SciTech Connect

    Rosas-Acosta, German; Wilson, Van G.

    2008-03-30

    Recent studies have demonstrated nuclear export by papillomavirus E1 proteins, but the requisite export sequence(s) for bovine papillomavirus (BPV) E1 were not defined. In this report we identify three functional nuclear export sequences (NES) present in BPV E1, with NES2 being the strongest in reporter assays. Nuclear localization of BPV1 E1 was modulated by over- or under-expression of CRM1, the major cellular exportin, and export was strongly reduced by the CRM1 inhibitor, Leptomycin B, indicating that E1 export occurs primarily through a CRM1-dependent process. Consistent with the in vivo functional results, E1 bound CRM1 in an in vitro pull-down assay. In addition, sumoylated E1 bound CRM1 more effectively than unmodified E1, suggesting that E1 export may be regulated by SUMO modification. Lastly, an E1 NES2 mutant accumulated in the nucleus to a greater extent than wild-type E1, yet was defective for viral origin replication in vivo. However, NES2 exhibited no intrinsic replication defect in an in vitro replication assay, implying that nucleocytoplasmic shuttling may be required to maintain E1 in a replication competent state.

  15. 40 CFR Figure E-1 to Subpart E of... - Designation Testing Checklist

    Code of Federal Regulations, 2010 CFR

    2010-07-01

    ... 40 Protection of Environment 5 2010-07-01 2010-07-01 false Designation Testing Checklist E Figure E-1 to Subpart E of Part 53 Protection of Environment ENVIRONMENTAL PROTECTION AGENCY (CONTINUED... Equivalent Methods for PM2.5 or PM10â2.5 Pt. 53, Subpt. E, Fig. E-1 Figure E-1 to Subpart E of Part...

  16. 26 CFR 48.4216(e)-1 - Exclusion of local advertising charges from sale price.

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    ... sale price. 48.4216(e)-1 Section 48.4216(e)-1 Internal Revenue INTERNAL REVENUE SERVICE, DEPARTMENT OF... Provisions Applicable to Manufacturers Taxes § 48.4216(e)-1 Exclusion of local advertising charges from sale price. (a) In general. Section 4216(e) deals with the treatment to be accorded charges made by...

  17. 26 CFR 48.4216(e)-1 - Exclusion of local advertising charges from sale price.

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ... sale price. 48.4216(e)-1 Section 48.4216(e)-1 Internal Revenue INTERNAL REVENUE SERVICE, DEPARTMENT OF... Provisions Applicable to Manufacturers Taxes § 48.4216(e)-1 Exclusion of local advertising charges from sale price. (a) In general. Section 4216(e) deals with the treatment to be accorded charges made by...

  18. 17 CFR 270.10e-1 - Death, disqualification, or bona fide resignation of directors.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... bona fide resignation of directors. 270.10e-1 Section 270.10e-1 Commodity and Securities Exchanges....10e-1 Death, disqualification, or bona fide resignation of directors. If a registered investment company, by reason of the death, disqualification, or bona fide resignation of any director, does not...

  19. Identification and characterization of an hnRNP E1 translational silencing motif

    PubMed Central

    Brown, Andrew S.; Mohanty, Bidyut K.; Howe, Philip H.

    2016-01-01

    Non-canonical transforming growth factor β (TGFβ) signaling through protein kinase B (Akt2) induces phosphorylation of heterogeneous nuclear ribonucleoprotein E1 (hnRNP E1) at serine-43 (p-hnRNP E1). This post-translational modification (PTM) of hnRNP E1 promotes its dissociation from a 3′ untranslated region (UTR) nucleic acid regulatory motif, driving epithelial to mesenchymal transition (EMT) and metastasis. We have identified an hnRNP E1 consensus-binding motif and genomically resolved a subset of genes in which it is contained. This study characterizes the binding kinetics of the consensus-binding motif and hnRNP E1, its various K-homology (KH) domains and p-hnRNP E1. Levels of p-hnRNP E1 are highly upregulated in metastatic cancer cells and low in normal epithelial tissue. We show a correlation between this PTM and levels of Akt2 and its activated form, phosphorylated serine-474 (p-Akt2). Using cellular progression models of metastasis, we observed a signature high level of Akt2, p-Akt2 and p-hnRNP E1 protein expression, coupled to a significantly reduced level of total hnRNP E1 in metastatic cells. Genes that are translationally silenced by hnRNP E1 and expressed by its dissociation are highly implicated in the progression of EMT and metastasis. This study provides insight into a non-canonical TGFβ signaling cascade that is responsible for inducing EMT by aberrant expression of hnRNP E1 silenced targets. The relevance of this system in metastatic progression is clearly shown in cellular models by the high abundance of p-hnRNP E1 and low levels of hnRNP E1. New insights provided by the resolution of this molecular mechanism provide targets for therapeutic intervention and give further insight into the role of the TGFβ microenvironment. PMID:27067543

  20. Subcellular localization of rat CYP2E1 impacts metabolic efficiency toward common substrates.

    PubMed

    Hartman, Jessica H; Martin, H Cass; Caro, Andres A; Pearce, Amy R; Miller, Grover P

    2015-12-01

    Cytochrome P450 2E1 (CYP2E1) detoxifies or bioactivates many low molecular-weight compounds. Most knowledge about CYP2E1 activity relies on studies of the enzyme localized to endoplasmic reticulum (erCYP2E1); however, CYP2E1 undergoes transport to mitochondria (mtCYP2E1) and becomes metabolically active. We report the first comparison of in vitro steady-state kinetic profiles for erCYP2E1 and mtCYP2E1 oxidation of probe substrate 4-nitrophenol and pollutants styrene and aniline using subcellular fractions from rat liver. For all substrates, metabolic efficiency changed with substrate concentration for erCYP2E1 reflected in non-hyperbolic kinetic profiles but not for mtCYP2E1. Hyperbolic kinetic profiles for the mitochondrial enzyme were consistent with Michaelis-Menten mechanism in which metabolic efficiency was constant. By contrast, erCYP2E1 metabolism of 4-nitrophenol led to a loss of enzyme efficiency at high substrate concentrations when substrate inhibited the reaction. Similarly, aniline metabolism by erCYP2E1 demonstrated negative cooperativity as metabolic efficiency decreased with increasing substrate concentration. The opposite was observed for erCYP2E1 oxidation of styrene; the sigmoidal kinetic profile indicated increased efficiency at higher substrate concentrations. These mechanisms and CYP2E1 levels in mitochondria and endoplasmic reticulum were used to estimate the impact of CYP2E1 subcellular localization on metabolic flux of pollutants. Those models showed that erCYP2E1 mainly carries out aniline metabolism at all aniline concentrations. Conversely, mtCYP2E1 dominates styrene oxidation at low styrene concentrations and erCYP2E1 at higher concentrations. Taken together, subcellular localization of CYP2E1 results in distinctly different enzyme activities that could impact overall metabolic clearance and/or activation of substrates and thus impact the interpretation and prediction of toxicological outcomes. PMID:26463279

  1. 17 CFR 274.127e-1 - Form N-27E-1, notice to periodic payment plan certificate holders of 18-month surrender rights...

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    .... Editorial Note: For Federal Register citations affecting Form N-27E-1, see the List of CFR Sections Affected... EXCHANGE COMMISSION (CONTINUED) FORMS PRESCRIBED UNDER THE INVESTMENT COMPANY ACT OF 1940 Forms for...

  2. Structural models of the membrane anchors of envelope glycoproteins E1 and E2 from pestiviruses

    SciTech Connect

    Wang, Jimin Li, Yue; Modis, Yorgo

    2014-04-15

    The membrane anchors of viral envelope proteins play essential roles in cell entry. Recent crystal structures of the ectodomain of envelope protein E2 from a pestivirus suggest that E2 belongs to a novel structural class of membrane fusion machinery. Based on geometric constraints from the E2 structures, we generated atomic models of the E1 and E2 membrane anchors using computational approaches. The E1 anchor contains two amphipathic perimembrane helices and one transmembrane helix; the E2 anchor contains a short helical hairpin stabilized in the membrane by an arginine residue, similar to flaviviruses. A pair of histidine residues in the E2 ectodomain may participate in pH sensing. The proposed atomic models point to Cys987 in E2 as the site of disulfide bond linkage with E1 to form E1–E2 heterodimers. The membrane anchor models provide structural constraints for the disulfide bonding pattern and overall backbone conformation of the E1 ectodomain. - Highlights: • Structures of pestivirus E2 proteins impose constraints on E1, E2 membrane anchors. • Atomic models of the E1 and E2 membrane anchors were generated in silico. • A “snorkeling” arginine completes the short helical hairpin in the E2 membrane anchor. • Roles in pH sensing and E1–E2 disulfide bond formation are proposed for E1 residues. • Implications for E1 ectodomain structure and disulfide bonding pattern are discussed.

  3. ColE1 plasmid incompatibility: localization and analysis of mutations affecting incompatibility.

    PubMed Central

    Hashimoto-Gotoh, T; Inselburg, J

    1979-01-01

    Deletion mutants of plasmid ColE1 that involve the replication origin and adjacent regions of the plasmid have been studied to determine the mechanism by which those mutations affect the expression of plasmid incompatibility. It was observed that (i) a region of ColE1 that is involved in the expression of plasmid incompatibility lies between base pairs -185 and -684; (ii) the integrity of at least part of the region of ColE1 DNA between base pairs -185 and -572 is essential for the expression of ColE1 incompatibility; (iii) the expression of incompatibility is independent of the ability of the ColE1 genome to replicate autonomously; (iv) plasmid incompatibility is affected by plasmid copy number; and (v) ColE1 plasmid-mediated DNA replication of the lambda phage-ColE1 chimera lambda imm434 Oam29 Pam3 ColE1 is inhibited by ColE1-incompatible but not by ColE1-compatible plasmids. Images PMID:378980

  4. Adenovirus type 5 E1A sensitizes hepatocellular carcinoma cells to gemcitabine.

    PubMed

    Lee, Wei-Ping; Tai, Dar-In; Tsai, Sun-Lung; Yeh, Chau-Ting; Chao, Yee; Lee, Shou-Dong; Hung, Mien-Chie

    2003-10-01

    Hepatocellular carcinoma (HCC) is resistant to conventional chemotherapy. A few clinical trials have shown that the cytidine analogue gemcitabine appears to have antitumor activity for HCC, but the overall survival times remain to be improved. In this study, we examined the synergistic effect of adenovirus type 5 E1A (E1A) and gemcitabine on HCC and found that E1A sensitized J5, J7, Huh7, and HepG2 HCC cells to gemcitabine. To further study the E1A-mediated chemosensitization, we established stable cell lines that expressed the E1A gene and then examined whether E1A could have proapoptotic activity while expressed in HCC cells. Our results clearly showed that E1A sensitized HCC cells to gemcitabine through induction of apoptosis. To study the underlying mechanism, we tested nuclear factor (NF)-kappaB activity and found that NF-kappaB was activated in HCC cells treated with gemcitabine but not in HCC cells that expressed E1A. Occurrence of apoptosis entails cleavage of poly (ADP-ribose) polymerase (PARP), a nuclear protein involved in DNA repair, genome stability, and maintenance of telomere length. Our study showed that gemcitabine enhanced PARP expression. However, E1A did not induce PARP cleavage but rather suppressed PARP expression at the transcriptional level. Further study showed that both NF-kappaB and PARP played protective roles in the prevention of E1A+gemcitabine-induced apoptosis. PMID:14559808

  5. [Induction of rat hepatic CYP2E1 expression by arecoline in vivo].

    PubMed

    Huang, Xiang-tao; Xiao, Run-mei; Wang, Ming-feng; Wang, Jun-jun; Chen, Yong

    2016-01-01

    The regulation mechanism of arecoline on rat hepatic CYP2E1 was studied in vivo. After oral administration of arecoline hydrobromide (AH; 4, 20 and 100 mg x kg(-1) x d(-1)) to rats for one week, the hepatic CYP2E1 mRNA level remained unchanged, but the hepatic CYP2E1 protein content was dose-dependently increased. Additionally, although the hepatic CYP2E1 activity was induced by AH treatment, the induction was attenuated with the increase in dosage. The results indicate that the effect of arecoline on rat hepaticdoes not involve transcriptional activation of the gene, but largely involves the stabilization of CYP2E1 protein against degradation or increased efficiency of CYP2E1 mRNA translation, and additionally involve the post- ranslational modification of CYP2E1 protein. Furthermore, the CYP2E1 response is fairly equal among the different species, the induction of rat hepatic CYP2E1 by arecoline suggests that there is a risk of metabolic interaction among the substrate drugs of CYP2E1 in betel-quid use human. PMID:27405178

  6. The frequency of cytochrome P450 2E1 polymorphisms in Black South Africans.

    PubMed

    Chelule, Paul K; Pegoraro, Rosemary J; Gqaleni, Nceba; Dutton, Michael F

    2006-01-01

    Polymorphisms in the promoter region of the Cytochrome P4502E1 (CYP2E1) gene reportedly modify the metabolic activity of CYP2E1 enzyme, and have been associated with increased susceptibility to squamous cell carcinoma (SCC) of the oesophagus in high prevalence areas such as China. To assess the frequency of these polymorphisms in Black South Africans, a population with a high incidence of oesophageal SCC, this study examined genomic DNA from 331 subjects for restriction fragment length polymorphisms in the CYP2E1 (RsaI and PstI digestion). The frequency of the CYP2E1 c1/c1 and c1/c3 genotypes was 95% and 5% respectively. The frequency of the CYP2E1 allele distribution was found to be markedly different between Chinese and South African populations; hence it is important to place racial differences into consideration when proposing allelic variants as genetic markers for cancer. PMID:17264406

  7. 26 CFR 1.1059(e)-1 - Non-pro rata redemptions.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... 26 Internal Revenue 11 2010-04-01 2010-04-01 true Non-pro rata redemptions. 1.1059(e)-1 Section 1... (CONTINUED) INCOME TAXES Special Rules § 1.1059(e)-1 Non-pro rata redemptions. (a) In general. Section 1059(d... 1059(e)(1). For example, if a redemption of stock is not pro rata as to all shareholders, any...

  8. 26 CFR 1.414(e)-1 - Definition of church plan.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... 26 Internal Revenue 5 2010-04-01 2010-04-01 false Definition of church plan. 1.414(e)-1 Section 1.414(e)-1 Internal Revenue INTERNAL REVENUE SERVICE, DEPARTMENT OF THE TREASURY (CONTINUED) INCOME TAX (CONTINUED) INCOME TAXES Pension, Profit-Sharing, Stock Bonus Plans, Etc. § 1.414(e)-1 Definition of church plan. (a) General rule. For the...

  9. Largazole and Its Derivatives Selectively Inhibit Ubiquitin Activating Enzyme (E1)

    PubMed Central

    Nasveschuk, Christopher G.; Wang, Wei; Quade, Bettina; Zhang, Gan; Kuchta, Robert D.; Phillips, Andrew J.; Liu, Xuedong

    2012-01-01

    Protein ubiquitination plays an important role in the regulation of almost every aspect of eukaryotic cellular function; therefore, its destabilization is often observed in most human diseases and cancers. Consequently, developing inhibitors of the ubiquitination system for the treatment of cancer has been a recent area of interest. Currently, only a few classes of compounds have been discovered to inhibit the ubiquitin-activating enzyme (E1) and only one class is relatively selective in E1 inhibition in cells. We now report that Largazole and its ester and ketone analogs selectively inhibit ubiquitin conjugation to p27Kip1 and TRF1 in vitro. The inhibitory activity of these small molecules on ubiquitin conjugation has been traced to their inhibition of the ubiquitin E1 enzyme. To further dissect the mechanism of E1 inhibition, we analyzed the effects of these inhibitors on each of the two steps of E1 activation. We show that Largazole and its derivatives specifically inhibit the adenylation step of the E1 reaction while having no effect on thioester bond formation between ubiquitin and E1. E1 inhibition appears to be specific to human E1 as Largazole ketone fails to inhibit the activation of Uba1p, a homolog of E1 in Schizosaccharomyces pombe. Moreover, Largazole analogs do not significantly inhibit SUMO E1. Thus, Largazole and select analogs are a novel class of ubiquitin E1 inhibitors and valuable tools for studying ubiquitination in vitro. This class of compounds could be further developed and potentially be a useful tool in cells. PMID:22279528

  10. Expression and characterization of a soluble rubella virus E1 envelope protein.

    PubMed

    Seto, N O; Gillam, S

    1994-10-01

    Individual specific antigenic rubella virus (RV) structural proteins are required for accurate serological diagnosis of acute and congenital rubella infections as well as rubella immune status. The RV envelope glycoprotein E1 is the major target antigen and plays an important role in viral-specific immune responses. The native virion is difficult to produce in large quantities and the protein subunits are also difficult to isolate without loss of antigenicity. The production of a soluble RV E1 (designated E1 delta Tm) using the baculovirus-insect cell expression system is described. In contrast to wild-type RV E1, the genetically engineered E1 delta Tm protein lacks a transmembrane anchor. It behaved as a secretory protein and was secreted abundantly from insect cells. Pulse-chase studies were used to examine the synthesis, glycosylation, and secretion of E1 delta Tm by the insect cells. The secreted E1 delta Tm protein was purified from serum-free medium by one-step immunochromatography. The purified E1 delta Tm protein retained full antigenicity and may be a convenient source of E1 protein for use in diagnostic assay and rubella vaccine development. PMID:7852960

  11. Identification and characterization of multiple conserved nuclear localization signals within adenovirus E1A

    SciTech Connect

    Marshall, Kris S.; Cohen, Michael J.; Fonseca, Greg J.; Todorovic, Biljana; King, Cason R.; Yousef, Ahmed F.; Zhang, Zhiying; Mymryk, Joe S.

    2014-04-15

    The human adenovirus 5 (HAdV-5) E1A protein has a well defined canonical nuclear localization signal (NLS) located at its C-terminus. We used a genetic assay in the yeast Saccharomyces cerevisiae to demonstrate that the canonical NLS is present and functional in the E1A proteins of each of the six HAdV species. This assay also detects a previously described non-canonical NLS within conserved region 3 and a novel active NLS within the N-terminal/conserved region 1 portion of HAdV-5 E1A. These activities were also present in the E1A proteins of each of the other five HAdV species. These results demonstrate that, despite substantial differences in primary sequence, HAdV E1A proteins are remarkably consistent in that they contain one canonical and two non-canonical NLSs. By utilizing independent mechanisms, these multiple NLSs ensure nuclear localization of E1A in the infected cell. - Highlights: • HAdV E1A uses multiple mechanisms for nuclear import. • We identified an additional non-canonical NLS in the N-terminal/CR1 portion of E1A. • The new NLS does not contact importin-alpha directly. • All NLSs are functionally conserved in the E1A proteins of all 6 HAdV species.

  12. Sex steroid hormones regulate constitutive expression of Cyp2e1 in female mouse liver

    PubMed Central

    Cheng, Jie; Gonzalez, Frank J.

    2013-01-01

    CYP2E1 is of paramount toxicological significance because it metabolically activates a large number of low-molecular-weight toxicants and carcinogens. In this context, factors that interfere with Cyp2e1 regulation may critically affect xenobiotic toxicity and carcinogenicity. The aim of this study was to investigate the role of female steroid hormones in the regulation of CYP2E1, as estrogens and progesterone are the bases of contraceptives and hormonal replacement therapy in menopausal women. Interestingly, a fluctuation in the hepatic expression pattern of Cyp2e1 was revealed in the different phases of the estrous cycle of female mice, with higher Cyp2e1 expression at estrus (E) and lower at methestrus (ME), highly correlated with that in plasma gonadal hormone levels. Depletion of sex steroids by ovariectomy repressed Cyp2e1 expression to levels similar to those detected in males and cyclic females at ME. Hormonal supplementation brought Cyp2e1 expression back to levels detected at E. The role of progesterone appeared to be more prominent than that of 17β-estradiol. Progesterone-induced Cyp2e1 upregulation could be attributed to inactivation of the insulin/PI3K/Akt/FOXO1 signaling pathway. Tamoxifen, an anti-estrogen, repressed Cyp2e1 expression potentially via activation of the PI3K/Akt/FOXO1 and GH/STAT5b-linked pathways. The sex steroid hormone-related changes in hepatic Cyp2e1 expression were highly correlated with those observed in Hnf-1α, β-catenin, and Srebp-1c. In conclusion, female steroid hormones are clearly involved in the regulation of CYP2E1, thus affecting the metabolism of a plethora of toxicants and carcinogenic agents, conditions that may trigger several pathologies or exacerbate the outcomes of various pathophysiological states. PMID:23548611

  13. Ubiquitin-dependent Proteasomal Degradation of Human Liver Cytochrome P450 2E1

    PubMed Central

    Wang, YongQiang; Guan, Shenheng; Acharya, Poulomi; Koop, Dennis R.; Liu, Yi; Liao, Mingxiang; Burlingame, Alma L.; Correia, Maria Almira

    2011-01-01

    Human liver CYP2E1 is a monotopic, endoplasmic reticulum-anchored cytochrome P450 responsible for the biotransformation of clinically relevant drugs, low molecular weight xenobiotics, carcinogens, and endogenous ketones. CYP2E1 substrate complexation converts it into a stable slow-turnover species degraded largely via autophagic lysosomal degradation. Substrate decomplexation/withdrawal results in a fast turnover CYP2E1 species, putatively generated through its futile oxidative cycling, that incurs endoplasmic reticulum-associated ubiquitin-dependent proteasomal degradation (UPD). CYP2E1 thus exhibits biphasic turnover in the mammalian liver. We now show upon heterologous expression of human CYP2E1 in Saccharomyces cerevisiae that its autophagic lysosomal degradation and UPD pathways are evolutionarily conserved, even though its potential for futile catalytic cycling is low due to its sluggish catalytic activity in yeast. This suggested that other factors (i.e. post-translational modifications or “degrons”) contribute to its UPD. Indeed, in cultured human hepatocytes, CYP2E1 is detectably ubiquitinated, and this is enhanced on its mechanism-based inactivation. Studies in Ubc7p and Ubc5p genetically deficient yeast strains versus corresponding isogenic wild types identified these ubiquitin-conjugating E2 enzymes as relevant to CYP2E1 UPD. Consistent with this, in vitro functional reconstitution analyses revealed that mammalian UBC7/gp78 and UbcH5a/CHIP E2-E3 ubiquitin ligases were capable of ubiquitinating CYP2E1, a process enhanced by protein kinase (PK) A and/or PKC inclusion. Inhibition of PKA or PKC blocked intracellular CYP2E1 ubiquitination and turnover. Here, through mass spectrometric analyses, we identify some CYP2E1 phosphorylation/ubiquitination sites in spatially associated clusters. We propose that these CYP2E1 phosphorylation clusters may serve to engage each E2-E3 ubiquitination complex in vitro and intracellularly. PMID:21209460

  14. Nr2e1 Deficiency Augments Palmitate-Induced Oxidative Stress in Beta Cells

    PubMed Central

    Shi, Xiaoli; Deng, Haohua; Dai, Zhe; Xu, Yancheng; Xiong, Xiaokan; Ma, Pei; Cheng, Jing

    2016-01-01

    Nuclear receptor subfamily 2 group E member 1 (Nr2e1) has been regarded as an essential regulator of the growth of neural stem cells. However, its function elsewhere is unknown. In the present study, we generated Nr2e1 knockdown MIN6 cells and studied whether Nr2e1 knockdown affected basal beta cell functions such as proliferation, cell death, and insulin secretion. We showed that knockdown of Nr2e1 in MIN6 cells resulted in increased sensitivity to lipotoxicity, decreased proliferation, a partial G0/G1 cell-cycle arrest, and higher rates of apoptosis. Moreover, Nr2e1 deficiency exaggerates palmitate-induced impairment in insulin secretion. At the molecular level, Nr2e1 deficiency augments palmitate-induced oxidative stress. Nr2e1 deficiency also resulted in decreases in antioxidant enzymes and expression level of Nrf2. Together, this study indicated a potential protective effect of Nr2e1 on beta cells, which may serve as a target for the development of novel therapies for diabetes. PMID:26649147

  15. 26 CFR 1.665(e)-1A - Preceding taxable year.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... 26 Internal Revenue 8 2011-04-01 2011-04-01 false Preceding taxable year. 1.665(e)-1A Section 1... (CONTINUED) INCOME TAXES (CONTINUED) Treatment of Excess Distributions of Trusts Applicable to Taxable Years Beginning on Or After January 1, 1969 § 1.665(e)-1A Preceding taxable year. (a) Definition—(1)...

  16. 26 CFR 1.665(e)-1A - Preceding taxable year.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... 26 Internal Revenue 8 2010-04-01 2010-04-01 false Preceding taxable year. 1.665(e)-1A Section 1... (CONTINUED) INCOME TAXES Treatment of Excess Distributions of Trusts Applicable to Taxable Years Beginning on Or After January 1, 1969 § 1.665(e)-1A Preceding taxable year. (a) Definition—(1) Domestic...

  17. 26 CFR 48.4216(e)-1 - Exclusion of local advertising charges from sale price.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... 26 Internal Revenue 16 2011-04-01 2011-04-01 false Exclusion of local advertising charges from sale price. 48.4216(e)-1 Section 48.4216(e)-1 Internal Revenue INTERNAL REVENUE SERVICE, DEPARTMENT OF THE TREASURY (CONTINUED) MISCELLANEOUS EXCISE TAXES MANUFACTURERS AND RETAILERS EXCISE TAXES Special Provisions Applicable to Manufacturers Taxes §...

  18. 26 CFR 1.263(e)-1 - Expenditures in connection with certain railroad rolling stock.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... by the Interstate Commerce Commission (49 CFR Part 1201), but only if (i) such unit exclusively moves... rolling stock. 1.263(e)-1 Section 1.263(e)-1 Internal Revenue INTERNAL REVENUE SERVICE, DEPARTMENT OF THE TREASURY (CONTINUED) INCOME TAX (CONTINUED) INCOME TAXES (CONTINUED) Items Not Deductible §...

  19. Resolvin E1 and chemokine-like receptor 1 mediate bone preservation.

    PubMed

    Gao, Li; Faibish, Dan; Fredman, Gabrielle; Herrera, Bruno S; Chiang, Nan; Serhan, Charles N; Van Dyke, Thomas E; Gyurko, Robert

    2013-01-15

    The polyunsaturated ω-3 fatty acid eicosapentaenoic acid-derived resolvin E1 (RvE1) enhances resolution of inflammation, prevents bone loss, and induces bone regeneration. Although the inflammation-resolving actions of RvE1 are characterized, the molecular mechanism of its bone-protective actions are of interest. To test the hypothesis that receptor-mediated events impact bone changes, we prepared transgenic mice overexpressing the RvE1 receptor chemokine-like receptor 1 (chemR23) on leukocytes. In zymosan-initiated peritonitis, neutrophil polymorphonuclear leukocyte infiltration in response to RvE1 was limited requiring log order lower doses in chemR23tg mice. Ligature-induced alveolar bone loss was diminished in chemR23tg mice. Local RvE1 treatment of uniform craniotomy in the parietal bone significantly accelerated regeneration of the bone defect. In in vitro bone cultures, RvE1 significantly enhanced expression of osteoprotegerin (OPG) without inducing change in receptor activator of NF-κB ligand levels, whereas the osteogenic markers alkaline phosphatase, bone sialoprotein, and Runt-related transcription factor 2 remained unchanged. These results indicate that RvE1 modulates osteoclast differentiation and bone remodeling by direct actions on bone, rescuing OPG production and restoring a favorable receptor activator of NF-κB ligand/OPG ratio, in addition to known anti-inflammatory and proresolving actions. PMID:23241890

  20. 26 CFR 1.1031(e)-1 - Exchange of livestock of different sexes.

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ... 26 Internal Revenue 11 2014-04-01 2014-04-01 false Exchange of livestock of different sexes. 1.1031(e)-1 Section 1.1031(e)-1 Internal Revenue INTERNAL REVENUE SERVICE, DEPARTMENT OF THE TREASURY... Exchange of livestock of different sexes. Section 1031(e) provides that livestock of different sexes...

  1. 26 CFR 1.1031(e)-1 - Exchange of livestock of different sexes.

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ... 26 Internal Revenue 11 2012-04-01 2012-04-01 false Exchange of livestock of different sexes. 1.1031(e)-1 Section 1.1031(e)-1 Internal Revenue INTERNAL REVENUE SERVICE, DEPARTMENT OF THE TREASURY... Exchange of livestock of different sexes. Section 1031(e) provides that livestock of different sexes...

  2. 26 CFR 1.1031(e)-1 - Exchange of livestock of different sexes.

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    ... 26 Internal Revenue 11 2013-04-01 2013-04-01 false Exchange of livestock of different sexes. 1.1031(e)-1 Section 1.1031(e)-1 Internal Revenue INTERNAL REVENUE SERVICE, DEPARTMENT OF THE TREASURY... Exchange of livestock of different sexes. Section 1031(e) provides that livestock of different sexes...

  3. 26 CFR 1.1031(e)-1 - Exchange of livestock of different sexes.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... 26 Internal Revenue 11 2010-04-01 2010-04-01 true Exchange of livestock of different sexes. 1.1031(e)-1 Section 1.1031(e)-1 Internal Revenue INTERNAL REVENUE SERVICE, DEPARTMENT OF THE TREASURY... livestock of different sexes. Section 1031(e) provides that livestock of different sexes are not property...

  4. 26 CFR 1.1031(e)-1 - Exchange of livestock of different sexes.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... 26 Internal Revenue 11 2011-04-01 2011-04-01 false Exchange of livestock of different sexes. 1.1031(e)-1 Section 1.1031(e)-1 Internal Revenue INTERNAL REVENUE SERVICE, DEPARTMENT OF THE TREASURY... Exchange of livestock of different sexes. Section 1031(e) provides that livestock of different sexes...

  5. 26 CFR 31.3121(e)-1 - State, United States, and citizen.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... 26 Internal Revenue 15 2011-04-01 2011-04-01 false State, United States, and citizen. 31.3121(e)-1... § 31.3121(e)-1 State, United States, and citizen. (a) When used in the regulations in this subpart, the..., the term “United States”, when used in a geographical sense, means the several states (including...

  6. 17 CFR 270.30e-1 - Reports to stockholders of management companies.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... 17 Commodity and Securities Exchanges 3 2010-04-01 2010-04-01 false Reports to stockholders of management companies. 270.30e-1 Section 270.30e-1 Commodity and Securities Exchanges SECURITIES AND EXCHANGE... stockholders of management companies. (a) Every registered management company shall transmit to...

  7. 26 CFR 1.509(e)-1 - Definition of gross investment income.

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    ... 26 Internal Revenue 7 2013-04-01 2013-04-01 false Definition of gross investment income. 1.509(e)-1 Section 1.509(e)-1 Internal Revenue INTERNAL REVENUE SERVICE, DEPARTMENT OF THE TREASURY... gross investment income. For the distinction between gross receipts and gross investment income, see §...

  8. CK2 Phosphorylation Inactivates DNA Binding by the Papillomavirus E1 and E2 Proteins

    PubMed Central

    Schuck, Stephen; Ruse, Cristian

    2013-01-01

    Papillomaviruses have complex life cycles that are understood only superficially. Although it is well established that the viral E1 and E2 proteins play key roles in controlling viral transcription and DNA replication, how these factors are regulated is not well understood. Here, we demonstrate that phosphorylation by the protein kinase CK2 controls the biochemical activities of the bovine papillomavirus E1 and E2 proteins by modifying their DNA binding activity. Phosphorylation at multiple sites in the N-terminal domain in E1 results in the loss of sequence-specific DNA binding activity, a feature that is also conserved in human papillomavirus (HPV) E1 proteins. The bovine papillomavirus (BPV) E2 protein, when phosphorylated by CK2 on two specific sites in the hinge, also loses its site-specific DNA binding activity. Mutation of these sites in E2 results in greatly increased levels of latent viral DNA replication, indicating that CK2 phosphorylation of E2 is a negative regulator of viral DNA replication during latent viral replication. In contrast, mutation of the N-terminal phosphorylation sites in E1 has no effect on latent viral DNA replication. We propose that the phosphorylation of the N terminus of E1 plays a role only in vegetative viral DNA replication, and consistent with such a role, caspase 3 cleavage of E1, which has been shown to be necessary for vegetative viral DNA replication, restores the DNA binding activity to phosphorylated E1. PMID:23637413

  9. Resolvin E1 inhibits dendritic cell migration in the skin and attenuates contact hypersensitivity responses.

    PubMed

    Sawada, Yu; Honda, Tetsuya; Hanakawa, Sho; Nakamizo, Satoshi; Murata, Teruasa; Ueharaguchi-Tanada, Yuri; Ono, Sachiko; Amano, Wataru; Nakajima, Saeko; Egawa, Gyohei; Tanizaki, Hideaki; Otsuka, Atsushi; Kitoh, Akihiko; Dainichi, Teruki; Ogawa, Narihito; Kobayashi, Yuichi; Yokomizo, Takehiko; Arita, Makoto; Nakamura, Motonobu; Miyachi, Yoshiki; Kabashima, Kenji

    2015-10-19

    Resolvin E1 (RvE1) is a lipid mediator derived from ω3 polyunsaturated fatty acids that exerts potent antiinflammatory roles in several murine models. The antiinflammatory mechanism of RvE1 in acquired immune responses has been attributed to attenuation of cytokine production by dendritic cells (DCs). In this study, we newly investigated the effect of RvE1 on DC motility using two-photon microscopy in a contact hypersensitivity (CHS) model and found that RvE1 impaired DC motility in the skin. In addition, RvE1 attenuated T cell priming in the draining lymph nodes and effector T cell activation in the skin, which led to the reduced skin inflammation in CHS. In contrast, leukotriene B4 (LTB4) induced actin filament reorganization in DCs and increased DC motility by activating Cdc42 and Rac1 via BLT1, which was abrogated by RvE1. Collectively, our results suggest that RvE1 attenuates cutaneous acquired immune responses by inhibiting cutaneous DC motility, possibly through LTB4-BLT1 signaling blockade. PMID:26438363

  10. Resolvin E1 inhibits dendritic cell migration in the skin and attenuates contact hypersensitivity responses

    PubMed Central

    Sawada, Yu; Hanakawa, Sho; Nakamizo, Satoshi; Murata, Teruasa; Ueharaguchi-Tanada, Yuri; Ono, Sachiko; Amano, Wataru; Nakajima, Saeko; Egawa, Gyohei; Tanizaki, Hideaki; Otsuka, Atsushi; Kitoh, Akihiko; Dainichi, Teruki; Ogawa, Narihito; Kobayashi, Yuichi; Yokomizo, Takehiko; Arita, Makoto; Nakamura, Motonobu; Miyachi, Yoshiki

    2015-01-01

    Resolvin E1 (RvE1) is a lipid mediator derived from ω3 polyunsaturated fatty acids that exerts potent antiinflammatory roles in several murine models. The antiinflammatory mechanism of RvE1 in acquired immune responses has been attributed to attenuation of cytokine production by dendritic cells (DCs). In this study, we newly investigated the effect of RvE1 on DC motility using two-photon microscopy in a contact hypersensitivity (CHS) model and found that RvE1 impaired DC motility in the skin. In addition, RvE1 attenuated T cell priming in the draining lymph nodes and effector T cell activation in the skin, which led to the reduced skin inflammation in CHS. In contrast, leukotriene B4 (LTB4) induced actin filament reorganization in DCs and increased DC motility by activating Cdc42 and Rac1 via BLT1, which was abrogated by RvE1. Collectively, our results suggest that RvE1 attenuates cutaneous acquired immune responses by inhibiting cutaneous DC motility, possibly through LTB4-BLT1 signaling blockade. PMID:26438363

  11. 26 CFR 1.665(e)-1A - Preceding taxable year.

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ... 26 Internal Revenue 8 2012-04-01 2012-04-01 false Preceding taxable year. 1.665(e)-1A Section 1... (CONTINUED) INCOME TAXES (CONTINUED) Treatment of Excess Distributions of Trusts Applicable to Taxable Years Beginning on Or After January 1, 1969 § 1.665(e)-1A Preceding taxable year. (a) Definition—(1)...

  12. 26 CFR 1.665(e)-1A - Preceding taxable year.

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ... 26 Internal Revenue 8 2014-04-01 2014-04-01 false Preceding taxable year. 1.665(e)-1A Section 1... (CONTINUED) INCOME TAXES (CONTINUED) Treatment of Excess Distributions of Trusts Applicable to Taxable Years Beginning on Or After January 1, 1969 § 1.665(e)-1A Preceding taxable year. (a) Definition—(1)...

  13. Distinct Features of Cap Binding by eIF4E1b Proteins

    PubMed Central

    Kubacka, Dorota; Miguel, Ricardo Núñez; Minshall, Nicola; Darzynkiewicz, Edward; Standart, Nancy; Zuberek, Joanna

    2015-01-01

    eIF4E1b, closely related to the canonical translation initiation factor 4E (eIF4E1a), cap-binding protein is highly expressed in mouse, Xenopus and zebrafish oocytes. We have previously characterized eIF4E1b as a component of the CPEB mRNP translation repressor complex along with the eIF4E-binding protein 4E-Transporter, the Xp54/DDX6 RNA helicase and additional RNA-binding proteins. eIF4E1b exhibited only very weak interactions with m7GTP-Sepharose and, rather than binding eIF4G, interacted with 4E-T. Here we undertook a detailed examination of both Xenopus and human eIF4E1b interactions with cap analogues using fluorescence titration and homology modeling. The predicted structure of eIF4E1b maintains the α + β fold characteristic of eIF4E proteins and its cap-binding pocket is similarly arranged by critical amino acids: Trp56, Trp102, Glu103, Trp166, Arg112, Arg157 and Lys162 and residues of the C-terminal loop. However, we demonstrate that eIF4E1b is 3-fold less well able to bind the cap than eIF4E1a, both proteins being highly stimulated by methylation at N7 of guanine. Moreover, eIF4E1b proteins are distinguishable from eIF4E1a by a set of conserved amino acid substitutions, several of which are located near to cap-binding residues. Indeed, eIF4E1b possesses several distinct features, namely, enhancement of cap binding by a benzyl group at N7 position of guanine, a reduced response to increasing length of the phosphate chain and increased binding to a cap separated by a linker from Sepharose, suggesting differences in the arrangement of the protein's core. In agreement, mutagenesis of the amino acids differentiating eIF4E1b from eIF4E1a reduces cap binding by eIF4E1a 2-fold, demonstrating their role in modulating cap binding. PMID:25463438

  14. Oxidative stress mediated toxicity exerted by ethanol-inducible CYP2E1

    SciTech Connect

    Wu Defeng; Cederbaum, Arthur I. . E-mail: arthur.cederbaum@mssm.edu

    2005-09-01

    Induction of CYP2E1 by ethanol is one of the central pathways by which ethanol generates a state of oxidative stress in hepatocytes. To study the biochemical and toxicological actions of CYP2E1, our laboratory established HepG2 cell lines which constitutively overexpress CYP2E1 and characterized these cells with respect to ethanol toxicity. Addition of ethanol or an unsaturated fatty acid such as arachidonic acid or iron was toxic to the CYP2E1-expressing cells but not control cells. This toxicity was associated with elevated lipid peroxidation and could be prevented by antioxidants and inhibitors of CYP2E1. Apoptosis occurred in the CYP2E1-expressing cells exposed to ethanol, arachidonic acid, or iron. Removal of GSH caused a loss of viability in the CYP2E1-expressing cells even in the absence of added toxin or pro-oxidant. This was associated with mitochondrial damage and decreased mitochondrial membrane potential. Low concentrations of iron and arachidonic acid synergistically interacted with CYP2E1 to produce cell toxicity, suggesting these nutrients may act as priming or sensitizing agents to alcohol-induced liver injury. Surprisingly, CYP2E1-expressing cells had elevated GSH levels, due to transcriptional activation of glutamate cysteine ligase. Similarly, levels of catalase, alpha-, and microsomal glutathione transferase were also increased, suggesting that upregulation of these antioxidant genes may reflect an adaptive mechanism to remove CYP2E1-derived oxidants. Using co-cultures, interaction between CYP2E1-derived diffusible mediators to activate collagen production in hepatic stellate cells was found. While it is likely that several mechanisms contribute to alcohol-induced liver injury, the linkage between CYP2E1-dependent oxidative stress, mitochondrial injury, stellate cell activation, and GSH homeostasis may contribute to the toxic action of ethanol on the liver. HepG2 cell lines overexpressing CYP2E1 may be a valuable model to characterize the

  15. The Equine Herpesvirus 2 E1 Open Reading Frame Encodes a Functional Chemokine Receptor

    PubMed Central

    Camarda, Grazia; Spinetti, Gaia; Bernardini, Giovanni; Mair, Catherine; Davis-Poynter, Nick; Capogrossi, Maurizio C.; Napolitano, Monica

    1999-01-01

    Several herpesviruses contain open reading frames (ORFs) that encode potential homologs of eucaryotic genes. Equine herpesvirus 2 (EHV-2) is a gammaherpesvirus related to other lymphotropic herpesviruses such as herpesvirus saimiri and Epstein-Barr virus. The E1 ORF of EHV-2, a G protein-coupled receptor homolog, shows 31 to 47% amino acid identity with known CC chemokine receptors. To investigate whether E1 may encode a functional receptor, we cloned the E1 ORF and expressed it in stably transfected cell lines. We report here the identification of the CC chemokine eotaxin as a functional ligand for the EHV-2 E1 receptor. Chemokines are likely to play a role in the regulation of immune functions in equine hosts during EHV-2 infection and, via interaction with E1, may affect viral replication and/or escape from immune responses. PMID:10559296

  16. Beyond Oncolytics: E1B55K-Deleted Adenovirus as a Vaccine Delivery Vector.

    PubMed

    Thomas, Michael A; Nyanhete, Tinashe; Tuero, Iskra; Venzon, David; Robert-Guroff, Marjorie

    2016-01-01

    Type 5 human adenoviruses (Ad5) deleted of genes encoding the early region 1B 55-kDa (E1B55K) protein including Onyx-015 (dl1520) and H101 are best known for their oncolytic potential. As a vaccine vector the E1B55K deletion may allow for the insertion of a transgene nearly 1,000 base pairs larger than now possible. This has the potential of extending the application for which the vectors are clinically known. However, the immune priming ability of E1B55K-deleted vectors is unknown, undermining our ability to gauge their usefulness in vaccine applications. For this reason, we created an E1B55K-deleted Ad5 vector expressing full-length single chain HIVBaLgp120 attached to a flexible linker and the first two domains of rhesus CD4 (rhFLSC) in exchange for the E3 region. In cell-based experiments the E1B55K-deleted vector promoted higher levels of innate immune signals including chemokines, cytokines, and the NKG2D ligands MIC A/B compared to an E1B55K wild-type vector expressing the same immunogen. Based on these results we evaluated the immune priming ability of the E1B55K-deleted vector in mice. The E1B55K-deleted vector promoted similar levels of Ad5-, HIVgp120, and rhFLSC-specific cellular and humoral immune responses as the E1B55K wild-type vector. In pre-clinical HIV-vaccine studies the wild-type vector has been employed as part of a very effective prime-boost strategy. This study demonstrates that E1B55K-deleted adenoviruses may serve as effective vaccine delivery vectors. PMID:27391605

  17. Role of CYP2E1 in thioacetamide-induced mouse hepatotoxicity

    SciTech Connect

    Kang, Jin Seok; Wanibuchi, Hideki; Morimura, Keiichirou; Wongpoomchai, Rawiwan; Chusiri, Yaowares; Gonzalez, Frank J.; Fukushima, Shoji

    2008-05-01

    Previous experiments showed that treatment of mice and rats with thioacetamide (TAA) induced liver cell damage, fibrosis and/or cirrhosis, associated with increased oxidative stress and activation of hepatic stellate cells. Some experiments suggest that CYP2E1 may be involved in the metabolic activation of TAA. However, there is no direct evidence on the role of CYP2E1 in TAA-mediated hepatotoxicity. To clarify this, TAA-induced hepatotoxicity was investigated using Cyp2e1-null mice. Male wild-type and Cyp2e1-null mice were treated with TAA (200 mg/kg of body weight, single, i.p.) at 6 weeks of age, and hepatotoxicity examined 24 and 48 h after TAA treatment. Relative liver weights of Cyp2e1-null mice were significantly different at 24 h compared to wild-type mice (p < 0.01). Serum levels of aspartate aminotransferase (AST), alanine aminotransferase (ALT), alkaline phosphatase (ALP) and lactate dehydrogenase (LDH) in Cyp2e1-null mice were significantly different at both time points compared to wild-type mice (p < 0.01). Histopathological examination showed Cyp2e1-null mice represented no hepatototoxic lesions, in clear contrast to severe centriobular necrosis, inflammation and hemorrhage at both time points in wild-type mice. Marked lipid peroxidation was also only limited to wild-type mice (p < 0.01). Similarly, TNF-{alpha}, IL-6 and glutathione peroxidase mRNA expression in Cyp2e1-null mice did not significantly differ from the control levels, contrasting with the marked alteration in wild-type mice (p < 0.01). Western blot analysis further revealed no increase in iNOS expression in Cyp2e1-null mice. These results reveal that CYP2E1 mediates TAA-induced hepatotoxicity in wild-type mice as a result of increased oxidative stress.

  18. Architecture of the hepatitis C virus E1 glycoprotein transmembrane domain studied by NMR.

    PubMed

    Zazrin, Hadas; Shaked, Hadassa; Chill, Jordan H

    2014-03-01

    Oligomerization of hepatitis C viral envelope proteins E1 and E2 is essential to virus fusion and assembly. Although interactions within the transmembrane (TM) domains of these glycoproteins have proven contributions to the E1/E2 heterodimerization process and consequent infectivity, there is little structural information on this entry mechanism. Here, as a first step towards our long-term goal of understanding the interaction between E1 and E2 TM-domains, we have expressed, purified and characterized E1-TM using structural biomolecular NMR methods. An MBP-fusion expression system yielded sufficient quantities of pure E1-TM, which was solubilized in two membrane-mimicking environments, SDS- and LPPG-micelles, affording samples amenable to NMR studies. Triple resonance assignment experiments and relaxation measurements provided information on the secondary structure and global fold of E1-TM in these environments. In SDS micelles E1-TM adopts a helical conformation, with helical stretches at residues 354-363 and 371-379 separated by a more flexible segment of residues 364-370. In LPPG micelles a helical conformation was observed for residues 354-377 with greater flexibility in the 366-367 dyad, suggesting LPPG provides a more native environment for the peptide. Replacement of key positively charged residue K370 with an alanine did not affect the secondary structure of E1-TM but did change the relative positioning within the micelle of the two helices. These results lay the foundation for structure determination of E1-TM and a molecular understanding of how E1-TM flexibility enhances its interaction with E2-TM during heterodimerization and membrane fusion. PMID:24192053

  19. Molecular mechanism of trichloroethylene-induced hepatotoxicity mediated by CYP2E1

    SciTech Connect

    Ramdhan, Doni Hikmat; Kamijima, Michihiro; Yamada, Naoyasu; Ito, Yuki; Yanagiba, Yukie; Nakamura, Daichi; Okamura, Ai; Ichihara, Gaku; Aoyama, Toshifumi; Gonzalez, Frank J.; Nakajima, Tamie

    2008-09-15

    Cytochrome P450 (CYP) 2E1 was suggested to be the major enzyme involved in trichloroethylene (TRI) metabolism and TRI-induced hepatotoxicity, although the latter molecular mechanism is not fully understood. The involvement of CYP2E1 in TRI-induced hepatotoxicity and its underlying molecular mechanism were studied by comparing hepatotoxicity in cyp2e1{sup +/+} and cyp2e1{sup -/-} mice. The mice were exposed by inhalation to 0 (control), 1000, or 2000 ppm of TRI for 8 h a day, for 7 days, and TRI-hepatotoxicity was assessed by measuring plasma alanine aminotransferase (ALT) and aspartate aminotransferase (AST) activities and histopathology. Urinary metabolites of trichloroethanol and trichloroacetic acid (TCA) were considerably greater in cyp2e1{sup +/+} compared to cyp2e1{sup -/-} mice, suggesting that CYP2E1 is the major P450 involved in the formation of these metabolites. Consistent with elevated plasma ALT and AST activities, cyp2e1{sup +/+} mice in the 2000 ppm group showed histopathological inflammation. TRI significantly upregulated PPAR{alpha}, which might function to inhibit NF{kappa}B p50 and p65 signalling. In addition, TRI-induced NF{kappa}B p52 mRNA, and significantly positive correlation between NF{kappa}B p52 mRNA expression and plasma ALT activity levels were observed, suggesting the involvement of p52 in liver inflammation. Taken together, the current study directly demonstrates that CYP2E1 was the major P450 involved in the first step of the TRI metabolism, and the metabolites produced may have two opposing roles: one inducing hepatotoxicity and the other protecting against the toxicity. Intermediate metabolite(s) from TRI to chloral hydrate produced by CYP2E1-mediated oxidation may be involved in the former, and TCA in the latter.

  20. Beyond Oncolytics: E1B55K-Deleted Adenovirus as a Vaccine Delivery Vector

    PubMed Central

    Thomas, Michael A.; Nyanhete, Tinashe; Tuero, Iskra; Venzon, David; Robert-Guroff, Marjorie

    2016-01-01

    Type 5 human adenoviruses (Ad5) deleted of genes encoding the early region 1B 55-kDa (E1B55K) protein including Onyx-015 (dl1520) and H101 are best known for their oncolytic potential. As a vaccine vector the E1B55K deletion may allow for the insertion of a transgene nearly 1,000 base pairs larger than now possible. This has the potential of extending the application for which the vectors are clinically known. However, the immune priming ability of E1B55K-deleted vectors is unknown, undermining our ability to gauge their usefulness in vaccine applications. For this reason, we created an E1B55K-deleted Ad5 vector expressing full-length single chain HIVBaLgp120 attached to a flexible linker and the first two domains of rhesus CD4 (rhFLSC) in exchange for the E3 region. In cell-based experiments the E1B55K-deleted vector promoted higher levels of innate immune signals including chemokines, cytokines, and the NKG2D ligands MIC A/B compared to an E1B55K wild-type vector expressing the same immunogen. Based on these results we evaluated the immune priming ability of the E1B55K-deleted vector in mice. The E1B55K-deleted vector promoted similar levels of Ad5-, HIVgp120, and rhFLSC-specific cellular and humoral immune responses as the E1B55K wild-type vector. In pre-clinical HIV-vaccine studies the wild-type vector has been employed as part of a very effective prime-boost strategy. This study demonstrates that E1B55K-deleted adenoviruses may serve as effective vaccine delivery vectors. PMID:27391605

  1. Cytochrome P450 2E1 (CYP2E1) regulates the response to oxidative stress and migration of breast cancer cells

    PubMed Central

    2013-01-01

    Introduction The cytochrome P450 (CYP) enzymes are a class of heme-containing enzymes involved in phase I metabolism of a large number of xenobiotics. The CYP family member CYP2E1 metabolises many xenobiotics and pro-carcinogens, it is not just expressed in the liver but also in many other tissues such as the kidney, the lung, the brain, the gastrointestinal tract and the breast tissue. It is induced in several pathological conditions including cancer, obesity, and type II diabetes implying that this enzyme is implicated in other biological processes beyond its role in phase I metabolism. Despite the detailed description of the role of CYP2E1 in the liver, its functions in other tissues have not been extensively studied. In this study, we investigated the functional significance of CYP2E1 in breast carcinogenesis. Methods Cellular levels of reactive oxygen species (ROS) were measured by H2DCFDA (2 2.9.2 2′,7′-dichlorodihydrofluorescein diacetate) staining and autophagy was assessed by tracing the cellular levels of autophagy markers using western blot assays. The endoplasmic reticulum stress and the unfolded protein response (UPR) were detected by luciferase assays reflecting the splicing of mRNA encoding the X-box binding protein 1 (XBP1) transcription factor and cell migration was evaluated using the scratch wound assay. Gene expression was recorded with standard transcription assays including luciferase reporter and chromatin immunoprecipitation. Results Ectopic expression of CYP2E1 induced ROS generation, affected autophagy, stimulated endoplasmic reticulum stress and inhibited migration in breast cancer cells with different metastatic potential and p53 status. Furthermore, evidence is presented indicating that CYP2E1 gene expression is under the transcriptional control of the p53 tumor suppressor. Conclusions These results support the notion that CYP2E1 exerts an important role in mammary carcinogenesis, provide a potential link between ethanol metabolism

  2. Orthogonal Ubiquitin Transfer through Engineered E1-E2 Cascades for Protein Ubiquitination

    PubMed Central

    Zhao, Bo; Bhuripanyo, Karan; Zhang, Keya; Kiyokawa, Hiroaki; Schindelin, Hermann; Yin, Jun

    2014-01-01

    SUMMARY Protein modification by ubiquitin (UB) controls diverse cellular processes. UB is conjugated to cellular proteins by sequential transfer through an E1-E2-E3 enzymatic cascade. The cross-activities of 2 E1s, 50 E2s and thousands of E3s encoded by the human genome make it difficult to identify the substrate proteins of a specific E3 enzyme in the cell. One way to solve this problem is to engineer an orthogonal UB transfer (OUT) cascade in which the engineered UB (xUB) is relayed by engineered E1, E2 and E3 enzymes (xE1, xE2, xE3) to modify the substrate proteins of a specific E3. Here, we use phage display and mutagenesis to construct xUB-xE1 and xE1-xE2 pairs that are orthogonal to the native E1 and E2 enzymes. Our work on engineering the UB transfer cascades will enable us to use OUT to map the signal transduction networks mediated by protein ubiquitination. PMID:23102221

  3. Intranuclear location of the adenovirus type 5 E1B 55-kilodalton protein.

    PubMed Central

    Smiley, J K; Young, M A; Flint, S J

    1990-01-01

    The intracellular location of the adenovirus type 5 E1B 55-kilodalton (kDa) protein, particularly the question of whether it is associated with nuclear pore complexes, was examined. Fractionation of adenovirus type 5-infected HeLa cell nuclei by an established procedure (N. Dwyer and G. Blobel, J. Cell. Biol. 70:581-591, 1976) yielded one population of E1B 55-kDa protein molecules released by digestion of nuclei with RNase A and a second population recovered in the pore complex-lamina fraction. Free and E1B 55-kDa protein-bound forms of the E4 34-kDa protein (P. Sarnow, C. A. Sullivan, and A. J. Levine, Virology 120:387-394, 1982) were largely recovered in the pore complex-lamina fraction. Nevertheless, the association of E1B 55-kDa protein molecules with this nuclear envelope fraction did not depend on interaction of the E1B 55-kDa protein with the E4 34-kDa protein. Comparison of the immunofluorescence patterns observed with antibodies recognizing the E1B 55-kDa protein or cellular pore complex proteins and of the behavior of these viral and cellular proteins during in situ fractionation suggests that the E1B 55-kDa protein does not become intimately or stably associated with pore complexes in adenovirus-infected cells. Images PMID:2143545

  4. Evidence for a tissue-specific induction of cutaneous CYP2E1 by dexamethasone.

    PubMed

    Sampol, E; Mirrione, A; Villard, P H; Piccerelle, P; Scoma, H; Berbis, P; Barra, Y; Durand, A; Lacarelle, B

    1997-06-27

    We studied in mouse the effect of topical application of dexamethasone or salicylic acid, on CYP2E1 and CYP3A expression (proteins and/or mRNA) in liver and skin. Dexamethasone was also administered by intraperitoneal injection. Topical application or intraperitoneal injection of dexamethasone increased cutaneous CYP2E1 (8 and 4-fold respectively) whereas the hepatic level of this isoform showed a slight decrease and hepatic CYP3A expression was increased (3-fold). Cutaneous CYP2E1 was increased (3-fold) after topical treatment by salicylic acid. This compound had no effect on hepatic CYP3A and CYP2E1 expression. Cutaneous CYP3A (protein and mRNA) was not detectable in all groups (control or treated animals). Dexamethasone and salicylic acid increased cutaneous CYP2E1 mRNA level (2.5 and 1.4-fold respectively). In conclusion, dexamethasone and salicylic acid induced cutaneous CYP2E1 protein and mRNA level. Cutaneous CYP2E1 induction by dexamethasone is a tissue-specific process. PMID:9207195

  5. E1A activates transcription of p73 and Noxa to induce apoptosis.

    PubMed

    Flinterman, Marcella; Guelen, Lars; Ezzati-Nik, Samira; Killick, Richard; Melino, Gerry; Tominaga, Kazuya; Mymryk, Joe S; Gäken, Joop; Tavassoli, Mahvash

    2005-02-18

    p73, a member of the p53 family of proteins, transcriptionally activates a number of genes involved in the control of cell cycle and apoptosis. Overexpression of p73 was detected in a large number of primary head and neck cancers, and in the established cell lines examined, these all contained inactivating p53 mutations. The significance of p73 overexpression in the pathogenesis of head and neck cancer is currently unclear. We have shown that the expression of adenovirus 5 E1A in a panel of head and neck cancer cell lines induces apoptosis independently of their p53 status. In this study we examined the role of p73 and its transcriptional targets in E1A-mediated induction of apoptosis. E1A expression resulted in significant activation of the TAp73 promoter but had no effect on the alternative, DeltaNp73 promoter. E1A also increased expression of endogenous TAp73 mRNA and protein. E1A mutants lacking the p300- and/or pRB-binding sites showed reduced ability to activate the TAp73 promoter. Additionally, mutations in the E2F1-binding sites in the TAp73 promoter impaired activation by E1A. Importantly, expression of the 13S isoform of E1A substantially induced the p53 apoptotic target Noxa in several p53-deficient cancer cell lines. Our results indicate that E1A activation of p73 and the p53 apoptotic target Noxa can occur in the absence of a functional p53. This activation is likely to play a key role in the mechanism of p53-independent apoptosis induced by E1A in some cancers and may provide an avenue for future cancer therapies. PMID:15572378

  6. Selective Time- and NADPH-Dependent Inhibition of Human CYP2E1 by Clomethiazole.

    PubMed

    Stresser, David M; Perloff, Elke S; Mason, Andrew K; Blanchard, Andrew P; Dehal, Shangara S; Creegan, Timothy P; Singh, Ritu; Gangl, Eric T

    2016-08-01

    The sedative clomethiazole (CMZ) has been used in Europe since the mid-1960s to treat insomnia and alcoholism. It has been previously demonstrated in clinical studies to reversibly inhibit human CYP2E1 in vitro and decrease CYP2E1-mediated elimination of chlorzoxazone. We have investigated the selectivity of CMZ inhibition of CYP2E1 in pooled human liver microsomes (HLMs). In a reversible inhibition assay of the major drug-metabolizing cytochrome P450 (P450) isoforms, CYP2A6 and CYP2E1 exhibited IC50 values of 24 µM and 42 µM, respectively with all other isoforms exhibiting values >300 µM. When CMZ was preincubated with NADPH and liver microsomal protein for 30 minutes before being combined with probe substrates, however, more potent inhibition was observed for CYP2E1 and CYP2B6 but not CYP2A6 or other P450 isoforms. The substantial increase in potency of CYP2E1 inhibition upon preincubation enables the use of CMZ to investigate the role of human CYP2E1 in xenobiotic metabolism and provides advantages over other chemical inhibitors of CYP2E1. The KI and kinact values obtained with HLM-catalyzed 6-hydroxylation of chlorzoxazone were 40 µM and 0.35 minute(-1), respectively, and similar to values obtained with recombinant CYP2E1 (41 µM, 0.32 minute(-1)). The KI and kinact values, along with other parameters, were used in a mechanistic static model to explain earlier observations of a profound decrease in the rate of chlorzoxazone elimination in volunteers despite the absence of detectable CMZ in blood. PMID:27149898

  7. Involvement of CYP 2E1 enzyme in ovotoxicity caused by 4-vinylcyclohexene and its metabolites.

    PubMed

    Rajapaksa, Kathila S; Cannady, Ellen A; Sipes, I Glenn; Hoyer, Patricia B

    2007-06-01

    4-Vinylcyclohexene (VCH) is bioactivated by hepatic CYP 2A and 2B to a monoepoxide (VCM) and subsequently to an ovotoxic diepoxide metabolite (VCD). Studies suggest that the ovary can directly bioactivate VCH via CYP 2E1. The current study was designed to evaluate the role of ovarian CYP 2E1 in VCM-induced ovotoxicity. Postnatal day 4 B6C3F(1) and CYP 2E1 wild-type (+/+) and null (-/-) mouse ovaries were cultured (15 days) with VCD (30 microM), 1,2-VCM (125-1000 microM), or vehicle. Twenty-eight days female CYP 2E1 +/+ and -/- mice were dosed daily (15 days; ip) with VCH, 1,2-VCM, VCD or vehicle. Following culture or in vivo dosing, ovaries were histologically evaluated. In culture, VCD decreased (p<0.05) primordial and primary follicles in ovaries from all three groups of mice. 1,2-VCM decreased (p<0.05) primordial follicles in B6C3F(1) and CYP 2E1 +/+ ovaries, but not in CYP 2E1 -/- ovaries in culture. 1,2-VCM did not affect primary follicles in any group of mouse ovaries. Conversely, following in vivo dosing, primordial and primary follicles were reduced (p<0.05) by VCD and VCM in CYP2E1 +/+ and -/-, and by VCH in +/+ mice. The data demonstrate that, whereas in vitro ovarian bioactivation of VCM requires CYP 2E1 enzyme, in vivo CYP 2E1 plays a minimal role. Thus, the findings support that hepatic metabolism dominates the contribution made by the ovary in bioactivation of VCM to its ovotoxic metabolite, VCD. This study also demonstrates the use of a novel ovarian culture system to evaluate ovary-specific metabolism of xenobiotics. PMID:17462685

  8. Involvement of CYP 2E1 enzyme in ovotoxicity caused by 4-vinylcyclohexene and its metabolites

    SciTech Connect

    Rajapaksa, Kathila S.; Cannady, Ellen A.; Sipes, I. Glenn; Hoyer, Patricia B. . E-mail: hoyer@u.arizona.edu

    2007-06-01

    4-Vinylcyclohexene (VCH) is bioactivated by hepatic CYP 2A and 2B to a monoepoxide (VCM) and subsequently to an ovotoxic diepoxide metabolite (VCD). Studies suggest that the ovary can directly bioactivate VCH via CYP 2E1. The current study was designed to evaluate the role of ovarian CYP 2E1 in VCM-induced ovotoxicity. Postnatal day 4 B6C3F{sub 1} and CYP 2E1 wild-type (+/+) and null (-/-) mouse ovaries were cultured (15 days) with VCD (30 {mu}M), 1,2-VCM (125-1000 {mu}M), or vehicle. Twenty-eight days female CYP 2E1 +/+ and -/- mice were dosed daily (15 days; ip) with VCH, 1,2-VCM, VCD or vehicle. Following culture or in vivo dosing, ovaries were histologically evaluated. In culture, VCD decreased (p < 0.05) primordial and primary follicles in ovaries from all three groups of mice. 1,2-VCM decreased (p < 0.05) primordial follicles in B6C3F{sub 1} and CYP 2E1 +/+ ovaries, but not in CYP 2E1 -/- ovaries in culture. 1,2-VCM did not affect primary follicles in any group of mouse ovaries. Conversely, following in vivo dosing, primordial and primary follicles were reduced (p < 0.05) by VCD and VCM in CYP2E1 +/+ and -/-, and by VCH in +/+ mice. The data demonstrate that, whereas in vitro ovarian bioactivation of VCM requires CYP 2E1 enzyme, in vivo CYP 2E1 plays a minimal role. Thus, the findings support that hepatic metabolism dominates the contribution made by the ovary in bioactivation of VCM to its ovotoxic metabolite, VCD. This study also demonstrates the use of a novel ovarian culture system to evaluate ovary-specific metabolism of xenobiotics.

  9. Origin of low-lying enhanced E1 strength in rare-Earth nuclei.

    PubMed

    Spieker, M; Pascu, S; Zilges, A; Iachello, F

    2015-05-15

    The experimental E1 strength distribution below 4 MeV in rare-earth nuclei suggests a local breaking of isospin symmetry. In addition to the octupole states, additional J^{π}=1^{-} states with enhanced E1 strength have been observed in rare-earth nuclei by means of (γ,γ') experiments. By reproducing the experimental results, the spdf interacting boson model calculations provide further evidence for the formation of an α cluster in medium-mass nuclei and might provide a new understanding of the origin of low-lying E1 strength. PMID:26024168

  10. Effects of orbital and spin current interference in E1 and M2 nuclear excitations

    SciTech Connect

    Goncharova, N. G.

    2015-12-15

    The interference of contributions from the orbital and spin currents to the E1 and M2 resonances is investigated. The results of the current interference analysis within the shell model are compared with the experimental data.

  11. Hepatitis C Virus Envelope Glycoprotein E1 Forms Trimers at the Surface of the Virion

    PubMed Central

    Falson, Pierre; Bartosch, Birke; Alsaleh, Khaled; Tews, Birke Andrea; Loquet, Antoine; Ciczora, Yann; Riva, Laura; Montigny, Cédric; Montpellier, Claire; Duverlie, Gilles; Pécheur, Eve-Isabelle; le Maire, Marc; Cosset, François-Loïc

    2015-01-01

    ABSTRACT In hepatitis C virus (HCV)-infected cells, the envelope glycoproteins E1 and E2 assemble as a heterodimer. To investigate potential changes in the oligomerization of virion-associated envelope proteins, we performed SDS-PAGE under reducing conditions but without thermal denaturation. This revealed the presence of SDS-resistant trimers of E1 in the context of cell-cultured HCV (HCVcc) as well as in the context of HCV pseudoparticles (HCVpp). The formation of E1 trimers was found to depend on the coexpression of E2. To further understand the origin of E1 trimer formation, we coexpressed in bacteria the transmembrane (TM) domains of E1 (TME1) and E2 (TME2) fused to reporter proteins and analyzed the fusion proteins by SDS-PAGE and Western blotting. As expected for strongly interacting TM domains, TME1–TME2 heterodimers resistant to SDS were observed. These analyses also revealed homodimers and homotrimers of TME1, indicating that such complexes are stable species. The N-terminal segment of TME1 exhibits a highly conserved GxxxG sequence, a motif that is well documented to be involved in intramembrane protein-protein interactions. Single or double mutations of the glycine residues (Gly354 and Gly358) in this motif markedly decreased or abrogated the formation of TME1 homotrimers in bacteria, as well as homotrimers of E1 in both HCVpp and HCVcc systems. A concomitant loss of infectivity was observed, indicating that the trimeric form of E1 is essential for virus infectivity. Taken together, these results indicate that E1E2 heterodimers form trimers on HCV particles, and they support the hypothesis that E1 could be a fusion protein. IMPORTANCE HCV glycoproteins E1 and E2 play an essential role in virus entry into liver cells as well as in virion morphogenesis. In infected cells, these two proteins form a complex in which E2 interacts with cellular receptors, whereas the function of E1 remains poorly understood. However, recent structural data suggest that E1

  12. Mechanistic analysis of local ori melting and helicase assembly by the papillomavirus E1 protein

    PubMed Central

    Schuck, Stephen; Stenlund, Arne

    2011-01-01

    Summary Preparation of DNA templates for replication requires opening of the duplex to expose single stranded (ss) DNA. The locally melted DNA is required for replicative DNA helicases to initiate unwinding. How local melting is generated in eukaryotic replicons is unknown, but initiator proteins from a handful of eukaryotic viruses can perform this function. Here we dissect the local melting process carried out by the papillomavirus E1 protein. We characterize the melting process kinetically and identify mutations in the E1 helicase and in the ori that arrest the local melting process. We show that a subset of these mutants have specific defects for melting of the center of the ori containing the binding sites for E1 and demonstrate that these mutants fail to untwist the ori DNA. This newfound understanding of how E1 generates local melting suggests possible mechanisms for local melting in other replicons. PMID:21884978

  13. Functional processing and secretion of Chikungunya virus E1 and E2 glycoproteins in insect cells

    PubMed Central

    2011-01-01

    Background Chikungunya virus (CHIKV) is a mosquito-borne, arthrogenic Alphavirus that causes large epidemics in Africa, South-East Asia and India. Recently, CHIKV has been transmitted to humans in Southern Europe by invading and now established Asian tiger mosquitoes. To study the processing of envelope proteins E1 and E2 and to develop a CHIKV subunit vaccine, C-terminally his-tagged E1 and E2 envelope glycoproteins were produced at high levels in insect cells with baculovirus vectors using their native signal peptides located in CHIKV 6K and E3, respectively. Results Expression in the presence of either tunicamycin or furin inhibitor showed that a substantial portion of recombinant intracellular E1 and precursor E3E2 was glycosylated, but that a smaller fraction of E3E2 was processed by furin into mature E3 and E2. Deletion of the C-terminal transmembrane domains of E1 and E2 enabled secretion of furin-cleaved, fully processed E1 and E2 subunits, which could then be efficiently purified from cell culture fluid via metal affinity chromatography. Confocal laser scanning microscopy on living baculovirus-infected Sf21 cells revealed that full-length E1 and E2 translocated to the plasma membrane, suggesting similar posttranslational processing of E1 and E2, as in a natural CHIKV infection. Baculovirus-directed expression of E1 displayed fusogenic activity as concluded from syncytia formation. CHIKV-E2 was able to induce neutralizing antibodies in rabbits. Conclusions Chikungunya virus glycoproteins could be functionally expressed at high levels in insect cells and are properly glycosylated and cleaved by furin. The ability of purified, secreted CHIKV-E2 to induce neutralizing antibodies in rabbits underscores the potential use of E2 in a subunit vaccine to prevent CHIKV infections. PMID:21762510

  14. 11 CFR 300.60 - Scope (2 U.S.C. 441i(e)(1)).

    Code of Federal Regulations, 2010 CFR

    2010-01-01

    ...) REGULATIONS NON-FEDERAL FUNDS Federal Candidates and Officeholders § 300.60 Scope (2 U.S.C. 441i(e)(1)). This subpart applies to: (a) Federal candidates; (b) Individuals holding Federal office (see 11 CFR 300.2(o... 11 Federal Elections 1 2010-01-01 2010-01-01 false Scope (2 U.S.C. 441i(e)(1)). 300.60 Section...

  15. 11 CFR 300.60 - Scope (2 U.S.C. 441i(e)(1)).

    Code of Federal Regulations, 2011 CFR

    2011-01-01

    ...) REGULATIONS NON-FEDERAL FUNDS Federal Candidates and Officeholders § 300.60 Scope (2 U.S.C. 441i(e)(1)). This subpart applies to: (a) Federal candidates; (b) Individuals holding Federal office (see 11 CFR 300.2(o... 11 Federal Elections 1 2011-01-01 2011-01-01 false Scope (2 U.S.C. 441i(e)(1)). 300.60 Section...

  16. Resolvin E1 regulates osteoclast fusion via DC-STAMP and NFATc1

    PubMed Central

    Zhu, Min; Van Dyke, Thomas E.; Gyurko, Robert

    2013-01-01

    Interactions between the immune and skeletal systems in inflammatory bone diseases are well appreciated, but the underlying molecular mechanisms that coordinate the resolution phase of inflammation and bone turnover have not been unveiled. Here we investigated the direct actions of the proresolution mediator resolvin E1 (RvE1) on bone-marrow-cell-derived osteoclasts in an in vitro murine model of osteoclast maturation and inflammatory bone resorption. Investigation of the actions of RvE1 treatment on the specific stages of osteoclast maturation revealed that RvE1 targeted late stages of osteoclast maturation to decrease osteoclast formation by 32.8%. Time-lapse vital microscopy and migration assays confirmed that membrane fusion of osteoclast precursors was inhibited. The osteoclast fusion protein DC-STAMP was specifically targeted by RvE1 receptor binding and was down-regulated by 65.4%. RvE1 did not affect the induction of the essential osteoclast transcription factor nuclear factor of activated T cells c1 (NFATc1) or its nuclear translocation; however, NFATc1 binding to the DC-STAMP promoter was significantly inhibited by 60.9% with RvE1 treatment as shown in electrophoresis mobility shift assay. Our findings suggest that proresolution mediators act directly on osteoclasts, in addition to down-regulation of inflammation, providing a novel mechanism for modulating osteoclast signaling in osteolytic inflammatory disease.—Zhu, M., Van Dyke, T. E., Gyurko, R. Resolvin E1 regulates osteoclast fusion via DC-STAMP and NFATc1. PMID:23629863

  17. 40 CFR Figure E-1 to Subpart E of... - Designation Testing Checklist

    Code of Federal Regulations, 2012 CFR

    2012-07-01

    ... Equivalent Methods for PM2.5 or PM10â2.5 Pt. 53, Subpt. E, Fig. E-1 Figure E-1 to Subpart E of Part 53... 40 CFR Part 53 or 40 CFR Part 50, Appendix L Verification Comments (Includes documentation of who...-7.4.10) Application Specification Tests Field Precision (§ 53.58) (L-5.1) Meets all Appendix...

  18. Response of an Escherichia coli-Bound Fluorescent Probe to Colicin E1

    PubMed Central

    Cramer, W. A.; Phillips, S. K.

    1970-01-01

    The fluorescent probe, 8-anilino-1-napthalenesulfonate (ANS) binds to Escherichia coli, showing an enhanced fluorescence. The interaction of colicin E1 with sensitive cells causes an increase of about 100% in the fluorescence of the bound ANS, and this change at equilibrium has an apparent “all-or-none” nature as a function of E1 multiplicity. Approximately 6 to 8% of the ANS is bound to the cells at equilibrium. The colicin E1-induced fluorescence increase can be attributed partly to an increase in ANS binding and partly to an increase in the fluorescence yield of the bound ANS. The kinetics of the E1-induced fluorescence increase in sensitive cells are very similar to those of the adenosine triphosphate decrease. The phosphorylation uncoupler p-trifluoromethoxy-carbonylcyanidephenylhydrazone also causes a large change in the fluorescence of bound ANS. Colicin E2 or E3 does not cause any fluorescence change, nor does colicin E1 cause fluorescence change with a colicinogenic strain. ANS appears to be a probe of structural or conformational change in the cell envelope that is closely associated with the colicin E1-induced adenosine triphosphate decrease. PMID:4923074

  19. ATF4 deficiency protects hepatocytes from oxidative stress via inhibiting CYP2E1 expression

    PubMed Central

    Wang, Chunxia; Li, Houkai; Meng, Qingshu; Du, Ying; Xiao, Fei; Zhang, Qian; Yu, Junjie; Li, Kai; Chen, Shanghai; Huang, Zhiying; Liu, Bin; Guo, Feifan

    2014-01-01

    Activating transcription factor (ATF) 4 is involved in the regulation of oxidative stress in fibroblasts and neurons. The role of ATF4 in hepatocytes, however, is unknown. The aim of this study was to investigate the role of ATF4 in hepatocytes in oxidative stress under a high-fat diet (HFD). Here, we showed that palmitate-stimulated reactive oxygen species (ROS) production and triglyceride (TG) accumulation is blocked by ATF4 deficiency in primary hepatocytes. Consistently, HFD-induced oxidative stress, TG accumulation and expression of cytochrome P450, family 2, subfamily, polypeptide 1 (CYP2E1) are also blocked by knocking down ATF4 expression in the mouse liver. This suggests that ATF4 might regulate oxidative stress viaCYP2E1 under an HFD. In addition, we observed that expression of CYP2E1 is indirectly regulated by ATF4 in a cAMP-responsive element binding protein (CREB)-dependent manner, which can directly activate the CYP2E1 promoter activity. Notably, ATF4-stimulated ROS production is inhibited in vivo by treatment with diallyl sulphide, a selective CYP2E1 inhibitor. Finally, we showed that ATF4 expression in the liver is responsible for the protective effects against HFD-induced CYP2E1 expression, oxidative stress, and TG accumulation. Taken together, these observations suggest that ATF4 is a novel regulator of oxidative stress as well as accumulation of TG in response to HFD. PMID:24373582

  20. E1A inhibits transforming growth factor-beta signaling through binding to Smad proteins.

    PubMed

    Nishihara, A; Hanai, J; Imamura, T; Miyazono, K; Kawabata, M

    1999-10-01

    Smads form a recently identified family of proteins that mediate intracellular signaling of the transforming growth factor (TGF)-beta superfamily. Smads bind to DNA and act as transcriptional regulators. Smads interact with a variety of transcription factors, and the interaction is likely to determine the target specificity of gene induction. Smads also associate with transcriptional coactivators such as p300 and CBP. E1A, an adenoviral oncoprotein, inhibits TGF-beta-induced transactivation, and the ability of E1A to bind p300/CBP is required for the inhibition. Here we determined the Smad interaction domain (SID) in p300 and found that two adjacent regions are required for the interaction. One of the regions is the C/H3 domain conserved between p300 and CBP, and the other is a nonconserved region. p300 mutants containing SID inhibit transactivation by TGF-beta in a dose-dependent manner. E1A inhibits the interaction of Smad3 with a p300 mutant that contains SID but lacks the E1A binding domain. We found that E1A interacts specifically with receptor-regulated Smads, suggesting a novel mechanism whereby E1A antagonizes TGF-beta signaling. PMID:10497242

  1. In vitro characterization of the NAD(+) synthetase NadE1 from Herbaspirillum seropedicae.

    PubMed

    Laskoski, Kerly; Santos, Adrian R S; Bonatto, Ana C; Pedrosa, Fábio O; Souza, Emanuel M; Huergo, Luciano F

    2016-05-01

    Nicotinamide adenine dinucleotide synthetase enzyme (NadE) catalyzes the amination of nicotinic acid adenine dinucleotide (NaAD) to form NAD(+). This reaction represents the last step in the majority of the NAD(+) biosynthetic routes described to date. NadE enzymes typically use either glutamine or ammonium as amine nitrogen donor, and the reaction is energetically driven by ATP hydrolysis. Given the key role of NAD(+) in bacterial metabolism, NadE has attracted considerable interest as a potential target for the development of novel antibiotics. The plant-associative nitrogen-fixing bacteria Herbaspirillum seropedicae encodes two putative NadE, namely nadE1 and nadE2. The nadE1 gene is linked to glnB encoding the signal transduction protein GlnB. Here we report the purification and in vitro characterization of H. seropedicae NadE1. Gel filtration chromatography analysis suggests that NadE1 is an octamer. The NadE1 activity was assayed in vitro, and the Michaelis-Menten constants for substrates NaAD, ATP, glutamine and ammonium were determined. Enzyme kinetic and in vitro substrate competition assays indicate that H. seropedicae NadE1 uses glutamine as a preferential nitrogen donor. PMID:26802007

  2. Specific uptake of radioiodinated fragment E1 by venous thrombi in pigs

    SciTech Connect

    Knight, L.C.; Olexa, S.A.; Malmud, L.S.; Budzynski, A.Z.

    1983-12-01

    Fragment E1, a product of plasmic digestion of cross-linked fibrin, binds specifically in vitro to polymerized fibrin but not to fibrinogen. Purified human Fragment E1 was radiolabeled with /sup 125/I or /sup 131/I by the Iodogen technique. The uptake of radioiodinated Fragment E1 in vitro into forming or preformed clots was demonstrated. Animal biodistribution studies of radioiodinated Fragment E1 showed its rapid removal from the circulation; radioactive catabolites did not reside long in any organ and were excreted in the urine. The uptake in vivo was evaluated in pigs with preexisting venous thrombi of various ages from 1 h up to 5 d at the time of intravenous systemic injection of the tracer. Radioiodinated fibrinogen was also injected into the same animals to compare the uptake of the two tracers. Thrombus-to-blood ratios for Fragment E1 averaged 43:1 (range 10-108) and 29:1 (range 8-107) in thrombi 1-6 h and 1-5 d old, respectively. In contrast, mean thrombus-to-blood ratios for fibrinogen were, in the same time intervals, 26:1 (range 17-41) and 2:1 (range 0.5-3.9), respectively. It is concluded that radioiodinated Fragment E1 is a specific marker of thrombi in vivo: its uptake by fresh thrombi is better than that of labeled fibrinogen and, in contrast to radioiodinated fibrinogen, this fragment is incorporated into old thrombi as well.

  3. Resolvin D1 and E1 promote resolution of inflammation in microglial cells in vitro.

    PubMed

    Rey, C; Nadjar, A; Buaud, B; Vaysse, C; Aubert, A; Pallet, V; Layé, S; Joffre, C

    2016-07-01

    Sustained inflammation in the brain together with microglia activation can lead to neuronal damage. Hence limiting brain inflammation and activation of microglia is a real therapeutic strategy for inflammatory disease. Resolvin D1 (RvD1) and resolvin E1 (RvE1) derived from n-3 long chain polyunsaturated fatty acids are promising therapeutic compounds since they actively turn off the systemic inflammatory response. We thus evaluated the anti-inflammatory activities of RvD1 and RvE1 in microglia cells in vitro. BV2 cells were pre-incubated with RvD1 or RvE1 before lipopolysaccharide (LPS) treatment. RvD1 and RvE1 both decreased LPS-induced proinflammatory cytokines (TNF-α, IL-6 and IL-1β) gene expression, suggesting their proresolutive activity in microglia. However, the mechanisms involved are distinct as RvE1 regulates NFκB signaling pathway and RvD1 regulates miRNAs expression. Overall, our findings support that pro-resolving lipids are involved in the resolution of brain inflammation and can be considered as promising therapeutic agents for brain inflammation. PMID:26718448

  4. Replication-associated activities of purified human papillomavirus type 11 E1 helicase.

    PubMed

    Rocque, W J; Porter, D J; Barnes, J A; Dixon, E P; Lobe, D C; Su, J L; Willard, D H; Gaillard, R; Condreay, J P; Clay, W C; Hoffman, C R; Overton, L K; Pahel, G; Kost, T A; Phelps, W C

    2000-03-01

    Replication of human papillomavirus type11 (HPV11) requires both the E1 and the E2 proteins. E1 is structurally and functionally similar to SV40 large T-antigen and is a DNA helicase/NTPase that binds to the origin of replication and initiates viral DNA replication. The biochemical characterization of HPV E1 is incompletely documented in the literature in part because of difficulties in expressing and purifying the protein. Herein, we report a method for the overexpression of full-length, untagged E1 (73.5 kDa) in baculovirus-infected Trichoplusia ni insect cells and the purification to homogeneity using a two-step procedure. The purified protein is a nonspecific NTPase that hydrolyzes ATP, dATP, UTP, or GTP equally well. Point mutations were made in the putative NTPase domain to verify that the activities observed were encoded by E1. Purified mutant D523N had negligible ATPase and helicase activities but retained DNA-binding activity. Sedimentation equilibrium ultracentrifugation and glycerol gradient centrifugation demonstrated that the wild-type protein is primarily a hexamer in its purified form. Secondary structure determination by circular dichroism revealed a large percentage of alpha-helical structure consistent with secondary structure predictions. These data define a fundamental set of biochemical and kinetic parameters for HPV E1 which are a critical prerequisite to future mechanistic studies of the enzyme. PMID:10686145

  5. Diabetes mellitus increases the in vivo activity of cytochrome P450 2E1 in humans

    PubMed Central

    Wang, Zaiqi; Hall, Stephen D; Maya, Juan F; Li, Lang; Asghar, Ali; Gorski, J C

    2003-01-01

    Aim Cytochrome P450 2E1 (CYP2E1) is thought to activate a number of protoxins, and has been implicated in the development of liver disease. Increased hepatic expression of CYP2E1 occurs in rat models of diabetes but it is unclear whether human diabetics display a similar up-regulation. This study was designed to test the hypothesis that human diabetics experience enhanced CYP2E1 expression. Methods The pharmacokinetics of a single dose of chlorzoxazone (500 mg), used as an index of hepatic CYP2E1 activity, was determined in healthy subjects (n = 10), volunteers with Type I (n = 13), and Type II (n = 8) diabetes mellitus. Chlorzoxazone and 6-hydroxychlorzoxazone in serum and urine were analysed by high-performance liquid chromatography. The expression of CYP2E1 mRNA in peripheral blood mononuclear cells was quantified by reverse transcriptase-polymerase chain reaction. Results The mean ± s.d. (90% confidence interval of the difference) chlorzoxazone area under the plasma concentration-time curve was significantly (P ≤ 0.05) reduced in obese Type II diabetics (15.7 ± 11.3 µg h ml−1; 9, 22) compared with healthy subjects (43.5 ± 16.9 µg h ml−1; 16, 40) and Type I diabetics (32.8 ± 9.2 µg h ml−1; 9, 25). There was a significant two-fold increase in the oral clearance of chlorzoxazone in obese Type II diabetics compared with healthy volunteers and Type I diabetics. The protein binding of chlorzoxazone was not significantly different between the three groups. In contrast, Type 1 diabetics and healthy volunteers demonstrated no difference in the oral clearance of chlorzoxazone. The urinary recovery of 6-hydroxychlorzoxazone as a percentage of the administered dose was not different between healthy, Type I and obese Type II diabetics. The elimination half-life of chlorzoxazone did not differ between the three groups. CYP2E1 mRNA was significantly elevated in Type I and obese Type II diabetics compared with healthy volunteers. The oral clearance of

  6. Autophagy Protects against CYP2E1/Chronic Ethanol-Induced Hepatotoxicity

    PubMed Central

    Lu, Yongke; Cederbaum, Arthur I.

    2015-01-01

    Autophagy is an intracellular pathway by which lysosomes degrade and recycle long-lived proteins and cellular organelles. The effects of ethanol on autophagy are complex but recent studies have shown that autophagy serves a protective function against ethanol-induced liver injury. Autophagy was found to also be protective against CYP2E1-dependent toxicity in vitro in HepG2 cells which express CYP2E1 and in vivo in an acute alcohol/CYPE1-dependent liver injury model. The goal of the current report was to extend the previous in vitro and acute in vivo experiments to a chronic ethanol model to evaluate whether autophagy is also protective against CYP2E1-dependent liver injury in a chronic ethanol-fed mouse model. Wild type (WT), CYP2E1 knockout (KO) or CYP2E1 humanized transgenic knockin (KI), mice were fed an ethanol liquid diet or control dextrose diet for four weeks. In the last week, some mice received either saline or 3-methyladenine (3-MA), an inhibitor of autophagy, or rapamycin, which stimulates autophagy. Inhibition of autophagy by 3-MA potentiated the ethanol-induced increases in serum transaminase and triglyceride levels in the WT and KI mice but not KO mice, while rapamycin prevented the ethanol liver injury. Treatment with 3-MA enhanced the ethanol-induced fat accumulation in WT mice and caused necrosis in the KI mice; little or no effect was found in the ethanol-fed KO mice or any of the dextrose-fed mice. 3-MA treatment further lowered the ethanol-decrease in hepatic GSH levels and further increased formation of TBARS in WT and KI mice, whereas rapamycin blunted these effects of ethanol. Neither 3-MA nor rapamycin treatment affected CYP2E1 catalytic activity or content or the induction CYP2E1 by ethanol. The 3-MA treatment decreased levels of Beclin-1 and Atg 7 but increased levels of p62 in the ethanol-fed WT and KI mice whereas rapamycin had the opposite effects, validating inhibition and stimulation of autophagy, respectively. These results suggest

  7. Cytochrome p450 2E1 polymorphisms and the risk of gastric cardia cancer

    PubMed Central

    Cai, Lin; Zheng, Zong-Li; Zhang, Zuo-Feng

    2005-01-01

    AIM: Genetic polymorphisms of drug-metabolizing enzymes have recently been shown to affect susceptibility to chemical carcinogenesis. Cytochrome P450 2E1 (CYP2E1) enzyme catalyzes the metabolism of many procarcinogens, such as N-nitrosamines and related compounds. The gene coding for this enzyme is polymorphic and thus may play a role in gastric cardia cancer (GCC) etiology. In this hospital-based case-control study, we evaluate the relationship between genetic polymorphisms of CYP2E1 and the risk of GCC. METHODS: The study subjects comprised 159 histologically confirmed GCC cases identified via hospital cancer registry and surgical records at five hospitals in Fuzhou, Fujian Province, China, between April and November 2001. Controls were 192 patients admitted to the same hospitals for nonmalignant conditions. The genotypes of CYP2E1 were detected by a PCR-based RFLP assay. The odds ratios were estimated by logistic regression analyses and were adjusted for potential confounding factors. RESULTS: The distribution of three genotypes of CYP2E1 in GCC cases and controls was significantly different (χ2 = 16.04, P<0.01). The frequency of the CYP2E1 (c1/c1) genotype in GCC cases and controls was 60.4% and 40.1%, respectively. The CYP2E1 (c1/c1) genotype was associated with an increased risk for GCC (the adjusted (OR) was 2.37, 95% confidence interval (CI): 1.52-3.70). Subjects who carried the CYP2E1 (c1/c1) genotype and were habitual smokers were at a significantly higher risk of developing GCC (OR = 4.68, 95%CI: 2.19-10.04) compared with those who had the CYP2E1 (c1/c2 or c2/c2) genotype and did not smoke. CONCLUSION: These results suggest that the CYP2E1 genotype may influence individual susceptibility to development of GCC, and that the risk increases significantly in smokers. PMID:15793883

  8. Redox Regulation of Human Estrogen Sulfotransferase (hSULT1E1)†

    PubMed Central

    Maiti, Smarajit; Zhang, Jimei; Chen, Guangping

    2007-01-01

    Sulfotransferases (SULTs) are enzymes that catalyze the sulfation of hydroxyl-containing compounds. Sulfation regulates hormone activities and detoxifies xenobiotics. Human estrogen sulfotransferase (hSULT1E1) catalyzes the sulfation of estrogens and regulates estrogen bioactivities. Oxidative regulation provides a biological mechanism for regulating enzyme activities in vivo. The oxidative regulation of human SULTs has not been reported. In this study, we used amino acid modification, manipulation of intracellular redox state, and site-directed mutagenesis to study the redox regulation of human SULTs and specifically the mechanism of hSULT1E1 inhibitory regulation by oxidized glutathione (GSSG). Of the four major human SULTs, hSULT1A1, hSULT1A3, and hSULT2A1 do not undergo redox regulation; hSULT1E1, on the other hand, can be redox regulated. GSSG inactivated hSULT1E1 activity in an efficient, time- and concentration-dependant manner. The co-enzyme adenosine 3′-phosphate 5′-phosphosulfate protected hSULT1E1 from GSSG-associated inactivation. A reduced glutathione (GSH) inducer (N-acetyl cysteine) significantly increased while a GSH depletor (buthionine sulfoxamine) significantly decreased hSULT1E1 activity, but both failed to affect the amount of hSULT1E1 protein in human hepatocyte carcinoma Hep G2 cells. Crystal structure suggested that no Cys residues exist near the active sites of hSULT1A1, hSULT1A3, and hSULT2A1, but Cys residues do exist within the active site of hSULT1E1. Site-directed mutagenesis demonstrated that Cys83 is critical for the redox regulation of hSULT1E1. This first report on the redox regulation of human SULTs suggests that the redox regulation of hSULT1E1 may interrupt the regulation and function of estrogens under various physiological and pathological conditions. PMID:17266938

  9. Requirement for the E1 Helicase C-Terminal Domain in Papillomavirus DNA Replication In Vivo

    PubMed Central

    Bergvall, Monika; Gagnon, David; Titolo, Steve; Lehoux, Michaël; D'Abramo, Claudia M.

    2016-01-01

    ABSTRACT The papillomavirus (PV) E1 helicase contains a conserved C-terminal domain (CTD), located next to its ATP-binding site, whose function in vivo is still poorly understood. The CTD is comprised of an alpha helix followed by an acidic region (AR) and a C-terminal extension termed the C-tail. Recent biochemical studies on bovine papillomavirus 1 (BPV1) E1 showed that the AR and C-tail regulate the oligomerization of the protein into a double hexamer at the origin. In this study, we assessed the importance of the CTD of human papillomavirus 11 (HPV11) E1 in vivo, using a cell-based DNA replication assay. Our results indicate that combined deletion of the AR and C-tail drastically reduces DNA replication, by 85%, and that further truncation into the alpha-helical region compromises the structural integrity of the E1 helicase domain and its interaction with E2. Surprisingly, removal of the C-tail alone or mutation of highly conserved residues within the domain still allows significant levels of DNA replication (55%). This is in contrast to the absolute requirement for the C-tail reported for BPV1 E1 in vitro and confirmed here in vivo. Characterization of chimeric proteins in which the AR and C-tail from HPV11 E1 were replaced by those of BPV1 indicated that while the function of the AR is transferable, that of the C-tail is not. Collectively, these findings define the contribution of the three CTD subdomains to the DNA replication activity of E1 in vivo and suggest that the function of the C-tail has evolved in a PV type-specific manner. IMPORTANCE While much is known about hexameric DNA helicases from superfamily 3, the papillomavirus E1 helicase contains a unique C-terminal domain (CTD) adjacent to its ATP-binding site. We show here that this CTD is important for the DNA replication activity of HPV11 E1 in vivo and that it can be divided into three functional subdomains that roughly correspond to the three conserved regions of the CTD: an alpha helix, needed

  10. The adenovirus E1A protein overrides the requirement for cellular ras in initiating DNA synthesis.

    PubMed Central

    Stacey, D W; Dobrowolski, S F; Piotrkowski, A; Harter, M L

    1994-01-01

    The adenovirus E1A protein can induce cellular DNA synthesis in growth-arrested cells by interacting with the cellular protein p300 or pRb. In addition, serum- and growth factor-dependent cells require ras activity to initiate DNA synthesis and recently we have shown that Balb/c 3T3 cells can be blocked in either early or late G1 following microinjection of an anti-ras antibody. In this study, the E1A 243 amino acid protein is shown through microinjection not only to shorten the G0 to S phase interval but, what is more important, to override the inhibitory effects exerted by the anti-ras antibody in either early or late G1. Specifically, whether E1A is co-injected with anti-ras into quiescent cells or injected 18 h following a separate injection of anti-ras after serum stimulation, it efficiently induces cellular DNA synthesis in cells that would otherwise be blocked in G0/G1. Moreover, injection of a mutant form of E1A that can no longer associate with p300 is just as efficient as wild-type E1A in stimulating DNA synthesis in cells whose ras activity has been neutralized by anti-ras. The results presented here show that E1A is capable of overriding the requirement of cellular ras activity in promoting the entry of cells into S phase. Moreover, the results suggest the possibility that pRb and/or pRb-related proteins may function in a ras-dependent pathway that enables E1A to achieve this activity. Images PMID:7813447

  11. Mutation in E1, the Ubiquitin Activating Enzyme, Reduces Drosophila Lifespan and Results in Motor Impairment

    PubMed Central

    Liu, Hsiu-Yu; Pfleger, Cathie M.

    2013-01-01

    Neurodegenerative diseases cause tremendous suffering for those afflicted and their families. Many of these diseases involve accumulation of mis-folded or aggregated proteins thought to play a causal role in disease pathology. Ubiquitinated proteins are often found in these protein aggregates, and the aggregates themselves have been shown to inhibit the activity of the proteasome. These and other alterations in the Ubiquitin Pathway observed in neurodegenerative diseases have led to the question of whether impairment of the Ubiquitin Pathway on its own can increase mortality or if ongoing neurodegeneration alters Ubiquitin Pathway function as a side-effect. To address the role of the Ubiquitin Pathway in vivo, we studied loss-of-function mutations in the Drosophila Ubiquitin Activating Enzyme, Uba1 or E1, the most upstream enzyme in the Ubiquitin Pathway. Loss of only one functional copy of E1 caused a significant reduction in adult lifespan. Rare homozygous hypomorphic E1 mutants reached adulthood. These mutants exhibited further reduced lifespan and showed inappropriate Ras activation in the brain. Removing just one functional copy of Ras restored the lifespan of heterozygous E1 mutants to that of wild-type flies and increased the survival of homozygous E1 mutants. E1 homozygous mutants also showed severe motor impairment. Our findings suggest that processes that impair the Ubiquitin Pathway are sufficient to cause early mortality. Reduced lifespan and motor impairment are seen in the human disease X-linked Infantile Spinal Muscular Atrophy, which is associated with mutation in human E1 warranting further analysis of these mutants as a potential animal model for study of this disease. PMID:23382794

  12. Multiple transcriptional regulatory domains in the human immunodeficiency virus type 1 long terminal repeat are involved in basal and E1A/E1B-induced promoter activity.

    PubMed Central

    Kliewer, S; Garcia, J; Pearson, L; Soultanakis, E; Dasgupta, A; Gaynor, R

    1989-01-01

    The human immunodeficiency virus (HIV) type 1 long terminal repeat (LTR) is the site of activation of the HIV tat protein. However, additional transactivators, such as the adenovirus E1A and herpesvirus ICPO proteins, have also been shown to be capable of activating the HIV LTR. Analysis of adenovirus mutants indicated that complete transactivation of the HIV LTR was dependent on both the E1A and E1B proteins. To determine which regions of the HIV LTR were important for complete E1A/E1B activation, a variety of oligonucleotide-directed mutations in HIV transcriptional regulatory domains were assayed both in vivo and in vitro. S1 nuclease analysis of RNA prepared after transfection of these HIV constructs into HeLa cells infected with wild-type adenovirus indicated that the enhancer, SP1, TATA, and a portion of the transactivation-responsive element were each required for complete E1A/E1B-mediated activation of the HIV LTR. These same promoter elements were required for both basal and E1A/E1B-induced levels of transcription in in vitro transcription reactions performed with cellular extracts prepared from cells infected with dl434, an E1A/E1B deletion mutant, or wild-type adenovirus. No mutations were found that reduced only E1A/E1B-induced expression without proportionally reducing basal levels of transcription, suggesting that E1A/E1B-mediated induction of the HIV LTR requires multiple promoter elements which are also required for basal transcriptional levels. Unlike activation by the tat protein, there was not a rigid dependence on maintenance of the transactivation-responsive stem base pairing for E1A/E1B-mediated activation either in vivo or in vitro, indicating that activation occurs by a mechanism distinct from that of tat induction. Images PMID:2529378

  13. Structural models of the membrane anchors of envelope glycoproteins E1 and E2 from pestiviruses.

    PubMed

    Wang, Jimin; Li, Yue; Modis, Yorgo

    2014-04-01

    The membrane anchors of viral envelope proteins play essential roles in cell entry. Recent crystal structures of the ectodomain of envelope protein E2 from a pestivirus suggest that E2 belongs to a novel structural class of membrane fusion machinery. Based on geometric constraints from the E2 structures, we generated atomic models of the E1 and E2 membrane anchors using computational approaches. The E1 anchor contains two amphipathic perimembrane helices and one transmembrane helix; the E2 anchor contains a short helical hairpin stabilized in the membrane by an arginine residue, similar to flaviviruses. A pair of histidine residues in the E2 ectodomain may participate in pH sensing. The proposed atomic models point to Cys987 in E2 as the site of disulfide bond linkage with E1 to form E1-E2 heterodimers. The membrane anchor models provide structural constraints for the disulfide bonding pattern and overall backbone conformation of the E1 ectodomain. PMID:24725935

  14. Dual E1 activation systems for ubiquitin differentially regulate E2 enzyme charging.

    PubMed

    Jin, Jianping; Li, Xue; Gygi, Steven P; Harper, J Wade

    2007-06-28

    Modification of proteins with ubiquitin or ubiquitin-like proteins (UBLs) by means of an E1-E2-E3 cascade controls many signalling networks. Ubiquitin conjugation involves adenylation and thioesterification of the carboxy-terminal carboxylate of ubiquitin by the E1-activating enzyme Ube1 (Uba1 in yeast), followed by ubiquitin transfer to an E2-conjugating enzyme through a transthiolation reaction. Charged E2s function with E3s to ubiquitinate substrates. It is currently thought that Ube1/Uba1 is the sole E1 for charging of E2s with ubiquitin in animals and fungi. Here we identify a divergent E1 in vertebrates and sea urchin, Uba6, which specifically activates ubiquitin but not other UBLs in vitro and in vivo. Human Uba6 and Ube1 have distinct preferences for E2 charging in vitro, and their specificity depends in part on their C-terminal ubiquitin-fold domains, which recruit E2s. In tissue culture cells, Uba6 is required for charging a previously uncharacterized Uba6-specific E2 (Use1), whereas Ube1 is required for charging the cell-cycle E2s Cdc34A and Cdc34B. Our data reveal unexpected complexity in the pathways that control the conjugation of ubiquitin, in which dual E1s orchestrate the charging of distinct cohorts of E2s. PMID:17597759

  15. Sub-meter Precision Lunar Figures from Chang'E-1 and SELENE Laser Altimetry

    NASA Astrophysics Data System (ADS)

    Baki Iz, H.; Shum, C. K.; Chen, Y. Q.; Dai, C. L.

    2010-05-01

    The selenocentric radial footprint radial distances of 8.8 million Chang'E-1, and 8.5 million SELENE laser altimetry measurements were used used to estimate improved triaxial, biaxial and spherical lunar figure parameters together with their geometric centers with respect to the center of mass of the Moon. The estimated equatorial semi-major, minor, and polar axes of the triaxial ellipsoid's shape parameters from the Chang'E-1 and SELENE solutions differ by 143 m, 3 m and 49 m and -186 m, -3 m, and -52 m in their geometric center positions with respect to the lunar center of mass. The new missions' laser altimetry measurements data reveals a more spherical lunar figure and are in better agreement with each other compared to the ULCN 2005's geometrically best fitting lunar figure. The RMS misclosures calculated for Chang'E-1 and SELENE data using all the estimated parameters were used to assess the goodness of fit of each solution. The estimates for geometrically best fitting solution from Chang'E-1's data give the smallest RMS misclosures for both Chang'E-1 and SELENE data. Overall, the spherical harmonic topographical models' RMS misclosures are larger than those best fitting solutions.

  16. Mitochondrial pyruvate dehydrogenase. Molecular cloning of the E1 alpha subunit and expression analysis.

    PubMed Central

    Grof, C P; Winning, B M; Scaysbrook, T P; Hill, S A; Leaver, C J

    1995-01-01

    A polymerase chain reaction-based approach was used to isolate cDNA clones encoding the E1 alpha subunit of the mitochondrial pyruvate dehydrogenase from higher plants. Putative full-length clones were identified on the basis of similarity to E1 alpha sequences from nonplant sources. Southern blot analysis revealed a small family of genes in potato (Solanum tuberosum L.), whereas in cucumber (Cucumis sativus) there are only one or two genes. Tissue-specific variation in the relative amounts of E1 alpha mRNA was observed in northern blot analysis of different potato tissues, with the highest steady-state transcript levels found in floral tissue. Measurement of pyruvate dehydrogenase activity in cucumber cotyledons showed that there is a transient increase to a maximum at 4 to 5 d postimbibition. Western blot analysis revealed that the amount of E1 alpha protein also peaks at this time. Steady-state transcript levels in germinating cucumber cotyledons also show transient accumulation, peaking 2 d postimbibition. These data are consistent with regulation of E1 alpha at the level of transcription and/or mRNA stability in postgerminative cucumber cotyledons. PMID:7659754

  17. Cooperative effects for CYP2E1 differ between styrene and its metabolites

    PubMed Central

    Hartman, Jessica H.; Boysen, Gunnar; Miller, Grover P.

    2014-01-01

    Cooperative interactions are frequently observed in the metabolism of drugs and pollutants by cytochrome P450s; nevertheless, the molecular determinants for cooperativity remain elusive. Previously, we demonstrated that steady-state styrene metabolism by CYP2E1 exhibits positive cooperativity.We hypothesized that styrene metabolites have lower affinity than styrene toward CYP2E1 and limited ability to induce cooperative effects during metabolism. To test the hypothesis, we determined the potency and mechanism of inhibition for styrene and its metabolites toward oxidation of 4-nitrophenol using CYP2E1 Supersomes® and human liver microsomes.Styrene inhibited the reaction through a mixed cooperative mechanism with high affinity for the catalytic site (67 μM) and lower affinity for the cooperative site (1100 μM), while increasing substrate turnover at high concentrations. Styrene oxide and 4-vinylphenol possessed similar affinity for CYP2E1. Styrene oxide behaved cooperatively like styrene, but 4-vinylphenol decreased turnover at high concentrations. Styrene glycol was a very poor competitive inhibitor. Among all compounds, there was a positive correlation with binding and hydrophobicity.Taken together, these findings for CYP2E1 further validate contributions of cooperative mechanisms to metabolic processes, demonstrate the role of molecular structure on those mechanisms and underscore the potential for heterotropic cooperative effects between different compounds. PMID:23327532

  18. Genetic mapping of a major site of phosphorylation in adenovirus type 2 E1A proteins

    SciTech Connect

    Tsukamotot, A.S.; Ponticelli, A.; Berk, A.J.; Gaynor, R.B.

    1986-07-01

    Adenovirus early region 1A (E1A) encodes two acidic phosphoproteins which are required for transactivation of viral transcription, efficient viral DNA replication in phase G/sub 0/-arrested human cells, and oncogenic transformation of rodent cells. Biochemical analysis of in vivo /sup 32/P-labeled adenovirus type 2 E1A proteins purified with monoclonal antibodies demonstrated that these proteins were phosphorylated at multiple serine residues. Two-dimensional phosphotryptic peptide maps of wild-type and mutant E1A proteins were used to locate a major site of E1A protein phosphorylation at serine-219 of the large E1A protein. Although this serine fell within a consensus sequence for phosphorylation by the cyclic AMP-dependent protein kinases, experiments with mutant CHO cells defective in these enzymes indicated that it was not. Oligonucleotide-directed mutagenesis was used to substitute an alanine for serine-219. This mutation prevented phosphorylation at this site. Nonetheless, the mutant was indistinguishable from the wild type for early gene transactivation, replication on G/sub 0/-arrested WI-38 cells, and transformation of cloned rat embryo fibroblast cells.

  19. Effect of BI-1 on insulin resistance through regulation of CYP2E1

    PubMed Central

    Lee, Geum-Hwa; Oh, Kyoung-Jin; Kim, Hyung-Ryong; Han, Hye-Sook; Lee, Hwa-Young; Park, Keun-Gyu; Nam, Ki-Hoan; Koo, Seung-Hoi; Chae, Han-Jung

    2016-01-01

    Diet-induced obesity is a major contributing factor to the progression of hepatic insulin resistance. Increased free fatty acids in liver enhances endoplasmic reticulum (ER) stress and production of reactive oxygen species (ROS), both are directly responsible for dysregulation of hepatic insulin signaling. BI-1, a recently studied ER stress regulator, was examined to investigate its association with ER stress and ROS in insulin resistance models. To induce obesity and insulin resistance, BI-1 wild type and BI-1 knock-out mice were fed a high-fat diet for 8 weeks. The BI-1 knock-out mice had hyperglycemia, was associated with impaired glucose and insulin tolerance under high-fat diet conditions. Increased activity of NADPH-dependent CYP reductase-associated cytochrome p450 2E1 (CYP2E1) and exacerbation of ER stress in the livers of BI-1 knock-out mice was also observed. Conversely, stable expression of BI-1 in HepG2 hepatocytes was shown to reduce palmitate-induced ER stress and CYP2E1-dependent ROS production, resulting in the preservation of intact insulin signaling. Stable expression of CYP2E1 led to increased ROS production and dysregulation of insulin signaling in hepatic cells, mimicking palmitate-mediated hepatic insulin resistance. We propose that BI-1 protects against obesity-induced hepatic insulin resistance by regulating CYP2E1 activity and ROS production. PMID:27576594

  20. Effect of BI-1 on insulin resistance through regulation of CYP2E1.

    PubMed

    Lee, Geum-Hwa; Oh, Kyoung-Jin; Kim, Hyung-Ryong; Han, Hye-Sook; Lee, Hwa-Young; Park, Keun-Gyu; Nam, Ki-Hoan; Koo, Seung-Hoi; Chae, Han-Jung

    2016-01-01

    Diet-induced obesity is a major contributing factor to the progression of hepatic insulin resistance. Increased free fatty acids in liver enhances endoplasmic reticulum (ER) stress and production of reactive oxygen species (ROS), both are directly responsible for dysregulation of hepatic insulin signaling. BI-1, a recently studied ER stress regulator, was examined to investigate its association with ER stress and ROS in insulin resistance models. To induce obesity and insulin resistance, BI-1 wild type and BI-1 knock-out mice were fed a high-fat diet for 8 weeks. The BI-1 knock-out mice had hyperglycemia, was associated with impaired glucose and insulin tolerance under high-fat diet conditions. Increased activity of NADPH-dependent CYP reductase-associated cytochrome p450 2E1 (CYP2E1) and exacerbation of ER stress in the livers of BI-1 knock-out mice was also observed. Conversely, stable expression of BI-1 in HepG2 hepatocytes was shown to reduce palmitate-induced ER stress and CYP2E1-dependent ROS production, resulting in the preservation of intact insulin signaling. Stable expression of CYP2E1 led to increased ROS production and dysregulation of insulin signaling in hepatic cells, mimicking palmitate-mediated hepatic insulin resistance. We propose that BI-1 protects against obesity-induced hepatic insulin resistance by regulating CYP2E1 activity and ROS production. PMID:27576594

  1. Epitope-mapped monoclonal antibodies against the HPV16E1--E4 protein.

    PubMed

    Doorbar, J; Ely, S; Coleman, N; Hibma, M; Davies, D H; Crawford, L

    1992-03-01

    The human papillomavirus (HPV) E1--E4 protein is the only nonstructural late protein encoded by the virus. We have isolated three hybridomas producing monoclonal antibodies to the E1--E4 protein of HPV16, which is the HPV type most frequently associated with cervical cancer. The three antibodies (TVG 401, 402, and 403) detect adjacent epitopes within the major seroreactive region of the molecule and show no reactivity against the E4 proteins of HPV1, HPV2, HPV4, or HPV6. The E1--E4 protein migrates as a 10K species on SDS-gel electrophoresis and forms cytoplasmic inclusion granules in infected cells in vitro similar in appearance to those produced by HPV1 in benign warts. In naturally occurring HPV16-induced tumors the E1--E4 protein was detected in the cytoplasm of cells in the upper layers of the lesion in areas in which HPV16 DNA replication was occurring, as determined by in situ hybridization. Although the epitopes recognized by these monoclonal antibodies survive brief fixation in 5% formaldehyde, reactivity was destroyed by prolonged fixation. These monoclonal antibodies represent the first against HPV16 E1--E4 and should complement those already available to E7 and L1 for the screening of frozen sections of clinical biopsies and will be of value in monitoring the progression of HPV infection from benign lesions to invasive cancer. PMID:1371027

  2. E1B and E4 oncoproteins of adenovirus antagonize the effect of apoptosis inducing factor

    SciTech Connect

    Turner, Roberta L.; Wilkinson, John C.; Ornelles, David A.

    2014-05-15

    Adenovirus inundates the productively infected cell with linear, double-stranded DNA and an abundance of single-stranded DNA. The cellular response to this stimulus is antagonized by the adenoviral E1B and E4 early genes. A mutant group C adenovirus that fails to express the E1B-55K and E4ORF3 genes is unable to suppress the DNA-damage response. Cells infected with this double-mutant virus display significant morphological heterogeneity at late times of infection and frequently contain fragmented nuclei. Nuclear fragmentation was due to the translocation of apoptosis inducing factor (AIF) from the mitochondria into the nucleus. The release of AIF was dependent on active poly(ADP-ribose) polymerase-1 (PARP-1), which appeared to be activated by viral DNA replication. Nuclear fragmentation did not occur in AIF-deficient cells or in cells treated with a PARP-1 inhibitor. The E1B-55K or E4ORF3 proteins independently prevented nuclear fragmentation subsequent to PARP-1 activation, possibly by altering the intracellular distribution of PAR-modified proteins. - Highlights: • E1B-55K or E4orf3 prevents nuclear fragmentation. • Nuclear fragmentation requires AIF and PARP-1 activity. • Adenovirus DNA replication activates PARP-1. • E1B-55K or E4orf3 proteins alter the distribution of PAR.

  3. Enantioselective Rhodium-Catalyzed Cycloisomerization of (E)-1,6-Enynes.

    PubMed

    Deng, Xu; Ni, Shao-Fei; Han, Zheng-Yu; Guan, Yu-Qing; Lv, Hui; Dang, Li; Zhang, Xu-Mu

    2016-05-17

    An enantioselective rhodium(I)-catalyzed cycloisomerization reaction of challenging (E)-1,6-enynes is reported. This novel process enables (E)-1,6-enynes with a wide range of functionalities, including nitrogen-, oxygen-, and carbon-tethered (E)-1,6-enynes, to undergo cycloisomerization with excellent enantioselectivity, in a high-yielding and operationally simple manner. Moreover, this Rh(I) -diphosphane catalytic system also exhibited superior reactivity and enantioselectivity for (Z)-1,6-enynes. A rationale for the striking reactivity difference between (E)- and (Z)-1,6-enynes using Rh(I) -BINAP and Rh(I) -TangPhos is outlined using DFT studies to provide the necessary insight for the design of new catalyst systems and the application to synthesis. PMID:27061132

  4. E1 and M1 γ-strength functions in 144Nd

    SciTech Connect

    Voinov, A. V.; Grimes, S. M.

    2015-12-14

    Both E1 and M1 γ-strength functions below the neutron separation energy were analyzed based on experimental data from 143Nd(n,γ)144Nd and 143Nd(n,γα)140Ce reactions. It is confirmed that the commonly adopted E1 model based on the temperature dependence of the width of the giant dipole resonance works well. The popular M1 strength function due to the spin-flip magnetic resonance located near the neutron binding energy is not capable of reproducing experimental data. As a result, the low-energy enhancement of the M1 strength or the energy-independent model of Weisskopf, both leading to the low-energy strength sizable to E1 one, fit experimental data best.

  5. Regulation of the effects of CYP2E1-induced oxidative stress by JNK signaling

    PubMed Central

    Schattenberg, Jörn M.; Czaja, Mark J.

    2014-01-01

    The generation of excessive amounts of reactive oxygen species (ROS) leads to cellular oxidative stress that underlies a variety of forms of hepatocyte injury and death including that from alcohol. Although ROS can induce cell damage through direct effects on cellular macromolecules, the injurious effects of ROS are mediated largely through changes in signal transduction pathways such as the mitogen-activated protein kinase c-Jun N-terminal kinase (JNK). In response to alcohol, hepatocytes have increased levels of the enzyme cytochrome P450 2E1 (CYP2E1) which generates an oxidant stress that promotes the development of alcoholic steatosis and liver injury. These effects are mediated in large part through overactivation of JNK that alters cell death pathways. Targeting the JNK pathway or its downstream effectors may be a useful therapeutic approach to the oxidative stress generated by CYP2E1 in alcoholic liver disease. PMID:25462060

  6. E1 and M1 γ-strength functions in 144Nd

    DOE PAGESBeta

    Voinov, A. V.; Grimes, S. M.

    2015-12-14

    Both E1 and M1 γ-strength functions below the neutron separation energy were analyzed based on experimental data from 143Nd(n,γ)144Nd and 143Nd(n,γα)140Ce reactions. It is confirmed that the commonly adopted E1 model based on the temperature dependence of the width of the giant dipole resonance works well. The popular M1 strength function due to the spin-flip magnetic resonance located near the neutron binding energy is not capable of reproducing experimental data. As a result, the low-energy enhancement of the M1 strength or the energy-independent model of Weisskopf, both leading to the low-energy strength sizable to E1 one, fit experimental data best.

  7. E 1 and M 1 γ -strength functions in 144Nd

    NASA Astrophysics Data System (ADS)

    Voinov, A. V.; Grimes, S. M.

    2015-12-01

    Both E 1 and M 1 γ -strength functions below the neutron separation energy were analyzed based on experimental data from 143Nd(n ,γ )144Nd and 143Nd(n ,γ α )140Ce reactions. It is confirmed that the commonly adopted E 1 model based on the temperature dependence of the width of the giant dipole resonance works well. The popular M 1 strength function due to the spin-flip magnetic resonance located near the neutron binding energy is not capable of reproducing experimental data. The low-energy enhancement of the M 1 strength or the energy-independent model of Weisskopf, both leading to the low-energy strength sizable to E 1 one, fit experimental data best.

  8. Two distinct regions of the BPV1 E1 replication protein interact with the activation domain of E2.

    PubMed

    Moscufo, N; Sverdrup, F; Breiding, D E; Androphy, E J

    1999-12-15

    Papillomavirus E1 and E2 proteins co-operation in viral DNA replication is mediated by protein-protein interactions that lead to formation of an E1-E2 complex. To identify the domains involved, portions of the two proteins were expressed as fusions to the DNA-binding protein LexA or the transactivation domain of VP16 and analyzed by the yeast two-hybrid system. The C-terminal 266 amino acids of BPV1 E1 (E1C266) interacted strongly with E2 in the yeast system and in a mammalian two-hybrid assay. VP16-E1C266 interacted with a region encompassing amino acids 1-200 of the transactivation domain of E2 that was fused to LexA. The interaction between E1 full length and E2 was clearly observed only when E1 was expressed as LexA-E1 chimera. In addition, we found that in the LexA context also the N-terminal region encompassing the first 340 amino acids of E1 (E1N340) interacted with E2 full length. The interactions of E1N340 and E1C266 with E2 were confirmed also by in vitro binding studies. These observations demonstrate that two distinct regions of E1 mediate the interaction with E2 in vivo. PMID:10581387

  9. Characterization of a novel thermostable β-glucosidase from a metagenomic library of termite gut.

    PubMed

    Wang, Qianfu; Qian, Changli; Zhang, Xiao-Zhou; Liu, Ning; Liu, Nin; Yan, Xing; Zhou, Zhihua

    2012-12-10

    A novel β-glucosidase-encoding gene, bgl-gs1, which was identified from a positive fosmid clone in a metagenomic library of the gut of Globitermes brachycerastes, [corrected] encodes a 455 amino acid polypeptide that contains a catalytic domain belonging to glycoside hydrolase family 1 (GH1). It was expressed in Escherichia coli BL21 (DE3) and the expression product showed a molecular mass of ∼51.7 kDa by SDS-PAGE. The optimal temperature and pH for the activity of the purified recombinant enzyme Bgl-gs1 with p-nitrophenyl-β-D-glucoside (pNPG) were 90°C and 6.0, respectively. The specific activities of Bgl-gs1 on pNPG and salicin were 110 and 14U/mg of protein, respectively, and its K(m) values were 0.18 and 2.59 mM, respectively. The residual activity of Bgl-gs1 was maintained above 70% after the recombinant enzyme was incubated at 75°C and pH 6.0 for 2h, and its half-life at 90°C was approximately 1h in the presence of 4mM pNPG. Bgl-gs1 showed synergistic effect with either a crude enzyme mixture of the fungal strain Trichoderma reesei Rut-C30 or a fusion protein (TcE1) created from the cellobiohydrolase cbh1 gene of T. reesei and endoglucanase from Acidothermus cellulolyticus; 87 and 137% increases in hydrolytic efficiency were noted on microcrystalline cellulose, respectively. These results suggest that the thermostable β-glucosidase Bgl-gs1 is a likely candidate for industrial applications. PMID:23040386

  10. Genetic study of the functional organization of the colicin E1 molecule.

    PubMed Central

    Suit, J L; Fan, M L; Kayalar, C; Luria, S E

    1985-01-01

    Colicin E1 fragments obtained by genetic manipulations of the ColE1 plasmid were tested for bactericidal activity, binding to bacterial cells, and reactions with a series of anticolicin monoclonal antibodies. Two of the fragments were also tested for ability to form channels in liposomal vesicles. The results are in agreement with studies from chemically and enzymatically derived colicin fragments, assigning the receptor binding activity to the central part of the molecule and the killing activity to a region near the carboxyl terminus. PMID:2579061

  11. CYP2E1-dependent elevation of serum cholesterol, triglycerides, and hepatic bile acids by isoniazid

    SciTech Connect

    Cheng, Jie; Krausz, Kristopher W.; Li, Feng; Ma, Xiaochao; Gonzalez, Frank J.

    2013-01-15

    Isoniazid is the first-line medication in the prevention and treatment of tuberculosis. Isoniazid is known to have a biphasic effect on the inhibition–induction of CYP2E1 and is also considered to be involved in isoniazid-induced hepatotoxicity. However, the full extent and mechanism of involvement of CYP2E1 in isoniazid-induced hepatotoxicity remain to be thoroughly investigated. In the current study, isoniazid was administered to wild-type and Cyp2e1-null mice to investigate the potential toxicity of isoniazid in vivo. The results revealed that isoniazid caused no hepatotoxicity in wild-type and Cyp2e1-null mice, but produced elevated serum cholesterol and triglycerides, and hepatic bile acids in wild-type mice, as well as decreased abundance of free fatty acids in wild-type mice and not in Cyp2e1-null mice. Metabolomic analysis demonstrated that production of isoniazid metabolites was elevated in wild-type mice along with a higher abundance of bile acids, bile acid metabolites, carnitine and carnitine derivatives; these were not observed in Cyp2e1-null mice. In addition, the enzymes responsible for bile acid synthesis were decreased and proteins involved in bile acid transport were significantly increased in wild-type mice. Lastly, treatment of targeted isoniazid metabolites to wild-type mice led to similar changes in cholesterol, triglycerides and free fatty acids. These findings suggest that while CYP2E1 is not involved in isoniazid-induced hepatotoxicity, while an isoniazid metabolite might play a role in isoniazid-induced cholestasis through enhancement of bile acid accumulation and mitochondria β-oxidation. -- Highlights: ► Isoniazid metabolites were elevated only in wild-type mice. ► Isoniazid caused no hepatotoxicity in wild-type and Cyp2e1-null mice. ► Isoniazid elevated serum cholesterol and triglycerides, and hepatic bile acids. ► Bile acid transporters were significantly decreased in isoniazid-treated mice.

  12. Substitution of specific cysteine residues in E1 glycoprotein of classical swine fever virus strain Brescia affects formation of E1-E2 heterodimers and alters virulence in swine

    Technology Transfer Automated Retrieval System (TEKTRAN)

    E1, along with E^rns and E2, is one of the three envelope glycoproteins of Classical Swine Fever Virus (CSFV). E1 and E2 are anchored to the virus envelope at their carboxyl termini and E^rns loosely associates with the viral envelope. In infected cells, E2 forms homodimers and heterodimers with E1,...

  13. 26 CFR 1.72(e)-1T - Treatment of distributions where substantially all contributions are employee contributions...

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... 72(o) are disregarded in their entirety (i.e., treated as neither employee contributions nor employer... all contributions are employee contributions (temporary). 1.72(e)-1T Section 1.72(e)-1T Internal... TAXES (CONTINUED) Items Specifically Included in Gross Income § 1.72(e)-1T Treatment of...

  14. 26 CFR 1.1033(e)-1 - Sale or exchange of livestock solely on account of drought.

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ... of drought. 1.1033(e)-1 Section 1.1033(e)-1 Internal Revenue INTERNAL REVENUE SERVICE, DEPARTMENT OF... § 1.1033(e)-1 Sale or exchange of livestock solely on account of drought. (a) The sale or exchange of... applicable if the sale or exchange of such livestock by the taxpayer is solely on account of drought....

  15. 26 CFR 1.1033(e)-1 - Sale or exchange of livestock solely on account of drought.

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    ... of drought. 1.1033(e)-1 Section 1.1033(e)-1 Internal Revenue INTERNAL REVENUE SERVICE, DEPARTMENT OF... § 1.1033(e)-1 Sale or exchange of livestock solely on account of drought. (a) The sale or exchange of... applicable if the sale or exchange of such livestock by the taxpayer is solely on account of drought....

  16. 26 CFR 1.1033(e)-1 - Sale or exchange of livestock solely on account of drought.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... of drought. 1.1033(e)-1 Section 1.1033(e)-1 Internal Revenue INTERNAL REVENUE SERVICE, DEPARTMENT OF... § 1.1033(e)-1 Sale or exchange of livestock solely on account of drought. (a) The sale or exchange of... applicable if the sale or exchange of such livestock by the taxpayer is solely on account of drought....

  17. 26 CFR 1.1033(e)-1 - Sale or exchange of livestock solely on account of drought.

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ... of drought. 1.1033(e)-1 Section 1.1033(e)-1 Internal Revenue INTERNAL REVENUE SERVICE, DEPARTMENT OF... § 1.1033(e)-1 Sale or exchange of livestock solely on account of drought. (a) The sale or exchange of... applicable if the sale or exchange of such livestock by the taxpayer is solely on account of drought....

  18. Cytochrome P450 2E1 inhibition prevents hepatic carcinogenesis induced by diethylnitrosamine in alcohol-fed rats

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Chronic alcohol ingestion increases hepatic cytochrome P450 2E1 (CYP2E1), which is associated with hepatocarcinogenesis. We investigated whether treatment with chlormethiazole (CMZ), a CYP2E1 inhibitor, protects against alcohol-associated hepatic carcinogenesis in rats. Rats were fed either an ethan...

  19. 26 CFR 1.72(e)-1T - Treatment of distributions where substantially all contributions are employee contributions...

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    ... all contributions are employee contributions (temporary). 1.72(e)-1T Section 1.72(e)-1T Internal... TAXES (CONTINUED) Items Specifically Included in Gross Income § 1.72(e)-1T Treatment of distributions... Reform Act (TRA) of 1984 change the law with regard to the treatment of non-annuity distributions...

  20. 26 CFR 1.72(e)-1T - Treatment of distributions where substantially all contributions are employee contributions...

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ... all contributions are employee contributions (temporary). 1.72(e)-1T Section 1.72(e)-1T Internal... TAXES (CONTINUED) Items Specifically Included in Gross Income § 1.72(e)-1T Treatment of distributions... Reform Act (TRA) of 1984 change the law with regard to the treatment of non-annuity distributions...

  1. 26 CFR 301.6223(e)-1 - Effect of Internal Revenue Service's failure to provide notice.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... years beginning prior to October 4, 2001, see § 301.6223(e)-1T contained in 26 CFR part 1, revised April...: Example 1. Partnership ABC has as one of its partners, A, a partnership with three partners, X, Y, and Z. ABC does not have more than 100 partners, and partnership A is entitled to notice under section...

  2. 26 CFR 301.6223(e)-1 - Effect of Internal Revenue Service's failure to provide notice.

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    ... years beginning prior to October 4, 2001, see § 301.6223(e)-1T contained in 26 CFR part 1, revised April...: Example 1. Partnership ABC has as one of its partners, A, a partnership with three partners, X, Y, and Z. ABC does not have more than 100 partners, and partnership A is entitled to notice under section...

  3. 26 CFR 301.6223(e)-1 - Effect of Internal Revenue Service's failure to provide notice.

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ... years beginning prior to October 4, 2001, see § 301.6223(e)-1T contained in 26 CFR part 1, revised April...: Example 1. Partnership ABC has as one of its partners, A, a partnership with three partners, X, Y, and Z. ABC does not have more than 100 partners, and partnership A is entitled to notice under section...

  4. 26 CFR 301.6223(e)-1 - Effect of Internal Revenue Service's failure to provide notice.

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ... years beginning prior to October 4, 2001, see § 301.6223(e)-1T contained in 26 CFR part 1, revised April...: Example 1. Partnership ABC has as one of its partners, A, a partnership with three partners, X, Y, and Z. ABC does not have more than 100 partners, and partnership A is entitled to notice under section...

  5. 26 CFR 301.6223(e)-1 - Effect of Internal Revenue Service's failure to provide notice.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... years beginning prior to October 4, 2001, see § 301.6223(e)-1T contained in 26 CFR part 1, revised April...: Example 1. Partnership ABC has as one of its partners, A, a partnership with three partners, X, Y, and Z. ABC does not have more than 100 partners, and partnership A is entitled to notice under section...

  6. NOVEL ASSAY TO ASSESS CYP-2E1-LIKE ACTIVITY IN THE JAPANESE MEDAKA (ORYZIAS LATIPES).

    EPA Science Inventory

    Liver microsomes and S-9 fraction of Japanese medaka (Oryzias latipes) metabolized the CYP2E1 specific substrate, p-nitrophenol (PNP), to a single hydroxylated product, 4-nitrocatechol. The use of liver S-9 fraction proved to be a viable alternative to liver microsomes and allowe...

  7. 26 CFR 1.1397E-1 - Qualified zone academy bonds.

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ... apply to bonds issued under section 1397E pursuant to allocations of the national qualified zone academy... 26 Internal Revenue 11 2012-04-01 2012-04-01 false Qualified zone academy bonds. 1.1397E-1 Section... academy bonds. (a) In general—(1) Overview. In general, a qualified zone academy bond (QZAB or QZABs) is...

  8. E1 Ubiquitin-Activating Enzyme UBA-1 Plays Multiple Roles throughout C. elegans Development

    PubMed Central

    Kulkarni, Madhura; Smith, Harold E.

    2008-01-01

    Poly-ubiquitination of target proteins typically marks them for destruction via the proteasome and provides an essential mechanism for the dynamic control of protein levels. The E1 ubiquitin-activating enzyme lies at the apex of the ubiquitination cascade, and its activity is necessary for all subsequent steps in the reaction. We have isolated a temperature-sensitive mutation in the Caenorhabditis elegans uba-1 gene, which encodes the sole E1 enzyme in this organism. Manipulation of UBA-1 activity at different developmental stages reveals a variety of functions for ubiquitination, including novel roles in sperm fertility, control of body size, and sex-specific development. Levels of ubiquitin conjugates are substantially reduced in the mutant, consistent with reduced E1 activity. The uba-1 mutation causes delays in meiotic progression in the early embryo, a process that is known to be regulated by ubiquitin-mediated proteolysis. The uba-1 mutation also demonstrates synthetic lethal interactions with alleles of the anaphase-promoting complex, an E3 ubiquitin ligase. The uba-1 mutation provides a sensitized genetic background for identifying new in vivo functions for downstream components of the ubiquitin enzyme cascade, and it is one of the first conditional mutations reported for the essential E1 enzyme in a metazoan animal model. PMID:18636104

  9. 26 CFR 301.6229(e)-1 - Information with respect to unidentified partner.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... 26 CFR part 1, revised April 1, 2001. ... 26 Internal Revenue 18 2010-04-01 2010-04-01 false Information with respect to unidentified....6229(e)-1 Information with respect to unidentified partner. (a) In general. A partner who is...

  10. The 8-Pyrrole-Benzothiazinones Are Noncovalent Inhibitors of DprE1 from Mycobacterium tuberculosis.

    PubMed

    Makarov, Vadim; Neres, João; Hartkoorn, Ruben C; Ryabova, Olga B; Kazakova, Elena; Šarkan, Michal; Huszár, Stanislav; Piton, Jérémie; Kolly, Gaëlle S; Vocat, Anthony; Conroy, Trent M; Mikušová, Katarína; Cole, Stewart T

    2015-08-01

    8-Nitro-benzothiazinones (BTZs), such as BTZ043 and PBTZ169, inhibit decaprenylphosphoryl-β-d-ribose 2'-oxidase (DprE1) and display nanomolar bactericidal activity against Mycobacterium tuberculosis in vitro. Structure-activity relationship (SAR) studies revealed the 8-nitro group of the BTZ scaffold to be crucial for the mechanism of action, which involves formation of a semimercaptal bond with Cys387 in the active site of DprE1. To date, substitution of the 8-nitro group has led to extensive loss of antimycobacterial activity. Here, we report the synthesis and characterization of the pyrrole-benzothiazinones PyrBTZ01 and PyrBTZ02, non-nitro-benzothiazinones that retain significant antimycobacterial activity, with MICs of 0.16 μg/ml against M. tuberculosis. These compounds inhibit DprE1 with 50% inhibitory concentration (IC50) values of <8 μM and present favorable in vitro absorption-distribution-metabolism-excretion/toxicity (ADME/T) and in vivo pharmacokinetic profiles. The most promising compound, PyrBTZ01, did not show efficacy in a mouse model of acute tuberculosis, suggesting that BTZ-mediated killing through DprE1 inhibition requires a combination of both covalent bond formation and compound potency. PMID:25987616

  11. The 8-Pyrrole-Benzothiazinones Are Noncovalent Inhibitors of DprE1 from Mycobacterium tuberculosis

    PubMed Central

    Makarov, Vadim; Neres, João; Hartkoorn, Ruben C.; Ryabova, Olga B.; Kazakova, Elena; Šarkan, Michal; Huszár, Stanislav; Piton, Jérémie; Kolly, Gaëlle S.; Vocat, Anthony; Conroy, Trent M.; Mikušová, Katarína

    2015-01-01

    8-Nitro-benzothiazinones (BTZs), such as BTZ043 and PBTZ169, inhibit decaprenylphosphoryl-β-d-ribose 2′-oxidase (DprE1) and display nanomolar bactericidal activity against Mycobacterium tuberculosis in vitro. Structure-activity relationship (SAR) studies revealed the 8-nitro group of the BTZ scaffold to be crucial for the mechanism of action, which involves formation of a semimercaptal bond with Cys387 in the active site of DprE1. To date, substitution of the 8-nitro group has led to extensive loss of antimycobacterial activity. Here, we report the synthesis and characterization of the pyrrole-benzothiazinones PyrBTZ01 and PyrBTZ02, non-nitro-benzothiazinones that retain significant antimycobacterial activity, with MICs of 0.16 μg/ml against M. tuberculosis. These compounds inhibit DprE1 with 50% inhibitory concentration (IC50) values of <8 μM and present favorable in vitro absorption-distribution-metabolism-excretion/toxicity (ADME/T) and in vivo pharmacokinetic profiles. The most promising compound, PyrBTZ01, did not show efficacy in a mouse model of acute tuberculosis, suggesting that BTZ-mediated killing through DprE1 inhibition requires a combination of both covalent bond formation and compound potency. PMID:25987616

  12. Defective human retinoblastoma protein identified by lack of interaction with the E1A oncoprotein.

    PubMed

    Paggi, M G; Martelli, F; Fanciulli, M; Felsani, A; Sciacchitano, S; Varmi, M; Bruno, T; Carapella, C M; Floridi, A

    1994-02-15

    Inactivating mutations of the retinoblastoma susceptibility gene (Rb) are involved in the pathogenesis of hereditary and sporadic retinoblastoma. Alterations in the Rb gene have also been found in several other human tumors occurring with epidemiological incidence higher than that of retinoblastoma. Four human malignant glioma cell lines were examined for abnormalities in the retinoblastoma gene product (pRb), using a procedure based on the interaction of pRb with an in vitro-translated adenovirus E1A oncoprotein. In the CRS-A2 cell line, derived from a glioblastoma multiforme, pRb did not bind with the in vitro-translated E1A protein. Restriction analysis of the CRS-A2 Rb gene and Rb mRNA expression provided patterns that could not be distinguished from the other glioma cell lines. Further investigation revealed the presence of a truncated pRb in the CRS-A2 cell line, due to a nucleotide insertion in the coding sequence at position 2550. In addition, this truncated Rb protein was undetectable in phosphorylated form. The binding assay with the in vitro-translated E1A was also used to study other cell lines with known mutations in the Rb gene. This method, which evaluates the interaction between in vitro-translated E1A and the pRb, is proposed as a rapid screening for detecting functional alterations in the retinoblastoma protein. PMID:8313367

  13. 26 CFR 301.6511(e)-1 - Special rules applicable to manufactured sugar.

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ... 26 Internal Revenue 18 2014-04-01 2014-04-01 false Special rules applicable to manufactured sugar... Assessment and Collection § 301.6511(e)-1 Special rules applicable to manufactured sugar. (a) Use as... the person entitled thereto. Such right accrues as of the date the manufactured sugar, or...

  14. 26 CFR 301.6511(e)-1 - Special rules applicable to manufactured sugar.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... 26 Internal Revenue 18 2011-04-01 2011-04-01 false Special rules applicable to manufactured sugar... Assessment and Collection § 301.6511(e)-1 Special rules applicable to manufactured sugar. (a) Use as... the person entitled thereto. Such right accrues as of the date the manufactured sugar, or...

  15. 26 CFR 301.6511(e)-1 - Special rules applicable to manufactured sugar.

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    ... 26 Internal Revenue 18 2013-04-01 2013-04-01 false Special rules applicable to manufactured sugar... Assessment and Collection § 301.6511(e)-1 Special rules applicable to manufactured sugar. (a) Use as... the person entitled thereto. Such right accrues as of the date the manufactured sugar, or...

  16. 26 CFR 301.6511(e)-1 - Special rules applicable to manufactured sugar.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... 26 Internal Revenue 18 2010-04-01 2010-04-01 false Special rules applicable to manufactured sugar... Assessment and Collection § 301.6511(e)-1 Special rules applicable to manufactured sugar. (a) Use as... the person entitled thereto. Such right accrues as of the date the manufactured sugar, or...

  17. 26 CFR 301.6511(e)-1 - Special rules applicable to manufactured sugar.

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ... 26 Internal Revenue 18 2012-04-01 2012-04-01 false Special rules applicable to manufactured sugar... Assessment and Collection § 301.6511(e)-1 Special rules applicable to manufactured sugar. (a) Use as... the person entitled thereto. Such right accrues as of the date the manufactured sugar, or...

  18. E-1 Dynamic Fluid-Flow Model Update: EASY/ROCETS Enhancement and Model Development Support

    NASA Technical Reports Server (NTRS)

    Follett, Randolph F.; Taylor, Robert P.

    1998-01-01

    This report documents the research conducted to update computer models for dynamic fluid flow simulation of the E-1 test stand subsystems at te NASA John C. Stennis Space Center.Work also involved significant upgrades to the capabilities of EASY/ROCKETS library through the inclusion of the NIST-12 thermodynamic property database and development of new control system modules.

  19. 26 CFR 301.6501(e)-1T - Omission from return (temporary).

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... and Collection § 301.6501(e)-1T Omission from return (temporary). (a) Income taxes—(1) General rule. (i) If the taxpayer omits from the gross income stated in the return of a tax imposed by subtitle A... the gross income so stated, the tax may be assessed, or a proceeding in court for the collection...

  20. 26 CFR 1.401(e)-1 - Definitions relating to plans covering self-employed individuals.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ..., Stock Bonus Plans, Etc. § 1.401(e)-1 Definitions relating to plans covering self-employed individuals...), (f), (g), (h), and (i), are also generally applicable to any plan covering a self-employed individual. However, in addition to such requirements and special rules, any plan covering a self-employed...

  1. Induction of CYP2E1 in non-alcoholic fatty liver diseases.

    PubMed

    Aljomah, Ghanim; Baker, Susan S; Liu, Wensheng; Kozielski, Rafal; Oluwole, Janet; Lupu, Benita; Baker, Robert D; Zhu, Lixin

    2015-12-01

    Mounting evidence supports a contribution of endogenous alcohol metabolism in the pathogenesis of non-alcoholic steatohepatitis (NASH). However, it is not known whether the expression of alcohol metabolism genes is altered in the livers of simple steatosis. There is also a current debate on whether fatty acids induce CYP2E1 in fatty livers. In this study, expression of alcohol metabolizing genes in the liver biopsies of simple steatosis patients was examined by quantitative real-time PCR (qRT-PCR), in comparison to biopsies of NASH livers and normal controls. Induction of alcohol metabolizing genes was also examined in cultured HepG2 cells treated with ethanol or oleic acid, by qRT-PCR and Western blots. We found that the mRNA expression of alcohol metabolizing genes including ADH1C, ADH4, ADH6, catalase and CYP2E1 was elevated in the livers of simple steatosis, to similar levels found in NASH livers. In cultured HepG2 cells, ethanol induced the expression of CYP2E1 mRNA and protein, but not ADH4 or ADH6; oleic acid did not induce any of these genes. These results suggest that elevated alcohol metabolism may contribute to the pathogenesis of NAFLD at the stage of simple steatosis as well as more severe stages. Our in vitro data support that CYP2E1 is induced by endogenous alcohol but not by fatty acids. PMID:26551085

  2. Enhanced E1 transitions and {alpha}-clustering in {sup 212}Po

    SciTech Connect

    Suzuki, Y.; Ohkubo, S.

    2011-05-06

    An {alpha}+{sup 208}Pb(0{sup +},3{sup -}) cluster model explains the recently observed enhanced E1 transitions from the new negative-parity levels to the yrast states in {sup 212}Po. Heavy and light nuclei present good examples of surface clustering and well-localized clustering.

  3. Identification and Mechanistic Studies of a Novel Ubiquitin E1 Inhibitor

    PubMed Central

    Ungermannova, Dana; Parker, Seth J.; Nasveschuk, Christopher G.; Chapnick, Douglas A.; Phillips, Andrew J.; Kuchta, Robert D.; Liu, Xuedong

    2012-01-01

    Protein degradation via the ubiquitin-proteasome pathway is important for a diverse number of cellular processes ranging from cell signaling to development. Disruption of the ubiquitin pathway occurs in a variety of human diseases, including several cancers and neurological disorders. Excessive proteolysis of tumor suppressor proteins, such as p27, occurs in numerous aggressive human tumors. To discover small-molecule inhibitors that potentially prevent p27 degradation, we developed a series of screening assays, including a cell-based screen of a small-molecule compound library and two novel nucleotide exchange assays. Several small-molecule inhibitors, including NSC624206, were identified and subsequently verified to prevent p27 ubiquitination in vitro. The mechanism of NSC624206 inhibition of p27 ubiquitination was further unraveled using the nucleotide exchange assays and shown to be due to antagonizing ubiquitin activating enzyme (E1). We determined that NSC624206 and PYR-41, a recently reported inhibitor of ubiquitin E1, specifically block ubiquitin-thioester formation but have no effect on ubiquitin adenylation. These studies reveal a novel E1 inhibitor that targets a specific step of the E1 activation reaction. NSC624206 could, therefore, be potentially useful for the control of excessive ubiquitin-mediated proteolysis in vivo. PMID:22274912

  4. 26 CFR 1.414(e)-1 - Definition of church plan.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... (CONTINUED) INCOME TAXES (CONTINUED) Pension, Profit-Sharing, Stock Bonus Plans, Etc. § 1.414(e)-1 Definition... is paid to, employees employed in connection with an unrelated trade or business. The determination... employees not employed in connection with an unrelated trade or business unless it fails to meet...

  5. Specific estrogen sulfotransferase (SULT1E1) substrates and molecular imaging probe candidates

    PubMed Central

    Cole, Graham B.; Keum, Gyochang; Liu, Jie; Small, Gary W.; Satyamurthy, Nagichettiar; Kepe, Vladimir; Barrio, Jorge R.

    2010-01-01

    This work focuses on the development of specific substrates for estrogen sulfotransferase (SULT1E1) to produce molecular imaging probes for this enzyme. SULT1E1 is a key enzyme in estrogen homeostasis, playing a central role in the prevention and development of human disease. In vitro sulfation assays showed alkyl and aryl substitutions to a fused heterocyclic system modeled after β-naphthol (βN), based on compounds that interact with the estrogen receptor, rendered several molecules with enhanced specificity for SULT1E1 over SULT1A1*1, SULT1A1*2, SULT1A3, and SULT2A1. Several 6-hydroxy-2-arylbenzothiazoles tested demonstrated excellent affinity—Vmax/Km ratios—and specificity for SULT1E1. Km values ranged from 0.12–2.36 μM. A strong correlation was observed between polarity of the 4′-sustituent on the 2-aryl moiety (Hammett σp) and the log(Vmax/Km) (r = 0.964). Substrate sensitivity is influenced by the acidity of the 6-phenolic group demonstrated by correlating its 1H NMR chemical shift (δOH) with the log(Vmax/Km) (r = 0.963). Acidity is mediated by the electron withdrawing capacity of the 4′-substituent outlined by the correlation of the C-2 13C NMR chemical shift (δC2) with the log(Vmax/Km) (r = 0.987). 2-[4-(Methylamino)phenyl]-6-hydroxybenzothiazole (2b) was radiolabeled with carbon-11 (11C-(2b)) and used in vivo for microPET scanning and tissue metabolite identification. High PET signal was paralleled with the presence of radiolabeled 11C-(2b)-6-O-sulfate and the SULT1E1 protein detected by western blot. Because this and other members of this family presenting specificity for SULT1E1 can be labeled with carbon-11 or fluorine-18, in vivo assays of SULT1E1 functional activity are now feasible in humans. PMID:20304798

  6. [Improved kidney function with intravenous prostaglandin E1 in patients with terminal heart failure].

    PubMed

    Wutte, M; Hülsmann, M; Berger, R; Rödler, S; Frey, B; Stanek, B; Pacher, R

    1998-07-31

    In end stage congestive heart failure activation of a series of compensatory mechanisms increase renal vascular resistance and impair renal function. Prostaglandin E1 is increasingly used in the treatment of severe heart failure for its vasodilating actions. In various experimental settings prostaglandin E analogues are known to improve renal function by modulating renal filtration pressure and redistribution of renal blood flow. However, prostaglandin E1 decreases systemic blood pressure and thus, also renal perfusion pressure, a fact by which renal function might be further compromized in heart failure patients. The aim of the study was to evaluate the effects of prostaglandin E1 on excretory renal function in patients with end stage heart failure and to prove the hypothesis, that the well known local actions of prostaglandins on renal microcirculation might outweigh the negative impact of an expected decrease in perfusion pressure. 25 patients with terminal congestive heart failure were investigated. 13 patients received prostaglandin E1 at a dose of 13.5 +/- 1.9 ng/kg/min in combination with constant rates of dopamine and dobutamine (group A), 12 patients received prostaglandin E1 at a dose of 10.3 +/- 1.7 ng/kg/min without catecholamines (group B). There was no significant difference in prostaglandin dosages between groups. Kidney function was assessed by measuring plasma creatinine and urea nitrogen, urinary output, creatinine clearance, osmotic and free water clearance at baseline and after 72 h of infusion therapy. Hemodynamic parameters were measured by using a balloon tipped pulmonary arterial catheter. Hemodynamic measurements during infusion showed a significant improvement in all patients. At the same time as expected mean arterial pressure decreased in both groups (p < 0.001). Nevertheless, in both groups a significant increase of creatinine clearance during infusion was observed (in group A from 45 ml/min to 78 ml/min., p < 0.05, in group B from 59

  7. Osteogenic gene expression of murine osteoblastic (MC3T3-E1) cells under cyclic tension

    NASA Astrophysics Data System (ADS)

    Kao, C. T.; Chen, C. C.; Cheong, U.-I.; Liu, S. L.; Huang, T. H.

    2014-08-01

    Low-level laser therapy (LLLT) can promote cell proliferation. The remodeling ability of the tension side of orthodontic teeth affects post-orthodontic stability. The purpose of the present study was to investigate the osteogenic effects of LLLT on osteoblast-like cells treated with a simulated tension system that provides a mechanical tension regimen. Murine osteoblastic (MC3T3-E1) cells were cultured in a Flexcell strain unit with programmed loads of 12% elongation at a frequency of 0.5 Hz for 24 and 48 h. The cultured cells were treated with a low-level diode laser using powers of 5 J and 10 J. The proliferation of MC3T3-E1 cells was determined using the Alamar Blue assay. The expression of osteogenic genes (type I collagen (Col-1), osteopontin (OPN), osteocalcin (OC), osteoprotegerin (OPG), receptor activator of nuclear factor kappa B ligand (RANKL), bone morphologic protein (BMP-2), and bone morphologic protein (BMP-4)) in MC3T3-E1 cells was analyzed using reverse transcription polymerase chain reaction (RT-PCR). The data were analyzed using one-way analysis of variance. The proliferation rate of tension-cultured MC3T3-E1 cells under 5 J and 10 J LLLT increased compared with that of the control group (p < 0.05). Prominent mineralization of the MC3T3-E1 cells was visible using a von Kossa stain in the 5 J LLLT group. Osteogenic genes (Col-1, OC, OPG and BMP-2) were significantly expressed in the MC3T3-E1 cells treated with 5 J and 10 J LLLT (p < 0.05). LLLT in tension-cultured MC3T3-E1 cells showed synergistic osteogenic effects, including increases in cell proliferation and Col-1, OPN, OC, OPG and BMP-2 gene expression. LLLT might be beneficial for bone remodeling on the tension side of orthodontics.

  8. Benzene metabolism by human liver microsomes in relation to cytochrome P450 2E1 activity.

    PubMed

    Seaton, M J; Schlosser, P M; Bond, J A; Medinsky, M A

    1994-09-01

    Low levels of benzene from sources including cigarette smoke and automobile emissions are ubiquitous in the environment. Since the toxicity of benzene probably results from oxidative metabolites, an understanding of the profile of biotransformation of low levels of benzene is critical in making a valid risk assessment. To that end, we have investigated metabolism of a low concentration of [14C]benzene (3.4 microM) by microsomes from human, mouse and rat liver. The extent of phase I benzene metabolism by microsomal preparations from 10 human liver samples and single microsomal preparations from both mice and rats was then related to measured activities of cytochrome P450 (CYP) 2E1. Measured CYP 2E1 activities, as determined by hydroxylation of p-nitrophenol, varied 13-fold (0.253-3.266 nmol/min/mg) for human samples. The fraction of benzene metabolized in 16 min ranged from 10% to 59%. Also at 16 min, significant amounts of oxidative metabolites were formed. Phenol was the main metabolite formed by all but two human microsomal preparations. In those samples, both of which had high CYP 2E1 activity, hydroquinone was the major metabolite formed. Both hydroquinone and catechol formation showed a direct correlation with CYP 2E1 activity over the range of activities present. A simulation model was developed based on a mechanism of competitive inhibition between benzene and its oxidized metabolites, and was fit to time-course data for three human liver preparations. Model calculations for initial rates of benzene metabolism ranging from 0.344 to 4.442 nmol/mg/min are directly proportional to measured CYP 2E1 activities. The model predicted the dependence of benzene metabolism on the measured CYP 2E1 activity in human liver samples, as well as in mouse and rat liver samples. These results suggest that differences in measured hepatic CYP 2E1 activity may be a major factor contributing to both interindividual and interspecies variations in hepatic metabolism of benzene

  9. CYP2E1 and NQO1 genotypes and bladder cancer risk in a Lebanese population.

    PubMed

    Basma, Hussein A; Kobeissi, Loulou H; Jabbour, Michel E; Moussa, Mohamad A; Dhaini, Hassan R

    2013-01-01

    Urinary bladder cancer incidence in Lebanon ranks among the highest in the world. Cytochrome P450 2E1 (CYP2E1), NAD(P)H quinone oxidoreductase1 (NQO1), and N-Acetyltransferase1 (NAT1), are drug-metabolizing enzymes (DMEs) involved in the metabolism of carcinogens, such as arylamines and heterocyclic amines, implicated in bladder cancer. The present study attempts to investigate the role of these DMEs genetic polymorphism in bladder cancer risk among Lebanese men. 54 cases and 106 controls were recruited from two hospitals in Beirut. An interview-based questionnaire was administered to assess suspected environmental and occupational risk factors. PCR-RFLP was performed on blood-based DNA samples to determine DMEs genotypes. Associations between bladder cancer and putative risk factors were measured using adjusted odds ratios (ORs) and their 95% confidence intervals (CIs). Results showed CYP2E1 c1/c1, NAT1*14A, and smoking, to be risk factors for bladder cancer. No significant differences in frequency distribution of the NQO1 genotypes were found in cases versus controls. The odds of carrying the CYP2E1 c1/c1 genotype were 4 times higher in cases compared to controls (OR=3.97, 95% CI: 0.48-32.7). The odds of carrying at least one NAT1*14A allele were 14 times higher in cases versus controls (OR=14.4, 95% CI: 1.016-204.9). Our study suggests CYP2E1 c1/c1, NAT1*14A, and smoking, as potential risk factors for bladder cancer in Lebanese. Further studies with larger samples must be conducted to confirm these findings. PMID:24319536

  10. CYP2E1 and NQO1 genotypes and bladder cancer risk in a Lebanese population

    PubMed Central

    Basma, Hussein A; Kobeissi, Loulou H; Jabbour, Michel E; Moussa, Mohamad A; Dhaini, Hassan R

    2013-01-01

    Urinary bladder cancer incidence in Lebanon ranks among the highest in the world. Cytochrome P450 2E1 (CYP2E1), NAD(P)H quinone oxidoreductase1 (NQO1), and N-Acetyltransferase1 (NAT1), are drug-metabolizing enzymes (DMEs) involved in the metabolism of carcinogens, such as arylamines and heterocyclic amines, implicated in bladder cancer. The present study attempts to investigate the role of these DMEs genetic polymorphism in bladder cancer risk among Lebanese men. 54 cases and 106 controls were recruited from two hospitals in Beirut. An interview-based questionnaire was administered to assess suspected environmental and occupational risk factors. PCR-RFLP was performed on blood-based DNA samples to determine DMEs genotypes. Associations between bladder cancer and putative risk factors were measured using adjusted odds ratios (ORs) and their 95% confidence intervals (CIs). Results showed CYP2E1 c1/c1, NAT1*14A, and smoking, to be risk factors for bladder cancer. No significant differences in frequency distribution of the NQO1 genotypes were found in cases versus controls. The odds of carrying the CYP2E1 c1/c1 genotype were 4 times higher in cases compared to controls (OR=3.97, 95% CI: 0.48-32.7). The odds of carrying at least one NAT1*14A allele were 14 times higher in cases versus controls (OR=14.4, 95% CI: 1.016-204.9). Our study suggests CYP2E1 c1/c1, NAT1*14A, and smoking, as potential risk factors for bladder cancer in Lebanese. Further studies with larger samples must be conducted to confirm these findings. PMID:24319536

  11. Structural basis of inhibition of Mycobacterium tuberculosis DprE1 by benzothiazinone inhibitors

    PubMed Central

    Batt, Sarah M.; Jabeen, Talat; Bhowruth, Veemal; Quill, Lee; Lund, Peter A.; Eggeling, Lothar; Alderwick, Luke J.; Fütterer, Klaus; Besra, Gurdyal S.

    2012-01-01

    Resistance against currently used antitubercular therapeutics increasingly undermines efforts to contain the worldwide tuberculosis (TB) epidemic. Recently, benzothiazinone (BTZ) inhibitors have shown nanomolar potency against both drug-susceptible and multidrug-resistant strains of the tubercle bacillus. However, their proposed mode of action is lacking structural evidence. We report here the crystal structure of the BTZ target, FAD-containing oxidoreductase Mycobacterium tuberculosis DprE1, which is essential for viability. Different crystal forms of ligand-free DprE1 reveal considerable levels of structural flexibility of two surface loops that seem to govern accessibility of the active site. Structures of complexes with the BTZ-derived nitroso derivative CT325 reveal the mode of inhibitor binding, which includes a covalent link to conserved Cys387, and reveal a trifluoromethyl group as a second key determinant of interaction with the enzyme. Surprisingly, we find that a noncovalent complex was formed between DprE1 and CT319, which is structurally identical to CT325 except for an inert nitro group replacing the reactive nitroso group. This demonstrates that binding of BTZ-class inhibitors to DprE1 is not strictly dependent on formation of the covalent link to Cys387. On the basis of the structural and activity data, we propose that the complex of DrpE1 bound to CT325 is a representative of the BTZ-target complex. These results mark a significant step forward in the characterization of a key TB drug target. PMID:22733761

  12. Heterologous expression of human cytochrome P450 2E1 in HepG2 cell line

    PubMed Central

    Zhuge, Jian; Luo, Ye; Yu, Ying-Nian

    2003-01-01

    AIM: Human cytochrome P-450 2E1 (CYP2E1) takes part in the biotransformation of ethanol, acetone, many small-molecule substrates and volatile anesthetics. CYP2E1 is involved in chemical activation of many carcinogens, procarcinogens, and toxicants. To assess the metabolic and toxicological characteristics of CYP2E1, we cloned CYP2E1 cDNA and established a HepG2 cell line stably expressing recombinant CYP 2E1. METHODS: Human CYP2E1 cDNA was amplified with reverse transcription-polymerase chain reaction (RT-PCR) from total RNAs extracted from human liver and cloned into pGEM-T vector. The cDNA segment was identified by DNA sequencing and subcloned into a mammalian expression vector pREP9. A transgenic cell line was established by transfecting the recombinant plasmid of pREP9-CYP2E1 to HepG2 cells. The expression of CYP2E1 mRNA was validated by RT-PCR. The enzyme activity of CYP2E1 catalyzing oxidation of 4-nitrophenol in postmitochondrial supernate (S9) fraction of the cells was determined by spectrophotometry. The metabolic activation of HepG2-CYP2E1 cells was assayed by N-nitrosodiethylamine (NDEA) cytotoxicity and micronucleus test. RESULTS: The cloned CYP2E1 cDNA segment was identical to that reported by Umeno et al (GenBank access No. J02843). HepG2-CYP2E1 cells expressed CYP2E1 mRNA and had 4-nitrophenol hydroxylase activity (0.162 ± 0.025 nmol·min-1·mg-1 S9 protein), which were undetectable in parent HepG2 cells. HepG2-CYP2E1 cells increased the cytotoxicity and micronucleus rate of NDEA in comparison with those of HepG2 cells. CONCLUSION: The cDNA of human CYP2E1 can be successfully cloned, and a cell line, HepG2-CYP2E1, which can efficiently express mRNA and has CYP2E1 activity, is established. The cell line is useful for testing the cytotoxicity, mutagenicity and metabolism of xenobiotics, which may possibly be activated or metabolized by CYP2E1. PMID:14669323

  13. Prenatal antidepressant exposure associated with CYP2E1 DNA methylation change in neonates

    PubMed Central

    Gurnot, Cécile; Martin-Subero, Ignacio; Mah, Sarah M; Weikum, Whitney; Goodman, Sarah J; Brain, Ursula; Werker, Janet F; Kobor, Michael S; Esteller, Manel; Oberlander, Tim F; Hensch, Takao K

    2015-01-01

    Some but not all neonates are affected by prenatal exposure to serotonin reuptake inhibitor antidepressants (SRI) and maternal mood disturbances. Distinguishing the impact of these 2 exposures is challenging and raises critical questions about whether pharmacological, genetic, or epigenetic factors can explain the spectrum of reported outcomes. Using unbiased DNA methylation array measurements followed by a detailed candidate gene approach, we examined whether prenatal SRI exposure was associated with neonatal DNA methylation changes and whether such changes were associated with differences in birth outcomes. Prenatal SRI exposure was first associated with increased DNA methylation status primarily at CYP2E1(βNon-exposed = 0.06, βSRI-exposed = 0.30, FDR = 0); however, this finding could not be distinguished from the potential impact of prenatal maternal depressed mood. Then, using pyrosequencing of CYP2E1 regulatory regions in an expanded cohort, higher DNA methylation status—both the mean across 16 CpG sites (P < 0.01) and at each specific CpG site (P < 0.05)—was associated with exposure to lower 3rd trimester maternal depressed mood symptoms only in the SRI-exposed neonates, indicating a maternal mood x SRI exposure interaction. In addition, higher DNA methylation levels at CpG2 (P = 0.04), CpG9 (P = 0.04) and CpG10 (P = 0.02), in the interrogated CYP2E1 region, were associated with increased birth weight independently of prenatal maternal mood, SRI drug exposure, or gestational age at birth. Prenatal SRI antidepressant exposure and maternal depressed mood were associated with altered neonatal CYP2E1 DNA methylation status, which, in turn, appeared to be associated with birth weight. PMID:25891251

  14. Fluxes and spectra of quasimonochromatic annihilation photons for studying E1 giant resonances in nuclei

    SciTech Connect

    Dzhilavyan, L. Z.

    2014-12-15

    The fluxes and spectra of quasimonochromatic photons originating from the in-flight annihilation of positrons interacting with electrons of targets are analyzed in the energy region characteristic of the excitation of E1 giant resonances in nuclei. Targets of small thickness and low atomic number are used. The dependences of the spectra on the energy and angle (and their scatter) for positrons incident to the target, on the collimation angle for photons, and on the target thickness are studied.

  15. Aminotriazole alleviates acetaminophen poisoning via downregulating P450 2E1 and suppressing inflammation.

    PubMed

    Jing, Yuping; Wu, Kunwei; Liu, Jiashuo; Ai, Qing; Ge, Pu; Dai, Jie; Jiang, Rong; Zhou, Dan; Che, Qian; Wan, Jingyuan; Zhang, Li

    2015-01-01

    Aminotriazole (ATZ) is commonly used as a catalase (CAT) inhibitor. We previously found ATZ attenuated oxidative liver injury, but the underlying mechanisms remain unknown. Acetaminophen (APAP) overdose frequently induces life-threatening oxidative hepatitis. In the present study, the potential hepatoprotective effects of ATZ on oxidative liver injury and the underlying mechanisms were further investigated in a mouse model with APAP poisoning. The experimental data indicated that pretreatment with ATZ dose- and time-dependently suppressed the elevation of plasma aminotransferases in APAP exposed mice, these effects were accompanied with alleviated histological abnormality and improved survival rate of APAP-challenged mice. In mice exposed to APAP, ATZ pretreatment decreased the CAT activities, hydrogen peroxide (H2O2) levels, malondialdehyde (MDA) contents, myeloperoxidase (MPO) levels in liver and reduced TNF-α levels in plasma. Pretreatment with ATZ also downregulated APAP-induced cytochrome P450 2E1 (CYP2E1) expression and JNK phosphorylation. In addition, posttreatment with ATZ after APAP challenge decreased the levels of plasma aminotransferases and increased the survival rate of experimental animals. Posttreatment with ATZ had no effects on CYP2E1 expression or JNK phosphorylation, but it significantly decreased the levels of plasma TNF-α. Our data indicated that the LD50 of ATZ in mice was 5367.4 mg/kg body weight, which is much higher than the therapeutic dose of ATZ in the present study. These data suggested that ATZ might be effective and safe in protect mice against APAP-induced hepatotoxicity, the beneficial effects might resulted from downregulation of CYP2E1 and inhibiton of inflammation. PMID:25884831

  16. Calculation of Radiative Corrections to E1 matrix elements in the Neutral Alkalis

    SciTech Connect

    Sapirstein, J; Cheng, K T

    2004-09-28

    Radiative corrections to E1 matrix elements for ns-np transitions in the alkali metal atoms lithium through francium are evaluated. They are found to be small for the lighter alkalis but significantly larger for the heavier alkalis, and in the case of cesium much larger than the experimental accuracy. The relation of the matrix element calculation to a recent decay rate calculation for hydrogenic ions is discussed, and application of the method to parity nonconservation in cesium is described.

  17. Ultrasound associated uptake of chitosan nanoparticles in MC3T3-E1 cells

    NASA Astrophysics Data System (ADS)

    Wu, Junyi

    Chitosan is a natural linear polysaccharide that has been well known for its applications in drug delivery system due to its unique physicochemical and biological properties. However, challenges still remain for it to become a fully realized therapeutic agent. In this study, we investigated the uptake of chitosan nanoparticles (CNP) under the ultrasound stimulation, using a model cell culture system (MC3T3-E1 mouse pre-osteoblasts). The CNP were fabricated by an ionic gelation method and were lyophilized prior to characterization and delivery to cells. Particle size and zeta potential were measured using Dynamic Light Scattering (DLS); the efficiency of chitosan complexation was measured using the ninhydrin assay. Cytotoxicity was examined by neutral red assay within 48 hours after delivery. The effect of ultrasound (US) on the efficiency of nanoparticle delivery to the MC3T3-E1 cells was examined at 1MHz and at either 1 or 2 W/cm2. Fluorescein isothiocyanate (FITC)-conjugated-CNP were used to visualize the internalized particles within the cytosol. The uptake of FITC-CNP exhibits a dose and time dependent effect, a strong FITC fluorescence was detected at the concentration of 500microg/mL under fluorescence microscope. Ultrasound assisted uptake of FITC-CNP performed a significant positive effect at 2W/cm2 with 60s of ultrasound exposure time. CNP displayed a slightly decrease in cell viability from 25microg/mL to 100microg/mL, while higher concentration of CNP facilitates the proliferation of MC3T3-E1 cells. Less than 10% of reduction in cell viability was observed for US at 1W/cm2 and 2W/cm2 with 30s and 60s of exposure time, which suggest a mild effect of US to MC3T3-E1 cell line.

  18. PPARgamma ligands suppress the feedback loop between E2F2 and cyclin-E1.

    PubMed

    Komatsu, Yoko; Ito, Ichiaki; Wayama, Mitsutoshi; Fujimura, Akiko; Akaogi, Kensuke; Machida, Hikaru; Nakajima, Yuka; Kuroda, Takao; Ohmori, Kazuji; Murayama, Akiko; Kimura, Keiji; Yanagisawa, Junn

    2008-05-23

    PPARgamma is a nuclear hormone receptor that plays a key role in the induction of peroxisome proliferation. A number of studies showed that PPARgamma ligands suppress cell cycle progression; however, the mechanism remains to be determined. Here, we showed that PPARgamma ligand troglitazone inhibited G1/S transition in colon cancer cells, LS174T. Troglitazone did not affect on either expression of CDK inhibitor (p18) or Wnt signaling pathway, indicating that these pathways were not involved in the troglitazone-dependent cell cycle arrest. GeneChip and RT-PCR analyses revealed that troglitazone decreased mRNA levels of cell cycle regulatory factors E2F2 and cyclin-E1 whose expression is activated by E2F2. Down-regulation of E2F2 by troglitazone results in decrease of cyclin-E1 transcription, which could inhibit phosphorylation of Rb protein, and consequently evoke the suppression of E2F2 transcriptional activity. Thus, we propose that troglitazone suppresses the feedback loop containing E2F2, cyclin-E1, and Rb protein. PMID:18355447

  19. Bursts of energetic electron induced large surface charging observed by Chang'E-1

    NASA Astrophysics Data System (ADS)

    Wang, X. Y.; Zhang, A. B.; Zhang, X. G.; Reme, H.; Kong, L. G.; Zhang, S. Y.; Yu, D. J.; Wang, S. J.; Zhu, G. W.

    2012-10-01

    A relationship between surface charging and bursts of energetic electron (BEE) event is presented in this paper. In a 200 km lunar polar orbit, during quiet time, 0.1-2.0 MeV BEE events were observed by High Energetic Particles Detectors (HPD) on board Chang'E-1, on December 22, 2007, when the spacecraft was within the inner terrestrial magnetosheath. At the same time, a large surface charging of ∼-5.4 kV was observed by Chang'E-1, which was evidenced by increasing the ions energy observed by Solar Wind Ion Detectors (SWIDs). We found that the surface charging is strongly correlated with BEE events, and the potentials of spacecraft surface charging was experientially expressed as U≈3.6×10-5·fT (kV). The BEE events did occur in the solar wind, geomagnetic tail and magnetosheath alternately, whereas the surface charging during the BEE events is in the magnetosheath or transition region of boundaries. Though the observed surface charging was fewer than the BEE events, it is expected that the occurrence of the charging events caused by the bursts of energetic electrons should be more frequent than the Chang'E-1 observations. Meanwhile, the spacecraft charging indicates the lunar surface can be charged to negative kilovolt-scale by the BEE events even in quiet times.

  20. Purification, crystallization and preliminary X-ray diffraction analysis of human enolase-phosphatase E1

    SciTech Connect

    Wang, Hui; Pang, Hai; Ding, Yi; Li, Yi; Wu, Xiao’ai; Rao, Zihe

    2005-05-01

    Recombinant human E1 enzyme has been crystallized using the hanging-drop vapour-diffusion method and diffraction-quality crystals were grown at 291 K using PEG 4000 as precipitant. Enolase-phosphatase E1 (MASA) is a bifunctional enzyme in the ubiquitous methionine-salvage pathway and catalyzes the continuous reaction of 2,3-diketo-5-methylthio-1-phosphopentane to yield the acireductone metabolite. Recombinant human E1 enzyme has been crystallized using the hanging-drop vapour-diffusion method and diffraction-quality crystals were grown at 291 K using PEG 4000 as precipitant. Diffraction data were collected to 1.7 Å resolution from SeMet-derivative crystals at 100 K using synchrotron radiation. The crystals belong to space group P2{sub 1}2{sub 1}2{sub 1}, with unit-cell parameters a = 54.02, b = 57.55, c = 87.32 Å. The structure was subsequently solved by the multi-wavelength anomalous diffraction (MAD) phasing method.

  1. Evaluation of E1A Double Mutant Oncolytic Adenovectors in Anti-Glioma Gene Therapy

    PubMed Central

    Ulasov, Ilya V.; Tyler, Matthew A.; Rivera, Angel A.; Nettelbeck, Dirk M.; Douglas, Joanne T.; Lesniak, Maciej S.

    2008-01-01

    Malignant glioma, in particular glioblastoma multiforme (GBM), represents one of the most devastating cancers currently known and existing treatment regimens do little to change patient prognosis. Conditionally replicating adenoviral vectors (CRAds) represent attractive experimental anti-cancer agents with potential for clinical application. However, early protein products of the wild type adenovirus backbone—such as E1A—limit CRAds’ replicative specificity. In this study, we evaluated the oncolytic potency and specificity of CRAds in which p300/CPB and/or pRb binding capacities of E1A were ablated to reduce non-specific replicative cytolysis. In vitro cytopathic assays, quantitative PCR analysis, Western blot, and flow cytometry studies demonstrate the superior anti-glioma efficacy of a double-mutated CRAd, Ad2/24CMV, which harbors mutations that reduce E1A binding to p300/CPB and pRb. When compared to its single-mutated and wild type counterparts, Ad2/24CMV demonstrated attenuated replication and cytotoxicity in representative normal human brain while displaying enhanced replicative cytotoxicity in malignant glioma. These results have implications for the development of double-mutated CRAd vectors for enhanced GBM therapy. PMID:18649343

  2. B(E1) Strengths from Coulomb excitation of 11Be

    SciTech Connect

    Summers, N C; Pain, S D; Orr, N A; Catford, W N; Angelique, J C; Ashwood, N I; Bouchat, V; Clarke, N M; Curtis, N; Freer, M; Fulton, B R; Hanappe, F; Labiche, M; Loucey, J L; Lemmon, R C; Mahboub, D; Ninane, A; Normand, G; Nunes, F M; Soic, N; Stuttge, L; Timis, C N; Thompson, I; Winfield, J S; Ziman, V

    2007-03-06

    The B(E1;1/2{sup +}{yields} 1/2{sup -}) strength for {sup 11}Be has been extracted from intermediate energy Coulomb excitation measurements, over a range of beam energies using a new reaction model, the extended continuum discretized coupled channels (XCDCC) method. In addition, a measurement of the excitation cross section for {sup 11}Be+{sup 208}Pb at 38.6 MeV/nucleon is reported. The B(E1) strength of 0.105(12) e{sup 2}fm{sup 2} derived from this measurement is consistent with those made previously at 60 and 64 MeV/nucleon, in contrast to an anomalously low result obtained at 43 MeV/nucleon. By coupling a multi-configuration description of the projectile structure with realistic reaction theory, the XCDCC model provides for the first time a fully quantum mechanical description of Coulomb excitation. The XCDCC calculations reveal that the excitation process involves significant contributions from nuclear, continuum, and higher-order effects. An analysis of the present and two earlier intermediate energy measurements yields a combined B(E1) strength of 0.105(7) e{sup 2}fm{sup 2}. This value is in good agreement with the value deduced independently from the lifetime of the 1/2{sup -} state in {sup 11}Be, and has a comparable precision.

  3. E1B and E4 Oncoproteins of Adenovirus Antagonize the Effect of Apoptosis Inducing Factor

    PubMed Central

    Turner, Roberta L.; Wilkinson, John C.; Ornelles, David A.

    2014-01-01

    Adenovirus inundates the productively infected cell with linear, double-stranded DNA and an abundance of single-stranded DNA. The cellular response to this stimulus is antagonized by the adenoviral E1B and E4 early genes. A mutant group C adenovirus that fails to express the E1B-55K and E4orf3 genes is unable to suppress the DNA-damage response. Cells infected with this double-mutant virus display significant morphological heterogeneity at late times of infection and frequently contain fragmented nuclei. Nuclear fragmentation was due to the translocation of apoptosis inducing factor (AIF) from the mitochondria into the nucleus. The release of AIF was dependent on active poly(ADP-ribose) polymerase-1 (PARP-1), which appeared to be activated by viral DNA replication. Nuclear fragmentation did not occur in AIF-deficient cells or in cells treated with a PARP-1 inhibitor. The E1B-55K or E4orf3 proteins independently prevented nuclear fragmentation subsequent to PARP-1 activation, possibly by altering the intracellular distribution of PAR-modified proteins. PMID:24889240

  4. [Intra-arterial administration of prostaglandin E1 in occlusive arterial diseases].

    PubMed

    Davidović, L; Vranes, M; Cernak, I; Kostić, D; Lovrić, A; Sagić, D; Lotina, S

    1992-01-01

    The authors present their result of a two-year follow-up of 106 patients to whom an intra-arterial perfusion of prostaglandin E1 was administered, as limb salvage procedure. The patients were in the IIIrd and IVth stage of occlusive diseases by Fountain, and surgical reconstructions were not possible. All patients were divided into five groups: A--diabetic angiopathy (5), B--distal form of atherosclerosis (40), C--diabetic angiopathy and atherosclerosis (45), D--Burger disease (10) and E--adjuvant therapy in reconstruction with poor run-off (6). The Doppler sonographic and angiographic measurements were performed. After transcutaneous (16 cases), or intraoperative (90 cases) introduction of the catheter into superficial of profunda femoral artery, a continuous intraarterial administration of prostaglandin E1 was carried out in a dose of 10 nanograms/kg body weight/minute (total dosage 3000 nanograms). The patients were controlled immediately after the treatment, as well as 1, 3, 6, 12, 24 and 36 months after the treatment. In efficiency of the treatment was estimated on the following basis: elimination of rest pain, healing of trophic ulceration and demarcation of gangrenous processes. Our late results of intra-arterial administration of prostaglandin E1 proved to be a very successful limb salvage procedure. The treatment was more successful in a connections between the upper knee arterial net and pedal arterial arches were preserved. PMID:1641706

  5. Instanton induced Yukawa couplings from distant E3 and E(-1) instantons

    NASA Astrophysics Data System (ADS)

    Goodsell, Mark D.; Witkowski, Lukas T.

    2016-01-01

    We calculate non-perturbative contributions to Yukawa couplings on D3-branes at orbifold singularities due to E3 and fractional E(-1) instantons which do not intersect the visible sector branes. While distant E3 instantons on bulk cycles typically contribute to Yukawa couplings, we find that distant fractional E(-1) can also give rise to new Yukawa couplings. However, fractional E(-1) instantons only induce Yukawa couplings if they are located at a singularity which shares a collapsed homologous two-cycle with the singularity supporting the visible sector. The non-perturbative contributions to Yukawa couplings exhibit a different flavour structure than the tree-level Yukawa couplings and, as a result, they can be sources of flavour violation. This is particularly relevant for schemes of moduli stabilisation which rely on superpotential contributions from E3 instantons, such as KKLT or the Large Volume Scenario. As a byproduct of our analysis, we shed some new light on the properties of annulus diagrams with matter field insertions in stringy instanton calculus.

  6. Protein binding sites involved in the assembly of the KplE1 prophage intasome.

    PubMed

    Panis, Gaël; Duverger, Yohann; Champ, Stéphanie; Ansaldi, Mireille

    2010-08-15

    The organization of the recombination regions of the KplE1 prophage in Escherichia coli K12 differs from that observed in the lambda prophage. Indeed, the binding sites characterized for the IntS integrase, the TorI recombination directionality factor (RDF) and the integration host factor (IHF) vary in number, spacing and orientation on the attL and attR regions. In this paper, we performed site-directed mutagenesis of the recombination sites to decipher if all sites are essential for the site-specific recombination reaction and how the KplE1 intasome is assembled. We also show that TorI and IntS form oligomers that are stabilized in the presence of their target DNA. Moreover, we found that IHF is the only nucleoid associated protein (NAP) involved in KplE1 recombination, although it is dispensable. This is consistent with the presence of only one functional IHF site on attR and none on attL. PMID:20494389

  7. Heterologous expression of mouse cytochrome P450 2e1 in V79 cells: construction and characterisation of the cell line and comparison with V79 cell lines stably expressing rat P450 2E1 and human P450 2E1.

    PubMed

    Bernauer, Ulrike; Glatt, Hansruedi; Heinrich-Hirsch, Barbara; Liu, Yungang; Muckel, Eva; Vieth, Barbel; Gundert-Remy, Ursula

    2003-01-01

    A V79 Chinese hamster cell line was constructed for stable expression of mouse cytochrome P450 2e1 (Cyp2e1), as an addition to the existing cell battery consisting of cell lines stably expressing rat CYP2E1 and human CYP2E1 (V79 Cell Battery). The aim was to establish a cell battery that offers the in vitro possibility of investigating species-specific differences in the toxicity and metabolism of chemicals representing substrates for CYP2E1. The newly established cell line (V79m2E1) effectively expressed Cyp2e1 in the catalytically active form. The expression of catalytically active CYP2E1 in V79m2E1 cells was maintained over several months in culture, as demonstrated by Western Blotting and chlorzoxazone (CLX) 6-hydroxylase activity. The cells exhibited CLX 6-hydroxylase activity with a Km of 27.8 microM/l and Vmax of 40 pmol/mg protein/minute, compared with a Km of 28.2/28.6 microM/l and a Vmax of 130/60 pmol/mg protein/minute from V79r2E1/V79h2E1 cells. Furthermore, the CYP2E1-dependent mutagenicity of N-nitrosodimethylamine could be demonstrated in the V79m2E1 cells. Therefore, the new cell battery permits the interspecies comparison of CYP2E1-dependent toxicity and of metabolism of chemicals between humans and the two major rodent species--the rat and the mouse--that are usually used in classical toxicity studies. PMID:16221041

  8. Chimeric Derivatives of Hepatitis B Virus Core Particles Carrying Major Epitopes of the Rubella Virus E1 Glycoprotein

    PubMed Central

    Skrastina, Dace; Petrovskis, Ivars; Petraityte, Rasa; Sominskaya, Irina; Ose, Velta; Liekniņa, Ilva; Bogans, Janis; Sasnauskas, Kestutis

    2013-01-01

    Three variants of the major rubella virus (RV) E1 protein virus-neutralizing epitope from position 214 to 285 were exposed on the hepatitis B virus (HBV) C-terminally truncated core (HBcΔ) in a virus-like particle (VLP) vector and were produced in Escherichia coli. All three chimeras demonstrated VLPs in bacterial cell lysates, but only HBcΔ-E1(245-285) demonstrated the correct VLP structure after purification. The other chimeras, HBcΔ-E1(214-285) and HBcΔ-E1(214-240), appeared after purification as non-VLP aggregates of 100 to 900 nm in diameter according to dynamic light scattering data. All three variants possessed the intrinsic antigenic activity of RV E1, since they were recognized by natural human anti-RV E1 antibodies and induced an anti-RV E1 response in mice. HBcΔ-E1(214-240) and HBcΔ-E1(245-285) can be regarded as prototypes for a putative RV vaccine because they were able to induce antibodies recognizing natural RV E1 protein in RV diagnostic kits. PMID:24006140

  9. Cytochrome P450 2E1 genetic polymorphism and gastric cancer in Changle, Fujian Province

    PubMed Central

    Cai, Lin; Yu, Shun-Zhang; Zhang, Zuo-Feng

    2001-01-01

    AIM: Genetic polymorphism in enzymes of carcinogen metabolism has been found to have the influence on the susceptibility to cancer. Cytochrome P450 2E1 (CYP2E1) is considered to play an important role in the metabolic activation of procarcinogens such as N-nitrosoamines and low molecular weight organic compounds. The purpose of this study is to determine whether CYP450 2E1 polymorphisms are associated with risks of gastric cancer. METHODS: We conducted a population based case-control study in Changle county, Fujian Province, a high-risk region of gastric cancer in China. Ninety-one incident gastric cancer patients and ninety-four healthy controls were included in our study. Datas including demographic characteristcs, diet intake, and alcohol and tobacco consumption of indivduals in our study were completed by a standardized questionnaire. PCR-RFLP revealed three genotypes:heterozygote (C1/C2) and two homozygotes (C1/C1 and C2/C2) in CYP2E1. RESULTS: The frequency of variant genotypes (C1/C2 and C2/C2) in gastric cancer cases and controls was 36.3% and 24.5%, respectively. The rare homozygous C2/C2 genotype was found in 6 indivduals in gastric cancer group (6.6%), whereas there was only one in the control group (1.1%). However, there was no statistically significan difference between the two groups (two-tailed Fisher’s exact test, P = 0.066). Indivduals in gastric cancer group were more likely to carry genotype C1/C2 (odds ratio, OR = 1.50) and C2/C2 (OR = 7.34) than indivduals in control group (χ² = 4.597, for trend P = 0.032). The frequencies of genotypes with the C2 allele (C1/C2 and C2/C2 genotypes) were compared with those of genotypes without C2 allele (C1/C1 genotype) among indivduals in gastric cancer group and control group according to the pattern of gastric cancer risk factors. The results show that indivduals who exposed to these gastric cancer risk factors and carry the C2 allele seemed to have a higher risk of developing gastric cancer. CONCLUSION

  10. Characterization of novel cytochrome P450 2E1 knockout rat model generated by CRISPR/Cas9.

    PubMed

    Wang, Xin; Tang, Yu; Lu, Jian; Shao, Yanjiao; Qin, Xuan; Li, Yongmei; Wang, Liren; Li, Dali; Liu, Mingyao

    2016-04-01

    A bacterial CRISPR-associated protein-9 nuclease (CRISPR/Cas9) from Streptococcus pyogenes has generated considerable excitement as a new tool to edit the targeted genome. Cytochrome P450 (CYP) 2E1 not only plays an important role in the xenobiotic metabolism and chemical toxicity, but also is involved in many kinds of diseases, such as alcoholic liver diseases and diabetes. Despite its importance, few animal models are used to predict CYP2E1 properties in physiology, pathology, as well as carcinogen activation. To establish a novel model for investigating the functions of CYP2E1 in vivo, this study has successfully generated the Cyp2e1 knockout (KO) rat model without detectable off-target effects using CRISPR/Cas9 system. The Cyp2e1 KO rats were viable and fertile and did not display any obvious physiological abnormities. The absent expression of CYP2E1 in KO rats also resulted in inactive behaviors in the metabolism of CYP2E1 substrates. The Cyp2e1 KO rats as a novel and available rodent animal model provide a powerful tool for the study of CYP2E1 in the chemical metabolism, toxicity, carcinogenicity, and its core factor in drug-drug interactions. PMID:26947455

  11. Human Papillomavirus Type 18 E1 Protein Is Translated from Polycistronic mRNA by a Discontinuous Scanning Mechanism

    PubMed Central

    Remm, Maido; Remm, Anu; Ustav, Mart

    1999-01-01

    Papillomaviruses are small double-stranded DNA viruses that replicate episomally in the nuclei of infected cells. The full-length E1 protein of papillomaviruses is required for the replication of viral DNA. The viral mRNA from which the human papillomavirus type 18 E1 protein is expressed is not known. We demonstrate that in eukaryotic cells, the E1 protein is expressed from polycistronic mRNA containing E6, E7, and E1 open reading frames (ORFs). The translation of adjacent E7 and E1 ORFs is not associated; it is performed by separate populations of ribosomes. The translation of the downstream E1 gene is preceded by ribosome scanning. Scanning happens at least at the 5′ end of the polycistronic mRNA and also approximately 100 bp in front of the E1 gene. Long areas in middle of the mRNA are bypassed by ribosomes, possibly by ribosomal “shunting.” Inactivation of short minicistrons in the upstream area of the E1 gene did not change the expression level of the E1 gene. PMID:10074156

  12. Characterization of papillomavirus E1 helicase mutants defective for interaction with the SUMO-conjugating enzyme Ubc9

    SciTech Connect

    Fradet-Turcotte, Amelie; Brault, Karine; Titolo, Steve; Howley, Peter M.; Archambault, Jacques

    2009-12-20

    The E1 helicase from BPV and HPV16 interacts with Ubc9 to facilitate viral genome replication. We report that HPV11 E1 also interacts with Ubc9 in vitro and in the yeast two-hybrid system. Residues in E1 involved in oligomerization (353-435) were sufficient for binding to Ubc9 in vitro, but the origin-binding and ATPase domains were additionally required in yeast. Nuclear accumulation of BPV E1 was shown previously to depend on its interaction with Ubc9 and sumoylation on lysine 514. In contrast, HPV11 and HPV16 E1 mutants defective for Ubc9 binding remained nuclear even when the SUMO pathway was inhibited. Furthermore, we found that K514 in BPV E1 and the analogous K559 in HPV11 E1 are not essential for nuclear accumulation of E1. These results suggest that the interaction of E1 with Ubc9 is not essential for its nuclear accumulation but, rather, depends on its oligomerization and binding to DNA and ATP.

  13. Downregulation of microRNA miR-520h by E1A Contributes to Anti-cancer Activity

    PubMed Central

    Su, Jen-Liang; Chen, Poshen B.; Chen, Ya-Huey; Chen, Shang-Chih; Chang, Yi-Wen; Jan, Yi-Hua; Cheng, Xiaoyun; Hsiao, Michael; Hung, Mien-Chie

    2010-01-01

    The leading cause of death in cancer patients is cancer metastasis, for which there is no effective treatment. MicroRNAs have been shown to play a significant role in cancer metastasis through regulation of gene expression. The Adenoviral type 5 E1A is associated with multiple tumor suppressing activities including the inhibition of metastasis, and E1A gene therapies have been tested in several clinical trials. However, the mechanisms involved in E1A-mediated tumor suppressing activities are not yet completely defined. Here we demonstrated that E1A down-regulated the expression of miRNA, miR-520h, which was critical for E1A-mediated cancer cell mobility and in vitro invasion activity. In addition, we identified a signal cascade, namely, E1A —| miRNA-520h —| PP2A/C —| IKK → NF-κB → Twist, in which E1A inhibited the expression of Twist through downregulation of miR-520h and the signal cascade. Our results indicated a functional link between miR-520h and tumorigenicity/invasive ability, and provided a new insight into the role of E1A-mediated miRNA regulation in tumor suppression. Therefore, the results identified a new cascade of E1A-mediated tumor suppression activity via downregulation of miRNA-520h expression. PMID:20501832

  14. Ethanol Induction of CYP2A5: Role of CYP2E1-ROS-Nrf2 Pathway

    PubMed Central

    Lu, Yongke; Zhang, Xu Hannah

    2012-01-01

    Chronic ethanol consumption was previously shown to induce CYP2A5 in mice, and this induction of CYP2A5 by ethanol was CYP2E1 dependent. In this study, the mechanisms of CYP2E1-dependent ethanol induction of CYP2A5 were investigated. CYP2E1 was induced by chronic ethanol consumption to the same degree in wild-type (WT) mice and CYP2A5 knockout (Cyp2a5 –/–) mice, suggesting that unlike the CYP2E1-dependent ethanol induction of CYP2A5, ethanol induction of CYP2E1 is not CYP2A5 dependent. Microsomal ethanol oxidation was about 25% lower in Cyp2a5 –/– mice compared with that in WT mice, suggesting that CYP2A5 can oxidize ethanol although to a lesser extent than CYP2E1 does. CYP2A5 was induced by short-term ethanol consumption in human CYP2E1 transgenic knockin (Cyp2e1 –/– KI) mice but not in CYP2E1 knockout (Cyp2e1 –/–) mice. The redox-sensitive transcription factor nuclear factor-erythroid 2-related factor 2 (Nrf2) was also induced by acute ethanol in Cyp2e1 –/– KI mice but not in Cyp2e1 –/– mice. Ethanol induction of CYP2A5 in Nrf2 knockout (Nrf2 –/–) mice was lower compared with that in WT mice, whereas CYP2E1 induction by ethanol was comparable in WT and Nrf2 –/– mice. Antioxidants (N-acetyl-cysteine and vitamin C), which blocked oxidative stress induced by chronic ethanol in WT mice and acute ethanol in Cyp2e1 –/– KI mice, also blunted the induction of CYP2A5 and Nrf2 by ethanol but not the induction of CYP2E1 by ethanol. These results suggest that oxidative stress induced by ethanol via induction of CYP2E1 upregulates Nrf2 activity, which in turn regulates ethanol induction of CYP2A5. Results obtained from primary hepatocytes, mice gavaged with binge ethanol or fed chronic ethanol, show that Nrf2-regulated ethanol induction of CYP2A5 protects against ethanol-induced steatosis. PMID:22552773

  15. Structure of the pyruvate dehydrogenase multienzyme complex E1 component from Escherichia coli at 1.85 A resolution.

    PubMed

    Arjunan, Palaniappa; Nemeria, Natalia; Brunskill, Andrew; Chandrasekhar, Krishnamoorthy; Sax, Martin; Yan, Yan; Jordan, Frank; Guest, John R; Furey, William

    2002-04-23

    The crystal structure of the recombinant thiamin diphosphate-dependent E1 component from the Escherichia coli pyruvate dehydrogenase multienzyme complex (PDHc) has been determined at a resolution of 1.85 A. The E. coli PDHc E1 component E1p is a homodimeric enzyme and crystallizes with an intact dimer in an asymmetric unit. Each E1p subunit consists of three domains: N-terminal, middle, and C-terminal, with all having alpha/beta folds. The functional dimer contains two catalytic centers located at the interface between subunits. The ThDP cofactors are bound in the "V" conformation in clefts between the two subunits (binding involves the N-terminal and middle domains), and there is a common ThDP binding fold. The cofactors are completely buried, as only the C2 atoms are accessible from solution through the active site clefts. Significant structural differences are observed between individual domains of E1p relative to heterotetrameric multienzyme complex E1 components operating on branched chain substrates. These differences may be responsible for reported alternative E1p binding modes to E2 components within the respective complexes. This paper represents the first structural example of a functional pyruvate dehydrogenase E1p component from any species. It also provides the first representative example for the entire family of homodimeric (alpha2) E1 multienzyme complex components, and should serve as a model for this class of enzymes. PMID:11955070

  16. Characterization of papillomavirus E1 helicase mutants defective for interaction with the SUMO-conjugating enzyme Ubc9.

    PubMed

    Fradet-Turcotte, Amélie; Brault, Karine; Titolo, Steve; Howley, Peter M; Archambault, Jacques

    2009-12-20

    The E1 helicase from BPV and HPV16 interacts with Ubc9 to facilitate viral genome replication. We report that HPV11 E1 also interacts with Ubc9 in vitro and in the yeast two-hybrid system. Residues in E1 involved in oligomerization (353-435) were sufficient for binding to Ubc9 in vitro, but the origin-binding and ATPase domains were additionally required in yeast. Nuclear accumulation of BPV E1 was shown previously to depend on its interaction with Ubc9 and sumoylation on lysine 514. In contrast, HPV11 and HPV16 E1 mutants defective for Ubc9 binding remained nuclear even when the SUMO pathway was inhibited. Furthermore, we found that K514 in BPV E1 and the analogous K559 in HPV11 E1 are not essential for nuclear accumulation of E1. These results suggest that the interaction of E1 with Ubc9 is not essential for its nuclear accumulation but, rather, depends on its oligomerization and binding to DNA and ATP. PMID:19836047

  17. Ubiquitination independent of E1 and E2 enzymes by bacterial effectors.

    PubMed

    Qiu, Jiazhang; Sheedlo, Michael J; Yu, Kaiwen; Tan, Yunhao; Nakayasu, Ernesto S; Das, Chittaranjan; Liu, Xiaoyun; Luo, Zhao-Qing

    2016-05-01

    Signalling by ubiquitination regulates virtually every cellular process in eukaryotes. Covalent attachment of ubiquitin to a substrate is catalysed by the E1, E2 and E3 three-enzyme cascade, which links the carboxy terminus of ubiquitin to the ε-amino group of, in most cases, a lysine of the substrate via an isopeptide bond. Given the essential roles of ubiquitination in the regulation of the immune system, it is not surprising that the ubiquitination network is a common target for diverse infectious agents. For example, many bacterial pathogens exploit ubiquitin signalling using virulence factors that function as E3 ligases, deubiquitinases or as enzymes that directly attack ubiquitin. The bacterial pathogen Legionella pneumophila utilizes approximately 300 effectors that modulate diverse host processes to create a permissive niche for its replication in phagocytes. Here we demonstrate that members of the SidE effector family of L. pneumophila ubiquitinate multiple Rab small GTPases associated with the endoplasmic reticulum. Moreover, we show that these proteins are capable of catalysing ubiquitination without the need for the E1 and E2 enzymes. A putative mono-ADP-ribosyltransferase motif critical for the ubiquitination activity is also essential for the role of the SidE family in intracellular bacterial replication in a protozoan host. The E1/E2-independent ubiquitination catalysed by these enzymes is energized by nicotinamide adenine dinucleotide, which activates ubiquitin by the formation of ADP-ribosylated ubiquitin. These results establish that ubiquitination can be catalysed by a single enzyme, the activity of which does not require ATP. PMID:27049943

  18. E1 of α-ketoglutarate dehydrogenase defends Mycobacterium tuberculosis against glutamate anaplerosis and nitroxidative stress

    PubMed Central

    Maksymiuk, Christina; Balakrishnan, Anand; Bryk, Ruslana; Rhee, Kyu Y.; Nathan, Carl F.

    2015-01-01

    Enzymes of central carbon metabolism (CCM) in Mycobacterium tuberculosis (Mtb) make an important contribution to the pathogen’s virulence. Evidence is emerging that some of these enzymes are not simply playing the metabolic roles for which they are annotated, but can protect the pathogen via additional functions. Here, we found that deficiency of 2-hydroxy-3-oxoadipate synthase (HOAS), the E1 component of the α-ketoglutarate (α-KG) dehydrogenase complex (KDHC), did not lead to general metabolic perturbation or growth impairment of Mtb, but only to the specific inability to cope with glutamate anaplerosis and nitroxidative stress. In the former role, HOAS acts to prevent accumulation of aldehydes, including growth-inhibitory succinate semialdehyde (SSA). In the latter role, HOAS can participate in an alternative four-component peroxidase system, HOAS/dihydrolipoyl acetyl transferase (DlaT)/alkylhydroperoxide reductase colorless subunit gene (ahpC)-neighboring subunit (AhpD)/AhpC, using α-KG as a previously undescribed source of electrons for reductase action. Thus, instead of a canonical role in CCM, the E1 component of Mtb’s KDHC serves key roles in situational defense that contribute to its requirement for virulence in the host. We also show that pyruvate decarboxylase (AceE), the E1 component of pyruvate dehydrogenase (PDHC), can participate in AceE/DlaT/AhpD/AhpC, using pyruvate as a source of electrons for reductase action. Identification of these systems leads us to suggest that Mtb can recruit components of its CCM for reactive nitrogen defense using central carbon metabolites. PMID:26430237

  19. C-E1 fusion protein synthesized by rubella virus DI RNAs maintained during serial passage

    SciTech Connect

    Tzeng, W.-P.; Frey, Teryl K. . E-mail: tfrey@gsu.edu

    2006-12-20

    Rubella virus (RUB) replicons are derivatives of the RUB infectious cDNA clone that retain the nonstructural open reading frame (NS-ORF) that encodes the replicase proteins but not the structural protein ORF (SP-ORF) that encodes the virion proteins. RUB defective interfering (DI) RNAs contain deletions within the SP-ORF and thus resemble replicons. DI RNAs often retain the 5' end of the capsid protein (C) gene that has been shown to modulate virus-specific RNA synthesis. However, when replicons either with or without the C gene were passaged serially in the presence of wt RUB as a source of the virion proteins, it was found that neither replicon was maintained and DI RNAs were generated. The majority DI RNA species contained in-frame deletions in the SP-ORF leading to a fusion between the 5' end of the C gene and the 3' end of the E1 glycoprotein gene. DI infectious cDNA clones were constructed and transcripts from these DI infectious cDNA clones were maintained during serial passage with wt RUB. The C-E1 fusion protein encoded by the DI RNAs was synthesized and was required for maintenance of the DI RNA during serial passage. This is the first report of a functional novel gene product resulting from deletion during DI RNA generation. Thus far, the role of the C-E1 fusion protein in maintenance of DI RNAs during serial passage remained elusive as it was found that the fusion protein diminished rather than enhanced DI RNA synthesis and was not incorporated into virus particles.

  20. Lysophosphatidic acid induces chemotaxis in MC3T3-E1 osteoblastic cells.

    PubMed

    Masiello, Lisa M; Fotos, Joseph S; Galileo, Deni S; Karin, Norman J

    2006-07-01

    Lysophosphatidic acid (LPA) is a bioactive lipid that has pleiotropic effects on a variety of cell types and enhances the migration of endothelial and cancer cells, but it is not known if this lipid can alter osteoblast motility. We performed transwell migration assays using MC3T3-E1 osteoblastic cells and found LPA to be a potent chemotactic agent. Quantitative time-lapse video analysis of osteoblast migration after wounds were introduced into cell monolayers indicated that LPA stimulated both migration velocity and the average migration distance per cell. LPA also elicited substantial changes in cell shape and actin cytoskeletal structure; lipid-treated cells contained fewer stress fibers and displayed long membrane processes that were enriched in F-actin. Quantitative RT-PCR analysis showed that MC3T3-E1 cells express all four known LPA-specific G-protein-coupled receptors (LPA1-LPA4) with a relative mRNA abundance of LPA1>LPA4>LPA2>LPA3. LPA-induced changes in osteoblast motility and morphology were antagonized by both pertussis toxin and Ki16425, a subtype-specific blocker of LPA1 and LPA3 receptor function. Cell migration in many cell types is linked to changes in intracellular Ca2+. Ki16425 also inhibited LPA-induced Ca2+ signaling in a dose-dependent manner, suggesting a link between LPA-induced Ca2+ transients and osteoblast chemotaxis. Our data show that LPA stimulates MC3T3-E1 osteoblast motility via a mechanism that is linked primarily to the G-protein-coupled receptor LPA1. PMID:16487757

  1. Calculation of energy levels, {ital E}1 transition amplitudes, and parity violation in francium

    SciTech Connect

    Dzuba, V.A.; Flambaum, V.V.; Sushkov, O.P.

    1995-05-01

    Many-body perturbation theory in the screened Coulomb interaction was used to calculate energy levels, {ital E}1 trransition amplitudes, and the parity-nonconserving (PNC) {ital E}1 amplitude of the 7{ital s}-8{ital s} transition in francium. The method takes into account the core-polarization effect, the second-order correlations, and the three dominating sequences of higher-order correlation diagrams: screening of the electron-electron interaction, particle-hole interaction, and the iterations of the self-energy operator. The result for the PNC amplitude for {sup 223}Fr is {ital E}1(7{ital s}-8{ital s})=(1.59{plus_minus}{similar_to}1%){times}10{sup {minus}10}{ital iea}{sub {ital B}}({minus}{ital Q}{sub {ital W}}/{ital N}), where {ital Q}{sub {ital W}} is the weak charge of the nucleus, {ital N}=136 is the number of neutrons, {ital e}={vert_bar}{ital e}{vert_bar} is the elementary charge, and {ital a}{sub {ital B}} is the Bohr radius. Our prediction for the position of the 8{ital s} energy level of Fr, which has not been measured yet, is 13 110 cm{sup {minus}1} below the limit of the continuous spectrum. The accuracy of the calculations was controlled by comparison with available experimental data and analogous calculations for cesium. It is estimated to be {similar_to}0.1% for the energy levels and {similar_to}1% for the transition amplitudes.

  2. Lysophosphatidic acid induces chemotaxis in MC3T3-E1 osteoblastic cells

    SciTech Connect

    Masiello, Lisa M.; Fotos, Joseph S.; Galileo, Deni S.; Karin, Norm J.

    2006-07-01

    Lysophosphatidic acid (LPA) is a bioactive lipid that has pleiotropic effects on a variety of cell types and enhances the migration of endothelial and cancer cells, but it is not known if this lipid can alter osteoblast motility. We performed transwell migration assays using MC3T3-E1 osteoblastic cells and found LPA to be a potent chemotactic agent. Quantitative time-lapse video analysis of osteoblast migration after wounds were introduced into cell monolayers indicated that LPA stimulated both migration velocity and the average migration distance per cell. LPA also elicited substantial changes in cell shape and actin cytoskeletal structure; lipid-treated cells contained fewer stress fibers and displayed long membrane processes that were enriched in F-actin. Quantitative RT-PCR analysis showed that MC3T3-E1 cells express all four known LPA-specific G protein-coupled receptors (LPA1-LPA4) with a relative mRNA abundance of LPA1 > LPA4 > LPA2 >> LPA3. LPA-induced changes in osteoblast motility and morphology were antagonized by both pertussis toxin and Ki16425, a subtype-specific blocker of LPA1 and LPA3 receptor function. Cell migration in many cell types is linked to changes in intracellular Ca2+. Ki16425 also inhibited LPA-induced Ca2+ signaling in a dose-dependent manner, suggesting a link between LPA-induced Ca2+ transients and osteoblast chemotaxis. Our data show that LPA stimulates MC3T3-E1 osteoblast motility via a mechanism that is linked primarily to the G protein-coupled receptor LPA1.

  3. A comparison of substrate dynamics in human CYP2E1 and CYP2A6

    SciTech Connect

    Harrelson, John P. . E-mail: harrelsonj@pacificu.edu; Henne, Kirk R.; Alonso, Darwin O.V.; Nelson, Sidney D.

    2007-01-26

    Considering the dynamic nature of CYPs, methods that reveal information about substrate and enzyme dynamics are necessary to generate predictive models. To compare substrate dynamics in CYP2E1 and CYP2A6, intramolecular isotope effect experiments were conducted, using deuterium labeled substrates: o-xylene, m-xylene, p-xylene, 2,6-dimethylnaphthalene, and 4,4'-dimethylbiphenyl. Competitive intermolecular experiments were also conducted using d{sub 0}- and d{sub 6}-labeled p-xylene. Both CYP2E1 and CYP2A6 displayed full isotope effect expression for o-xylene oxidation and almost complete suppression for dimethylbiphenyl. Interestingly (k {sub H}/k {sub D}){sub obs} for d{sub 3}-p-xylene oxidation ((k {sub H}/k {sub D}){sub obs} = 6.04 and (k {sub H}/k {sub D}){sub obs} = 5.53 for CYP2E1 and CYP2A6, respectively) was only slightly higher than (k {sub H}/k {sub D}){sub obs} for d{sub 3}-dimethylnaphthalene ((k {sub H}/k {sub D}){sub obs} = 5.50 and (k {sub H}/k {sub D}){sub obs} = 4.96, respectively). One explanation is that in some instances (k {sub H}/k {sub D}){sub obs} values are generated by the presence of two substrates-bound simultaneously to the CYP. Speculatively, if this explanation is valid, then intramolecular isotope effect experiments should be useful in the mechanistic investigation of P450 cooperativity.

  4. Cooperativity in CYP2E1 metabolism of acetaminophen and styrene mixtures.

    PubMed

    Hartman, Jessica H; Letzig, Lynda G; Roberts, Dean W; James, Laura P; Fifer, E Kim; Miller, Grover P

    2015-10-01

    Risk assessment for exposure to mixtures of drugs and pollutants relies heavily on in vitro characterization of their bioactivation and/or metabolism individually and extrapolation to mixtures assuming no interaction. Herein, we demonstrated that in vitro CYP2E1 metabolic activation of acetaminophen and styrene mixtures could not be explained through the Michaelis-Menten mechanism or any models relying on that premise. As a baseline for mixture studies with styrene, steady-state analysis of acetaminophen oxidation revealed a biphasic kinetic profile that was best described by negative cooperativity (Hill coefficient=0.72). The best-fit mechanism for this relationship involved two binding sites with differing affinities (Ks=830μM and Kss=32mM). Introduction of styrene inhibited that reaction less than predicted by simple competition and thus provided evidence for a cooperative mechanism within the mixture. Likewise, acetaminophen acted through a mixed-type inhibition mechanism to impact styrene epoxidation. In this case, acetaminophen competed with styrene for CYP2E1 (Ki=830μM and Ksi=180μM for catalytic and effector sites, respectively) and resulted in cooperative impacts on binding and catalysis. Based on modeling of in vivo clearance, cooperative interactions between acetaminophen and styrene resulted in profoundly increased styrene activation at low styrene exposure levels and therapeutic acetaminophen levels. Current Michaelis-Menten based toxicological models for mixtures such as styrene and acetaminophen would fail to detect this concentration-dependent relationship. Hence, future studies must assess the role of alternate CYP2E1 mechanisms in bioactivation of compounds to improve the accuracy of interpretations and predictions of toxicity. PMID:26225832

  5. GALILÉE-1: a validation and processing system for ENDF-6 and GND evaluations

    NASA Astrophysics Data System (ADS)

    Coste-Delclaux, Mireille; Jouanne, Cédric; Moreau, Frédéric; Mounier, Claude

    2016-03-01

    GALILÉE-1 is the new validation and processing system for evaluated data, developed at CEA. This system can handle evaluations stored either in the ENDF-6 format or in the new General Nuclear Data (GND) format. It consists of various components respectively dedicated to read/write the evaluated data whatever the format is, to diagnose inconsistencies in the evaluated data and to provide continuous-energy and multigroup data as well as probability tables for transport and depletion codes. All these components are written in C++ language and share the same objects. This paper describes the state of progress of the various parts of the system and gives some illustrations.

  6. The emission spectroscopy of the e1Π-a1Δ system of VN molecule

    NASA Astrophysics Data System (ADS)

    Fan, Qunchao; Hu, Shi; Sun, Weiguo; Wang, Qi

    2013-10-01

    Vanadium nitride is an important metallurgical additive and a good electrode material molecule. It is necessary to understand its intrinsic microstructure for better applications. Four groups of known experimental transition data of low-lying rotational quantum states and the analytical formula developed recently by Sun group are used to study the Ree, Pee, Rff and Pff-branch emission spectra of the (0, 0) band of the e1Π-a1Δ system of VN molecule respectively. The results not only reproduce all known experimental spectral lines accurately, but also generate valid data of the unknown spectral lines up to J = 80 that may not be available experimentally.

  7. Vinculin expression in MC3T3-E1 cells in response to mechanical stimulus

    PubMed Central

    Cora-Cruz, J.J.; Diffoot-Carlo, N.; Sundaram, P.A.

    2015-01-01

    Loading frequency is known to influence the expression of the focal adhesions of the adherent cells. A small cyclical tensile force was transmitted to mouse pre-osteoblast MC3T3-E1 cells through PDMS substrates of varying stiffness. Changes in cell behavior with respect to proliferation and characteristics of focal adhesions were quantified through immunofluorescence labeling of vinculin. Amount of inactive vinculin was higher on substrates subjected to cyclic stimulation when compared with the results of the static substrates, whereas the number and area of focal adhesion points underwent a reduction. Inactive vinculin appears as a cloud in the cytoplasm in the vicinity of the nucleus. PMID:26858974

  8. Calculation of radiative corrections to E1 matrix elements in the neutral alkali metals

    SciTech Connect

    Sapirstein, J.; Cheng, K.T.

    2005-02-01

    Radiative corrections to E1 matrix elements for ns-np transitions in the alkali-metal atoms lithium through francium are evaluated. They are found to be small for the lighter alkali metals but significantly larger for the heavier alkali metals, and in the case of cesium much larger than the experimental accuracy. The relation of the matrix element calculation to a recent decay rate calculation for hydrogenic ions is discussed, and application of the method to parity nonconservation in cesium is described.

  9. Bortezomib alleviates drug-induced liver injury by regulating CYP2E1 gene transcription

    PubMed Central

    PARK, WOO-JAE; KIM, SO-YEON; KIM, YE-RYUNG; PARK, JOO-WON

    2016-01-01

    Acute liver failure, i.e., the fatal deterioration of liver function, is the most common indication that emergency liver transplantation is necessary. Moreover, in the USA, drug-induced liver injury (DILI), including acetaminophen (APAP)-induced hepatotoxicity, is the main cause of acute liver failure. Matching a donor for liver transplantation is extremely difficult, and thus the development of a novel therapy for DILI is urgently needed. Following recent approval by the FDA of the proteasomal inhibitor bortezomib, its therapeutic effects on various human diseases, including solid and hematologic malignancies, have been validated. However, the specific action of proteasomal inhibition in cases of DILI had not been elucidated prior to this study. To examine the effects of proteasomal inhibition in DILI experimentally, male C56Bl/6 mice were injected with 1 mg bortezomib/kg before APAP treatment. Bortezomib not only alleviated APAP-induced hepatotoxicity in a time- and dose-dependent manner, it also alleviated CCl4- and thioacetamide-induced hepatotoxicity. We also noted that bortezomib significantly reduced cytochrome P450 2E1 (CYP2E1) expression and activity in the liver, which was accompanied by the induction of endoplasmic reticulum (ER) stress. In addition, bortezomib decreased hepatocyte nuclear factor-1α-induced promoter activation of CYP2E1 in Hep3B cells. By contrast, another proteasome inhibitor, MG132, did not cause ER stress and did not markedly affect CYP2E1 enzyme activity. Liver injury induced by APAP was aggravated by MG132, possibly via elevation of connexin 32 expression. This study suggests that proteasome inhibition has different effects in cases of DILI depending on the specific inhibitor being used. Furthermore, results from the mouse model indicated that bortezomib, but not MG132, was effective in alleviating DILI. ER stress induced by proteasome inhibition has previously been shown to exert various effects on DILI patients, and thus each

  10. Chikungunya virus with E1-A226V mutation causing two outbreaks in 2010, Guangdong, China

    PubMed Central

    2013-01-01

    Background CHIKV is a mosquito-borne emerging pathogen that has a major health impact in humans in tropical zones around the globe. A new variant of the virus, E1-A226V caused a large outbreak in the Indian Ocean islands and India from 2004–2007. CHIKV outbreak was initially reported in Dongguan region of Guangdong in 2010 in China, another smaller CHIKV outbreak was found in Yangjiang region of Guangdong two weeks later. The viral agent causing the two outbreaks was inferred to be the new E1-A226V variant and Yangjiang CHIKV might be introduced from Dongguan. To confirm the hypothesis and determine the origin of CHIKV causing the outbreaks, we described Yangjiang outbreak in this study, and the molecular characterization of CHIKV from Yangjiang and Dongguang outbreaks were analyzed. Results 27 clinical cases of CHIK fever were reported in outbreak in Yangjiang region. Sera sample from 12 clinical cases were collected from the outbreak, and nucleic acid and antibody tests for CHIKV were performed using Real-time RT-PCR and indirect immunofluorescence. Positive samples of Real-time RT-PCR were subjected to viral isolation. The results showed 3/12 samples positive for Real-time RT-PCR. 7/12 and 4/12 samples were positive for IgM and IgG against CHIKV respectively, two virus strains were isolated. Four viral genomes from Dongguan and Yangjiang were sequenced, characterized and phylogeneticly analyzed. Phylogenetic analysis revealed that the four seqeunced viruses had the closest relationship (99.4~99.6% identify) with the Singapore 2008 isolate belonging to the Indian ocean clade. A common mutation at the site of the E1-A226V was observed among four viruses. Four and three aa substitutions were detected in the CHIKV sequence from the Dongguan and Yangjiang outbreak strains respectively. Conclusion CHIKV with an E1-A226V mutation that originated from Southeast Asia isolates caused two outbreaks in China in 2010, and originated from two different infectious sources

  11. Adenovirus type 5 early region 4 is responsible for E1A-induced p53-independent apoptosis.

    PubMed Central

    Marcellus, R C; Teodoro, J G; Wu, T; Brough, D E; Ketner, G; Shore, G C; Branton, P E

    1996-01-01

    In the absence of E1B, the 289- and 243-residue E1A products of human adenovirus type 5 induce p53-dependent apoptosis. However, our group has shown recently that the 289-residue E1A protein is also able to induce apoptosis by a p53-independent mechanism (J. G. Teodoro, G. C. Shore, and P. E. Branton, Oncogene 11:467-474, 1995). Preliminary results suggested that p53-independent cell death required expression of one or more additional adenovirus early gene products. Here we show that both the E1B 19-kDa protein and cellular Bcl-2 inhibit or significantly delay p53-independent apoptosis. Neither early region E2 or E3 appeared to be necessary for such cell death. Analysis of a series of E1A mutants indicated that mutations in the transactivation domain and other regions of E1A correlated with E1A-mediated transactivation of E4 gene expression. Furthermore, p53-deficient human SAOS-2 cells infected with a mutant which expresses E1B but none of the E4 gene products remained viable for considerably longer times than those infected with wild-type adenovirus type 5. In addition, an adenovirus vector lacking both E1 and E4 was unable to induce DNA degradation and cell killing in E1A-expressing cell lines. These data showed that an E4 product is essential for E1A-induced p53-independent apoptosis. PMID:8709247

  12. The cognition-enhancing activity of E1R, a novel positive allosteric modulator of sigma-1 receptors

    PubMed Central

    Zvejniece, L; Vavers, E; Svalbe, B; Vilskersts, R; Domracheva, I; Vorona, M; Veinberg, G; Misane, I; Stonans, I; Kalvinsh, I; Dambrova, M

    2014-01-01

    Background and Purpose Here, we describe the in vitro and in vivo effects of (4R,5S)-2-(5-methyl-2-oxo-4-phenyl-pyrrolidin-1-yl)-acetamide (E1R), a novel positive allosteric modulator of sigma-1 receptors. Experimental Approach E1R was tested for sigma receptor binding activity in a [3H](+)-pentazocine assay, in bradykinin (BK)-induced intracellular Ca2+ concentration ([Ca2+]i) assays and in an electrically stimulated rat vas deferens model. E1R's effects on cognitive function were tested using passive avoidance (PA) and Y-maze tests in mice. A selective sigma-1 receptor antagonist (NE-100), was used to study the involvement of the sigma-1 receptor in the effects of E1R. The open-field test was used to detect the effects of E1R on locomotion. Key Results Pretreatment with E1R enhanced the selective sigma-1 receptor agonist PRE-084's stimulating effect during a model study employing electrically stimulated rat vasa deferentia and an assay measuring the BK-induced [Ca2+]i increase. Pretreatment with E1R facilitated PA retention in a dose-related manner. Furthermore, E1R alleviated the scopolamine-induced cognitive impairment during the PA and Y-maze tests in mice. The in vivo and in vitro effects of E1R were blocked by treatment with the selective sigma-1 receptor antagonist NE-100. E1R did not affect locomotor activity. Conclusion and Implications E1R is a novel 4,5-disubstituted derivative of piracetam that enhances cognition and demonstrates efficacy against scopolamine-induced cholinergic dysfunction in mice. These effects are attributed to its positive modulatory action on the sigma-1 receptor and this activity may be relevant when developing new drugs for treating cognitive symptoms related to neurodegenerative diseases. PMID:24490863

  13. Bax inhibitor 1 regulates ER-stress-induced ROS accumulation through the regulation of cytochrome P450 2E1.

    PubMed

    Kim, Hyung-Ryong; Lee, Geum-Hwa; Cho, Eun Yi; Chae, Soo-Wan; Ahn, Taeho; Chae, Han-Jung

    2009-04-15

    This study investigated the molecular mechanism by which Bax inhibitor 1 (BI1) abrogates the accumulation of reactive oxygen species (ROS) in the endoplasmic reticulum (ER). Electron uncoupling between NADPH-dependent cytochrome P450 reductase (NPR) and cytochrome P450 2E1 (P450 2E1) is a major source of ROS on the ER membrane. ER stress produced ROS accumulation and lipid peroxidation of the ER membrane, but BI1 reduced this accumulation. Under ER stress, expression of P450 2E1 in control cells was upregulated more than in BI1-overexpressing cells. In control cells, inhibiting P450 2E1 through chemical or siRNA approaches suppressed ROS accumulation, ER membrane lipid peroxidation and the resultant cell death after ER stress. However, it had little effect in BI1-overexpressing cells. In addition, BI1 knock down also increased ROS accumulation and expression of P450 2E1. In a reconstituted phospholipid membrane containing purified BI1, NPR and P450 2E1, BI1 dose-dependently decreased the production of ROS. BI1 bound to NPR with higher affinity than P450 2E1. Furthermore, BI1 overexpression reduced the interaction of NPR and P450 2E1, and decreased the catalytic activity of P450 2E1, suggesting that the flow of electrons from NPR to P450 2E1 can be modulated by BI1. In summary, BI1 reduces the accumulation of ROS and the resultant cell death through regulating P450 2E1. PMID:19339548

  14. The Human Adenovirus Type 5 E4orf6/E1B55K E3 Ubiquitin Ligase Complex Can Mimic E1A Effects on E2F.

    PubMed

    Dallaire, Frédéric; Schreiner, Sabrina; Blair, G Eric; Dobner, Thomas; Branton, Philip E; Blanchette, Paola

    2016-01-01

    The human adenovirus E4orf6/E1B55K E3 ubiquitin ligase is well known to promote viral replication by degrading an increasing number of cellular proteins that inhibit the efficient production of viral progeny. We report here a new function of the adenovirus 5 (Ad5) viral ligase complex that, although at lower levels, mimics effects of E1A products on E2F transcription factors. When expressed in the absence of E1A, the E4orf6 protein in complex with E1B55K binds E2F, disrupts E2F/retinoblastoma protein (Rb) complexes, and induces hyperphosphorylation of Rb, leading to induction of viral and cellular DNA synthesis as well as stimulation of early and late viral gene expression and production of viral progeny of E1/E3-defective adenovirus vectors. These new and previously undescribed functions of the E4orf6/E1B55K E3 ubiquitin ligase could play an important role in promoting the replication of wild-type viruses. IMPORTANCE During the course of work on the adenovirus E3 ubiquitin ligase formed by the viral E4orf6 and E1B55K proteins, we found, very surprisingly, that expression of these species was sufficient to permit low levels of replication of an adenovirus vector lacking E1A, the central regulator of infection. E1A products uncouple E2F transcription factors from Rb repression complexes, thus stimulating viral gene expression and cell and viral DNA synthesis. We found that the E4orf6/E1B55K ligase mimics these functions. This finding is of significance because it represents an entirely new function for the ligase in regulating adenovirus replication. PMID:27303679

  15. The Human Adenovirus Type 5 E4orf6/E1B55K E3 Ubiquitin Ligase Complex Can Mimic E1A Effects on E2F

    PubMed Central

    Dallaire, Frédéric; Schreiner, Sabrina; Blair, G. Eric; Dobner, Thomas; Branton, Philip E.

    2015-01-01

    ABSTRACT The human adenovirus E4orf6/E1B55K E3 ubiquitin ligase is well known to promote viral replication by degrading an increasing number of cellular proteins that inhibit the efficient production of viral progeny. We report here a new function of the adenovirus 5 (Ad5) viral ligase complex that, although at lower levels, mimics effects of E1A products on E2F transcription factors. When expressed in the absence of E1A, the E4orf6 protein in complex with E1B55K binds E2F, disrupts E2F/retinoblastoma protein (Rb) complexes, and induces hyperphosphorylation of Rb, leading to induction of viral and cellular DNA synthesis as well as stimulation of early and late viral gene expression and production of viral progeny of E1/E3-defective adenovirus vectors. These new and previously undescribed functions of the E4orf6/E1B55K E3 ubiquitin ligase could play an important role in promoting the replication of wild-type viruses. IMPORTANCE During the course of work on the adenovirus E3 ubiquitin ligase formed by the viral E4orf6 and E1B55K proteins, we found, very surprisingly, that expression of these species was sufficient to permit low levels of replication of an adenovirus vector lacking E1A, the central regulator of infection. E1A products uncouple E2F transcription factors from Rb repression complexes, thus stimulating viral gene expression and cell and viral DNA synthesis. We found that the E4orf6/E1B55K ligase mimics these functions. This finding is of significance because it represents an entirely new function for the ligase in regulating adenovirus replication. PMID:27303679

  16. First Selenodetic Result from Chang'E-1 Lunar Orbiter Mission

    NASA Astrophysics Data System (ADS)

    Ping, Jinsong; Huang, Qian; Yan, Jianguo; Shi, Xian; Wang, Guangli; Sha, Kai; Tang, Geshi; Chen, Ming; Cao, Jianfeng

    The first Chinese lunar orbiter, Chang'E-1, was sent to the circle polar orbit of the moon on Nov. 5. During the first month mission checkout period and the following normal mission period, it has been tracked using Range and RR and VLBI method. The tracking data may contribute to both of the OD and the POD of the mission. At the same time, the laser altimeter has been operated to measure the topography of the moon with very high accuracy and spatial resolution. Using the domestic tracking data, POD of Chang'E-1 mission was carried out. It is expected to merge the tracking data with other missions so as to improve the lunar global gravity field. By combining the POD data and attitude information as well as other necessary telemetry messages, the topography of the moon was renewed. Based on the renewed lunar DEM information and the lunar gravity field, the lunar crastial sphere and the Moho surface have been estimated using a downward continuation analyzing method. It may contribute to the selenodetic researches by combining with the historic data.

  17. Poly(L-lactide) crystallization topography directs MC3T3-E1 cells response.

    PubMed

    Li, Wenqiang; Lu, Lu; Jiao, Yanpeng; Zhang, Chaowen; Zhou, Changren

    2016-09-01

    Biomaterial surface topography significantly influences cellular form and function. Using poly(L-lactic acid) films with normal spherulites, banded spherulites, and amorphous surfaces as model substrates, we conducted a systematic assessment of the role for polymer crystallization induced surface morphologies on cell growth and contact guidance. Microscopy and image analysis showed that the MC3T3-E1 cells spread out in a random fashion on the amorphous substrate. At 24 h post-seeding, MC3T3-E1 cells on both types of spherulite surfaces were elongated and aligned along the spherulite radius direction. For the banded spherulite surface with radial stripes and coupling annular grooves, the cell orientation and cell nuclear localization were related to the grooves structure. With increasing time, this orientation preference was weaker. These results demonstrate that the patterning of polymer crystallization structure provide important signals for guiding cells to exhibit characteristic orientation and morphology especially in the early stages of regeneration. PMID:27376548

  18. Collective vector method for calculation of E1 moments in atomic transition arrays

    SciTech Connect

    Bloom, S.D.; Goldberg, A.

    1985-10-01

    The CV (collective vector) method for calculating E1 moments for a transition array is described and applied in two cases, herein denoted Z26A and Z26B, pertaining to two different configurations of iron VI. The basic idea of the method is to create a CV from each of the parent (''initial state'') state-vectors of the transition array by application of the E1 operator. The moments of each of these CV's, referred to the parent energy, are then the rigorous moments for that parent, requiring no state decomposition of the manifold of daughter state-vectors. Since, in cases of practical interest, the daughter manifold can be orders of magnitude larger in size than the parent manifold, this makes possible the calculation of many moments higher than the second in situations hitherto unattainable via standard methods. The combination of the moments of all the parents, with proper statistical weighting, then yields the transition array moments from which the transition strength distribution can be derived by various procedures. We describe two of these procedures: (1) The well-known GC (Gram-Charlier) expansion in terms of Hermite polynomials, (2) The Lanczos algorithm or Stieltjes imaging method, also called herein the delta expansion. Application is made in the cases of Z26A (50 lines) and Z26B (5523 lines) and the relative merits and shortcomings of the two procedures are discussed. 10 refs., 15 figs., 2 tabs.

  19. Construction of an adenovirus type 7a E1A- vector.

    PubMed Central

    Abrahamsen, K; Kong, H L; Mastrangeli, A; Brough, D; Lizonova, A; Crystal, R G; Falck-Pedersen, E

    1997-01-01

    A strategy for constructing replication-defective adenovirus vectors from non-subgroup C viruses has been successfully demonstrated with adenovirus type 7 strain a (Ad7a) as the prototype. An E1A-deleted Ad7a reporter virus expressing the chloramphenicol acetyltransferase (CAT) gene from the cytomegalovirus promoter enhancer was constructed with DNA fragments isolated from Ad7a, an Ad7a recombination reporter plasmid, and the 293 cell line. The Ad7a-CAT virus particle transduces A549 cells as efficiently as Ad5-based vectors. Intravenous infections in a murine model indicate that the Ad7a-CAT virus infects a variety of tissues, with maximal levels of CAT gene expression found in the liver. The duration of Ad7a-CAT transgene expression in the liver was maximally maintained 2 weeks postinfection, with a decline to baseline activity by the week 4 postinfection. Ad7a-CAT represents the first example of a non-subgroup C E1A- adenovirus gene transfer vector. PMID:9343264

  20. Active sites of two orthologous cytochromes P450 2E1: Differences revealed by spectroscopic methods

    SciTech Connect

    Anzenbacherova, Eva; Hudecek, Jiri; Murgida, Daniel; Hildebrandt, Peter; Marchal, Stephane; Lange, Reinhard; Anzenbacher, Pavel . E-mail: anzen@tunw.upol.cz

    2005-12-09

    Cytochromes P450 2E1 of human and minipig origin were examined by absorption spectroscopy under high hydrostatic pressure and by resonance Raman spectroscopy. Human enzyme tends to denature to the P420 form more easily than the minipig form; moreover, the apparent compressibility of the heme active site (as judged from a redshift of the absorption maximum with pressure) is greater than that of the minipig counterpart. Relative compactness of the minipig enzyme is also seen in the Raman spectra, where the presence of planar heme conformation was inferred from band positions characteristic of the low-spin heme with high degree of symmetry. In this respect, the CYP2E1 seems to be another example of P450 conformational heterogeneity as shown, e.g., by Davydov et al. for CYP3A4 [Biochem. Biophys. Res. Commun. 312 (2003) 121-130]. The results indicate that the flexibility of the CYP active site is likely one of its basic structural characteristics.

  1. CD36 is a co-receptor for hepatitis C virus E1 protein attachment

    PubMed Central

    Cheng, Jun-Jun; Li, Jian-Rui; Huang, Meng-Hao; Ma, Lin-Lin; Wu, Zhou-Yi; Jiang, Chen-Chen; Li, Wen-Jing; Li, Yu-Huan; Han, Yan-Xing; Li, Hu; Chen, Jin-Hua; Wang, Yan-Xiang; Song, Dan-Qing; Peng, Zong-Gen; Jiang, Jian-Dong

    2016-01-01

    The cluster of differentiation 36 (CD36) is a membrane protein related to lipid metabolism. We show that HCV infection in vitro increased CD36 expression in either surface or soluble form. HCV attachment was facilitated through a direct interaction between CD36 and HCV E1 protein, causing enhanced entry and replication. The HCV co-receptor effect of CD36 was independent of that of SR-BI. CD36 monoclonal antibodies neutralized the effect of CD36 and reduced HCV replication. CD36 inhibitor sulfo-N-succinimidyl oleate (SSO), which directly bound CD36 but not SR-BI, significantly interrupted HCV entry, and therefore inhibited HCV replication. SSO’s antiviral effect was seen only in HCV but not in other viruses. SSO in combination with known anti-HCV drugs showed additional inhibition against HCV. SSO was considerably safe in mice. Conclusively, CD36 interacts with HCV E1 and might be a co-receptor specific for HCV entry; thus, CD36 could be a potential drug target against HCV. PMID:26898231

  2. A cascading activity-based probe sequentially targets E1-E2-E3 ubiquitin enzymes.

    PubMed

    Mulder, Monique P C; Witting, Katharina; Berlin, Ilana; Pruneda, Jonathan N; Wu, Kuen-Phon; Chang, Jer-Gung; Merkx, Remco; Bialas, Johanna; Groettrup, Marcus; Vertegaal, Alfred C O; Schulman, Brenda A; Komander, David; Neefjes, Jacques; El Oualid, Farid; Ovaa, Huib

    2016-07-01

    Post-translational modifications of proteins with ubiquitin (Ub) and ubiquitin-like modifiers (Ubls), orchestrated by a cascade of specialized E1, E2 and E3 enzymes, control a wide range of cellular processes. To monitor catalysis along these complex reaction pathways, we developed a cascading activity-based probe, UbDha. Similarly to the native Ub, upon ATP-dependent activation by the E1, UbDha can travel downstream to the E2 (and subsequently E3) enzymes through sequential trans-thioesterifications. Unlike the native Ub, at each step along the cascade, UbDha has the option to react irreversibly with active site cysteine residues of target enzymes, thus enabling their detection. We show that our cascading probe 'hops' and 'traps' catalytically active Ub-modifying enzymes (but not their substrates) by a mechanism diversifiable to Ubls. Our founder methodology, amenable to structural studies, proteome-wide profiling and monitoring of enzymatic activity in living cells, presents novel and versatile tools to interrogate Ub and Ubl cascades. PMID:27182664

  3. Buserelin acetate microparticle dispersion effects drug release and plasma E(1) levels.

    PubMed

    Usami, Makiko; Misawa, Kazumasa; Yagi, Naomi; Sekikawa, Hitoshi; Nabeshima, Toshitaka

    2007-07-18

    We investigated the effect of different dispersion methods on release behavior and efficacy onset following microparticle administration of buserelin acetate (BA) sustained-release injection. In this in vitro release study, the initial dispersion of BA increased with increased stirring speed (p<0.01). Stability of BA was studied over 7 days after BA release. The initial BA release rate was higher (p<0.01) after a 1-min vibration dispersion method (VDM) using a test tube mixer (2000 rpm) compared with the standard dispersion method (SDM) by hand. Without shaking, powder aggregation was observed, and BA release was lower than in either the SDM or VDM methods. In this study using 4-week-old Sprague-Dawley female rats, the initial plasma estrone (E(1)) concentrations were lower (p<0.05) in the VDM method than in the SDM method. Observations by optical microscope and scanning microscope showed no change in microparticle shape or distribution of size induced by SDM, VDM or the ultrasonication dispersion method. These results suggest that different dispersion methods do not change the shape and distribution of microparticle size, but clearly change the BA release rate and the transition in plasma E(1) concentrations that can affect drug efficacy. PMID:17398044

  4. Hepatitis C Virus E1 and E2 Proteins Used as Separate Immunogens Induce Neutralizing Antibodies with Additive Properties

    PubMed Central

    Beaumont, Elodie; Roch, Emmanuelle; Chopin, Lucie; Roingeard, Philippe

    2016-01-01

    Various strategies involving the use of hepatitis C virus (HCV) E1 and E2 envelope glycoproteins as immunogens have been developed for prophylactic vaccination against HCV. However, the ideal mode of processing and presenting these immunogens for effective vaccination has yet to be determined. We used our recently described vaccine candidate based on full-length HCV E1 or E2 glycoproteins fused to the heterologous hepatitis B virus S envelope protein to compare the use of the E1 and E2 proteins as separate immunogens with their use as the E1E2 heterodimer, in terms of immunogenetic potential and the capacity to induce neutralizing antibodies. The specific anti-E1 and anti-E2 antibody responses induced in animals immunized with vaccine particles harboring the heterodimer were profoundly impaired with respect to those in animals immunized with particles harboring E1 and E2 separately. Moreover, the anti-E1 and anti-E2 antibodies had additive neutralizing properties that increase the cross-neutralization of heterologous strains of various HCV genotypes, highlighting the importance of including both E1 and E2 in the vaccine for an effective vaccination strategy. Our study has important implications for the optimization of HCV vaccination strategies based on HCV envelope proteins, regardless of the platform used to present these proteins to the immune system. PMID:26966906

  5. 26 CFR 1.1033(e)-1 - Sale or exchange of livestock solely on account of drought.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... of drought. 1.1033(e)-1 Section 1.1033(e)-1 Internal Revenue INTERNAL REVENUE SERVICE, DEPARTMENT OF... Sale or exchange of livestock solely on account of drought. (a) The sale or exchange of livestock... if the sale or exchange of such livestock by the taxpayer is solely on account of drought....

  6. Partition of E1A proteins between soluble and structural fractions of adenovirus-infected and -transformed cells.

    PubMed Central

    Chatterjee, P K; Flint, S J

    1986-01-01

    The partition of E1A proteins between soluble and structural framework fractions of human cells infected or transformed by subgroup C adenoviruses was investigated by using gentle cell fractionation conditions. A polyclonal antibody raised against a trpE-E1A fusion protein (K.R. Spindler, D.S.E. Rosser, and A. J. Berk, J. Virol. 132-141, 1984) synthesized in Escherichia coli was used to measure the steady-state levels of E1A proteins recovered in the various fractions by immunoblotting. The relative concentration of E1A proteins recovered in the soluble fraction of adenovirus type 2-infected cells was at least fivefold greater than the relative concentration in the corresponding fraction of transformed 293 cells. The observed distribution of E1A proteins was not altered by the sulfhydryl-blocking reagent N-ethylmaleimide. E1A proteins were recovered in nuclear matrix, chromatin, and cytoskeleton fractions after further fractionation of the structural framework fraction. However, the E1A protein species that could be identified by one-dimensional gel electrophoresis were not uniformly distributed among the subcellular fractions examined. The results obtained when fractionation was performed in the presence of the oxidation catalysts Cu2+ or (ortho-phenanthroline)2 Cu2+ indicate that E1A proteins can be efficiently cross-linked, via disulfide bonds, to the structural framework of both adenovirus-infected and adenovirus-transformed cells. Images PMID:3023654

  7. Cytochrome P4502E1 inhibitor, chlormethiazole, decreases lipopolysaccharide-induced inflammation in rat Kupffer cells with ethanol treatment

    Technology Transfer Automated Retrieval System (TEKTRAN)

    To investigate the role of Cytochrome P4502E1 in sensitizing Kupffer cells to lipopolysaccharide (LPS)-mediated inflammation after ethanol induction. Sprague-Dawley rats were fed a liquid ethanol diet, control diet or ethanol diet supplemented with CYP2E1 inhibitor, chlormethiazole (CMZ), for 4'week...

  8. RORα switches transcriptional mode of ERRγ that results in transcriptional repression of CYP2E1 under ethanol-exposure

    PubMed Central

    Han, Yong-Hyun; Kim, Don-Kyu; Na, Tae-Young; Ka, Na-Lee; Choi, Hueng-Sik; Lee, Mi-Ock

    2016-01-01

    Increased cytochrome P450 2E1 (CYP2E1) expression is the main cause of oxidative stress, which exacerbates alcoholic liver diseases (ALDs). Estrogen-related receptor gamma (ERRγ) induces CYP2E1 expression and contributes to enhancing alcohol-induced liver injury. Retinoic acid-related orphan receptor alpha (RORα) has antioxidative functions; however, potential cross-talk between ERRγ and RORα in the regulation of CYP2E1 has not been studied. We report that RORα suppressed ERRγ-mediated CYP2E1 expression. A physical interaction of RORα with ERRγ at the ERRγ−response element in the CYP2E1 promoter was critical in this suppression. At this site, coregulator recruitment of ERRγ was switched from coactivator p300 to the nuclear receptor corepressor 1 in the presence of RORα. Cross-talk between ERRγ and RORα was demonstrated in vivo, in that administration of JC1–40, a RORα activator, significantly decreased both CYP2E1 expression and the signs of liver injury in ethanol-fed mice, and this was accompanied by coregulator switching. Thus, this non-classical RORα pathway switched the transcriptional mode of ERRγ, leading to repression of alcohol-induced CYP2E1 expression, and this finding may provide a new therapeutic strategy against ALDs. PMID:26464440

  9. 26 CFR 1.72(e)-1T - Treatment of distributions where substantially all contributions are employee contributions...

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ... 26 Internal Revenue 2 2014-04-01 2014-04-01 false Treatment of distributions where substantially all contributions are employee contributions (temporary). 1.72(e)-1T Section 1.72(e)-1T Internal Revenue INTERNAL REVENUE SERVICE, DEPARTMENT OF THE TREASURY (CONTINUED) INCOME TAX (CONTINUED) INCOME TAXES (CONTINUED) Items Specifically Included...

  10. Immunoelectron microscopic localization of the ubiquitin-activating enzyme E1 in HepG2 cells.

    PubMed Central

    Schwartz, A L; Trausch, J S; Ciechanover, A; Slot, J W; Geuze, H

    1992-01-01

    As the first enzyme in the ubiquitin system the ubiquitin-activating enzyme E1 plays a pivotal role in all pathways of protein ubiquitination. In an effort to learn more about the cell biology of this pathway, we have purified the 110-kDa enzyme to homogeneity and generated a panel of distinct monoclonal antibodies to it. Using quantitative electron microscopic immunolocalization with these anti-E1 monoclonal antibodies, we find that E1 is abundant both within the cytoplasm and nucleus. Within the cytoplasm, E1 was found throughout the cytoplasmic volume as well as enriched along the cytoplasmic face of the rough endoplasmic reticulum and associated with the dense material along the desmosomal junctions. E1 was also found associated with the cytoplasmic surface of endosomal/lysosomal vacuoles. Interestingly, E1 was also found within the mitochondria. The lumen of rough endoplasmic reticulum, Golgi complex, endosomes, and lysosomes were negative. The specific localization of E1 to distinct subcellular organelles suggests that E1 may play multiple physiological roles within the cell. Images PMID:1376922

  11. Functional analysis of the C-terminal region of human adenovirus E1A reveals a misidentified nuclear localization signal

    SciTech Connect

    Cohen, Michael J.; King, Cason R.; Dikeakos, Jimmy D.; Mymryk, Joe S.

    2014-11-15

    The immortalizing function of the human adenovirus 5 E1A oncoprotein requires efficient localization to the nucleus. In 1987, a consensus monopartite nuclear localization sequence (NLS) was identified at the C-terminus of E1A. Since that time, various experiments have suggested that other regions of E1A influence nuclear import. In addition, a novel bipartite NLS was recently predicted at the C-terminal region of E1A in silico. In this study, we used immunofluorescence microscopy and co-immunoprecipitation analysis with importin-α to verify that full nuclear localization of E1A requires the well characterized NLS spanning residues 285–289, as well as a second basic patch situated between residues 258 and 263 ({sup 258}RVGGRRQAVECIEDLLNEPGQPLDLSCKRPRP{sup 289}). Thus, the originally described NLS located at the C-terminus of E1A is actually a bipartite signal, which had been misidentified in the existing literature as a monopartite signal, altering our understanding of one of the oldest documented NLSs. - Highlights: • Human adenovirus E1A is localized to the nucleus. • The C-terminus of E1A contains a bipartite nuclear localization signal (NLS). • This signal was previously misidentified to be a monopartite NLS. • Key basic amino acid residues within this sequence are highly conserved.

  12. CYP2E1 impairs GLUT4 gene expression and function: NRF2 as a possible mediator.

    PubMed

    Armoni, M; Harel, C; Ramdas, M; Karnieli, E

    2014-06-01

    Impaired GLUT4 function/expression in insulin target tissues is well-documented in diabetes and obesity. Cytochrome P450 isoform 2E1 (CYP2E1) induces oxidative stress, leading to impaired insulin action. CYP2E1 knockout mice are protected against high fat diet-induced insulin resistance and obesity; however the molecular mechanisms are still unclear. We examined whether CYP2E1 impairs GLUT4 gene expression and function in adipose and muscle cells. CYP2E1 overexpression in skeletal muscle-derived L6 cells inhibited insulin-stimulated Glut4 translocation and 2-deoxyglucose uptake, with the latter inhibition being blocked by vitamin E. CYP2E1 overexpression in L6 and primary rat adipose (PRA) cells suppressed GLUT4 gene expression at promoter and mRNA levels, whereas CYP2E1 silencing had opposite effects. In PRA, CYP2E1-induced suppression of GLUT4 expression was blocked by chlormethiazole (CYP2E1-specific inhibitor) and the antioxidants vitamin E and N-acetyl-l-cysteine. CYP2E1 effect was mediated by the transcription factor NF-E2-related factor 2 (NRF2), as evident from its complete reversal by a coexpressed dominant-negative, but not wild-type NRF2. GLUT4 transcription was suppressed by NRF2 overexpression, and enhanced by NRF2 silencing. Promoter and ChIP analysis showed a direct and specific binding of NRF2 to a 58-326 GLUT4 promoter region that was required to maintain CYP2E1 suppression; this binding was enhanced by CYP2E1 overexpression. We suggest a mechanism for CYP2E1 action that involves: a) suppression of GLUT4 gene expression that is mediated by NRF2; b) impairment of insulin-stimulated Glut4 translocation and function. CYP2E1 and NRF2 are introduced as negative regulators of GLUT4 expression and function in insulin-sensitive cells. PMID:24500986

  13. E1A Blocks Hyperphosphorylation of p130 and p107 without Affecting the Phosphorylation Status of the Retinoblastoma Protein

    PubMed Central

    Parreño, Matilde; Garriga, Judit; Limón, Ana; Mayol, Xavier; Beck, George R.; Moran, Elizabeth; Graña, Xavier

    2000-01-01

    The phosphorylation status of the pRB family of growth suppressor proteins is regulated in a cell cycle entry-, progression-, and exit-dependent manner in normal cells. We have shown previously that p130, a member of this family, exhibits patterns of phosphorylated forms associated with various cell growth and differentiation stages. However, human 293 cells, which are transformed cells that express the adenoviral oncoproteins E1A and E1B, exhibit an abnormal pattern of p130 phosphorylated forms. Here we report that, unlike pRB, the phosphorylation status of both p130 and p107 is not modulated during the cell cycle in 293 cells as it is in other cells. Conditional overexpression of individual G1/S cyclins in 293 cells does not alter the phosphorylation status of p130, suggesting that the expression of E1A and/or E1B blocks hyperphosphorylation of p130. In agreement with these observations, transient cotransfection of vectors expressing E1A 12S, but not E1B, in combination with pocket proteins into U-2 OS cells blocks hyperphosphorylation of both p130 and p107. However, the phosphorylation status of pRB is not altered by cotransfection of E1A 12S vectors. Moreover, MC3T3-E1 preosteoblasts stably expressing E1A 12S also exhibit a block in hyperphosphorylation of endogenous p130 and p107. Direct binding of E1A to p130 and p107 is not required for the phosphorylation block since E1A 12S mutants defective in binding to the pRB family also block hyperphosphorylation of p130 and p107. Our data reported here identify a novel function of E1A, which affects p130 and p107 but does not affect pRB. Since E1A does not bind the hyperphosphorylated forms of p130, this function of E1A might prevent the existence of “free” hyperphosphorylated p130, which could act as a CDK inhibitor. PMID:10708433

  14. Phylogeography of E1b1b1b-M81 haplogroup and analysis of its subclades in Morocco.

    PubMed

    Reguig, Ahmed; Harich, Nourdin; Barakat, Abdelhamid; Rouba, Hassan

    2014-01-01

    In this study we analyzed 295 unrelated Berber-speaking men from northern, central, and southern Morocco to characterize frequency of the E1b1b1b-M81 haplogroup and to refine the phylogeny of its subclades: E1b1b1b1-M107, E1b1b1b2-M183, and E1b1b1b2a-M165. For this purpose, we typed four biallelic polymorphisms: M81, M107, M183, and M165. A large majority of the Berber-speaking male lineages belonged to the Y-chromosomal E1b1b1b-M81 haplogroup. The frequency ranged from 79.1% to 98.5% in all localities sampled. E1b1b1b2-M183 was the most dominant subclade in our samples, ranging from 65.1% to 83.1%. In contrast, the E1b1b1b1-M107 and E1b1b1b2a-M165 subclades were not found in our samples. Our results suggest a predominance of the E1b1b1b-M81 haplogroup among Moroccan Berber-speaking males with a decreasing gradient from south to north. The most prevalent subclade in this haplogroup was E1b1b1b2-M183, for which diffferences among these three groups were statistically significant between central and southern groups. PMID:25397701

  15. MicroRNA-16 Modulates HuR Regulation of Cyclin E1 in Breast Cancer Cells

    PubMed Central

    Guo, Xun; Connick, Melanie C.; Vanderhoof, Jennifer; Ishak, Mohammad-Ali; Hartley, Rebecca S.

    2015-01-01

    RNA binding protein (RBPs) and microRNAs (miRNAs or miRs) are post-transcriptional regulators of gene expression that are implicated in development of cancers. Although their individual roles have been studied, the crosstalk between RBPs and miRNAs is under intense investigation. Here, we show that in breast cancer cells, cyclin E1 upregulation by the RBP HuR is through specific binding to regions in the cyclin E1 mRNA 3' untranslated region (3'UTR) containing U-rich elements. Similarly, miR-16 represses cyclin E1, dependent on its cognate binding sites in the cyclin E1 3'UTR. Evidence in the literature indicates that HuR can regulate miRNA expression and recruit or dissociate RNA-induced silencing complexes (RISC). Despite this, miR-16 and HuR do not affect the other’s expression level or binding to the cyclin E1 3'UTR. While HuR overexpression partially blocks miR-16 repression of a reporter mRNA containing the cyclin E1 3'UTR, it does not block miR-16 repression of endogenous cyclin E1 mRNA. In contrast, miR-16 blocks HuR-mediated upregulation of cyclin E1. Overall our results suggest that miR-16 can override HuR upregulation of cyclin E1 without affecting HuR expression or association with the cyclin E1 mRNA. PMID:25830480

  16. Inhibition of Human Papillomavirus DNA Replication by an E1-Derived p80/UAF1-Binding Peptide

    PubMed Central

    Lehoux, Michaël; Fradet-Turcotte, Amélie; Lussier-Price, Mathieu; Omichinski, James G.

    2012-01-01

    The papillomavirus E1 helicase is recruited by E2 to the viral origin, where it assembles into a double hexamer that orchestrates replication of the viral genome. We previously identified the cellular WD40 repeat-containing protein p80/UAF1 as a novel interaction partner of E1 from anogenital human papillomavirus (HPV) types. p80 was found to interact with the first 40 residues of HPV type 31 (HPV31) E1, and amino acid substitutions within this domain abrogated the maintenance of the viral episome in keratinocytes. In this study, we report that these p80-binding substitutions reduce by 70% the ability of E1 to support transient viral DNA replication without affecting its interaction with E2 and assembly at the origin in vivo. Microscopy studies revealed that p80 is relocalized from the cytoplasm to discrete subnuclear foci by E1 and E2. Chromatin immunoprecipitation assays further revealed that p80 is recruited to the viral origin in an E1- and E2-dependent manner. Interestingly, overexpression of a 40-amino-acid-long p80-binding peptide, derived from HPV31 E1, was found to inhibit viral DNA replication by preventing the recruitment of endogenous p80 to the origin. Mutant peptides defective for p80 interaction were not inhibitory, demonstrating the specificity of this effect. Characterization of this E1 peptide by nuclear magnetic resonance (NMR) showed that it is intrinsically disordered in solution, while mapping studies indicated that the WD repeats of p80 are required for E1 interaction. These results provide additional evidence for the requirement for p80 in anogenital HPV DNA replication and highlight the potential of E1-p80 interaction as a novel antiviral target. PMID:22278251

  17. Inhibition of human papillomavirus DNA replication by an E1-derived p80/UAF1-binding peptide.

    PubMed

    Lehoux, Michaël; Fradet-Turcotte, Amélie; Lussier-Price, Mathieu; Omichinski, James G; Archambault, Jacques

    2012-04-01

    The papillomavirus E1 helicase is recruited by E2 to the viral origin, where it assembles into a double hexamer that orchestrates replication of the viral genome. We previously identified the cellular WD40 repeat-containing protein p80/UAF1 as a novel interaction partner of E1 from anogenital human papillomavirus (HPV) types. p80 was found to interact with the first 40 residues of HPV type 31 (HPV31) E1, and amino acid substitutions within this domain abrogated the maintenance of the viral episome in keratinocytes. In this study, we report that these p80-binding substitutions reduce by 70% the ability of E1 to support transient viral DNA replication without affecting its interaction with E2 and assembly at the origin in vivo. Microscopy studies revealed that p80 is relocalized from the cytoplasm to discrete subnuclear foci by E1 and E2. Chromatin immunoprecipitation assays further revealed that p80 is recruited to the viral origin in an E1- and E2-dependent manner. Interestingly, overexpression of a 40-amino-acid-long p80-binding peptide, derived from HPV31 E1, was found to inhibit viral DNA replication by preventing the recruitment of endogenous p80 to the origin. Mutant peptides defective for p80 interaction were not inhibitory, demonstrating the specificity of this effect. Characterization of this E1 peptide by nuclear magnetic resonance (NMR) showed that it is intrinsically disordered in solution, while mapping studies indicated that the WD repeats of p80 are required for E1 interaction. These results provide additional evidence for the requirement for p80 in anogenital HPV DNA replication and highlight the potential of E1-p80 interaction as a novel antiviral target. PMID:22278251

  18. Heterogeneity of adenovirus type 5 E1A proteins: multiple serine phosphorylations induce slow-migrating electrophoretic variants but do not affect E1A-induced transcriptional activation or transformation.

    PubMed Central

    Richter, J D; Slavicek, J M; Schneider, J F; Jones, N C

    1988-01-01

    The 289-amino-acid product encoded by the adenovirus E1A 13S mRNA has several pleiotropic activities, including transcriptional activation, transcriptional repression, and when acting in concert with certain oncogene products, cell transformation. In all cell types in which E1A has been introduced (except bacteria), E1A protein is extensively posttranslationally modified to yield several isoelectric and molecular weight variants. The most striking variant is one that has a retarded mobility, by about Mr = 2,000, in sodium dodecyl sulfate gels. We have investigated the nature of this modification and have assessed its importance for E1A activity. Phosphorylation is responsible for the altered mobility of E1A, since acid phosphatase treatment eliminates the higher apparent molecular weight products. By using several E1A deletion mutants, we show that at least two seryl residues, residing between residues 86 and 120 and 224 and 289, are the sites of phosphorylation and that each phosphorylation can independently induce the mobility shift. However, E1A mutants lacking these seryl residues transcriptionally activate the adenovirus E3 and E2A promoters and transform baby rat kidney cells to near wild-type levels. Images PMID:2835499

  19. Molecular basis of maple syrup urine disease: Novel mutations at the E1[alpha] locus that impair E1([alpha][sub 2][beta][sub 2]) assembly or decrease steady-state E1[alpha] mRNA levels of branched-chain [alpha]-keto acid dehydrogenase complex

    SciTech Connect

    Chuang, J.L.; Fisher, C.R.; Chuang, D.T.; Cox, R.P. )

    1994-08-01

    The authors report the occurrence of three novel mutations in the E1[alpha] (BCKDHA) locus of the branched-chain [alpha]-keto acid dehydrogenase (BCKAD) complex that cause maple syrup urine disease (MSUD). An 8-bp deletion in exon 7 is present in one allele of a compound-heterozygous patient (GM-649). A single C nucleotide insertion in exon 2 occurs in one allele of an intermediate-MSUD patient (Lo). The second allele of patient Lo carries an A-to-G transition in exon 9 of the E1[alpha] gene. This missense mutation changes Tyr-368 to Cys (Y368C) in the E1[alpha] subunit. Both the 8-bp deletion and the single C insertion generate a downstream nonsense codon. Both mutations appear to be associated with a low abundance of the mutant E1[alpha] mRNA, as determined by allele-specific oligonucleotide probing. Transfection studies strongly suggest that the Y368C substitution in the E1[alpha] subunit impairs its proper assembly with the normal E1[beta]. Unassembled as well as misassembled E1[alpha] and E1[beta] subunits are degraded in the cell. 32 refs., 8 figs.

  20. Combined System of Activated Sludge and Ozonation for the Treatment of Kraft E1 Effluent

    PubMed Central

    Assalin, Marcia Regina; dos Santos Almeida, Edna; Durán, Nelson

    2009-01-01

    The treatment of paper mill effluent for COD, TOC, total phenols and color removal was investigated using combined activated sludge-ozonation processes and single processes. The combined activated sludge-O3/pH 10 treatment was able to remove around 80% of COD, TOC and color from Kraft E1 effluent. For the total phenols, the efficiency removal was around 70%. The ozonation post treatment carried out at pH 8.3 also showed better results than the single process. The COD, TOC, color and total phenols removal efficiency obtained were 75.5, 59.1, 77 and 52.3%, respectively. The difference in the concentrations of free radical produced by activated sludge-O3/pH 10 and activated sludge-O3/pH 8.3 affected mainly the TOC and total phenol removal values. PMID:19440438

  1. Preoperative cervical dilatation by vaginal pessaries containing prostaglandin E1 analogue.

    PubMed

    Chen, J K; Elder, M G

    1983-09-01

    A double-blind trial comparing use of placebo with use of a single intravaginal pessary of 1 mg 16,16-dimethyl-trans-delta 2-prostaglandin E1 methyl ester, inserted approximately three hours before first-trimester surgical abortion, was carried out. There were 54 women each in the study and control groups. A spring gauge was used to record the force required to dilate the cervix. With the use of the prostaglandin analogue, operative time, blood loss, and the need for additional mechanical dilatation of the cervix were significantly reduced compared with the control group (P less than .0001). This reduction greatly facilitates the operation and should reduce the incidence of cervical trauma. PMID:6348613

  2. The use of laser altimetry data in Chang'E-1 precision orbit determination

    NASA Astrophysics Data System (ADS)

    Chang, Sheng-Qi; Huang, Yong; Li, Pei-Jia; Hu, Xiao-Gong; Fan, Min

    2016-09-01

    Accurate altimetric measurement not only can be applied to the calculation of a topography model but also can be used to improve the quality of the orbit reconstruction in the form of crossovers. Altimetry data from the Chang'E-1 (CE-1) laser altimeter are analyzed in this paper. The differences between the crossover constraint equation in the form of height discrepancies and in the form of minimum distances are mainly discussed. The results demonstrate that the crossover constraint equation in the form of minimum distances improves the CE-1 orbit precision. The overlap orbit performance has increased ∼ 30% compared to the orbit using only tracking data. External assessment using the topography model also shows orbit improvement. The results will be helpful for recomputing ephemeris and improving the CE-1 topography model.

  3. Ion selectivity of colicin E1: II. Permeability to organic cations.

    PubMed

    Bullock, J O; Kolen, E R; Shear, J L

    1992-05-01

    Channels formed by colicin E1 in planar lipid bilayers have large diameters and conduct both cations and anions. The rates at which ions are transported, however, are relatively slow, and the relative anion-to-cation selectivity is modulated over a wide range by the pH of the bathing solutions. We have examined the permeability of these channels to cationic probes having a variety of sizes, shapes, and charge distributions. All of the monovalent probes were found to be permeant, establishing a minimum diameter at the narrowest part of the pore of approximately 9 A. In contrast to this behavior, all of the polyvalent organic cations were shown to be impermeant. This simple exclusionary rule is interpreted as evidence that, when steric restrictions require partial dehydration of an ion, the structure of the channel is able to provide a substitute electrostatic environment for only one charged group at time. PMID:1380089

  4. Behavioural and autonomic induction of prostaglandin E-1 fever in squirrel monkeys.

    PubMed Central

    Crawshaw, L I; Stitt, J T

    1975-01-01

    1. Prostaglandins E1 (PGE1) was injected into the preoptic/anterior hypothalamic (PO/AH) area of the squirrel monkey. 2. Increases in rectal temperature (Tre) produced by PGE1 injections of 20 ng to 500 ng were dose-dependent. 3. When ambient temperature (Ta) was below the thermoneutral zone, increases in Tre were produced entirely by increases in metabolic rate. With Ta at the upper end of the thermoneutral zone, increases in Tre were produced by vasoconstriction in addition to lesser increases in metabolic rate. 4. During sessions of behavioural temperature regulation, PGE1 injections were followed by the selection of a higher Ta, increased skin temperature and subsequent increases in Tre. 5. PGE1 injections produce dose-dependent increases in Tre which are similar regardless of ambient temperature or whether behavioural or autonomic means are utilized to raise the heat content of the body. PMID:804544

  5. A human papilloma virus type 11 transcript encoding an E1--E4 protein.

    PubMed

    Nasseri, M; Hirochika, R; Broker, T R; Chow, L T

    1987-08-01

    The human papilloma virus (HPV) associated with a genital wart (condyloma acuminatum) was determined to be type 11. The majority of the viral DNA molecules were monomeric circles present in the cells at high copy number, as demonstrated by one- and two-dimensional agarose gell electrophoretic separation followed by Southern blot analysis. A cDNA library in phage lambda gt11 was constructed from poly(A)-selected mRNA recovered from the tissue. Recombinant clones corresponding to the most abundant 1.2-kb viral mRNA species detected by Northern blot hybridization and by electron microscopic analysis of R loops were isolated and their nucleotide sequence was determined. Comparison to the prototype HPV-11 DNA sequence revealed that this message consisted of two exons. The promotor-proximal exon spanned nucleotides 716 through 847 and the distal exon included nucleotides 3325 through 4390 or 4392. The mRNAs were alternatively polyadenylated after either of these latter two sites, in both cases following a G and preceding a U residue. Fourteen or sixteen bases upstream from the poly(A) was the hexanucleotide AGUAAA, which apparently serves as the signal for cleavage and polyadenylation of the nascent message. The splice donor and acceptor sites conformed to the usual /GU. . .AG/pattern. The exons joined open reading frame (ORF) E1, which contributed the initiation codon and four additional triplets, to ORF E4, which specified 85 amino acids to encode a protein of 10,022 Da. The cDNA also contained the ORFs E5a and E5b toward the 3' end. The complete sequence of the cDNA revealed three single-base changes from the prototype HPV-11, two resulting in altered amino acids in E4. Neither affects the coding potential of the overlapping E2 ORF. The function of the E1--E4 protein is unknown. PMID:2887066

  6. Communication between Thiamin Cofactors in the Escherichia coli Pyruvate Dehydrogenase Complex E1 Component Active Centers

    PubMed Central

    Nemeria, Natalia S.; Arjunan, Palaniappa; Chandrasekhar, Krishnamoorthy; Mossad, Madouna; Tittmann, Kai; Furey, William; Jordan, Frank

    2010-01-01

    Kinetic, spectroscopic, and structural analysis tested the hypothesis that a chain of residues connecting the 4′-aminopyrimidine N1′ atoms of thiamin diphosphates (ThDPs) in the two active centers of the Escherichia coli pyruvate dehydrogenase complex E1 component provides a signal transduction pathway. Substitution of the three acidic residues (Glu571, Glu235, and Glu237) and Arg606 resulted in impaired binding of the second ThDP, once the first active center was filled, suggesting a pathway for communication between the two ThDPs. 1) Steady-state kinetic and fluorescence quenching studies revealed that upon E571A, E235A, E237A, and R606A substitutions, ThDP binding in the second active center was affected. 2) Analysis of the kinetics of thiazolium C2 hydrogen/deuterium exchange of enzyme-bound ThDP suggests half-of-the-sites reactivity for the E1 component, with fast (activated site) and slow exchanging sites (dormant site). The E235A and E571A variants gave no evidence for the slow exchanging site, indicating that only one of two active sites is filled with ThDP. 3) Titration of the E235A and E237A variants with methyl acetylphosphonate monitored by circular dichroism suggested that only half of the active sites were filled with a covalent predecarboxylation intermediate analog. 4) Crystal structures of E235A and E571A in complex with ThDP revealed the structural basis for the spectroscopic and kinetic observations and showed that either substitution affects cofactor binding, despite the fact that Glu235 makes no direct contact with the cofactor. The role of the conserved Glu571 residue in both catalysis and cofactor orientation is revealed by the combined results for the first time. PMID:20106967

  7. Melatonin Suppresses Autophagy Induced by Clinostat in Preosteoblast MC3T3-E1 Cells

    PubMed Central

    Yoo, Yeong-Min; Han, Tae-Young; Kim, Han Sung

    2016-01-01

    Microgravity exposure can cause cardiovascular and immune disorders, muscle atrophy, osteoporosis, and loss of blood and plasma volume. A clinostat device is an effective ground-based tool for simulating microgravity. This study investigated how melatonin suppresses autophagy caused by simulated microgravity in preosteoblast MC3T3-E1 cells. In preosteoblast MC3T3-E1 cells, clinostat rotation induced a significant time-dependent increase in the levels of the autophagosomal marker microtubule-associated protein light chain (LC3), suggesting that autophagy is induced by clinostat rotation in these cells. Melatonin treatment (100, 200 nM) significantly attenuated the clinostat-induced increases in LC3 II protein, and immunofluorescence staining revealed decreased levels of both LC3 and lysosomal-associated membrane protein 2 (Lamp2), indicating a decrease in autophagosomes. The levels of phosphorylation of mammalian target of rapamycin (p-mTOR) (Ser2448), phosphorylation of extracellular signal-regulated kinase (p-ERK), and phosphorylation of serine-threonine protein kinase (p-Akt) (Ser473) were significantly reduced by clinostat rotation. However, their expression levels were significantly recovered by melatonin treatment. Also, expression of the Bcl-2, truncated Bid, Cu/Zn- superoxide dismutase (SOD), and Mn-SOD proteins were significantly increased by melatonin treatment, whereas levels of Bax and catalase were decreased. The endoplasmic reticulum (ER) stress marker GRP78/BiP, IRE1α, and p-PERK proteins were significantly reduced by melatonin treatment. Treatment with the competitive melatonin receptor antagonist luzindole blocked melatonin-induced decreases in LC3 II levels. These results demonstrate that melatonin suppresses clinostat-induced autophagy through increasing the phosphorylation of the ERK/Akt/mTOR proteins. Consequently, melatonin appears to be a potential therapeutic agent for regulating microgravity-related bone loss or osteoporosis. PMID:27070587

  8. Joint Tracking of Chang'E-1 with VLBI and USB

    NASA Astrophysics Data System (ADS)

    Hu, Xiaogong

    Chinese lunar exploration mission Chang'E-I made use of a Chinese Unified S-Band (USB) system and a network of four Very Long Baseline Interferometry (VLBI) antennas to meet the orbit determination/predication requirements of spacecraft tracking and scientific data analysis, which for the first time handled telemetry and control for a spacecraft at a distance of about 380,000 km. Chang'E-1 provided a perfect chance to quantify the contributions of VLBI to orbit determination/predication, and to test and evaluate the performance of the USB-VLBI joint system. We investigate in this paper the quality of the data and analyze the precision of orbit determination with different data arcs and data combinations during the 2-week journey from Earth to Chang'E-1's lunar mission orbit, using GEODYN II orbit determination software. The residuals of VLBI delay is about 3 ns (RMS, root-mean-squares), the residuals of VLBI delay-rate is about 0.6 ps/s, the residuals of ranging is about 1 2 m and Doppler is about 1 cm/s. Three flight phases are invistigated, namely 3 phasing orbits near Earth, the translunar trajectory and the lunar catputed orbits. We found including of VLBI data substantially improved orbit precision for short data arcs, therefore VLBI played an important role in assessing spacecraft manuvore performance. Given the differential nature of VLBI observables and their errors mostly from BBC's nonlinear phase-frequency responses, it appears for longer data arcs the VLBI contribution is relatively minor. We conclude that for Chang'E-I, the inclusion of VLBI data improved substantially the performance of orbit determination and prediction, even though the VLBI data acquisition and correlation imposes a major burden on data processing.

  9. Antibiotic resistance due to an unusual ColE1-type replicon plasmid in Aeromonas salmonicida.

    PubMed

    Vincent, Antony T; Emond-Rheault, Jean-Guillaume; Barbeau, Xavier; Attéré, Sabrina A; Frenette, Michel; Lagüe, Patrick; Charette, Steve J

    2016-06-01

    Aeromonas salmonicida subsp. salmonicida is a fish pathogen known to have a rich plasmidome. In the present study, we discovered an isolate of this bacterium bearing an additional unidentified small plasmid. After having sequenced the DNA of that isolate by next-generation sequencing, it appeared that the new small plasmid is a ColE1-type replicon plasmid, named here pAsa7. This plasmid bears a functional chloramphenicol-acetyltransferase-encoding gene (cat-pAsa7) previously unknown in A. salmonicida and responsible for resistance to chloramphenicol. A comparison of pAsa7 with pAsa2, the only known ColE1-type replicon plasmid usually found in A. salmonicida subsp. salmonicida, revealed that even if both plasmids share a high structural similarity, it is still unclear if pAsa7 is a derivative of pAsa2 since they showed several mutations at the nucleotide level. Transcriptomic analysis revealed that the cat-pAsa4 gene, another chloramphenicol-acetyltransferase-encoding gene, found on the large plasmid pAsa4, was significantly more transcribed than cat-pAsa7. This was correlated with a higher chloramphenicol resistance for isolates bearing pAsa4 compared with the one having pAsa7. Finally, a phylogenetic analysis showed that both CAT-pAsa4 and CAT-pAsa7 proteins were in different clusters. The clustering was supported by the identity of residues involved in the catalytic site. In addition, to give a better understanding of the large drug-resistance panel of A. salmonicida, this study reinforces the hypothesis that A. salmonicida subsp. salmonicida is a considerable reservoir for mobile genetic elements such as plasmids. PMID:27028891

  10. Human ubiquitin-activating enzyme, E1. Indication of potential nuclear and cytoplasmic subpopulations using epitope-tagged cDNA constructs.

    PubMed

    Handley-Gearhart, P M; Stephen, A G; Trausch-Azar, J S; Ciechanover, A; Schwartz, A L

    1994-12-30

    The ubiquitin-activating enzyme E1 catalyzes the first step in the ubiquitin conjugation pathway. Previously, we have cloned and sequenced the cDNA for human E1. Expression of the E1 cDNA in the ts20 cell line, which harbors a thermolabile E1, abrogated the phenotypic defects associated with this line. However, little is known of the cell biology of the E1 protein or the nature of the E1 doublet. Thus, we constructed epitope-tagged E1 cDNAs in which the HA monoclonal antibody epitope tag sequence (from influenza hemagglutinin and recognized by the 12CA5 monoclonal antibody) was fused to the amino terminus of E1. Because the amino-terminal amino acid sequence of E1 is unknown, three constructs were made in which the HA tag was placed at each of the first three ATGs in the open reading frame (HA-1E1, HA-2E1, and HA-3E1). Western analysis of HeLa cells transfected with the constructs revealed that HA-1E1 closely comigrated with the upper band of the E1 doublet, and HA-2E1 comigrated with the lower band of the E1 doublet; HA-3E1 appeared smaller than either of the E1 bands. Metabolic labeling with 32P and immunoprecipitation with anti-HA antibody revealed that only the HA-1E1 protein product is phosphorylated; polyclonal anti-E1 antibody showed that only the upper band of the endogenous E1 doublet is phosphorylated. Each of the constructs was able to rescue the mutant phenotype of the ts20 cell line. Immunofluorescence studies showed that HA-2E1 and HA-3E1 were distributed in the cytoplasm with both negative and positive nuclei. This pattern of distribution has also been observed when immunostaining with a monoclonal antibody to E1 (1C5). However, the staining pattern associated with a polyclonal anti-E1 antibody (JJJ) is characterized by positive staining cytoplasm and nuclei in all cells. The HA-1E1 construct exhibited apparently exclusive nuclear distribution in HeLa cells. The difference between the staining patterns of the polyclonal and monoclonal anti-E1

  11. The Transmembrane Domain of Hepatitis C Virus Glycoprotein E1 Is a Signal for Static Retention in the Endoplasmic Reticulum

    PubMed Central

    Cocquerel, Laurence; Duvet, Sandrine; Meunier, Jean-Christophe; Pillez, André; Cacan, René; Wychowski, Czeslaw; Dubuisson, Jean

    1999-01-01

    Hepatitis C virus (HCV) glycoproteins E1 and E2 assemble to form a noncovalent heterodimer which, in the cell, accumulates in the endoplasmic reticulum (ER). Contrary to what is observed for proteins with a KDEL or a KKXX ER-targeting signal, the ER localization of the HCV glycoprotein complex is due to a static retention in this compartment rather than to its retrieval from the cis-Golgi region. A static retention in the ER is also observed when E2 is expressed in the absence of E1 or for a chimeric protein containing the ectodomain of CD4 in fusion with the transmembrane domain (TMD) of E2. Although they do not exclude the presence of an intracellular localization signal in E1, these data do suggest that the TMD of E2 is an ER retention signal for HCV glycoprotein complex. In this study chimeric proteins containing the ectodomain of CD4 or CD8 fused to the C-terminal hydrophobic sequence of E1 were shown to be localized in the ER, indicating that the TMD of E1 is also a signal for ER localization. In addition, these chimeric proteins were not processed by Golgi enzymes, indicating that the TMD of E1 is responsible for true retention in the ER, without recycling through the Golgi apparatus. Together, these data suggest that at least two signals (TMDs of E1 and E2) are involved in ER retention of the HCV glycoprotein complex. PMID:10074109

  12. Localization of the adenovirus E1Aa protein, a positive-acting transcriptional factor, in infected cells infected cells.

    PubMed Central

    Feldman, L T; Nevins, J R

    1983-01-01

    The function of the adenovirus E1Aa protein (the product of the 13S E1A mRNA) during a productive viral infection is to activate transcription of the six early viral transcription units. To study the mechanism of action of this protein, a peptide which was 13 amino acids long and had a sequence unique to the protein product of the adenovirus 13S E1A mRNA (pE1Aa) was coupled to keyhole limpet hemocyanin and used to raise an antibody in rabbits. The resulting antiserum was specific to this protein and did not react with the protein product of the 12S E1A mRNA, which shares considerable sequence with the E1Aa protein. This antiserum was used to probe for the E1Aa protein in situ by indirect immunofluorescence and in extracts of infected HeLa cells. We found that the protein was associated with large cellular structures both in the nucleus and in the cytoplasm. The nuclear form of the protein was analyzed further and was found to purify with the nuclear matrix. Images PMID:6346057

  13. The Chang'E-1 orbiter plays a distinctive role in China's first successful selenodetic lunar mission

    NASA Astrophysics Data System (ADS)

    Ping, Jinsong; Su, Xiaoli; Huang, Qian; Yan, Jianguo

    2011-12-01

    The first Chinese lunar orbiter Chang'E-1 is a successful mission with many fruitful results obtained in various disciplines. The scientific data acquired by the Chang'E-1 payloads can benefit studies of the lunar origin and evolution, as well as other relevant research areas, after careful validation of the data. Among the new results, the Chang'E-1 selenodetic products are continually uncovering characteristics of the lunar surface, undersurface and inner structure. Successful lunar orbiters such as the Clementine, Lunar Prospector, KAGUYA/SELENE, Chang'E-1, Lunar Reconnaissance Orbiter and GRAIL have been revealing, with increasing clarity, global selenodetic characteristics with state-of-the-art fine resolution and high precision. In particular, the Chang'E-1 plays an important distinctive role in selenodetic exploration through enhancing lunar topography and gravity models. The gravity model has been successfully improved with a factor of two after applying the Chang'E-1 long-wavelength tracking data. Using the new models, some medium-scale lunar surface characteristics such as basins and volcanoes have been identified. Furthermore, the old mascon basins of Bouguer, gravity anomaly and craters have been discovered with the Chang'E-1 selenodetic data.

  14. Adenovirus E1A downregulates cJun- and JunB-mediated transcription by targeting their coactivator p300.

    PubMed Central

    Lee, J S; See, R H; Deng, T; Shi, Y

    1996-01-01

    Transcription factors and cofactors play critical roles in cell growth and differentiation. Alterations of their activities either through genetic mutations or by viral oncoproteins often result in aberrant cell growth and tumorigenesis. The transcriptional cofactor p300 has recently been shown to be complexed with transcription factors YY1 and CREB. Adenovirus E1A oncoproteins target these transcription complexes via physical interactions with p300, resulting in alterations of transcription mediated by these transcription factors. Here we show that p300 is also critical for repression by E1A of the activities of cJun and JunB, two members of the AP-1 transcriptional complexes. This repressive effect of E1A is dependent on the p300-binding domain of E1A and can be relieved by overexpression of p300. These results suggest that p300 serves as a mediator protein for downregulation of AP-1 activity by E1A. This hypothesis was further supported by the following observations: (i) in the absence of E1A, overexpression of p300 stimulated transcription both through an AP-1 site present in the collagenase promoter and through Jun proteins in GAL4 fusion protein-based assays; and (ii) overexpression of a mutant p300 lacking the E1A-interacting domain reduced the responsiveness of Jun-dependent transcription to E1A repression. As predicted from the functional results, p300 physically interacted with the Jun proteins. These findings thus established that p300 is a cofactor for cJun and JunB. We propose that p300 is a common mediator protein through which E1A gains control over multiple transcriptional regulatory pathways in the host cells. PMID:8754832

  15. The multiple roles of cyclin E1 in controlling cell cycle progression and cellular morphology of Trypanosoma brucei.

    PubMed

    Gourguechon, Stéphane; Savich, Jason M; Wang, Ching C

    2007-05-11

    Regulation of eukaryotic cell cycle progression requires sequential activation and inactivation of cyclin-dependent kinases. Previous RNA interference (RNAi) experiments in Trypanosoma brucei indicated that cyclin E1, cdc2-related kinase (CRK)1 and CRK2 are involved in regulating G1/S transition, whereas cyclin B2 and CRK3 play a pivotal role in controlling the G2/M checkpoint. To search for potential interactions between the other cyclins and CRKs that may not have been revealed by the RNAi assays, we used the yeast two-hybrid system and an in vitro glutathione-S-transferase pulldown assay and observed interactions between cyclin E1 and CRK1, CRK2 and CRK3. Cyclins E1-E4 are homologues of yeast Pho80 cyclin. But yeast complementation assays indicated that none of them possesses a Pho80-like function. Analysis of cyclin E1+CRK1 and cyclin E1+CRK2 double knockdowns in the procyclic form of T. brucei indicated that the cells were arrested more extensively in the G1 phase beyond the cumulative effect of individual knockdowns. But BrdU incorporation was impaired significantly only in cyclin E1+CRK1-depleted cells, whereas a higher percentage of cyclin E1+CRK2 knockdown cells assumed a grossly elongated posterior end morphology. A double knockdown of cyclin E1 and CRK3 arrested cells in G2/M much more efficiently than if only CRK3 was depleted. Taken together, these data suggest multiple functions of cyclin E1: it forms a complex with CRK1 in promoting G1/S phase transition; it forms a complex with CRK2 in controlling the posterior morphogenesis during G1/S transition; and it forms a complex with CRK3 in promoting passage across the G2/M checkpoint in the trypanosome. PMID:17376478

  16. THE MULTIPLE ROLES OF CYCLIN E1 IN CONTROLLING CELL CYCLE PROGRESSION AND CELLULAR MORPHOLOGY OF TRYPANOSOMA BRUCEI

    PubMed Central

    Gourguechon, Stéphane; Savich, Jason M.; Wang, Ching C.

    2009-01-01

    SUMMARY Regulation of eukaryotic cell cycle progression requires sequential activation and inactivation of cyclin-dependent kinases. Previous RNA interference (RNAi) experiments in Trypanosoma brucei indicated that cyclin E1, cdc2-related kinase (CRK)1 and CRK2 are involved in regulating G1/S transition, whereas cyclin B2 and CRK3 play a pivotal role in controlling the G2/M checkpoint. To search for potential interactions between the other cyclins and CRKs that may not have been revealed by the RNAi assays, we used the yeast two-hybrid system and an in vitro GST pulldown assay and observed interactions between cyclin E1 and CRK1, CRK2 and CRK3. Cyclins E1-E4 are homologues of yeast Pho80 cyclin. But yeast complementation assays indicated that none of them possesses a Pho80-like function. Analysis of cyclin E1+CRK1 and cyclin E1+CRK2 double knockdowns in the procyclic form of T. brucei indicated that the cells were arrested more extensively in the G1 phase beyond the cumulative effect of individual knockdowns. But BrdU incorporation was significantly impaired only in cyclin E1+CRK1 depleted cells, whereas a higher percentage of cyclin E1+CRK2 knockdown cells assumed a grossly elongated posterior end morphology. A double knockdown of cyclin E1 and CRK3 arrested cells in G2/M much more efficiently than if CRK3 was depleted alone. Taken together, these data suggest multiple functions of cyclin E1. It forms a complex with CRK1 in promoting G1/S phase transition, with CRK2 in controlling the posterior morphogenesis during G1/S transition and with CRK3 in promoting passage across the G2/M checkpoint in the trypanosome. PMID:17376478

  17. Chronic administration of caderofloxacin, a new fluoroquinolone, increases hepatic CYP2E1 expression and activity in rats

    PubMed Central

    Liu, Li; Miao, Ming-xing; Zhong, Ze-yu; Xu, Ping; Chen, Yang; Liu, Xiao-dong

    2016-01-01

    Aim: Caderofloxacin is a new fluoroquinolone that is under phase III clinical trials in China. Here we examined the effects of caderofloxacin on rat hepatic cytochrome P450 (CYP450) isoforms as well as the potential of caderofloxacin interacting with co-administered drugs. Methods: Male rats were treated with caderofloxacin (9 mg/kg, ig) once or twice daily for 14 consecutive days. The effects of caderofloxacin on CYP3A, 2D6, 2C19, 1A2, 2E1 and 2C9 were evaluated using a “cocktail” of 6 probes (midazolam, dextromethorphan, omeprazole, theophylline, chlorzoxazone and diclofenac) injected on d 0 (prior to caderofloxacin exposure) and d 15 (after caderofloxacin exposure). Hepatic microsomes from the caderofloxacin-treated rats were used to assess CYP2E1 activity and chlorzoxazone metabolism. The expression of CYP2E1 mRNA and protein in hepatic microsomes was analyzed with RT-PCR and Western blotting, respectively. Results: Fourteen-day administration of caderofloxacin significantly increased the activity of hepatic CYP2E1, leading to enhanced metabolism of chlorzoxazone. In vitro microsomal study confirmed that CYP2E1 was a major metabolic enzyme involved in chlorzoxazone metabolism, and the 14-d administration of caderofloxacin significantly increased the activity of CYP2E1 in hepatic microsomes, resulting in increased formation of 6-hydroxychlorzoxazone. Furthermore, the 14-d administration of caderofloxacin significantly increased the expression of CYP2E1 mRNA and protein in liver microsomes, which was consistent with the pharmacokinetic results. Conclusion: Fourteen-day administration of caderofloxacin can induce the expression and activity of hepatic CYP2E1 in rats. When caderofloxacin is administered, a potential drug-drug interaction mediated by CYP2E1 induction should be considered. PMID:26838075

  18. The regulation of cytochrome P450 2E1 during LPS-induced inflammation in the rat

    SciTech Connect

    Abdulla, Dalya; Goralski, Kerry B.; Renton, Kenneth W. . E-mail: Ken.Renton@dal.ca

    2006-10-01

    It is well known that inflammatory and infectious conditions differentially regulate cytochrome P450 (P450)-mediated drug metabolism in the liver. We have previously outlined a potential pathway for the downregulation in hepatic cytochrome P450 following LPS-mediated inflammation in the CNS (Abdulla, D., Goralski, K.B., Garcia Del Busto Cano, E., Renton, K.W., 2005. The signal transduction pathways involved in hepatic cytochrome P450 regulation in the rat during an LPS-induced model of CNS inflammation. Drug Metab. Dispos). The purpose of this study was to outline the effects of LPS-induced peripheral and central nervous system inflammation on hepatic cytochrome P450 2E1 (CYP2E1) in vivo, an enzyme that plays an important role in various physiological and pathological states. We report an increase in hepatic mRNA expression of CYP2E1 that occurred as early as 2-3 h following either the intraperitoneal (i.p.) injection of 5 mg/kg LPS or i.c.v. administration of 25 {mu}g of LPS. This increase in CYP2E1 mRNA expression was sustained for 24 h. In sharp contrast to the increase in hepatic CYP2E1 mRNA, we observed a significant reduction in the catalytic activity of this enzyme 24 h following either the i.c.v. or i.p. administration of LPS. Cycloheximide or actinomycin-D did not change the LPS-mediated downregulation in hepatic CYP2E1 catalytic activity. Our results support the idea that LPS acts at two different levels to regulate hepatic CYP2E1: a transcriptional level to increase CYP2E1 mRNA expression and a post-transcriptional level to regulate CYP2E1 protein and activity.

  19. Characterization of recombinant HPV6 and 11 E1 helicases: effect of ATP on the interaction of E1 with E2 and mapping of a minimal helicase domain.

    PubMed

    White, P W; Pelletier, A; Brault, K; Titolo, S; Welchner, E; Thauvette, L; Fazekas, M; Cordingley, M G; Archambault, J

    2001-06-22

    To better characterize the enzymatic activities required for human papillomavirus (HPV) DNA replication, the E1 helicases of HPV types 6 and 11 were produced using a baculovirus expression system. The purified wild type proteins and a version of HPV11 E1 lacking the N-terminal 71 amino acids, which was better expressed, were found to be hexameric over a wide range of concentrations and to have helicase and ATPase activities with relatively low values for K(m)(ATP) of 12 microm for HPV6 E1 and 6 microm for HPV11 E1. Interestingly, the value of K(m)(ATP) was increased 7-fold in the presence of the E2 transactivation domain. In turn, ATP was found to perturb the co-operative binding of E1 and E2 to DNA. Mutant and truncated versions of in vitro translated E1 were used to identify a minimal ATPase domain composed of the C-terminal 297 amino acids. This fragment was expressed, purified, and found to be fully active in ATP hydrolysis, single-stranded DNA binding, and unwinding assays, despite lacking the minimal origin-binding domain. PMID:11304544

  20. ColE1-Plasmid Production in Escherichia coli: Mathematical Simulation and Experimental Validation

    PubMed Central

    Freudenau, Inga; Lutter, Petra; Baier, Ruth; Schleef, Martin; Bednarz, Hanna; Lara, Alvaro R.; Niehaus, Karsten

    2015-01-01

    Plasmids have become very important as pharmaceutical gene vectors in the fields of gene therapy and genetic vaccination in the past years. In this study, we present a dynamic model to simulate the ColE1-like plasmid replication control, once for a DH5α-strain carrying a low copy plasmid (DH5α-pSUP 201-3) and once for a DH5α-strain carrying a high copy plasmid (DH5α-pCMV-lacZ) by using ordinary differential equations and the MATLAB software. The model includes the plasmid replication control by two regulatory RNA molecules (RNAI and RNAII) as well as the replication control by uncharged tRNA molecules. To validate the model, experimental data like RNAI- and RNAII concentration, plasmid copy number (PCN), and growth rate for three different time points in the exponential phase were determined. Depending on the sampled time point, the measured RNAI- and RNAII concentrations for DH5α-pSUP 201-3 reside between 6 ± 0.7 and 34 ± 7 RNAI molecules per cell and 0.44 ± 0.1 and 3 ± 0.9 RNAII molecules per cell. The determined PCNs averaged between 46 ± 26 and 48 ± 30 plasmids per cell. The experimentally determined data for DH5α-pCMV-lacZ reside between 345 ± 203 and 1086 ± 298 RNAI molecules per cell and 22 ± 2 and 75 ± 10 RNAII molecules per cell with an averaged PCN of 1514 ± 1301 and 5806 ± 4828 depending on the measured time point. As the model was shown to be consistent with the experimentally determined data, measured at three different time points within the growth of the same strain, we performed predictive simulations concerning the effect of uncharged tRNA molecules on the ColE1-like plasmid replication control. The hypothesis is that these tRNA molecules would have an enhancing effect on the plasmid production. The in silico analysis predicts that uncharged tRNA molecules would indeed increase the plasmid DNA production. PMID:26389114

  1. Regulation of hepatic sulfotransferase (SULT) 1E1 expression and effects on estrogenic activity in cystic fibrosis (CF)☆

    PubMed Central

    Falany, Charles N.; He, Dongning; Li, Li; Falany, Josie L.; Wilborn, Teresa W.; Kocarek, Thomas A.; Runge-Morris, Melissa

    2013-01-01

    Cystic fibrosis (CF) is a major genetic disease in Caucasians affecting 1 in 2500 newborns. Hepatobiliary pathology is a major cause of morbidity and mortality in CF second only to pulmonary disease. SULT1E1 activity is significantly elevated, generally 20–30-fold, in hepatocytes of mouse models of CF. SULT1E1 is responsible for the inactivation of β-estradiol (E2) at physiological concentrations via conjugation with sulfonate. The increase in SULT1E1 activity results in the alteration of E2-regulated protein expression in CF mouse liver. To investigate the mechanism by which the absence of CFTR in human cholangiocytes induces SULT1E1 expression in hepatocytes, a membrane-separated human MMNK-1 cholangiocyte and human HepG2 hepatocyte co-culture system was developed. The cystic fibrosis transmembrane conductance regulator (CFTR) is expressed in bile duct cholangiocytes but not hepatocytes, whereas SULT1E1 is expressed in hepatocytes but not cholangiocytes. CFTR expression in MMNK-1 cells was inhibited with siRNA by >90% as determined by immunoblot and immunohistochemical analysis. Control and CFTR-siRNA-MMNK-1 cells were co-cultured with HepG2 cells in a Transwell membrane-separated system. After 8 h of co-culture, HepG2 cells were removed from exposure to MMNK-1 cells and placed in fresh medium. After 24–48 h, expression of SULT1E1 and selected E2-regulated proteins was analyzed in the HepG2 cells. Results demonstrated that SULT1E1 message and activity were selectively induced in HepG2 cells co-cultured with CFTR-deficient MMNK-1 cells. The expression of E2-regulated proteins (IGF-1, GST-P1 and carbonic anhydrase II) was also altered in response to decreased E2 levels. Thus, the loss of CFTR activity in cholangiocytes stimulates the expression of SULT1E1 in hepatocytes by a paracrine mechanism. SULT1E1 expression in HepG2 cells is inducible by sterol mediated liver-X-receptor (LXR) activation although not by progestins that induce SULT1E1 in the endometrium

  2. CYP2E1 epigenetic regulation in chronic, low-level toluene exposure: Relationship with oxidative stress and smoking habit

    SciTech Connect

    Jiménez-Garza, Octavio; Baccarelli, Andrea A.; Byun, Hyang-Min; Márquez-Gamiño, Sergio; Barrón-Vivanco, Briscia Socorro

    2015-08-01

    Background: CYP2E1 is a versatile phase I drug-metabolizing enzyme responsible for the biotransformation of most volatile organic compounds, including toluene. Human toluene exposure increases CYP2E1 mRNA and modifies its activity in leucocytes; however, epigenetic implications of this interaction have not been investigated. Goal: To determine promoter methylation of CYP2E1 and other genes known to be affected by toluene exposure. Methods: We obtained venous blood from 24 tannery workers exposed to toluene (mean levels: 10.86 +/− 7 mg/m{sup 3}) and 24 administrative workers (reference group, mean levels 0.21 +/− 0.02 mg/m{sup 3}) all of them from the city of León, Guanajuato, México. After DNA extraction and bisulfite treatment, we performed PCR-pyrosequencing in order to measure methylation levels at promoter region of 13 genes. Results: In exposed group we found significant correlations between toluene airborne levels and CYP2E1 promoter methylation (r = − .36, p < 0.05), as well as for IL6 promoter methylation levels (r = .44, p < 0.05). Moreover, CYP2E1 promoter methylation levels where higher in toluene-exposed smokers compared to nonsmokers (p = 0.009). We also observed significant correlations for CYP2E1 promoter methylation with GSTP1 and SOD1 promoter methylation levels (r = − .37, p < 0.05 and r = − .34, p < 0.05 respectively). Conclusion: These results highlight the importance of considering CYP2E1 epigenetic modifications, as well as its interactions with other genes, as key factors for unraveling the sub cellular mechanisms of toxicity exerted by oxidative stress, which can initiate disease process in chronic, low-level toluene exposure. People co-exposed to toluene and tobacco smoke are in higher risk due to a possible CYP2E1 repression. - Highlights: • We investigated gene-specific methylation in persons chronically exposed to toluene. • In a previous study, a reduced CYP2E1 activity was observed in these participants. • CYP2E1

  3. Cellular localization studies on human estrogen sulfotransferase SULT1E1 in human embryonic kidney 293 cells.

    PubMed

    Kapoor, Ruchita; Nimmagadda, Deepthi; Sheng, Jonathan J

    2007-01-01

    Human cytosolic sulfotransferase SULT1E1 catalyzes the sulfation of endogenous estrogens as well as xenobiotic estrogen-like chemicals. This reaction increases the water solubility of the molecule, which may affect its cellular distribution and biological activity. This could alter estrogen signaling to the estrogen receptor in human estrogen receptor-positive cells. The current work characterized the cellular distribution of SULT1E1 in the human embryonic kidney 293 (HEK293) cell line using green fluorescent protein (GFP) tagging and immunochemistry methods. The GFP-tagged recombinant SULT1E1 protein was expressed and localized in the cytoplasm of HEK293 cells. By using a commercial anti-SULT1E1 peptide antibody, a 35.7-kDa protein was detected in HEK293 cells via Western blot. The molecular mass of the protein detected suggested that it may be related to native SULT1E1 protein. However, reverse transcription-polymerase chain reaction (RT-PCR) with gene-specific primers could not confirm the presence of the SULT1E1 transcript in the total RNA sample of HEK293 cells. The discrepancy between protein and transcript data could be due to the instability of SULT1E1 mRNA or the specificity of the anti-SULT1E1 antibody used. In the present work, RT-PCR analysis with gene-specific primers also identified a transcript fragment of human estrogen-related receptor gamma. Future studies on the functional relationship between estrogen-related receptors and sulfotransferases are expected to provide additional insights into the physiological and toxicological roles of human estrogen sulfotransferases. PMID:17035602

  4. Human CYP2E1-dependent mutagenicity of mono- and dichlorobiphenyls in Chinese hamster (V79)-derived cells.

    PubMed

    Zhang, Chiteng; Lai, Yanmei; Jin, Guifang; Glatt, Hansruedi; Wei, Qinzhi; Liu, Yungang

    2016-02-01

    Polychlorinated biphenyls (PCBs) are a group of persistent organic pollutants with confirmed carcinogenicity to humans. Metabolic activation of lower chlorinated PCBs to genotoxic metabolites may involve hydroxylation and further oxidation, and some hydroxylated metabolites may be sulfo-conjugated. However, the genotoxicity of individual PCB compounds is largely unknown. In this study, 15 mono- and dichlorobiphenyls were investigated for genotoxicity using the micronucleus and Hprt mutagenicity assays in a Chinese hamster V79-derived cell line expressing both human cytochrome P450 (CYP) 2E1 and human sulfotransferase (SULT) 1A1 (V79-hCYP2E1-hSULT1A1). All tested compounds were inactive in both assays in V79 control cells. However, eight dichlorobiphenyls strongly induced micronuclei and other congeners were weakly positive for this endpoint in V79-hCYP2E1-hSULT1A1 cells. The effects of each PCB in V79-hCYP2E1-hSULT1A1 cells were abolished or reduced in the presence of a CYP2E1 inhibitor (1-aminobenzotriazole), or enhanced by pretreatment of the cells with (CYP2E1-inducing) ethanol, while the genotoxicity was not significantly affected by a SULT1 inhibitor (pentachlorophenol). As representative dichlorobiphenyls, PCB 5, 10, 8 and 11 (2,3-, 2,5-, 2,4'- and 3,3'-dichlorobiphenyl, respectively) strongly induced Hprt gene mutations in V79-hCYP2E1-hSULT1A1 cells in a concentration-dependent manner. This is the first indication that human CYP2E1 is capable of converting a series of dichlorobiphenyls to strong mutagens. PMID:26547025

  5. Sites of disruption within E1 and E2 genes of HPV16 and association with cervical dysplasia.

    PubMed

    Tsakogiannis, D; Gortsilas, P; Kyriakopoulou, Z; Ruether, I G A; Dimitriou, T G; Orfanoudakis, G; Markoulatos, P

    2015-11-01

    Integration of HPV16 DNA into the host chromosome usually disrupts the E1 and/or E2 genes. The present study investigated the disruption of E1, E2 genes in a total of eighty four HPV16-positive precancerous and cervical cancer specimens derived from Greek women (seventeen paraffin-embedded cervical biopsies and sixty seven Thin Prep samples). Complete E2 and E1 genes were amplified using three and nine overlapping primer sets respectively, in order to define the sites of disruption. Extensive mapping analysis revealed that disruption/deletion events within E2 gene occurred in high grade and cervical cancer samples (x(2) test, P < 0.01), while no evidence of E2 gene disruption was documented among low grade cervical intraepithelial neoplasias. In addition, disruptions within the E1 gene occur both in high and low grade cervical intraepithelial neoplasia. This leads to the assumption that in low grade cervical intraepithelial neoplasias only E1 gene disruption was involved (Fisher's exact test, P < 0.05), while in high grade malignancies and cervical cancer cases deletions in both E1 and E2 genes occurred. Furthermore, the most prevalent site of disruption of E1 gene was located between nucleotides 1059 and 1323, while the most prevalent deleted region of the E2 gene was located between nucleotides 3172 and 3649 (E2 hinge region). Therefore, it is proposed that each population has its own profile of frequencies and sites of disruptions and extensive mapping analysis of E1 and E2 genes is mandatory in order to determine suitable markers for HPV16 DNA integration analysis in distinct populations. PMID:25959607

  6. Modulation of p53-mediated transcriptional repression and apoptosis by the adenovirus E1B 19K protein.

    PubMed Central

    Sabbatini, P; Chiou, S K; Rao, L; White, E

    1995-01-01

    BRK cell lines that stably express adenovirus E1A and a murine temperature-sensitive p53 undergo apoptosis when p53 assumes the wild-type conformation. Expression of the E1B 19,000-molecular-weight (19K) protein rescues cells from this p53-mediated apoptosis and diverts cells to a growth-arrested state. As p53 likely functions as a tumor suppressor by regulating transcription, the ability of the E1B 19K protein to regulate p53-mediated transactivation and transcriptional repression was investigated. In promoter-reporter assays the E1B 19K did not block p53-mediated transactivation but did alleviate p53-mediated transcriptional repression. E1B 19K expression permitted efficient transcriptional activation of the p21/WAF-1/cip-1 mRNA by p53, consistent with maintenance of the growth arrest function of p53. The E1B 19K protein is thereby unique among DNA virus-transforming proteins that target p53 for inactivation in that it selectively modulates the transcriptional properties of p53. The E1B 19K protein also rescued cells from apoptosis induced by inhibitors of transcription and protein synthesis. This suggests that cell death may result from the inhibition of expression of survival factors which function to maintain cell viability. p53 may induce apoptosis through generalized transcriptional repression. In turn, the E1B 19K protein may prevent p53-mediated apoptosis by alleviating p53-mediated transcriptional repression. PMID:7823921

  7. A repeat sequence causes competition of ColE1-type plasmids.

    PubMed

    Lin, Mei-Hui; Fu, Jen-Fen; Liu, Shih-Tung

    2013-01-01

    Plasmid pSW200 from Pantoea stewartii contains 41 copies of 15-bp repeats and has a replicon that is homologous to that of ColE1. Although deleting the repeats (pSW207) does not change the copy number and stability of the plasmid. The plasmid becomes unstable and is rapidly lost from the host when a homoplasmid with the repeats (pSW201) is present. Deleting the repeats is found to reduce the transcriptional activity of RNAIp and RNAIIp by about 30%, indicating that the repeats promote the transcription of RNAI and RNAII, and how the RNAI that is synthesized by pSW201 inhibits the replication of pSW207. The immunoblot analysis herein demonstrates that RNA polymerase β subunit and σ(70) in the lysate from Escherichia coli MG1655 bind to a biotin-labeled DNA probe that contains the entire sequence of the repeat region. Electrophoretic mobility shift assay also reveals that purified RNA polymerase shifts a DNA probe that contains four copies of the repeats. These results thus obtained reveal that RNA polymerase holoenzyme binds to the repeats. The repeats also exchange RNA polymerase with RNAIp and RNAIIp in vitro, revealing the mechanism by which the transcription is promoted. This investigation elucidates a mechanism by which a plasmid prevents the invasion of an incompatible plasmid and maintains its stability in the host cell during evolution. PMID:23613898

  8. Chang’E-1 gamma ray spectrometer and preliminary radioactive results on the lunar surface

    NASA Astrophysics Data System (ADS)

    Zhu, Meng-Hua; Ma, Tao; Chang, Jin

    2010-10-01

    The Chang'E-1(CE-1) spacecraft took a gamma-ray spectrometer (hereafter, CGRS) to detect the element distributions on the lunar surface in a circular, 200 km altitude, polar orbit with approximately 2 h periodicity. CGRS consists of two large CsI(Tl) crystals as the main and anticoincidence detectors. The large CsI crystal of CGRS has a higher detector effective area than other lunar gamma ray spectrometers. For its 1-year mission, gamma ray spectra including many peaks of major elements and trace elements on the lunar surface have been measured by CGRS. Global measurement within 0.55-0.75 MeV is given here to describe the distribution of radioactive composition (e.g., uranium and thorium) on the lunar surface. Although CGRS has a lower energy resolution that cannot separate the uranium peak from others in this energy region, 609 keV uranium gamma ray line dominates the shape of the spectrum in this energy region. Therefore, the radioactive map can indirectly describe the uranium distribution on the lunar surface. The radioactive map shows that the higher radiation is concentrated in the Procellarum KREEP Terrene (PKT) on the nearside with an oval shape. The secondary high-radiation is located in South Pole-Aitken (SPA) basin. Lunar highlands have lower concentration. The relationship between radiation and topography displays different linear correlations for lunar highlands and SPA basin, which imply the different processes for these two regions.

  9. Experimental and computational studies on zwitterionic (E)-1-((4-phenoxyphenyliminio)methyl)naphthalen-2-olate

    NASA Astrophysics Data System (ADS)

    Alpaslan, Gökhan; Macit, Mustafa; Erdönmez, Ahmet; Büyükgüngör, Orhan

    2011-06-01

    The Schiff base compound (E)-1-((4-phenoxyphenyliminio)methyl)naphthalen-2-olate has been synthesized and characterized by IR, UV-Vis, and X-ray single-crystal determination. Molecular geometry of the title compound in the ground state have been calculated using the density functional method (DFT) with 6-31G(d,p) basis set and compared with the experimental data. The calculated results show that the optimized geometry can well reproduce the crystal structure. By using TD-DFT method, electronic absorption spectra of the title compound have been predicted and a good agreement with the TD-DFT method and the experimental ones is determined. Molecular orbital coefficient analyses reveal that the electronic transitions are mainly assigned to n → p∗ and p → p∗ electronic transitions. To investigate the tautomeric stability, optimization calculations at B3LYP/6-31G(d,p) level were performed for the NH and OH forms of the title compound. Calculated results reveal that the OH form is more stable than NH form. In addition, molecular electrostatic potential and non-linear optical properties of the title compound were performed at B3LYP/6-31G(d,p) level of theory.

  10. Vibrational studies on (E)-1-((pyridine-2-yl)methylene)semicarbazide using experimental and theoretical method

    NASA Astrophysics Data System (ADS)

    Subashchandrabose, S.; Ramesh Babu, N.; Saleem, H.; Syed Ali Padusha, M.

    2015-08-01

    The (E)-1-((pyridine-2-yl)methylene)semicarbazide (PMSC) was synthesized. The experimental and theoretical study on molecular structure and vibrational spectra were carried out. The FT-IR (400-4000 cm-1), FT-Raman (50-3500 cm-1) and UV-Vis (200-500 nm) spectra of PMSC were recorded. The geometric structure, conformational analysis, vibrational wavenumbers of PMSC in the ground state have been calculated using B3LYP method of 6-311++G(d,p) basis set. The complete vibrational assignments were made on the basis of TED, calculated by SQM method. The Non-linear optical activity was measured by means of first order hyperpolarizability calculation and π-electrons of conjugative bond in the molecule. The intra-molecular charge transfer, mode hyperconjugative interaction and molecular stabilization energies were calculated. The band gap energies between occupied and unoccupied molecular orbitals were analyzed; it proposes lesser band gap with more reactivity. To understand the electronic properties of this molecule the Mulliken charges were also calculated.

  11. Activity of immobilised rat hepatic microsomal CYP2E1 using alumina membrane as a support.

    PubMed

    Tanvir, Shazia; Morandat, Sandrine; Frederic, Nadaud; Adenier, Hervé; Pulvin, Sylviane

    2009-11-30

    Porous alumina membranes are attractive materials for the construction of biosensors and also have utility for the production of immobilised enzyme bioreactors. Microsomes from rat liver were adsorbed onto alumina membrane activated by silane. Microsomal membranes were pumped through the channels where they became immobilised by binding to amine groups on the surface of the alumina membrane. In an effort to gain a quantitative understanding of the effects of microsomal film growth on enzyme activity, we compared the para-nitrophenol (pNP) hydroxylase activity of the microsomes by varying the amount of microsomes fixed in alumina microchannels. The alumina membrane was placed in a fluidic device at a fast flow that afforded short residence time (seconds) to obtain transformation of pNP to 4-nitrocatechol (pNC), which was detected by LC-MS/MS. This enabled the use of this bioreactor where CYP2E1 activity is low and tissue sources are limiting. The microsomes, successfully immobilised on the alumina membranes, were used to produce stable biocatalytic reactors that can be used repeatedly over a period of 2 months. PMID:19703600

  12. Discovery of low-lying E1 and M1 strengths in {sup 232}Th

    SciTech Connect

    Adekola, A. S.; Hammond, S. L.; Hill, A.; Karwowski, H. J.; Angell, C. T.; Howell, C. R.; Kwan, E.; Kelley, J. H.

    2011-03-15

    Properties of low-energy dipole states in {sup 232}Th have been investigated with the nuclear resonance fluorescence technique. The present work used monoenergetic {gamma}-ray beams at energies of 2-4 MeV from the high-intensity {gamma}-ray source at Triangle Universities Nuclear Laboratory. Over 40 transitions corresponding to deexcitation to the ground state and first excited state were observed for the first time. Excitation energies, integrated cross sections, decay widths, branching ratios, and transition strengths for those states in {sup 232}Th were determined and compared with quasiparticle random-phase-approximation calculations. A large number of E1 transitions were observed for the first time in actinide nuclei with summed strength of 3.28(69)x10{sup -3} e{sup 2} fm{sup 2}. The observed summed M1 strength of 4.26(63){mu}{sub N}{sup 2} is in good agreement with the other actinides and with the systematics of the scissors mode in deformed rare-earth nuclei.

  13. Mechanism of the Lower Esophageal Sphincter Relaxation ACTION OF PROSTAGLANDIN E1 AND THEOPHYLLINE

    PubMed Central

    Goyal, Raj K.; Rattan, Satish

    1973-01-01

    The intravenous injection of prostaglandin E1 (PGE1) causes a dose-dependent relaxation of the lower esophageal sphincter (LES) in the intact, lightly anesthetized opossum. The action of PGE1 is not inhibited by the drugs that produce muscarinic or nicotinic cholinergic antagonism or alpha and beta adrenergic antagonism in the doses that inhibited the action of respective agonists. Moreover, this action is not affected by exogenous gastrin pentapeptide. The action of PGE1 on the LES is mimicked by isoproterenol, theophylline ethylenediamine, and dibutyryl cyclic AMP. Both theophylline, a phosphodiesterase inhibitor, and isoproterenol, an adenyl cyclase stimulator, added to the action of PGE1. On the other hand, adenyl cyclase inhibitor nicotinic acid, as well as phosphodiesterase stimulator, imidazole inhibited its action. Further, both nicotinic acid and imidazole inhibited the degree of LES relaxation produced by esophageal distension. These studies suggest that intracellular cyclic AMP may act as the “second messenger” in the regulation of the lower esophageal sphincter relaxation. Images PMID:4346007

  14. Modulation of Osteogenesis in MC3T3-E1 Cells by Different Frequency Electrical Stimulation

    PubMed Central

    Wang, Yu; Cui, Haitao; Wu, Zhenxu; Wu, Naipeng; Wang, Zongliang; Chen, Xuesi; Wei, Yen; Zhang, Peibiao

    2016-01-01

    Electrical stimulation (ES) is therapeutic to many bone diseases, from promoting fracture regeneration to orthopedic intervention. The application of ES offers substantial therapeutic potential, while optimal ES parameters and the underlying mechanisms responsible for the positive clinical impact are poorly understood. In this study, we assembled an ES cell culture and monitoring device. Mc-3T3-E1 cells were subjected to different frequency to investigate the effect of osteogenesis. Cell proliferation, DNA synthesis, the mRNA levels of osteosis-related genes, the activity of alkaline phosphatase (ALP), and intracellular concentration of Ca2+ were thoroughly evaluated. We found that 100 Hz could up-regulate the mRNA levels of collagen I, collagen II and Runx2. On the contrary, ES could down-regulate the mRNA levels of osteopontin (OPN). ALP activity assay and Fast Blue RR salt stain showed that 100 Hz could accelerate cells differentiation. Compared to the control group, 100 Hz could promote cell proliferation. Furthermore, 1 Hz to 10 Hz could improve calcium deposition in the intracellular matrix. Overall, these results indicate that 100Hz ES exhibits superior potentialities in osteogenesis, which should be beneficial for the clinical applications of ES for the treatment of bone diseases. PMID:27149625

  15. Cytoplasmic pH influences cytoplasmic calcium in MC3T3-E1 osteoblast cells

    NASA Technical Reports Server (NTRS)

    Lin, H. S.; Hughes-Fulford, M.; Kumegawa, M.; Pitts, A. C.; Snowdowne, K. W.

    1993-01-01

    We found that the cytoplasmic concentration of calcium (Cai) of MC3T3-E1 osteoblasts was influenced by the type of pH buffer we used in the perfusing medium, suggesting that intracellular pH (pHi) might influence Cai. To study this effect, the Cai and pHi were monitored as we applied various experimental conditions known to change pHi. Exposure to NH4Cl caused a transient increase in both pHi and Cai without a change in extracellular pH (pHo). Decreasing pHo and pHi by lowering the bicarbonate concentration of the medium decreased Cai, and increasing pHi by the removal of 5% CO2 increased Cai. Clamping pHi to known values with 10 microM nigericin, a potassium proton ionophore, also influenced Cai: acid pHi lowered Cai, whereas alkaline pHi increased it. The rise in Cai appears to be very sensitive to the extracellular concentration of calcium, suggesting the existence of a pH-sensitive calcium influx mechanism. We conclude that physiologic changes in pH could modulate Cai by controlling the influx of calcium ions and could change the time course of the Cai transient associated with hormonal activation.

  16. Enhanced metabolism of halogenated hydrocarbons in transgenic plants containing mammalian cytochrome P450 2E1

    PubMed Central

    Doty, Sharon Lafferty; Shang, Tanya Q.; Wilson, Angela M.; Tangen, Jeff; Westergreen, Aram D.; Newman, Lee A.; Strand, Stuart E.; Gordon, Milton P.

    2000-01-01

    Chlorinated solvents, especially trichloroethylene (TCE), are the most widespread groundwater contaminants in the United States. Existing methods of pumping and treating are expensive and laborious. Phytoremediation, the use of plants for remediation of soil and groundwater pollution, is less expensive and has low maintenance; however, it requires large land areas and there are a limited number of suitable plants that are known to combine adaptation to a particular environment with efficient metabolism of the contaminant. In this work, we have engineered plants with a profound increase in metabolism of the most common contaminant, TCE, by introducing the mammalian cytochrome P450 2E1. This enzyme oxidizes a wide range of important pollutants, including TCE, ethylene dibromide, carbon tetrachloride, chloroform, and vinyl chloride. The transgenic plants had a dramatic enhancement in metabolism of TCE of up to 640-fold as compared with null vector control plants. The transgenic plants also showed an increased uptake and debromination of ethylene dibromide. Therefore, transgenic plants with this enzyme could be used for more efficient remediation of many sites contaminated with halogenated hydrocarbons. PMID:10841534

  17. Two-quasiparticle isomer, E1 hindrances and residual interactions in {sup 172}Tm

    SciTech Connect

    Hughes, R. O.; Lane, G. J.; Dracoulis, G. D.; Kibedi, T.; Nieminen, P.; Watanabe, H.

    2008-04-15

    The structure of the neutron-rich nucleus {sup 172}Tm has been studied using incomplete fusion of {sup 7}Li on an {sup 170}Er target at 30 MeV. A 190-{mu}s isomer at an excitation energy of 476 keV was identified using chopped beams and {gamma}-ray spectroscopy. The isomer decays with very inhibited E1 transitions to the rotational bands based on the parallel and antiparallel couplings of the {nu}5/2{sup -}[512] x {pi}1/2{sup +}[411] configuration, the latter (K{sup {pi}}=2{sup -}) being the ground state. The isomeric state has been assigned J{sup {pi}}=6{sup +}, arising from the energetically favored (parallel) coupling of the {nu}5/2{sup -}[512] x {pi}7/2{sup -}[523] configuration. The proton-neutron residual interaction was deduced for the configuration of the isomeric state and is found to agree with previous empirical studies.

  18. Type II ligands as chemical auxiliaries to favor enzymatic transformations by P450 2E1.

    PubMed

    Ménard, Amélie; Fabra, Camilo; Huang, Yue; Auclair, Karine

    2012-11-26

    The remarkable ability of P450 enzymes to oxidize inactivated C-H bonds and the high substrate promiscuity of many P450 isoforms have inspired us and others to investigate their use as biocatalysts. Our lab has pioneered a chemical-auxiliary approach to control the promiscuity of P450 3A4 and provide product predictability. The recent realization that type II ligands are sometimes also P450 substrates has prompted the design of a new generation of chemical auxiliaries with type II binding properties. This approach takes advantage of the high affinity of type II ligands for the active site of these enzymes. Although type II ligands typically block P450 activity, we report here that type II ligation can be harnessed to achieve just the opposite, that is, to favor biocatalysis and afford predictable oxidation of small hydrocarbon substrates with P450 2E1. Moreover, the observed predictability was rationalized by molecular docking. We hope that this approach might find future use with other P450 isoforms and yield complimentary products. PMID:23129539

  19. Genetic and structural analysis of the ColE1 Rop (Rom) protein.

    PubMed Central

    Castagnoli, L; Scarpa, M; Kokkinidis, M; Banner, D W; Tsernoglou, D; Cesareni, G

    1989-01-01

    Repressor of primer (Rop) is a small dimeric protein that participates in the mechanism that controls the copy number of plasmid of the ColE1 family by increasing the affinity between two complementary RNAs. The Rop dimer is a bundle of four tightly packed alpha-helices that are held together by hydrophobic interactions. We have systematically altered, by site directed mutagenesis, most of the solvent exposed amino acids of the Rop bundle and we have identified the alterations that cause a decrease of the activity of the regulatory molecule. We conclude that Rop folding is rather insensitive to amino acid substitutions and to other mutations as drastic as deletions and insertions. Looking along the 2-fold symmetry axis the amino acid side chains whose alterations affect the function of Rop are all located on one side of the molecule. Furthermore they are clustered at the extremities of the alpha-helix bundle, the only exception being the aromatic ring of Phe-14. Images PMID:2721494

  20. Rates of E1, E2, M1, and M2 transitions in Ni II

    NASA Astrophysics Data System (ADS)

    Cassidy, C. M.; Hibbert, A.; Ramsbottom, C. A.

    2016-03-01

    Aims: We present rates for all E1, E2, M1, and M2 transitions among the 295 fine-structure levels of the configurations 3d9, 3d84s, 3d74s2, 3d84p, and 3d74s4p, determined through an extensive configuration interaction calculation. Methods: The CIV3 code developed by Hibbert and coworkers is used to determine for these levels configuration interaction wave functions with relativistic effects introduced through the Breit-Pauli approximation. Results: Two different sets of calculations have been undertaken with different 3d and 4d functions to ascertain the effect of such variation. The main body of the text includes a representative selection of data, chosen so that key points can be discussed. Some analysis to assess the accuracy of the present data has been undertaken, including comparison with earlier calculations and the more limited range of experimental determinations. The full set of transition data is given in the supplementary material as it is very extensive. Conclusions: We believe that the present transition data are the best currently available. Full Table 4 and Tables 5-8 are only available at the CDS via anonymous ftp to http://cdsarc.u-strasbg.fr (ftp://130.79.128.5) or via http://cdsarc.u-strasbg.fr/viz-bin/qcat?J/A+A/587/A107

  1. Ziram Causes Dopaminergic Cell Damage by Inhibiting E1 Ligase of the Proteasome*

    PubMed Central

    Chou, Arthur P.; Maidment, Nigel; Klintenberg, Rebecka; Casida, John E.; Li, Sharon; Fitzmaurice, Arthur G.; Fernagut, Pierre-Olivier; Mortazavi, Farzad; Chesselet, Marie-Francoise; Bronstein, Jeff M.

    2008-01-01

    The etiology of Parkinson disease (PD) is unclear but may involve environmental toxins such as pesticides leading to dysfunction of the ubiquitin proteasome system (UPS). Here, we measured the relative toxicity of ziram (a UPS inhibitor) and analogs to dopaminergic neurons and examined the mechanism of cell death. UPS (26 S) activity was measured in cell lines after exposure to ziram and related compounds. Dimethyl- and diethyldithiocarbamates including ziram were potent UPS inhibitors. Primary ventral mesencephalic cultures were exposed to ziram, and cell toxicity was assessed by staining for tyrosine hydroxylase (TH) and NeuN antigen. Ziram caused a preferential damage to TH+ neurons and elevated α-synuclein levels but did not increase aggregate formation. Mechanistically, ziram altered UPS function through interfering with the targeting of substrates by inhibiting ubiquitin E1 ligase. Sodium dimethyldithiocarbamate administered to mice for 2 weeks resulted in persistent motor deficits and a mild reduction in striatal TH staining but no nigral cell loss. These results demonstrate that ziram causes selective dopaminergic cell damage in vitro by inhibiting an important degradative pathway implicated in the etiology of PD. Chronic exposure to widely used dithiocarbamate fungicides may contribute to the development of PD, and elucidation of its mechanism would identify a new potential therapeutic target. PMID:18818210

  2. Block adjustment of Chang'E-1 images based on rational function model

    NASA Astrophysics Data System (ADS)

    Liu, Bin; Liu, Yiliang; Di, Kaichang; Sun, Xiliang

    2014-05-01

    Chang'E-1(CE-1) is the first lunar orbiter of China's lunar exploration program. The CCD camera carried by CE-1 has acquired stereo images covering the entire lunar surface. Block adjustment and 3D mapping using CE-1 images are of great importance for morphological and other scientific research of the Moon. Traditional block adjustment based on rigorous sensor model is complicated due to a large number of parameters and possible correlations among them. To tackle this problem, this paper presents a block adjustment method using Rational Function Model (RFM). The RFM parameters are generated based on rigorous sensor model using virtual grid of control points. Afterwards, the RFM based block adjustment solves the refinement parameters through a least squares solution. Experimental results using CE-1 images located in Sinus Irdium show that the RFM can fit the rigorous sensor model with a high precision of 1% pixel level. Through the RFM-based block adjustment, the back-projection residuals in image space can be reduced from around 1.5 pixels to sub-pixel., indicating that RFM can replace rigorous sensor model for geometric processing of lunar images.

  3. MC3T3-E1 Cell Response to Pure Titanium, Zirconia and Nano-Hydroxyapatite

    NASA Astrophysics Data System (ADS)

    Lee, Dong-Hwan; Han, Jung-Suk; Yang, Jae-Ho; Lee, Jai-Bong; Kim, Dae-Joon

    Titanium, zirconia and HAp were known as good biocompatible materials for tissue engineering. Osteblastic cell response is influence by the surface topography of material and its chemical composition as well. To evaluate the influence of different chemical compositions on osteoblast-like cells the specimens were polished until they have almost identical surface roughness. The commercially pure titanium, zirconia/alumina composite and nano-sized hydroxyapatite (HAp) specimens synthesized by hydrothermal method were used to evaluate the cell attachment, proliferation and differentiation. Confocal laser microscopy was used measurement of surface roughness, and flourescence microscopy and SEM were used to evaluate initial cell attachment and morphology after 3 hours. MTS assay was performed for cell proliferation after 1, 3, 7 days and ALP assay was used for cell differentiation after 7, 10, 14 days of cell culture period. Surface topography of nano-HAp specimen was almost identical compared with those of titanium and zirconia specimen. Under this condition, proliferation and differentiation of MC3T3-E1 cells was not significantly different with those on titanium and zirconia specimen. However, cells on Nano-HAp specimen showed quicker and more active cellular reaction for attachment when measured by the expression of adhesion proteins through confocal laser microscopy. The results suggested that the new nano-sized HAp can be applied as a suitable material for skeletal tissue engineering.

  4. Search for E1 strength in 62,64Fe around the threshold

    NASA Astrophysics Data System (ADS)

    Wieland, O.; Bracco, A.; Camera, F.; Avigo, R.; PRESPEC Collaboration; AGATA Collaboration

    2015-02-01

    The structure and nature of the pygmy dipole resonance (PDR) states below and above the neutron threshold is a recent open problem, particularly in exotic, neutron rich nuclei. Present experimental observations give only limited information on this subject. New experiments using different methods are needed. A recent measurement at the GSI laboratories on 62,64Fe with the PRESPEC (2014) setup, following a past experiment with the RISING (2005) setup on 68Ni, will contribute to solve the open questions. The setup located at GSI consists of the segmented HPGe detector array AGATA, scintillators (HECTOR), an Energy Loss / Total Energy time of flight measuring detector system called LYCCA and the fragment separator (FRS) apparatus. The experiment is based on relativistic Coulomb excitation together with the detection of the incoming and outgoing particles event by event. The detection of the produced y-rays in the reactions, provides insight into the problem of the electric dipole response and E1 strength distribution around particle separation threshold.

  5. Dem Extraction from CHANG'E-1 Lam Data by Surface Skinning Technology

    NASA Astrophysics Data System (ADS)

    Zhang, X.-B.; Zhang, W.-M.

    2011-08-01

    DEM is a digital model or 3-D representation of a terrain's surface and it is created from terrain elevation data. The main models for DEM extraction based on Lidar data or Laser Altimeter data currently use the idea that point cloud is scattered, such as regular grid model, TIN model and contour model. Essentially, in these above methods, the discrete points are interpolated into regular grid data and irregular grid data. In fact, point cloud generated by Laser Altimeter is not totally scattered, but have some regularity. In this paper, to utilize this regularity, the proposed method adopts surface skinning technology to generate DEM from Chang'E-1 Laser Altimeter data. The surface skinning technology is widely used in the field of mechanical engineering. Surface skinning is the process of passing a smooth surface through a set of curves called sectional curves, which, in general, may not be compatible. In the process of generating section line, a need for attention is that it needs to use curvature method to get a set of characteristic points, and these feature points were used to subdivide segment; the next step is generating several curves on some key places. These curves describe the shape of the curved surface. The last step is to generate a curved surface that through these curves. The result shows that, this idea is feasible, useful and it provides a novel way to generate accurate DEM.

  6. Lunar absolute reflectance as observed by Chang'E-1 Imaging Interferometer

    NASA Astrophysics Data System (ADS)

    Zhang, Jiang; Ling, ZongCheng; Liu, JianZhong; Wu, ZhongChen; Li, Bo; Ni, YuHeng

    2015-08-01

    Lunar absolute reflectance, which describes the fraction of solar radiation reflected by the Moon, is fundamental for the Chang'E-1 Imaging Interferometer (IIM) to map lunar mineralogical and elemental distributions. Recent observations made by the Spectral Irradiance Monitor (SIM) onboard the Solar Radiation and Climate Experiment (SORCE) spacecraft indicate that temporal variation in the solar radiation might have non-negligible influence on reflectance calculation, and the SIM measurements are different from the two previously used solar irradiances, i.e., ATLAS3 and Newkur. To provide reliable science results, we examined solar irradiance variability with the SIM daily observations, derived lunar absolute reflectances from the IIM 2A radiance with the SIM, ATLAS3 and Newkur data, and compared them with the Chandrayaan-1 Moon Mineralogy Mapper (M3), the Robotic Lunar Observatory (ROLO) and the Kaguya Multispectral Imager (MI) results. The temporal variability of the SIM solar irradiance is 0.25%-1.1% in the IIM spectral range, and less than 0.2% during the IIM observations. Nevertheless, the differences between the SIM measurements and the ATLAS3 and Newkur data can respectively rise up to 8% and 5% at particular IIM bands, resulting in discrepancy between which might affect compositional mapping. The IIM absolute reflectance we derived for the Moon using the SIM data, except for the last two bands, is consistent with the ROLO and the MI observations, although it is lower.

  7. Adenovirus E1A coding sequences that enable ras and pmt oncogenes to transform cultured primary cells.

    PubMed Central

    Zerler, B; Moran, B; Maruyama, K; Moomaw, J; Grodzicker, T; Ruley, H E

    1986-01-01

    Plasmids expressing partial adenovirus early region 1A (E1A) coding sequences were tested for activities which facilitate in vitro establishment (immortalization) of primary baby rat kidney cells and which enable the T24 Harvey ras-related oncogene and the polyomavirus middle T antigen (pmt) gene to transform primary baby rat kidney cells. E1A cDNAs expressing the 289- and 243-amino acid proteins expressed both E1A transforming functions. Mutant hrA, which encodes a 140-amino acid protein derived from the amino-terminal domain shared by the 289- and 243-amino acid proteins, enabled ras (but not pmt) to transform and facilitated in vitro establishment to a low, but detectable, extent. These studies suggest that E1A functions which collaborate with ras oncogenes and those which facilitate establishment are linked. Furthermore, E1A transforming functions are not associated with activities of the 289-amino acid E1A proteins required for efficient transcriptional activation of viral early region promoters. Images PMID:3022137

  8. Competitive Inhibition of Lysine Acetyltransferase 2B by a Small Motif of the Adenoviral Oncoprotein E1A.

    PubMed

    Shi, Shasha; Liu, Ke; Chen, Yanheng; Zhang, Shijun; Lin, Juanyu; Gong, Chenfang; Jin, Quanwen; Yang, Xiang-Jiao; Chen, Ruichuan; Ji, Zhiliang; Han, Aidong

    2016-07-01

    The adenovirus early region 1A (E1A) oncoprotein hijacks host cells via direct interactions with many key cellular proteins, such as KAT2B, also known as PCAF (p300/CBP associated factor). E1A binds the histone acetyltransferase (HAT) domain of KAT2B to repress its transcriptional activation. However, the molecular mechanism by which E1A inhibits the HAT activity is not known. Here we demonstrate that a short and relatively conserved N-terminal motif (cNM) in the intrinsically disordered E1A protein is crucial for KAT2B interaction, and inhibits its HAT activity through a direct competition with acetyl-CoA, but not its substrate histone H3. Molecular modeling together with a series of mutagenesis experiments suggests that the major helix of E1A cNM binds to a surface of the acetyl-CoA pocket of the KAT2B HAT domain. Moreover, transient expression of the cNM peptide is sufficient to inhibit KAT2B-specific H3 acetylation H3K14ac in vivo Together, our data define an essential motif cNM in N-terminal E1A as an acetyl-CoA entry blocker that directly associates with the entrance of acetyl-CoA binding pocket to block the HAT domain access to its cofactor. PMID:27143356

  9. In Silico Prediction of Human Sulfotransferase 1E1 Activity Guided by Pharmacophores from Molecular Dynamics Simulations.

    PubMed

    Rakers, Christin; Schumacher, Fabian; Meinl, Walter; Glatt, Hansruedi; Kleuser, Burkhard; Wolber, Gerhard

    2016-01-01

    Acting during phase II metabolism, sulfotransferases (SULTs) serve detoxification by transforming a broad spectrum of compounds from pharmaceutical, nutritional, or environmental sources into more easily excretable metabolites. However, SULT activity has also been shown to promote formation of reactive metabolites that may have genotoxic effects. SULT subtype 1E1 (SULT1E1) was identified as a key player in estrogen homeostasis, which is involved in many physiological processes and the pathogenesis of breast and endometrial cancer. The development of an in silico prediction model for SULT1E1 ligands would therefore support the development of metabolically inert drugs and help to assess health risks related to hormonal imbalances. Here, we report on a novel approach to develop a model that enables prediction of substrates and inhibitors of SULT1E1. Molecular dynamics simulations were performed to investigate enzyme flexibility and sample protein conformations. Pharmacophores were developed that served as a cornerstone of the model, and machine learning techniques were applied for prediction refinement. The prediction model was used to screen the DrugBank (a database of experimental and approved drugs): 28% of the predicted hits were reported in literature as ligands of SULT1E1. From the remaining hits, a selection of nine molecules was subjected to biochemical assay validation and experimental results were in accordance with the in silico prediction of SULT1E1 inhibitors and substrates, thus affirming our prediction hypotheses. PMID:26542807

  10. Structure of Hepatitis C Virus Envelope Glycoprotein E1 Antigenic Site 314-324 in Complex with Antibody IGH526.

    PubMed

    Kong, Leopold; Kadam, Rameshwar U; Giang, Erick; Ruwona, Tinashe B; Nieusma, Travis; Culhane, Jeffrey C; Stanfield, Robyn L; Dawson, Philip E; Wilson, Ian A; Law, Mansun

    2015-08-14

    Hepatitis C virus (HCV) is a positive-strand RNA virus within the Flaviviridae family. The viral "spike" of HCV is formed by two envelope glycoproteins, E1 and E2, which together mediate viral entry by engaging host receptors and undergoing conformational changes to facilitate membrane fusion. While E2 can be readily produced in the absence of E1, E1 cannot be expressed without E2 and few reagents, including monoclonal antibodies (mAbs), are available for study of this essential HCV glycoprotein. A human mAb to E1, IGH526, was previously reported to cross-neutralize different HCV isolates, and therefore, we sought to further characterize the IGH526 neutralizing epitope to obtain information for vaccine design. We found that mAb IGH526 bound to a discontinuous epitope, but with a major component corresponding to E1 residues 314-324. The crystal structure of IGH526 Fab with this E1 glycopeptide at 1.75Å resolution revealed that the antibody binds to one face of an α-helical peptide. Single mutations on the helix substantially lowered IGH526 binding but did not affect neutralization, indicating either that multiple mutations are required or that additional regions are recognized by the antibody in the context of the membrane-associated envelope oligomer. Molecular dynamics simulations indicate that the free peptide is flexible in solution, suggesting that it requires stabilization for use as a candidate vaccine immunogen. PMID:26135247

  11. Novel approach to differentiate subclades of varicella-zoster virus genotypes E1 and E2 in Germany.

    PubMed

    Schmidt-Chanasit, Jonas; Olschläger, Stephan; Bialonski, Alexandra; Heinemann, Patrick; Bleymehl, Karoline; Gross, Gerd; Günther, Stephan; Ulrich, Rainer G; Doerr, Hans Wilhelm

    2009-11-01

    Varicella-zoster virus (VZV) is the causative agent of chicken pox (varicella) in children and reactivation of VZV in elderly or immunocompromised persons can cause shingles (zoster). A subclade differentiation of the most prevalent VZV genotypes E1 and E2 in Germany was not possible with the current genotyping methods in use, but is highly important to understand the VZV molecular evolution in more detail and especially to follow up the routes of infection. Therefore the objective of this study was to develop a simple PCR-based method for differentiation of E1 and E2 subclades. Viral DNA was isolated from vesicle fluid samples of six selected German zoster patients and used to amplify nine complete open reading frames (ORFs) of the VZV genome by different PCR assays. Phylogenetic analysis was performed by a Bayesian approach. Based on the analysis of a total of nine ORFs, a 7482 bp stretch consisting of ORFs 5, 37 and 62 contained informative sites for identification of novel subclades E1a, E2a and E2b for VZV genotypes E1 and E2. Specific single nucleotide polymorphisms (SNPs) were demonstrated for subclades E2a and E2b within the ORFs 5, 37 and 62, whereas a subclade E1a-specific SNP was found in ORF 56. The classification of E1 and E2 subclades may facilitate a more exact and in-depth monitoring of the molecular evolution of VZV in Germany in the future. PMID:19712712

  12. FER-1, an enhancer of the ferritin H gene and a target of E1A-mediated transcriptional repression.

    PubMed Central

    Tsuji, Y; Akebi, N; Lam, T K; Nakabeppu, Y; Torti, S V; Torti, F M

    1995-01-01

    Ferritin, the major intracellular iron storage protein of eucaryotic cells, is regulated during inflammation and malignancy. We previously reported that transcription of the H subunit of ferritin (ferritin H) is negatively regulated by the adenovirus E1A oncogene in mouse NIH 3T3 fibroblasts (Y. Tsuji, E. Kwak, T. Saika, S. V. Torti, and F. M. Torti, J. Biol. Chem. 268:7270-7275, 1993). To elucidate the mechanism of transcriptional repression of the ferritin H gene by E1A, a series of deletions in the 5' flanking region of the mouse ferritin H gene were constructed, fused to the chloramphenicol acetyltransferase (CAT) gene, and transiently cotransfected into NIH 3T3 cells with an E1A expression plasmid. The results indicate that the E1A-responsive region is located approximately 4.1 kb 5' to the transcription initiation site of the ferritin H gene. Further analyses revealed that a 37-bp region, termed FER-1, is the target of E1A-mediated repression. This region also serves as an enhancer, augmenting ferritin H transcription independently of position and orientation. FER-1 was dissected into two component elements, i.e., a 22-bp dyad symmetry element and a 7-bp AP1-like sequence. Insertion of these DNA sequences into a ferritin H-CAT chimeric gene lacking an E1A-responsive region indicated that (i) the 22-bp dyad symmetry sequence by itself has no enhancer activity, (ii) the AP1-like sequence has moderate enhancer activity which is repressed by E1A, and (iii) the combination of the dyad symmetry element and the AP1-like sequence is required for maximal enhancer activity and repression by E1A. Gel retardation assays and cotransfection experiments with c-fos and c-jun expression vectors suggested that members of the Fos and Jun families bind to the AP1-like element of FER-1 and contribute to its regulation. In addition, gel retardation assays showed that E1A reduces the ability of nuclear proteins to bind to the AP1-like sequence without affecting the levels of

  13. Molecular and enzymatic characterization of alkaline lipase from Bacillus amyloliquefaciens E1PA isolated from lipid-rich food waste.

    PubMed

    Saengsanga, Thanakorn; Siripornadulsil, Wilailak; Siripornadulsil, Surasak

    2016-01-01

    Bacillus amyloliquefaciens E1PA is a lipase-producing strain that was originally isolated from lipid-rich food waste, and the production of its lipase was found to be induced by vegetable oils. The E1PA lipase was successfully expressed and secreted in a heterologous Escherichia coli host and was ultimately purified. The conserved pentapeptide motif Ala-His-Ser-Met-Gly was observed at positions 108-112. The purified recombinant lipase was stable over a pH range of 4.0-11.0 at 40 °C and exhibited maximal activity at pH 10. The recombinant E1PA lipase hydrolyzed a wide range of acyl esters (C4-C18). However, the highest activity (3.5 units mg(-1)) was observed when the p-nitrophenyl ester of myristate (C14) was used as a substrate. Compared to the lipases produced by Bacillus spp., the E1PA lipase displayed a structural molecular mass excluding the leader sequence (19.22 kDa) and a pI (9.82) that were similar to those reported for B. amyloliquefaciens lipases and lipase subfamily I.4 but that were quite distinct from those of lipase subfamily I.5 (approximately 43 kDa, pI 6). These results suggested that Bacillus lipases are closely related. Although the recombinant E1PA lipase digested only certain oils, the wild-type E1PA lipase degraded a variety of oils, including blended and re-used cooking oils. The recombinant and wild-type forms of the E1PA lipase were able to digest heterogeneous lipid-rich food waste at similar levels; this result suggests that this lipase can function even when it solely consists of its structural enzyme component. The enzyme exhibited lipid hydrolysis ability as either an intracellular domain of the recombinant protein or an extracellular domain secreted by the E1PA strain. However, the recombinant lipase showed higher activity than the wild-type E1PA lipase, indicating that the recombinant protein from E. coli possessed effective lipase activity. Thus, the inducible alkaline E1PA lipase exhibited the ability to act on a broad spectrum

  14. Construction of a colicin E1-R factor composite plasmid in vitro: means for amplification of deoxyribonucleic acid.

    PubMed Central

    Tanaka, T; Weisblum, B

    1975-01-01

    A composite plasmid has been constructed in vitro from colicin E1 factor (mass of 4.2 megadaltons [Md]) and nontransmissible resistance factor RSF 1010 (mass, 5.5. Md) deoxyribonucleic acids (DNAs) by the sequential action of Escherichia coli endonuclease (RI (Eco RI) and T4 phage DNA ligase on the covalently closed circular forms of the constituents. The composite plasmid was selected and amplified in vivo by sequential transformation of E. coli C600 with the ligated mixture and selection of transformants in medium containing streptomycin plus colicin E1, followed by amplification in the presence of chloramphenicol and purification of the extracted plasmid by dye-buoyant density gradient centrifugation in ethidium bromide-cesium chloride solution. Treatment of the composite plasmid with Eco RI yielded two fragments with mobilities corresponding to the linear forms of the parental plasmids, whereas Serratia marscesens endonuclease R (SmaR), which introduces a single scission in the colicin E1 factor but not in RSF 1010, convErted the composite plasmid to a single linear molecule (mass, 9.7 Md). Sequential degradation of colicin E1 factor with Sma R and Eco RI produced two fragments with masses of 3.5 and 0.7 Md; sequential degradation of RSF 1010 produced only one fragment (due to the cleavage with Eco RI), and sequential degradation of the composite plasmid produced the expected three fragments--an RSF 1010 Eco RI linear and the two expected products from the colicin E1 factor moiety. The composite plasmid conferred on the host cell resistance to streptomycin, sulfonamides, and colicin E1, but colicin E1 itself was not synthesized. In contrast, colicin E1 was synthesized by cells containing simultaneously both colicin E1 factor and RSF 1010 as separate entities. In the presence of chloramphenicol, the composite plasmid continued to replicate for 6 h. whereas replication of RSF 1010 and chromosomal DNA stopped within 2 h. Continued replication in the presence of

  15. Dechlorination and Decolorization of Organics in Bleach Plant E-1 Effluent by Photochemical Processes

    NASA Astrophysics Data System (ADS)

    Xie, Tianyan

    1994-01-01

    Photochemical study of the dechlorination of four model compounds, 4,5-dichloroguaiacol, 2,4,6-trichlorophenol, 2,3,4,5-tetrachlorophenol, and tetrachloroguaiacol in aqueous solutions under UV radiation was conducted using ArF (193 nm) and KrF (248 nm) excimer laser to explore the response of chlorinated phenolics present in the E_1 effluent from conventional chlorine bleaching of softwood kraft pulp towards photo-oxidation processes. Kinetic study show that the overall dechlorination reaction follow the first order rate law. The factors affecting the dechlorination were investigated. The quantum yield of chloride ion formation was found to be dependent on pH of the reaction mixture, and orignal chlorine content of the compounds. The effect of the substituents on the aromatic ring on the reactivity of the compounds was studied. The mechanism for the dechlorination was proposed involving homolytic photo-dissociation, heterolytic cleavage of carbon-chlorine bonds and substitution reactions of hydroxyl radicals. It was found that the dechlorination under formation to chloride is influenced by the amount of organically bound chlorine in the starting material. Dechlorination reaction favors high pH. Guaiacols more easily undergo dechlorination than phenols. Four fractions of high relative molecular-mass chloro-organics or polychlorinated oxylignin (PCOL) were isolated from an E_1 effluent by combination of ultrafiltration, and purified by repeated precipitation. The fractions were analysed by classical functional group analysis and spectrophotometric methods. The analytical data indicated that the major structural differences between PCOL fractions and kraft lignin preparations are with regard to the content of founctional groups such as carboxyl content, methoxyl and hydroxyl contents. In addition, IR, ^1H and ^{13 }C NMR spectral analyses revealed an almost complete absence of absorption attributable to aromatic structures in PCOLs. These results and others led to the

  16. Anik-E1 and E2 satellite failures of January 1994 revisited

    NASA Astrophysics Data System (ADS)

    Lam, H.-L.; Boteler, D. H.; Burlton, B.; Evans, J.

    2012-10-01

    The consecutive failures of the geosynchronous Anik-E1 communication satellite on January 20, 1994, and Anik-E2 about nine hours later on January 21 (both incidents occurred on January 20 local time) received considerable publicity because the malfunctions of the satellites disrupted television and computer data transmissions across Canada, as well as telephone services to remote northern communities for hours. This often-cited event is revisited here with materials not covered before. Using publicly available information, Anik-E failure details, media coverage, recovery effort and cost incurred are first presented. This is then followed by scrutiny of space weather conditions pertinent to the occurrences of the Anik-E upsets. We trace the space weather episode's inception on the Sun, propagation through interplanetary medium, and manifestation in magnetic field variations as well as in energetic electron flux increases, and its eventual impact on the Anik-Es. The genesis of the energetic electron enhancements that have been blamed for the satellite malfunctions is thus traceable via high-speed solar wind stream with Alfven wave fluctuations to a longitudinally wide coronal hole on the Sun. Furthermore, strong magnetic pulsations preceding electron flux peaks indicate Pc5 ULF (Ultra Low Frequency) waves as a probable acceleration mechanism for the energetic electron flux enhancement that resulted in the internal charging of the Anik-Es. The magnetic fluctuations may even be possible triggers for the subsequent discharge that caused the satellites to malfunction. This incident illustrates that satellite operators should be on alert for elevated high-energy electron environment that is above established thresholds, as specifications in satellite design may not render a satellite immune from internal charging.

  17. Mechanism of Effect of Prostaglandin E1 on Renal Water Excretion

    PubMed Central

    Berl, T.; Schrier, R. W.

    1973-01-01

    The present study examined the effect of prostaglandin E1 (PGE1) on renal water excretion in the anesthetized dog. Renal perfusion pressure was kept constant by adjustment of a suprarenal aortic clamp. In seven experiments the intravenous administration of PGE1 (7 μg/min) significantly increased urinary osmolality from 76 to 381 mosmol (P < 0.001) and decreased free water clearance from 2.2 to - 0.02 ml/min (P < 0.001). These effects promptly were reversed with cessation of the infusion. This antidiuretic effect occurred both in innervated and denervated kidneys and was not associated with changes in glomerular filtration rate, renal vascular resistance, or solute excretion rate. In 10 experiments in hypophysectomized dogs no effect of intravenous PGE1 on free water clearance and urinary osmolality was observed. The intrarenal administration of PGE1 (1 μg/min) to six water-loaded and two hypophysectomized dogs caused no systemic vascular changes and increased rather than decreased free water clearance (2.83 to 4.08 ml/min, P < 0.001). No significant change in urinary osmolality occurred. Glomerular filtration rate was not altered by the intrarenal infusion, but reversible changes in solute excretion rate and renal vascular resistance occurred. These results thus indicate that the antidiuresis associated with intravenous PGE1 is mediated primarily by the release of vasopressin rather than alterations in renal hemodynamics or solute excretion. The diuretic effect of intrarenal PGE1 occurs in the absence of vasopressin and is most likely mediated primarily by increased distal delivery of tubular fluid to the diluting segment of the nephron rather than changes in water permeability of the renal tubular epithelium. PMID:4683884

  18. Characterization of the Self-Cleaving Effector Protein NopE1 of Bradyrhizobium japonicum ▿

    PubMed Central

    Schirrmeister, Jana; Friedrich, Lars; Wenzel, Mandy; Hoppe, Markus; Wolf, Christine; Göttfert, Michael; Zehner, Susanne

    2011-01-01

    NopE1 is a type III-secreted protein of the symbiont Bradyrhizobium japonicum which is expressed in nodules. In vitro it exhibits self-cleavage in a duplicated domain of unknown function (DUF1521) but only in the presence of calcium. Here we show that either domain is self-sufficient for cleavage. An exchange of the aspartic acid residue at the cleavage site with asparagine prevented cleavage; however, cleavage was still observed with glutamic acid at the same position, indicating that a negative charge at the cleavage site is sufficient. Close to each cleavage site, an EF-hand-like motif is present. A replacement of one of the conserved aspartic acid residues with alanine prevented cleavage at the neighboring site. Except for EDTA, none of several protease inhibitors blocked cleavage, suggesting that a known protease-like mechanism is not involved in the reaction. In line with this, the reaction takes place within a broad pH and temperature range. Interestingly, magnesium, manganese, and several other divalent cations did not induce cleavage, indicating a highly specific calcium-binding site. Based on results obtained by blue-native gel electrophoresis, it is likely that the uncleaved protein forms a dimer and that the fragments of the cleaved protein oligomerize. A database search reveals that the DUF1521 domain is present in proteins encoded by Burkholderia phytofirmans PsNJ (a plant growth-promoting betaproteobacterium) and Vibrio coralliilyticus ATCC BAA450 (a pathogenic gammaproteobacterium). Obviously, this domain is more widespread in proteobacteria, and it might contribute to the interaction with hosts. PMID:21642459

  19. Nitric Oxide-Induced Autophagy in MC3T3-E1 Cells is Associated with Cytoprotection via AMPK Activation

    PubMed Central

    Yang, Jung Yoon; Park, Min Young; Park, Sam Young; Yoo, Hong Il; Kim, Min Seok; Kim, Jae Hyung

    2015-01-01

    Nitric oxide (NO) is important in the regulation of bone remodeling, whereas high concentration of NO promotes cell death of osteoblast. However, it is not clear yet whether NO-induced autophagy is implicated in cell death or survival of osteoblast. The present study is aimed to examine the role of NO-induced autophagy in the MC3T3-E1 cells and their underlying molecular mechanism. The effect of sodium nitroprusside (SNP), an NO donor, on the cytotoxicity of the MC3T3-E1 cells was determined by MTT assay and expression of apoptosis or autophagy associated molecules was evaluated by western blot analysis. The morphological observation of autophagy and apoptosis by acridine orange stain and TUNEL assay were performed, respectively. Treatment of SNP decreased the cell viability of the MC3T3-E1 cells in dose- and time-dependent manner. SNP increased expression levels of p62, ATG7, Beclin-1 and LC3-II, as typical autophagic markers and augmented acidic autophagolysosomal vacuoles, detected by acridine orange staining. However, pretreatment with 3-methyladenine (3MA), the specific inhibitor for autophagy, decreased cell viability, whereas increased the cleavage of PARP and caspase-3 in the SNP-treated MC3T3-E1 cells. AMP-activated protein kinase (AMPK), a major autophagy regulatory kinase, was activated in SNP-treated MC3T3-E1 cells. In addition, pretreatment with compound C, an inhibitor of AMPK, decreased cell viability, whereas increased the number of apoptotic cells, cleaved PARP and caspase-3 levels compared to those of SNP-treated MC3T3-E1 cells. Taken together, it is speculated that NO-induced autophagy functions as a survival mechanism via AMPK activation against apoptosis in the MC3T3-E1 cells. PMID:26557017

  20. Structural Dissection of a Gating Mechanism Preventing Misactivation of Ubiquitin by NEDD8’s E1

    SciTech Connect

    Souphron,J.; Waddell, M.; Paydar, A.; Tokgöz-Gromley, Z.; Roussel, M.; Schulman, B.

    2008-01-01

    Post-translational covalent modification by ubiquitin and ubiquitin-like proteins (UBLs) is a major eukaryotic mechanism for regulating protein function. In general, each UBL has its own E1 that serves as the entry point for a cascade. The E1 first binds the UBL and catalyzes adenylation of the UBL's C-terminus, prior to promoting UBL transfer to a downstream E2. Ubiquitin's Arg 72, which corresponds to Ala72 in the UBL NEDD8, is a key E1 selectivity determinant: swapping ubiquitin and NEDD8 residue 72 identity was shown previously to swap their E1 specificity. Correspondingly, Arg190 in the UBA3 subunit of NEDD8's heterodimeric E1 (the APPBP1-UBA3 complex), which corresponds to a Gln in ubiquitin's E1 UBA1, is a key UBL selectivity determinant. Here, we dissect this specificity with biochemical and X-ray crystallographic analysis of APPBP1-UBA3-NEDD8 complexes in which NEDD8's residue 72 and UBA3's residue 190 are substituted with different combinations of Ala, Arg, or Gln. APPBP1-UBA3's preference for NEDD8's Ala72 appears to be indirect, due to proper positioning of UBA3's Arg190. By contrast, our data are consistent with direct positive interactions between ubiquitin's Arg72 and an E1's Gln. However, APPBP1-UBA3's failure to interact with a UBL having Arg72 is not due to a lack of this favorable interaction, but rather arises from UBA3's Arg190 acting as a negative gate. Thus, parallel residues from different UBL pathways can utilize distinct mechanisms to dictate interaction selectivity, and specificity can be amplified by barriers that prevent binding to components of different conjugation cascades.

  1. Immunological Characterization of a Modified Vaccinia Virus Ankara Vector Expressing the Human Papillomavirus 16 E1 Protein

    PubMed Central

    Remy-Ziller, Christelle; Germain, Claire; Spindler, Anita; Hoffmann, Chantal; Silvestre, Nathalie; Rooke, Ronald; Bonnefoy, Jean-Yves

    2014-01-01

    Women showing normal cytology but diagnosed with a persistent high-risk human papillomavirus (HR-HPV) infection have a higher risk of developing high-grade cervical intraepithelial neoplasia and cervical cancer than noninfected women. As no therapeutic management other than surveillance is offered to these women, there is a major challenge to develop novel targeted therapies dedicated to the treatment of these patients. As such, E1 and E2 antigens, expressed early in the HPV life cycle, represent very interesting candidates. Both proteins are necessary for maintaining coordinated viral replication and gene synthesis during the differentiation process of the epithelium and are essential for the virus to complete its normal and propagative replication cycle. In the present study, we evaluated a new active targeted immunotherapeutic, a modified vaccinia virus Ankara (MVA) vector containing the E1 sequence of HPV16, aimed at inducing cellular immune responses with the potential to help and clear persistent HPV16-related infection. We carried out an extensive comparative time course analysis of the cellular immune responses induced by different schedules of immunization in C57BL/6 mice. We showed that multiple injections of MVA-E1 allowed sustained HPV16 E1-specific cellular immune responses in vaccinated mice and had no impact on the exhaustion phenotype of the generated HPV16 E1-specific CD8+ T cells, but they led to the differentiation of multifunctional effector T cells with high cytotoxic capacity. This study provides proof of concept that an MVA expressing HPV16 E1 can induce robust and long-lasting E1-specific responses and warrants further development of this candidate. PMID:24307238

  2. Immunological characterization of a modified vaccinia virus Ankara vector expressing the human papillomavirus 16 E1 protein.

    PubMed

    Remy-Ziller, Christelle; Germain, Claire; Spindler, Anita; Hoffmann, Chantal; Silvestre, Nathalie; Rooke, Ronald; Bonnefoy, Jean-Yves; Préville, Xavier

    2014-02-01

    Women showing normal cytology but diagnosed with a persistent high-risk human papillomavirus (HR-HPV) infection have a higher risk of developing high-grade cervical intraepithelial neoplasia and cervical cancer than noninfected women. As no therapeutic management other than surveillance is offered to these women, there is a major challenge to develop novel targeted therapies dedicated to the treatment of these patients. As such, E1 and E2 antigens, expressed early in the HPV life cycle, represent very interesting candidates. Both proteins are necessary for maintaining coordinated viral replication and gene synthesis during the differentiation process of the epithelium and are essential for the virus to complete its normal and propagative replication cycle. In the present study, we evaluated a new active targeted immunotherapeutic, a modified vaccinia virus Ankara (MVA) vector containing the E1 sequence of HPV16, aimed at inducing cellular immune responses with the potential to help and clear persistent HPV16-related infection. We carried out an extensive comparative time course analysis of the cellular immune responses induced by different schedules of immunization in C57BL/6 mice. We showed that multiple injections of MVA-E1 allowed sustained HPV16 E1-specific cellular immune responses in vaccinated mice and had no impact on the exhaustion phenotype of the generated HPV16 E1-specific CD8⁺ T cells, but they led to the differentiation of multifunctional effector T cells with high cytotoxic capacity. This study provides proof of concept that an MVA expressing HPV16 E1 can induce robust and long-lasting E1-specific responses and warrants further development of this candidate. PMID:24307238

  3. Mouse osteoblastic cell line (MC3T3-E1) expresses extracellular calcium (Ca2+o)-sensing receptor and its agonists stimulate chemotaxis and proliferation of MC3T3-E1 cells

    NASA Technical Reports Server (NTRS)

    Yamaguchi, T.; Chattopadhyay, N.; Kifor, O.; Butters, R. R. Jr; Sugimoto, T.; Brown, E. M.; O'Malley, B. W. (Principal Investigator)

    1998-01-01

    The calcium-sensing receptor (CaR) is a G protein-coupled receptor that plays key roles in extracellular calcium ion (Ca2+o) homeostasis in parathyroid gland and kidney. Osteoblasts appear at sites of osteoclastic bone resorption during bone remodeling in the "reversal" phase following osteoclastic resorption and preceding bone formation. Bone resorption produces substantial local increases in Ca2+o that could provide a signal for osteoblasts in the vicinity, leading us to determine whether such osteoblasts express the CaR. In this study, we used the mouse osteoblastic, clonal cell line MC3T3-E1. Both immunocytochemistry and Western blot analysis, using an antiserum specific for the CaR, detected CaR protein in MC3T3-E1 cells. We also identified CaR transcripts in MC3T3-E1 cells by Northern analysis using a CaR-specific riboprobe and by reverse transcription-polymerase chain reaction with CaR-specific primers, followed by nucleotide sequencing of the amplified products. Exposure of MC3T3-E1 cells to high Ca2+o (up to 4.8 mM) or the polycationic CaR agonists, neomycin and gadolinium (Gd3+), stimulated both chemotaxis and DNA synthesis in MC3T3-E1 cells. Therefore, taken together, our data strongly suggest that the osteoblastic cell line MC3T3-E1 possesses both CaR protein and mRNA very similar, if not identical, to those in parathyroid and kidney. Furthermore, the CaR in these osteoblasts could play a key role in regulating bone turnover by stimulating the proliferation and migration of such cells to sites of bone resorption as a result of local release of Ca2+o.

  4. Human Papillomavirus Type 31b E1 and E2 Transcript Expression Correlates with Vegetative Viral Genome Amplification

    PubMed Central

    Ozbun, Michelle A.; Meyers, Craig

    2013-01-01

    Human papillomavirus (HPV) genome replication is dependent on the expression of E1 and E2 proteins. The organotypic (raft) culture system was used to investigate changes in viral early gene expression and vegetative genome replication during the complete life cycle of HPV type 31b (HPV31b). We have previously shown the synthesis of HPV31b viral particles as early as 10 days of growth of CIN-612 9E raft tissues (Ozbun, M. A., and Meyers, C (1997) J. Virol. 71, 5161–5172). In the present study, we investigated the structures and temporal expression levels of HPV31b G1 and E2 transcripts, as well as the replication of the viral genome during the viral life cycle. The amplification state of the HPV31b genome was maximal at 10 days of raft tissue growth. Furthermore, the expression levels of E1 and E2 RNAs correlated with vegetative viral DNA replication. Levels of E1- and E2-specific transcripts were dissimilar throughout the viral life cycle. E2 RNA levels remained relatively constant, whereas E1 RNA levels were upregulated during the maximal amplification of viral genomes and the biosynthesis of virions. These data indicate that E1 may be the major regulator of viral genome amplification in preparation for DNA packaging and virion morphogenesis. PMID:9721231

  5. Endophytic Bacillus subtilis Strain E1R-J Is a Promising Biocontrol Agent for Wheat Powdery Mildew

    PubMed Central

    Gong, Yufei; Huo, Yunxia; Han, Qingmei; Kang, Zhensheng; Huang, Lili

    2015-01-01

    In this study, the biocontrol efficacies of 14 endophytic bacterial strains were tested against Blumeria graminis f. sp. tritici (Bgt) in pot experiments under greenhouse conditions. Bacillus subtilis strain E1R-j significantly reduced disease index and exhibited the best control (90.97%). When different formulations of E1R-j were sprayed 24 h before Bgt inoculation, fermentation liquid without bacterial cell and crude protein suspension displayed the similar effects; and they reduced disease index more than bacterial cell suspension (109 cfu mL−1) and fermentation liquid without protein. The control effects were not significantly different between 1011 and 109 cfu mL−1 of bacterial cell suspension but were higher than 107 cfu mL−1. Further observations showed that conidial germination and appressorial formation of Bgt were retarded by spraying E1R-j 24 h before Bgt inoculation. Compared with the water check, conidial germination and appressorial formation were decreased by 43.3% and 42.7%, respectively. In the treatment with E1R-j, the number of houstoria significantly reduced and the speed of mycelial extension was slowed down in the wheat leaves. Scanning electron microscopy observation revealed that E1R-j significantly suppressed the conidial germination and caused rupture and deformation of germ tubes. On the surface of wheat leaves, mycelia and conidiophores became shrinking. PMID:25759819

  6. Interaction of the Dr1 inhibitory factor with the TATA binding protein is disrupted by adenovirus E1A.

    PubMed Central

    Kraus, V B; Inostroza, J A; Yeung, K; Reinberg, D; Nevins, J R

    1994-01-01

    Past experiments have shown that the adenovirus E1A12S product activates the hsp70 promoter, dependent on the TATA element and dependent on N-terminal E1A sequences. Other experiments have identified a factor termed Dr1 that interacts with and inhibits the transcriptional activity of the TATA-binding protein (TBP). We now find that the E1A12S protein can disrupt the interaction of the Dr1 factor with the TATA-specific TBP factor, allowing the productive interaction of TBP with TFIIA. This E1A-mediated disruption is dependent on N-terminal sequences that are also essential for the TATA-dependent trans-activation of the hsp70 promoter. Moreover, we also find that Dr1 expression in transfected cells can inhibit transcription from the hsp70 promoter and that this can be overcome by coexpression of the wild-type E1A protein, dependent on N-terminal sequences. We conclude that the activation of hsp70 through the TATA element may be mechanistically similar to the activation of the E2 promoter via E2F, in each case involving a release of a transcription factor from an inactive complex. Images PMID:8022773

  7. CYP2E1 gene rs6413420 polymorphism was first found in the Bouyei ethnic group of China

    PubMed Central

    Liu, Wei; Zhou, Li; Wang, Hongju; Zheng, Bo; Wu, Desheng; Yang, Xifei; Liu, Jianjun

    2014-01-01

    Background: China is a multinational country. The relationship between gene polymorphisms of xenobiotic metabolizing enzymes and national ethnicity has not previously investigated among Chinese people. The aim of this study was to investigate distributions of CYP1A1 and CYP2E1 gene polymorphisms in five ethnic groups of China. Methods: 829 blood samples were collected from five ethnic groups (Han, Shui, Miao, Zhuang, Bouyei). Taqman-MGB probe was used in Real-time PCR to test the gene polymorphisms of CYP1A1 (rs1048943 and rs4646903) and CYP2E1 (rs2031920 and rs6413420). We further validate the SNP genotyping results through DNA sequencing. Results: The genotype distribution of all four SNPs was in accordance with Hardy-Weinberg equilibrium except the genotype distribution of rs4646903 in Han and Bouyei ethnic groups (p=0.013 and 0.0005, respectively). CYP2E1 gene rs6413420 polymorphism was first found in the Bouyei ethnic group in China. The results of DNA sequencing were entirely in line with the SNP genotyping assay. Conclusions: The CYP1A1 and CYP2E1 genetic polymorphisms were different in different ethnic groups in China. CYP2E1 gene rs6413420 polymorphism was first found in the Bouyei ethnic group of China. PMID:25419409

  8. Interaction Studies of Resolvin E1 Analog (RX-10045) with Efflux Transporters

    PubMed Central

    Cholkar, Kishore; Trinh, Hoang M.; Vadlapudi, Aswani Dutt; Wang, Zhiying; Pal, Dhananjay

    2015-01-01

    Abstract Purpose: Screening interactions of a resolvin E1 analog (RX-10045) with efflux transporters (P-glycoprotein [P-gp], multidrug resistance-associated protein [MRP2], and breast cancer-resistant protein [BCRP]). Methods: Madin-Darby canine kidney cells transfected with P-gp, MRP2, and BCRP genes were selected for this study. [3H]-Digoxin, [3H]-vinblastine and [3H]-abacavir were selected as model substrates for P-gp, MRP2, and BCRP. Uptake and permeability studies across cell monolayer in both apical to basal (AP–BL) and BL–AP of these substrates were conducted in the presence of specific efflux pump inhibitors and RX-10045. Cell viability studies were conducted with increasing concentrations of RX-10045. Results: Uptake studies showed a higher accumulation in the presence of inhibitors (GF120918 and ketoconazole for P-gp; MK571 for MRP2; and β-estradiol for BCRP) as well as RX-10045. Similarly, dose-dependent inhibition studies demonstrated higher accumulation of various substrates ([3H]-digoxin, [3H]-vinblastine, and [3H]-abacavir) in the presence of RX-10045. IC50 values of dose-dependent inhibition of RX-10045 for P-gp, MRP2, and BCRP were 239±11.2, 291±79.2, and 300±42 μM, respectively. Cell viability assay indicated no apparent toxicity up to 350 μM concentration. Enhanced permeability for model substrates was observed in the presence of RX-10045. Uptake studies in human corneal epithelial cells suggest that RX-10045 is a strong inhibitor of organic cation transporter-1 (OCT-1). Conclusions: In summary, the resolvin analog (RX-10045) was identified as a substrate/inhibitor for efflux transporters (MRP2 and BCRP). Also, RX-10045 appears to be a strong inhibitor/substrate of OCT-1. Novel formulation strategies such as nanoparticles, nanomicelles, and liposomes for circumventing efflux barriers and delivering higher drug concentrations leading to a higher therapeutic efficacy may be employed. PMID:25844889

  9. Tetra Primer ARMS PCR Optimization to Detect Single Nucleotide Polymorphisms of the CYP2E1 Gene.

    PubMed

    Suhda, Saihas; Paramita, Dewi Kartikawati; Fachiroh, Jajah

    2016-01-01

    Single nucleotide polymorphism (SNP) detection has been used extensively for genetic association studies of diseases including cancer. For mass, yet accurate and more economic SNP detection we have optimized tetra primer amplification refractory mutation system polymerase chain reaction (ARMS PCR) to detect three SNPs in the cytochrome P450 2E1 (CYP2E1) gene locus; i.e. rs3813865, rs2070672 and rs3813867. The optimization system strategies used were (1) designing inner and outer primers; (2) determining of their optimum primer concentration ratios; and (3) determining of the optimum PCR annealing temperature. The tetra primer ARMS PCR result could be directly observed using agarose gel electrophoresis. The method succesfully determined three SNPs in CYP2E1 locus, the results being consistent with validation using DNA sequencing and restriction fragment length polymorphisms (RFLP). PMID:27509930

  10. Identification of inhibitory component in cinnamon--O-methoxycinnamaldehyde inhibits CYP1A2 and CYP2E1-.

    PubMed

    Hasegawa, Atsushi; Yoshino, Masaki; Nakamura, Hiroyoshi; Ishii, Itsuko; Watanabe, Toshiko; Kiuchi, Masahiro; Ishikawa, Tsutomu; Ohmori, Shigeru; Kitada, Mitsukazu

    2002-01-01

    The Cinnamomi Cortex and Ephedra Herba were found to more strongly inhibit aminopyrine N-demethylation in rat liver microsomes compared to other constituents included in Sho-seiryu-to. The component inhibiting drug oxidations catalyzed by CYP1A2 and CYP2E1 was isolated from Cinnamomi Cortex, and was identified as o-methoxycinnamaldehyde (OMCA). When phenacetin and 4-nitrophenol were used as probe substrates for CYP1A2 and CYP2E1, respectively, the OMCA was shown to be a competitive inhibitor against CYP1A2 while it was a mixed type inhibitor against CYP2E1. The inhibitory effect of OMCA on 4-nitrophenol 2-hydroxylation (K(i)=6.3 microM) was somewhat potent compared to that observed on phenacetin O-deethylation (K(i)=13.7 microM) in rat liver microsomes. PMID:15618674

  11. The adenovirus E1A repression domain disrupts the interaction between the TATA binding protein and the TATA box in a manner reversible by TFIIB.

    PubMed Central

    Song, C Z; Loewenstein, P M; Toth, K; Tang, Q; Nishikawa, A; Green, M

    1997-01-01

    The human adenovirus E1A 243 amino acid oncoprotein possesses a transcription repression function that appears to be linked with its ability to induce cell cycle progression and to inhibit cell differentiation. The molecular mechanism of E1A repression has been poorly understood. Recently, we reported that the TATA binding protein (TBP) is a cellular target of E1A repression. Here we demonstrate that the interaction between TBP and the E1A repression domain is direct and specific. The TBP binding domain within E1A 243R maps to E1A N-terminal residues approximately 1 to 35 and is distinct from the TBP binding domain within conserved region 3 unique to the E1A 289R transactivator. An E1A protein fragment consisting of only the E1A N-terminal 80 amino acids (E1A 1-80) and containing the E1A repression function was found to block the interaction between TBP and the TATA box element as shown by gel mobility and DNase protection analysis. Interestingly, a preformed TBP-TATA box promoter complex can be dissociated by E1A 1-80. Further, TFIIB can prevent E1A disruption of TBP-TATA box interaction. TFIIB, like TBP, can overcome E1A repression of transcription in vitro. The ability of the E1A repression domain to block TBP interaction with the TATA box and the ability of TFIIB to reverse E1A disruption of the TBP-TATA box complex implies a mechanism for E1A repression distinct from those of known cellular repressors that target TBP. PMID:9121468

  12. Cytochrome P450 2E1 inhibition prevents hepatic carcinogenesis induced by diethylnitrosamine in alcohol-fed rats

    PubMed Central

    Ye, Qinyuan; Lian, Fuzhi; Chavez, Pollyanna R.G.; Chung, Jayong; Ling, Wenhua; Qin, Hua; Seitz, Helmut K.

    2012-01-01

    Chronic alcohol ingestion increases hepatic cytochrome P450 2E1 (CYP2E1), which is associated with hepatocarcinogenesis. We investigated whether treatment with chlormethiazole (CMZ), a CYP2E1 inhibitor, protects against alcohol-associated hepatic carcinogenesis in rats. Rats were fed either an ethanol liquid diet or a non-ethanol liquid diet, with or without CMZ for one and ten months. A single intraperitoneal injection of diethylnitrosamine (DEN, 20 mg/kg) was given to initiate hepatic carcinogenesis. CYP2E1 expression, inflammatory proteins, cell proliferation, protein-bound 4-HNE, etheno-DNA adducts, 8-hydroxy-2'-deoxyguanosine (8-OHdG), retinoid concentrations, and hepatic carcinogenesis were examined. Ethanol feeding for 1 month with DEN resulted in significantly increased hepatic CYP2E1 levels and increased nuclear accumulation of NF-κB protein and TNF-α expression, which were associated with increased cyclin D1 expression and p-GST positive altered hepatic foci. All of these changes induced by ethanol feeding were significantly inhibited by the one month CMZ treatment. At 10-months of treatment, hepatocellular adenomas were detected in ethanol-fed rats only, but neither in control rats nor in animals receiving ethanol and CMZ. The 8-OHdG formation was found to be significantly increased in ethanol fed animals and normalized with CMZ treatment. In addition, alcohol-reduced hepatic retinol and retinoic acid concentrations were restored by CMZ treatment to normal levels in the rats at 10 months of treatment. These data demonstrate that the inhibition of ethanol-induced CYP2E1 as a key pathogenic factor can counteract the tumor-promoting action of ethanol by decreasing TNF-α expression, NF-κB activation, and oxidative DNA damage as well as restoring normal hepatic levels of retinoic acid in DEN-treated rats. PMID:23543859

  13. Incomplete LPS Core-Specific Felix01-Like Virus vB_EcoM_VpaE1.

    PubMed

    Šimoliūnas, Eugenijus; Vilkaitytė, Monika; Kaliniene, Laura; Zajančkauskaitė, Aurelija; Kaupinis, Algirdas; Staniulis, Juozas; Valius, Mindaugas; Meškys, Rolandas; Truncaitė, Lidija

    2015-12-01

    Bacteriophages represent a valuable source for studying the mechanisms underlying virus-host interactions. A better understanding of the host-specificity of viruses at the molecular level can promote various phage applications, including bacterial diagnostics, antimicrobial therapeutics, and improve methods in molecular biology. In this study, we describe the isolation and characterization of a novel coliphage, vB_EcoM_VpaE1, which has different host specificity than its relatives. Morphology studies, coupled with the results of genomic and proteomic analyses, indicate that vB_EcoM_VpaE1 belongs to the newly proposed genus Felix01likevirus in the family Myoviridae. The genus Felix01likevirus comprises a group of highly similar phages that infect O-antigen-expressing Salmonella and Escherichia coli (E. coli) strains. Phage vB_EcoM_VpaE1 differs from the rest of Felix01-like viruses, since it infects O-antigen-deficient E. coli strains with an incomplete core lipopolysaccharide (LPS). We show that vB_EcoM_VpaE1 can infect mutants of E. coli that contain various truncations in their LPS, and can even recognize LPS that is truncated down to the inner-core oligosaccharide, showing potential for the control of rough E. coli strains, which usually emerge as resistant mutants upon infection by O-Ag-specific phages. Furthermore, VpaE1 can replicate in a wide temperature range from 9 to 49 °C, suggesting that this virus is well adapted to harsh environmental conditions. Since the structural proteins of such phages tend to be rather robust, the receptor-recognizing proteins of VpaE1 are an attractive tool for application in glycan analysis, bacterial diagnostics and antimicrobial therapeutics. PMID:26633460

  14. Cytochrome P450-2E1 promotes aging-related hepatic steatosis, apoptosis and fibrosis through increased nitroxidative stress.

    PubMed

    Abdelmegeed, Mohamed A; Choi, Youngshim; Ha, Seung-Kwon; Song, Byoung-Joon

    2016-02-01

    The role of ethanol-inducible cytochrome P450-2E1 (CYP2E1) in promoting aging-dependent hepatic disease is unknown and thus was investigated in this study. Young (7 weeks) and aged female (16 months old) wild-type (WT) and Cyp2e1-null mice were used in this study to evaluate age-dependent changes in liver histology, steatosis, apoptosis, fibrosis and many nitroxidative stress parameters. Liver histology showed that aged WT mice exhibited markedly elevated hepatocyte vacuolation, ballooning degeneration, and inflammatory cell infiltration compared to all other groups. These changes were accompanied with significantly higher hepatic triglyceride and serum cholesterol in aged WT mice although serum ALT and insulin resistance were not significantly altered. Aged WT mice showed the highest rates of hepatocyte apoptosis and hepatic fibrosis. Further, the highest levels of hepatic hydrogen peroxide, lipid peroxidation, protein carbonylation, nitration, and oxidative DNA damage were observed in aged WT mice. These increases in the aged WT mice were accompanied by increased levels of mitochondrial nitroxidative stress and alteration of mitochondrial complex III and IV proteins in aged WT mice, although hepatic ATP levels seems to be unchanged. In contrast, the aging-related nitroxidative changes were very low in aged Cyp2e1-null mice. These results suggest that CYP2E1 is important in causing aging-dependent hepatic steatosis, apoptosis and fibrosis possibly through increasing nitroxidative stress and that CYP2E1 could be a potential target for translational research in preventing aging-related liver disease. PMID:26703967

  15. Cytochrome P450 2E1 inhibition prevents hepatic carcinogenesis induced by diethylnitrosamine in alcohol-fed rats.

    PubMed

    Ye, Qinyuan; Lian, Fuzhi; Chavez, Pollyanna R G; Chung, Jayong; Ling, Wenhua; Qin, Hua; Seitz, Helmut K; Wang, Xiang-Dong

    2012-12-01

    Chronic alcohol ingestion increases hepatic cytochrome P450 2E1 (CYP2E1), which is associated with hepatocarcinogenesis. We investigated whether treatment with chlormethiazole (CMZ), a CYP2E1 inhibitor, protects against alcohol-associated hepatic carcinogenesis in rats. Rats were fed either an ethanol liquid diet or a non-ethanol liquid diet, with or without CMZ for one and ten months. A single intraperitoneal injection of diethylnitrosamine (DEN, 20 mg/kg) was given to initiate hepatic carcinogenesis. CYP2E1 expression, inflammatory proteins, cell proliferation, protein-bound 4-HNE, etheno-DNA adducts, 8-hydroxy-2'-deoxyguanosine (8-OHdG), retinoid concentrations, and hepatic carcinogenesis were examined. Ethanol feeding for 1 month with DEN resulted in significantly increased hepatic CYP2E1 levels and increased nuclear accumulation of NF-κB protein and TNF-α expression, which were associated with increased cyclin D1 expression and p-GST positive altered hepatic foci. All of these changes induced by ethanol feeding were significantly inhibited by the one month CMZ treatment. At 10-months of treatment, hepatocellular adenomas were detected in ethanol-fed rats only, but neither in control rats nor in animals receiving ethanol and CMZ. The 8-OHdG formation was found to be significantly increased in ethanol fed animals and normalized with CMZ treatment. In addition, alcohol-reduced hepatic retinol and retinoic acid concentrations were restored by CMZ treatment to normal levels in the rats at 10 months of treatment. These data demonstrate that the inhibition of ethanol-induced CYP2E1 as a key pathogenic factor can counteract the tumor-promoting action of ethanol by decreasing TNF-α expression, NF-κB activation, and oxidative DNA damage as well as restoring normal hepatic levels of retinoic acid in DEN-treated rats. PMID:23543859

  16. Occupational Toluene Exposure Induces Cytochrome P450 2E1 mRNA Expression in Peripheral Lymphocytes

    PubMed Central

    Mendoza-Cantú, Ania; Castorena-Torres, Fabiola; de León, Mario Bermúdez; Cisneros, Bulmaro; López-Carrillo, Lizbeth; Rojas-García, Aurora E.; Aguilar-Salinas, Alberto; Manno, Maurizio; Albores, Arnulfo

    2006-01-01

    Print workers are exposed to organic solvents, of which the systemic toxicant toluene is a main component. Toluene induces expression of cytochrome P450 2E1 (CYP2E1), an enzyme involved in its own metabolism and that of other protoxicants, including some procarcinogens. Therefore, we investigated the association between toluene exposure and the CYP2E1 response, as assessed by mRNA content in peripheral lymphocytes or the 6-hydroxychlorzoxazone (6OH-CHZ)/chlorzoxazone (CHZ) quotient (known as CHZ metabolic ratio) in plasma, and the role of genotype (5′-flanking region RsaI/PstI polymorphic sites) in 97 male print workers. The geometric mean (GM) of toluene concentration in the air was 52.80 ppm (10–760 ppm); 54% of the study participants were exposed to toluene concentrations that exceeded the maximum permissible exposure level (MPEL). The GM of urinary hippuric acid at the end of a work shift (0.041 g/g creatinine) was elevated relative to that before the shift (0.027 g/g creatinine; p < 0.05). The GM of the CHZ metabolic ratio was 0.33 (0–9.3), with 40% of the subjects having ratios below the GM. However, the average CYP2E1 mRNA level in peripheral lymphocytes was 1.07 (0.30–3.08), and CYP2E1 mRNA levels within subjects correlated with the toluene exposure ratio (environmental toluene concentration:urinary hippuric acid concentration) (p = 0.014). Genotype did not alter the association between the toluene exposure ratio and mRNA content. In summary, with further validation, CYP2E1 mRNA content in peripheral lymphocytes could be a sensitive and noninvasive biomarker for the continuous monitoring of toluene effects in exposed persons. PMID:16581535

  17. Evaluation of hepatic cytochrome P4502E1 in the species-dependent bioactivation of 4-vinylcyclohexene.

    PubMed

    Fontaine, S M; Hoyer, P B; Sipes, I G

    2001-07-13

    4-Vinyl-1-cyclohexene (VCH), is converted by multiple forms of cytochrome P450 (CYP) to two monoepoxides (4-vinyl-1-cyclohexene 1,2-epoxide [VCH-1,2-epoxide], 4-vinyl-1-cyclohexene 7,8-epoxide [VCH-7,8-epoxide]), and 4-vinyl-1-cyclohexene diepoxide (VCD). A greater degree of formation of these epoxides by female B6C3F1 mice as compared to Fischer 344 rats correlates with the ovarian toxicity observed only in the mice. Understanding which isoforms of CYP are involved in VCH bioactivation will better explain the species-dependent ovotoxicity of VCH. Present studies focus on the role of CYP2E1, as this isoform is responsible for the bioactivation of several structurally related small molecular weight compounds, including 1,3-butadiene. Hepatic microsomes prepared from either mice or rats pretreated with the CYP inducer acetone demonstrated 2-fold increases in the formation of VCH-1,2-epoxide. However, incubations with microsomes from cyp2e1-deficient mice compared to those from wild type mice revealed no differences in the rates of bioactivation of VCH to the monoepoxides. Since repeated exposure to VCH is required for VCH-induced ovotoxicity, rodents were dosed with VCH for 5 or 10 d to observe effects on the hepatic concentration of CYP2E1 and/or associated activities. VCH pretreatment failed to increase the concentration of CYP2E1 or CYP2E1 activity in either species, as measured by immunoblotting analysis and p-nitrophenol hydroxylation. Based on these data, it is concluded that CYP2E1 does not play a role in the species differences between mice and rats in the bioactivation of VCH following repeated exposure to VCH. Other isoforms, such as those in CYP2A and CYP2B subfamilies, are likely involved in VCH bioactivation. PMID:11488405

  18. Incomplete LPS Core-Specific Felix01-Like Virus vB_EcoM_VpaE1

    PubMed Central

    Šimoliūnas, Eugenijus; Vilkaitytė, Monika; Kaliniene, Laura; Zajančkauskaitė, Aurelija; Kaupinis, Algirdas; Staniulis, Juozas; Valius, Mindaugas; Meškys, Rolandas; Truncaitė, Lidija

    2015-01-01

    Bacteriophages represent a valuable source for studying the mechanisms underlying virus-host interactions. A better understanding of the host-specificity of viruses at the molecular level can promote various phage applications, including bacterial diagnostics, antimicrobial therapeutics, and improve methods in molecular biology. In this study, we describe the isolation and characterization of a novel coliphage, vB_EcoM_VpaE1, which has different host specificity than its relatives. Morphology studies, coupled with the results of genomic and proteomic analyses, indicate that vB_EcoM_VpaE1 belongs to the newly proposed genus Felix01likevirus in the family Myoviridae. The genus Felix01likevirus comprises a group of highly similar phages that infect O-antigen-expressing Salmonella and Escherichia coli (E. coli) strains. Phage vB_EcoM_VpaE1 differs from the rest of Felix01-like viruses, since it infects O-antigen-deficient E. coli strains with an incomplete core lipopolysaccharide (LPS). We show that vB_EcoM_VpaE1 can infect mutants of E. coli that contain various truncations in their LPS, and can even recognize LPS that is truncated down to the inner-core oligosaccharide, showing potential for the control of rough E. coli strains, which usually emerge as resistant mutants upon infection by O-Ag-specific phages. Furthermore, VpaE1 can replicate in a wide temperature range from 9 to 49 °C, suggesting that this virus is well adapted to harsh environmental conditions. Since the structural proteins of such phages tend to be rather robust, the receptor-recognizing proteins of VpaE1 are an attractive tool for application in glycan analysis, bacterial diagnostics and antimicrobial therapeutics. PMID:26633460

  19. Determination of human papillomavirus 16 physical status through E1/E6 and E2/E6 ratio analysis.

    PubMed

    Tsakogiannis, Dimitris; Kyriakopoulou, Zaharoula; Ruether, Irina Georgia Anna; Amoutzias, Grigoris D; Dimitriou, Tilemachos G; Diamantidou, Valentina; Kotsovassilis, Constantin; Markoulatos, Panayotis

    2014-12-01

    Human papillomavirus (HPV) 16 genome integration into the host chromosome is a crucial event during the life cycle of the virus and a major step towards carcinogenesis. The integration of HPV16 DNA promotes a constitutive high expression level of E6 and E7 oncoproteins, resulting in the extensive proliferation of the infected epithelial cells. In the present report the physical status of the HPV16 genome was studied, through determination of E1/E6 and E2/E6 DNA copy number ratios in 61 cervical samples of low- and high-grade malignancy and 8 cervical cancer samples, all of them associated with HPV16 infection. The selection of E1, E2 and E6 amplification target regions was performed according to the most prevalent deleted/disrupted sites of E1 and E2 genes. For this target selection we also considered the most conserved regions of E1, E2 and E6 genes among the same HPV16 isolates that were recently reported by our group. The analysis of HPV16 DNA form revealed a significant association among the mixed DNA forms in low-grade and high-grade malignancies, (χ(2), P<0.01). The comparative analysis of E1/E6 and E2/E6 in the same cervical samples provides an accurate picture of HPV16 DNA form and may reveal whether different HPV16 DNA integrants coexist in the same cervical sample or not. This study proposes that E1/E6 and E2/E6 ratios determine with accuracy the HPV16 DNA integration pattern and may predict multiple integration events in the examined sample, thus providing significant information about the progression of cervical dysplasia. PMID:25212758

  20. High glucose induces autophagy of MC3T3-E1 cells via ROS-AKT-mTOR axis.

    PubMed

    Wang, Xiaoju; Feng, Zhengping; Li, Jiling; Chen, Lixue; Tang, Weixue

    2016-07-01

    In the present study, we investigate the function of ROS-AKT-mTOR axis on the apoptosis, proliferation and autophagy of MC3T3-E1 cells, and the proliferation of MC3T3-E1 cells after autophagy inhibition under high glucose conditions. MC3T3-E1 cells cultured in vitro were divided into the following groups: normal control group, N-acetylcysteine (NAC) group, 11.0 mM high glucose group, 11.0 mM high glucose + NAC group, 22.0 mM high glucose group, 22.0 mM high glucose + NAC group, CQ group, 22.0 mM high glucose + CQ group, 3-MA group and 3-MA + 22.0 mM high glucose group. ROS production was measured by DCFH-DA fluorescent probe. Cell proliferation was measured by MTT assay. Cells in different groups were stained with Annexin V-FITC/PI, and then apoptosis rate was detected by flow cytometry. Nucleus morphology was observed under fluorescence microscope after being incubated with Honchest33258. Protein expression was measured using Western blotting and immunofluorescence. Cell apoptosis and proliferation in high glucose group were increased and decreased, respectively, in a dose-dependent manner. Autophagy was significantly induced in high glucose group, even though different concentration of glucose induced autophagy in different stages of autophagy. ROS production in MC3T3-E1 cells was remarkably increased in high glucose group, but not in a dose-dependent manner. NAC, as an antioxidant, reduced ROS production and ameliorated cell apoptosis, proliferation abnormity and autophagy caused by high glucose. Expression of p-AKT and p-mTOR proteins were dramatically decreased in high glucose group, and NAC reversed their expression. In addition, 3-MA, an inhibitor of autophagy, significantly decreased the proliferation of MC3T3-E1 cells. When cocultured with 22.0 mM glucose that induced autophagy, proliferation of MC3T3-E1 cells was not affected compared to 22.0 mM high glucose group. Our present findings reveal that high glucose affects apoptosis

  1. Ultrafast carrier relaxation through Auger recombination in the topological insulator B i1.5S b0.5T e1.7S e1.3

    NASA Astrophysics Data System (ADS)

    Onishi, Yoshito; Ren, Zhi; Segawa, Kouji; Kaszub, Wawrzyniec; Lorenc, Maciej; Ando, Yoichi; Tanaka, Koichiro

    2015-02-01

    Ultrafast carrier dynamics have great significance for our understanding of the transport properties of the surface state in topological insulator (TI) materials. We report midinfrared pump-probe measurements on the intrinsic TI material B i1.5S b0.5T e1.7S e1.3 and show that the change in photoinduced reflectivity can be decomposed into a fast negative part and a slow positive part. Calculations of the dielectric function made at various carrier temperatures and densities reveal that the fast negative component corresponds to the disappearance of the phase-space filling effect due to hot carriers around the probe energy and the decay component corresponds to the recombination of carriers near the band edge. The ratio of the fast negative component to the slow positive component is larger in the excitations conducted at the higher carrier densities, which suggests that the carrier temperature increases through Auger recombination. A qualitative analysis using rate equations reinforces this assumption, so we conclude that Auger recombination is the main cause of the population relaxation at carrier densities higher than 1018c m-3 and that we determined the Auger coefficient for B i1.5S b0.5T e1.7S e1.3 as C =0.4 ×10-26c m6/s .

  2. Construction of a cellulase hyper-expression system in Trichoderma reesei by promoter and enzyme engineering

    PubMed Central

    2012-01-01

    Background Trichoderma reesei is the preferred organism for producing industrial cellulases. However, a more efficient heterologous expression system for enzymes from different organism is needed to further improve its cellulase mixture. The strong cbh1 promoter of T. reesei is frequently used in heterologous expression, however, the carbon catabolite repressor CREI may reduce its strength by binding to the cbh1 promoter at several binding sites. Another crucial point to enhance the production of heterologous enzymes is the stability of recombinant mRNA and the prevention of protein degradation within the endoplasmic reticulum, especially for the bacteria originated enzymes. In this study, the CREI binding sites within the cbh1 promoter were replaced with the binding sites of transcription activator ACEII and the HAP2/3/5 complex to improve the promoter efficiency. To further improve heterologous expression efficiency of bacterial genes within T. reesei, a flexible polyglycine linker and a rigid α-helix linker were tested in the construction of fusion genes between cbh1 from T. reesei and e1, encoding an endoglucanase from Acidothermus cellulolyticus. Results The modified promoter resulted in an increased expression level of the green fluorescent protein reporter by 5.5-fold in inducing culture medium and 7.4-fold in repressing culture medium. The fusion genes of cbh1 and e1 were successfully expressed in T. reesei under the control of promoter pcbh1m2. The higher enzyme activities and thermostability of the fusion protein with rigid linker indicated that the rigid linker might be more suitable for the heterologous expression system in T. reesei. Compared to the parent strain RC30-8, the FPase and CMCase activities of the secreted enzyme mixture from the corresponding transformant R1 with the rigid linker increased by 39% and 30% at 60°C, respectively, and the reduced sugar concentration in the hydrolysate of pretreated corn stover (PCS) was dramatically

  3. The new ENSEMBLES E1 mitigation scenario for future climate simulations

    NASA Astrophysics Data System (ADS)

    Royer, J.-F.; Lowe, J.; Johns, T.; van Vuuren, D.; Stehfest, E.; Denoblet-Ducoudré, N.; Boucher, O.; Rognerud, B.; Huebener, H.

    2009-04-01

    that equivalent to 450-ppm of CO2 concentration. This new stabilisation scenario (E1) has been developed with the latest version of the integrated impact assessment model IMAGE at Netherlands Environmental Assessment Agency (MNP), with improvements in the land use representation of the A1B scenario. Though the coupled climate models are directly forced by the concentrations of the well-mixed greenhouse gases (CO2, CH4, N2O, CFCs and other minor species) produced by the IMAGE model, it has however been necessary to make further adaptations, by using additional modelling tools, in order to produce the other forcing data needed by the GCMs. The 3-dimensional atmospheric fields for anthropogenic sulphate aerosols concentrations have been computed by a chemistry-transport model using the emissions from the IMAGE scenarios. The ozone concentrations have been computed by the chemistry-transport model from the University of Oslo. The land use maps over the period 1850-2000 have been derived from the LUCID project based on a combination of the crop dataset of Ramankutty and Foley and pasture from the HYDE dataset. They have been then merged with the land-use maps produced by the IMAGE scenarios in order to derive adapted land-use maps over the 21st century. The different forcings and some results of the "stream 2" ENSEMBLES scenarios are described and illustrated in this presentation.

  4. Analysis of the selective advantage conferred by a C-E1 fusion protein synthesized by rubella virus DI RNAs

    SciTech Connect

    Claus, Claudia; Tzeng, W.-P.; Liebert, Uwe Gerd; Frey, Teryl K.

    2007-12-05

    During serial passaging of rubella virus (RUB) in cell culture, the dominant species of defective-interfering RNA (DI) generated contains an in-frame deletion between the capsid protein (C) gene and E1 glycoprotein gene resulting in production of a C-E1 fusion protein that is necessary for the maintenance of the DI [Tzeng, W.P., Frey, T.K. (2006). C-E1 fusion protein synthesized by rubella virus DI RNAs maintained during serial passage. Virology 356 198-207.]. A BHK cell line stably expressing the RUB structural proteins was established which was used to package DIs into virus particles following transfection with in vitro transcripts from DI infectious cDNA constructs. Packaging of a DI encoding an in-frame C-GFP-E1 reporter fusion protein corresponding to the C-E1 fusion protein expressed in a native DI was only marginally more efficient than packaging of a DI encoding GFP, indicating that the C-E1 fusion protein did not function by enhancing packaging. However, infection with the DI encoding the C-GFP-E1 fusion protein (in the absence of wt RUB helper virus) resulted in formation of clusters of GFP-positive cells and the percentage of GFP-positive cells in the culture following infection remained relatively constant. In contrast, a DI encoding GFP did not form GFP-positive clusters and the percentage of GFP-positive cells declined by roughly half from 2 to 4 days post-infection. Cluster formation and sustaining the percentage of infected (GFP-positive) cells required the C part of the fusion protein, including the downstream but not the upstream of two arginine clusters (both of which are associated with RNA binding and association with mitochondrial p32 protein) and the E1 part through the transmembrane sequence, but not the C-terminal cytoplasmic tail. Among a collection of mutant DI constructs, cluster formation and sustaining infected cell percentage correlated with maintenance during serial passage with wt RUB. We hypothesize that cluster formation and

  5. 26 CFR 1.404(e)-1 - Contributions on behalf of a self-employed individual to or under a pension, annuity, or profit...

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ...; application of section 404(a) (8), (9), and (10) and section 404 (e) and (f). 1.404(e)-1 Section 1.404(e)-1...) INCOME TAXES (CONTINUED) Pension, Profit-Sharing, Stock Bonus Plans, Etc. § 1.404(e)-1 Contributions on... requirements of section 401; application of section 404(a) (8), (9), and (10) and section 404 (e) and (f)....

  6. 26 CFR 1.6655(e)-1 - Time and manner for making election under the Omnibus Budget Reconciliation Act of 1993.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... Omnibus Budget Reconciliation Act of 1993. 1.6655(e)-1 Section 1.6655(e)-1 Internal Revenue INTERNAL...) Additions to the Tax, Additional Amounts, and Assessable Penalties § 1.6655(e)-1 Time and manner for making election under the Omnibus Budget Reconciliation Act of 1993. (a) Description. Section 6655(e)(2)(C),...

  7. 26 CFR 1.6655(e)-1 - Time and manner for making election under the Omnibus Budget Reconciliation Act of 1993.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... Omnibus Budget Reconciliation Act of 1993. 1.6655(e)-1 Section 1.6655(e)-1 Internal Revenue INTERNAL... the Tax, Additional Amounts, and Assessable Penalties § 1.6655(e)-1 Time and manner for making election under the Omnibus Budget Reconciliation Act of 1993. (a) Description. Section 6655(e)(2)(C),...

  8. 26 CFR 1.1402(e)(1)-1 - Election by ministers, members of religious orders, and Christian Science practitioners for self...

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ... orders, and Christian Science practitioners for self-employment coverage. 1.1402(e)(1)-1 Section 1.1402(e)(1)-1 Internal Revenue INTERNAL REVENUE SERVICE, DEPARTMENT OF THE TREASURY (CONTINUED) INCOME TAX (CONTINUED) INCOME TAXES (CONTINUED) Tax on Self-Employment Income § 1.1402(e)(1)-1 Election by...

  9. 26 CFR 1.1402(e)(1)-1 - Election by ministers, members of religious orders, and Christian Science practitioners for self...

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... orders, and Christian Science practitioners for self-employment coverage. 1.1402(e)(1)-1 Section 1.1402(e)(1)-1 Internal Revenue INTERNAL REVENUE SERVICE, DEPARTMENT OF THE TREASURY (CONTINUED) INCOME TAX (CONTINUED) INCOME TAXES (CONTINUED) Tax on Self-Employment Income § 1.1402(e)(1)-1 Election by...

  10. 26 CFR 1.1402(e)(1)-1 - Election by ministers, members of religious orders, and Christian Science practitioners for self...

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ... orders, and Christian Science practitioners for self-employment coverage. 1.1402(e)(1)-1 Section 1.1402(e)(1)-1 Internal Revenue INTERNAL REVENUE SERVICE, DEPARTMENT OF THE TREASURY (CONTINUED) INCOME TAX (CONTINUED) INCOME TAXES (CONTINUED) Tax on Self-Employment Income § 1.1402(e)(1)-1 Election by...

  11. 26 CFR 1.1402(e)(1)-1 - Election by ministers, members of religious orders, and Christian Science practitioners for self...

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... orders, and Christian Science practitioners for self-employment coverage. 1.1402(e)(1)-1 Section 1.1402(e)(1)-1 Internal Revenue INTERNAL REVENUE SERVICE, DEPARTMENT OF THE TREASURY (CONTINUED) INCOME TAX (CONTINUED) INCOME TAXES Tax on Self-Employment Income § 1.1402(e)(1)-1 Election by ministers, members...

  12. 26 CFR 1.1402(e)(1)-1 - Election by ministers, members of religious orders, and Christian Science practitioners for self...

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    ... orders, and Christian Science practitioners for self-employment coverage. 1.1402(e)(1)-1 Section 1.1402(e)(1)-1 Internal Revenue INTERNAL REVENUE SERVICE, DEPARTMENT OF THE TREASURY (CONTINUED) INCOME TAX (CONTINUED) INCOME TAXES (CONTINUED) Tax on Self-Employment Income § 1.1402(e)(1)-1 Election by...

  13. 26 CFR 1.404(e)-1A - Contributions on behalf of a self-employed individual to or under a qualified pension, annuity...

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    ... individual to or under a qualified pension, annuity, or profit-sharing plan. 1.404(e)-1A Section 1.404(e)-1A...) INCOME TAXES (CONTINUED) Pension, Profit-Sharing, Stock Bonus Plans, Etc. § 1.404(e)-1A Contributions on... behalf of self-employed individuals described in subsections (a) (8) and (9), (e), and (f) of section...

  14. 26 CFR 1.404(e)-1 - Contributions on behalf of a self-employed individual to or under a pension, annuity, or profit...

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    ...; application of section 404(a) (8), (9), and (10) and section 404 (e) and (f). 1.404(e)-1 Section 1.404(e)-1...) INCOME TAXES (CONTINUED) Pension, Profit-Sharing, Stock Bonus Plans, Etc. § 1.404(e)-1 Contributions on... requirements of section 401; application of section 404(a) (8), (9), and (10) and section 404 (e) and (f)....

  15. 26 CFR 1.404(e)-1A - Contributions on behalf of a self-employed individual to or under a qualified pension, annuity...

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ... individual to or under a qualified pension, annuity, or profit-sharing plan. 1.404(e)-1A Section 1.404(e)-1A...) INCOME TAXES (CONTINUED) Pension, Profit-Sharing, Stock Bonus Plans, Etc. § 1.404(e)-1A Contributions on... behalf of self-employed individuals described in subsections (a) (8) and (9), (e), and (f) of section...

  16. 26 CFR 1.404(e)-1 - Contributions on behalf of a self-employed individual to or under a pension, annuity, or profit...

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ...; application of section 404(a) (8), (9), and (10) and section 404 (e) and (f). 1.404(e)-1 Section 1.404(e)-1...) INCOME TAXES (CONTINUED) Pension, Profit-Sharing, Stock Bonus Plans, Etc. § 1.404(e)-1 Contributions on... requirements of section 401; application of section 404(a) (8), (9), and (10) and section 404 (e) and (f)....

  17. 26 CFR 1.404(e)-1 - Contributions on behalf of a self-employed individual to or under a pension, annuity, or profit...

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ...; application of section 404(a) (8), (9), and (10) and section 404 (e) and (f). 1.404(e)-1 Section 1.404(e)-1...) INCOME TAXES (CONTINUED) Pension, Profit-Sharing, Stock Bonus Plans, Etc. § 1.404(e)-1 Contributions on... requirements of section 401; application of section 404(a) (8), (9), and (10) and section 404 (e) and (f)....

  18. 26 CFR 1.404(e)-1A - Contributions on behalf of a self-employed individual to or under a qualified pension, annuity...

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ... individual to or under a qualified pension, annuity, or profit-sharing plan. 1.404(e)-1A Section 1.404(e)-1A...) INCOME TAXES (CONTINUED) Pension, Profit-Sharing, Stock Bonus Plans, Etc. § 1.404(e)-1A Contributions on... behalf of self-employed individuals described in subsections (a) (8) and (9), (e), and (f) of section...

  19. 26 CFR 1.417(e)-1T - Restrictions and valuations of distributions from plans subject to sections 401(a)(11) and 417...

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... 26 Internal Revenue 5 2010-04-01 2010-04-01 false Restrictions and valuations of distributions from plans subject to sections 401(a)(11) and 417. (Temporary) 1.417(e)-1T Section 1.417(e)-1T Internal... TAXES Pension, Profit-Sharing, Stock Bonus Plans, Etc. § 1.417(e)-1T Restrictions and valuations...

  20. A sequence that affects the copy number and stability of pSW200 and ColE1.

    PubMed

    Wu, Ying-Chung; Liu, Shih-Tung

    2010-07-01

    Pantoea stewartii SW2 contains 13 plasmids. One of these plasmids, pSW200, has a replicon that resembles that of ColE1. This study demonstrates that pSW200 contains a 9-bp UP element, 5'-AAGATCTTC, which is located immediately upstream of the -35 box in the RNAII promoter. A transcriptional fusion study reveals that substituting this 9-bp sequence reduces the activity of the RNAII promoter by 78%. The same mutation also reduced the number of plasmid copies from 13 to 5, as well as the plasmid stability. When a similar sequence in a ColE1 derivative, pYCW301, is mutated, the copy number of the plasmid also declines from 34 to 16 per cell. Additionally, inserting this 9-bp sequence stabilizes an unstable pSW100 derivative, pSW142K, which also contains a replicon resembling that of ColE1, indicating the importance of this sequence in maintaining the stability of the plasmid. In conclusion, the 9-bp sequence upstream of the -35 box in the RNAII promoter is required for the efficient synthesis of RNAII and maintenance of the stability of the plasmids in the ColE1 family. PMID:20494993

  1. IGT Stack No. 6 (SDG{ampersand}E-1) Test Plan and Component Specification Document: Topical report, March 1996

    SciTech Connect

    1997-12-31

    The purpose of Stack-6 (SDG{ampersand}E-1) is to scale up and demonstrate the long term performance and endurance characteristics of the IMHEX stack design and the Generation No. 2 cell components (improved pore matching electrodes) in a 20 cell subscale stack test.

  2. 26 CFR 25.2523(e)-1 - Marital deduction; life estate with power of appointment in donee spouse.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... 26 Internal Revenue 14 2011-04-01 2010-04-01 true Marital deduction; life estate with power of... Deductions § 25.2523(e)-1 Marital deduction; life estate with power of appointment in donee spouse. (a) In... (whether or not in trust) and the spouse is entitled for life to all the income from a specific portion...

  3. 26 CFR 25.2523(e)-1 - Marital deduction; life estate with power of appointment in donee spouse.

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    ... 26 Internal Revenue 14 2013-04-01 2013-04-01 false Marital deduction; life estate with power of... Deductions § 25.2523(e)-1 Marital deduction; life estate with power of appointment in donee spouse. (a) In... (whether or not in trust) and the spouse is entitled for life to all the income from a specific portion...

  4. 26 CFR 25.2523(e)-1 - Marital deduction; life estate with power of appointment in donee spouse.

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ... 26 Internal Revenue 14 2014-04-01 2013-04-01 true Marital deduction; life estate with power of... Deductions § 25.2523(e)-1 Marital deduction; life estate with power of appointment in donee spouse. (a) In... (whether or not in trust) and the spouse is entitled for life to all the income from a specific portion...

  5. 26 CFR 25.2523(e)-1 - Marital deduction; life estate with power of appointment in donee spouse.

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ... 26 Internal Revenue 14 2012-04-01 2012-04-01 false Marital deduction; life estate with power of... Deductions § 25.2523(e)-1 Marital deduction; life estate with power of appointment in donee spouse. (a) In... (whether or not in trust) and the spouse is entitled for life to all the income from a specific portion...

  6. 26 CFR 25.2523(e)-1 - Marital deduction; life estate with power of appointment in donee spouse.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... 26 Internal Revenue 14 2010-04-01 2010-04-01 false Marital deduction; life estate with power of... Deductions § 25.2523(e)-1 Marital deduction; life estate with power of appointment in donee spouse. (a) In... (whether or not in trust) and the spouse is entitled for life to all the income from a specific portion...

  7. 26 CFR 301.6503(e)-1 - Suspension of running of period of limitation; certain powers of appointment.

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    ... 26 Internal Revenue 18 2013-04-01 2013-04-01 false Suspension of running of period of limitation... Limitations Limitations on Assessment and Collection § 301.6503(e)-1 Suspension of running of period of... complied with, the running of the period of limitation for assessment or collection of any estate...

  8. 26 CFR 301.6503(e)-1 - Suspension of running of period of limitation; certain powers of appointment.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... 26 Internal Revenue 18 2010-04-01 2010-04-01 false Suspension of running of period of limitation... Limitations Limitations on Assessment and Collection § 301.6503(e)-1 Suspension of running of period of... complied with, the running of the period of limitation for assessment or collection of any estate...

  9. 26 CFR 301.6503(e)-1 - Suspension of running of period of limitation; certain powers of appointment.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... 26 Internal Revenue 18 2011-04-01 2011-04-01 false Suspension of running of period of limitation... Limitations Limitations on Assessment and Collection § 301.6503(e)-1 Suspension of running of period of... complied with, the running of the period of limitation for assessment or collection of any estate...

  10. 26 CFR 301.6503(e)-1 - Suspension of running of period of limitation; certain powers of appointment.

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ... 26 Internal Revenue 18 2012-04-01 2012-04-01 false Suspension of running of period of limitation... Limitations Limitations on Assessment and Collection § 301.6503(e)-1 Suspension of running of period of... complied with, the running of the period of limitation for assessment or collection of any estate...

  11. 26 CFR 301.6503(e)-1 - Suspension of running of period of limitation; certain powers of appointment.

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ... 26 Internal Revenue 18 2014-04-01 2014-04-01 false Suspension of running of period of limitation... Limitations Limitations on Assessment and Collection § 301.6503(e)-1 Suspension of running of period of... complied with, the running of the period of limitation for assessment or collection of any estate...

  12. On the ability of prostaglandin E1, and arachidonic acid to modulate experimentally induced oedema in the rat paw.

    PubMed Central

    Lewis, A J; Nelson, D J; Sugrue, M F

    1975-01-01

    1 Prostaglandins E1 and E2 but not prostaglandin F2alpha, arachidonic acid or linolenic acid, produced slight oedema when injected into the rat hindpaw. 2 Prostaglandin E1 potentiated hindpaw oedema produced by carrageenan, kaolin, bradykinin and trypsin but not that produced by 5-hydroxytryptamine (5-HT), histamine, dextran B or compound 48/80. Carrageenan- and bradykinin-induced paw oedemas were also potentiated by prostaglandin E2. Arachidonic acid potentiated responses to carrageenan and kaolin but not responses to bradykinin, trypsin, 5-HT, histamine, dextran B or compound 48/80. Linolenic acid did not potentiate hindpaw oedema induced by carrageenan. 3 Potentiation of carrageenan-induced oedema by prostaglandin E1 was not diminished by pretreatment with indomethacin, hydrocortisone or cyproheptadine. However, arachidonic acid potentiation of carrageenan oedema was reduced by pretreatment with non-steroidal anti-inflammatory drugs but not by anti-inflammatory steroids or by paracetamol. 4 The enhancement of the response to carrageenan and kaolin by prostaglandins E1, E2 and arachidonic acid is discussed in terms of kinin mediation. PMID:1182349

  13. Registration of DGE-3, a durum wheat disomic substitution line 1E(1B) involving a wheatgrass chromosome

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Durum wheat (Triticum turgidum L., 2n = 4x = 28; AABB genomes) alien disomic substitution 1E(1B) line DGE-3 (PI 665473) was developed by the U.S. Department of Agriculture – Agricultural Research Service, Northern Crop Science Lab, Cereal Crops Research Unit, Fargo, ND and released in 2012. It was ...

  14. How the MccB bacterial ancestor of ubiquitin E1 initiates biosynthesis of the microcin C7 antibiotic

    SciTech Connect

    Regni, Catherine A.; Roush, Rebecca F.; Miller, Darcie J.; Nourse, Amanda; Walsh, Christopher T.; Schulman, Brenda A.

    2009-09-11

    The 39-kDa Escherichia coli enzyme MccB catalyses a remarkable posttranslational modification of the MccA heptapeptide during the biosynthesis of microcin C7 (MccC7), a 'Trojan horse' antibiotic. The approximately 260-residue C-terminal region of MccB is homologous to ubiquitin-like protein (UBL) activating enzyme (E1) adenylation domains. Accordingly, MccB-catalysed C-terminal MccA-acyl-adenylation is reminiscent of the E1-catalysed activation reaction. However, unlike E1 substrates, which are UBLs with a C-terminal di-glycine sequence, MccB's substrate, MccA, is a short peptide with an essential C-terminal Asn. Furthermore, after an intramolecular rearrangement of MccA-acyl-adenylate, MccB catalyses a second, unique reaction, producing a stable phosphoramidate-linked analogue of acyl-adenylated aspartic acid. We report six-crystal structures of MccB in apo, substrate-, intermediate-, and inhibitor-bound forms. Structural and kinetic analyses reveal a novel-peptide clamping mechanism for MccB binding to heptapeptide substrates and a dynamic-active site for catalysing dual adenosine triphosphate-consuming reactions. The results provide insight into how a distinctive member of the E1 superfamily carries out two-step activation for generating the peptidyl-antibiotic MccC7.

  15. 11 CFR 102.12 - Designation of principal campaign committee (2 U.S.C. 432(e) (1) and (3)).

    Code of Federal Regulations, 2011 CFR

    2011-01-01

    ... 11 Federal Elections 1 2011-01-01 2011-01-01 false Designation of principal campaign committee (2 U.S.C. 432(e) (1) and (3)). 102.12 Section 102.12 Federal Elections FEDERAL ELECTION COMMISSION GENERAL REGISTRATION, ORGANIZATION, AND RECORDKEEPING BY POLITICAL COMMITTEES (2 U.S.C. 433) § 102.12 Designation of principal campaign committee (2...

  16. Association of mitochondrial deoxyribonucleic acid mutation with polymorphism in CYP2E1 gene in oral carcinogenesis

    PubMed Central

    Pandey, Rahul; Mehrotra, Divya; Catapano, Carlo; Choubey, Vimal; Sarin, Rajiv; Mahdi, Abbas Ali; Singh, Stuti

    2012-01-01

    Background Oral carcinogenesis is a complex process affected by genetic as well as environmental factors. CYP2E1 gene is involved in metabolism of number of compounds and carcinogens. Its normal functioning is required for homeostasis of free radical. Mitochondrial deoxyribonucleic acid (mtDNA) is 10–100 times more susceptible to damage than nuclear DNA. Mitochondrial DNA large scale deletions are well documented in oral cancer. However, the relationship between CYP2E1 gene polymorphisms and mtDNA damage is still not documented in literature. Materials and Methods Case–control study involving 50 subjects was carried out. Deoxyribonucleic acid extraction was done from study subject tissue samples. Restriction fragment length polymorphism (RFLP) and polymerase chain reaction (PCR) amplification was done to confirm CYP2E1 gene polymorphisms. The PCR amplification was done for mtDNA 4977 bp deletion. Statistical analysis was carried out using SPSS version 11.5 with χ2 tests. Results c1c1 and DD polymorphisms are prevalent in North Indian population having oral cancer. These polymorphisms are significantly associated with mtDNA 4977 bp deletion. Conclusion Mitochondrial DNA damage induced by wild CYP2E1 forms and imperfect DNA repair in mtDNA may act synergistically to greatly enhance oral cancer risk. PMID:25756024

  17. Induction of CYP2E1 in testes of isoniazid-treated rats as possible cause of testicular disorders.

    PubMed

    Shayakhmetova, Ganna M; Bondarenko, Larysa B; Voronina, Alla K; Anisimova, Svitlana I; Matvienko, Anatoliy V; Kovalenko, Valentina M

    2015-04-16

    Isoniazid is reported to be the most reliable and cost-effective remedy for tuberculosis treatment and prophylaxis among first line anti-tuberculosis drugs. Conventionally, the most common and best studied adverse effect of isoniazid is hepatotoxicity, but as for testicular toxicity the problem has not yet explored extensively. The aim of the study was to identify in vivo influence of isoniazid on induction of testicular cytochrome Р-450 2Е1 (CYP2E1) mRNA expression and enzymatic activity, testes DNA fragmentation, serum total testosterone level, and spermatogenesis indices. The significant induction of CYP2E1 was demonstrated in rat's testes following isoniazid administration, specifically CYP2E1 mRNA expression and p-nitrophenolhydroxylase activity was increased in 28 and 7 times as compared with control, respectively. These changes were accompanied by activating of testicular GST in 32%, changing in levels and character of DNA fragmentation, as well as damaging of the spermatogenic epithelium, decreasing in serum testosterone content (1.62 fold), sperm count (19%), and losing of fertility in comparison with untreated males. We assume that in testes of isoniazid-treated rats CYP2E1 may act as a trigger in generating of reactive oxygen species and other toxic metabolites which subsequently mediates DNA damage, spermatogenesis disturbances, and altered male fertilizing capacity. PMID:25683034

  18. Registration of DGE-2, a durum wheat disomic alien substitution line 1E(1A) involving a diploid wheatgrass chromosome

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The durum wheat (Triticum turgidum L., 2n = 2x = 28; AABB genomes) alien disomic substitution 1E(1A) line DGE-2 (PI 663216) was developed by the USDA–ARS, Cereal Crops Research Unit, Northern Crop Science Laboratory, Fargo, North Dakota and released in 2011. DGE-2 has 2n = 28 chromosomes, which are...

  19. Mobilization properties of small ColE1-like plasmids carrying kanamycin resistance gene isolated from Salmonella enterica serotypes

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Background: Previously we isolated and characterized various groups of small kanamycin resistance (KanR) ColE1-like plasmids from different serotypes of Salmonella enterica isolates. These plasmids all carried the aph(3)-I gene encoding the aminoglycoside phosphotransferase responsible for the kanam...

  20. An amino acid substitution in the pyruvate dehydrogenase E1{alpha} gene, affecting mitochondrial import of the precursor protein

    SciTech Connect

    Takakubo, F.; Thorburn, D.R.; Dahl, H.H.M.

    1995-10-01

    A mutation in the mitochondrial targeting sequence was characterized in a male patient with X chromosome-linked pyruvate dehydrogenase E1{alpha} deficiency. The mutation was a base substitution of G by C at nucleotide 134 in the mitochondrial targeting sequence of the PDHA1 gene, resulting in an arginine-to-proline substitution at codon 10 (R10P). Pyruvate dehydrogenase activity in cultured skin fibroblasts was 28% of the control value, and immunoblot analysis revealed a decreased level of pyruvate dehydrogenase E1{alpha}immunoreactivity. Chimeric constructs in which the normal and mutant pyruvate dehydrogenase E1{alpha} targeting sequences were attached to the mitochondrial matrix protein ornithine transcarbamylase were synthesized in a cell free translation system, and mitochondrial import of normal and mutant proteins was compared in vitro. The results show that ornithine transcarbamylase targeted by the mutant pyruvate dehydrogenase E1{alpha} sequence was translocated into the mitochondrial matrix at a reduced rate, suggesting that defective import is responsible for the reduced pyruvate dehydrogenase level in mitochondria. The mutation was also present in an affected brother and the mildly affected mother. The clinical presentations of this X chromosome-linked disorder in affected family members are discussed. To our knowledge, this is the first report of an amino acid substitution in a mitochondrial targeting sequence resulting in a human genetic disease. 58 refs., 5 figs., 1 tab.

  1. Salmonid alphavirus glycoprotein E2 requires low temperature and E1 for virion formation and induction of protective immunity.

    PubMed

    Hikke, Mia C; Braaen, Stine; Villoing, Stephane; Hodneland, Kjartan; Geertsema, Corinne; Verhagen, Lisa; Frost, Petter; Vlak, Just M; Rimstad, Espen; Pijlman, Gorben P

    2014-10-29

    Salmonid alphavirus (SAV; also known as Salmon pancreas disease virus; family Togaviridae) causes pancreas disease and sleeping disease in Atlantic salmon and rainbow trout, respectively, and poses a major burden to the aquaculture industry. SAV infection in vivo is temperature-restricted and progeny virus is only produced at low temperatures (10-15 °C). Using engineered SAV replicons we show that viral RNA replication is not temperature-restricted suggesting that the viral structural proteins determine low-temperature dependency. The processing/trafficking of SAV glycoproteins E1 and E2 as a function of temperature was investigated via baculovirus vectors in Sf9 insect cells and by transfection of CHSE-214 fish cells with DNA constructs expressing E1 and E2. We identified SAV E2 as the temperature determinant by demonstrating that membrane trafficking and surface expression of E2 occurs only at low temperature and only in the presence of E1. Finally, a vaccination-challenge model in Atlantic salmon demonstrates the biological significance of our findings and shows that SAV replicon DNA vaccines encoding E2 elicit protective immunity only when E1 is co-expressed. This is the first study that identifies E2 as the critical determinant of SAV low-temperature dependent virion formation and defines the prerequisites for induction of a potent immune response in Atlantic salmon by DNA vaccination. PMID:25269093

  2. Effects of herbal products and their constituents on human cytochrome P450(2E1) activity.

    PubMed

    Raner, Gregory M; Cornelious, Sean; Moulick, Kamalika; Wang, Yingqing; Mortenson, Ashley; Cech, Nadja B

    2007-12-01

    Ethanolic extracts from fresh Echinacea purpurea and Spilanthes acmella and dried Hydrastis canadensis were examined with regard to their ability to inhibit cytochrome P450(2E1) mediated oxidation of p-nitrophenol in vitro. In addition, individual constituents of these extracts, including alkylamides from E. purpurea and S. acmella, caffeic acid derivatives from E. purpurea, and several of the major alkaloids from H. canadensis, were tested for inhibition using the same assay. H. canadensis (goldenseal) was a strong inhibitor of the P450(2E1), and the inhibition appeared to be related to the presence of the alkaloids berberine, hydrastine and canadine in the extract. These compounds inhibited 2E1 with K(I) values ranging from 2.8 microM for hydrastine to 18 microM for berberine. The alkylamides present in E. purpurea and S. acmella also showed significant inhibition at concentrations as low as 25 microM, whereas the caffeic acid derivatives had no effect. Commercial green tea preparations, along with four of the individual tea catechins, were also examined and were found to have no effect on the activity of P450(2E1). PMID:17658211

  3. Ghrelin inhibits the apoptosis of MC3T3-E1 cells through ERK and AKT signaling pathway

    SciTech Connect

    Liang, Qiu-Hua; Liu, Yuan; Wu, Shan-Shan; Cui, Rong-Rong; Yuan, Ling-Qing Liao, Er-Yuan

    2013-11-01

    Ghrelin is a 28-amino-acid peptide that acts as a natural endogenous ligand of the growth hormone secretagogue receptor (GHSR) and strongly stimulates the release of growth hormone from the hypothalamus–pituitary axis. Previous studies have identified the important physiological effects of ghrelin on bone metabolism, such as regulating proliferation and differentiation of osteoblasts, independent of GH/IGF-1 axis. However, research on effects and mechanisms of ghrelin on osteoblast apoptosis is still rare. In this study, we identified expression of GHSR in MC3T3-E1 cells and determined the effects of ghrelin on the apoptosis of osteoblastic MC3T3-E1 cells and the mechanism involved. Our data demonstrated that ghrelin inhibited the apoptosis of osteoblastic MC3T3-E1 cells induced by serum deprivation, as determined by terminal deoxynucleotidyl transferase-mediated deoxyribonucleotide triphosphate nick end-labeling (TUNEL) and ELISA assays. Moreover, ghrelin upregulated Bcl-2 expression and downregulated Bax expression in a dose-dependent manner. Our study also showed decreased activated caspase-3 activity under the treatment of ghrelin. Further study suggested that ghrelin stimulated the phosphorylation of ERK and AKT. Pretreatment of cells with the ERK inhibitor PD98059, PI3K inhibitor LY294002, and GHSR-siRNA blocked the ghrelin-induced activation of ERK and AKT, respectively; however, ghrelin did not stimulate the phosphorylation of p38 or JNK. PD90859, LY294002 and GHSR-siRNA attenuated the anti-apoptosis effect of ghrelin in MC3T3-E1 cells. In conclusion, ghrelin inhibits the apoptosis of osteoblastic MC3T3-E1 cells induced by serum deprivation, which may be mediated by activating the GHSR/ERK and GHSR/PI3K/AKT signaling pathways. - Highlights: • We explored the effects of ghrelin on serum deprivation-induced MC3T3-E1 cells apoptosis. • Both ELISA and TUNEL were used to detect the apoptosis. • The receptor of ghrelin, GHSR, was expressed in MC3T3-E1

  4. Analysis of the Selective Advantage Conferred by a C-E1 Fusion Protein Synthesized by Rubella Virus DI RNAs

    PubMed Central

    Claus, Claudia; Tzeng, Wen-Pin; Liebert, Uwe Gerd; Frey, Teryl K.

    2009-01-01

    During serial passaging of rubella virus (RUB) in cell culture, the dominant species of defective-interfering RNA (DI) generated contains an in-frame deletion between the capsid protein (C) gene and E1 glycoprotein gene resulting in production of a C-E1 fusion protein that is necessary for maintenance of the DI (Tzeng & Frey, 2006). A BHK cell line stably expressing the RUB structural proteins was established which was used to package DI’s into virus particles following transfection with in vitro transcripts from DI infectious cDNA constructs. Packaging of a DI encoding an in-frame C-GFP-E1 reporter fusion protein corresponding to the C-E1 fusion protein expressed in a native DI was only marginally more efficient than packaging of a DI encoding GFP, indicating that the C-E1 fusion protein did not function by enhancing packaging. However, infection with the DI encoding the C-GFP-E1 fusion protein (in the absence of wt RUB helper virus) resulted in formation of clusters of GFP-positive cells and the percentage of GFP-positive cells in the culture following infection remained relatively constant. In contrast, a DI encoding GFP did not form GFP-positive clusters and the percentage of GFP-positive cells declined by roughly half from two to four days post-infection. Cluster formation and sustaining the percentage of infected (GFP-positive) cells required the C part of the fusion protein, including the downstream but not the upstream of two arginine clusters (both of which are associated with RNA binding and association with mitochondrial p32 protein) and the E1 part through the transmembrane sequence, but not the C-terminal cytoplasmic tail. Among a collection of mutant DI constructs, cluster formation and sustaining infected cell percentage correlated with maintenance during serial passage with wt RUB. We hypothesize that cluster formation and sustaining infected cell percentage increases the likelihood of co-infection by a DI and wt RUB during serial passage thus

  5. Crystal Structure of UBA2[superscript ufd]-Ubc9: Insights into E1-E2 Interactions in Sumo Pathways

    SciTech Connect

    Wang, Jing; Taherbhoy, Asad M.; Hunt, Harold W.; Seyedin, Steven N.; Miller, David W.; Miller, Darcie J.; Huang, Danny T.; Schulman, Brenda A.

    2012-04-30

    Canonical ubiquitin-like proteins (UBLs) such as ubiquitin, Sumo, NEDD8, and ISG15 are ligated to targets by E1-E2-E3 multienzyme cascades. The Sumo cascade, conserved among all eukaryotes, regulates numerous biological processes including protein localization, transcription, DNA replication, and mitosis. Sumo conjugation is initiated by the heterodimeric Aos1-Uba2 E1 enzyme (in humans called Sae1-Uba2), which activates Sumo's C-terminus, binds the dedicated E2 enzyme Ubc9, and promotes Sumo C-terminal transfer between the Uba2 and Ubc9 catalytic cysteines. To gain insights into details of E1-E2 interactions in the Sumo pathway, we determined crystal structures of the C-terminal ubiquitin fold domain (ufd) from yeast Uba2 (Uba2{sup ufd}), alone and in complex with Ubc9. The overall structures of both yeast Uba2{sup ufd} and Ubc9 superimpose well on their individual human counterparts, suggesting conservation of fundamental features of Sumo conjugation. Docking the Uba2{sup ufd}-Ubc9 and prior full-length human Uba2 structures allows generation of models for steps in Sumo transfer from Uba2 to Ubc9, and supports the notion that Uba2 undergoes remarkable conformational changes during the reaction. Comparisons to previous structures from the NEDD8 cascade demonstrate that UBL cascades generally utilize some parallel E1-E2 interaction surfaces. In addition, the structure of the Uba2{sup ufd}-Ubc9 complex reveals interactions unique to Sumo E1 and E2. Comparison with a previous Ubc9-E3 complex structure demonstrates overlap between Uba2 and E3 binding sites on Ubc9, indicating that loading with Sumo and E3-catalyzed transfer to substrates are strictly separate steps. The results suggest mechanisms establishing specificity and order in Sumo conjugation cascades.

  6. Hepatitis C Virus (HCV) Envelope Glycoproteins E1 and E2 Contain Reduced Cysteine Residues Essential for Virus Entry*

    PubMed Central

    Fraser, Johanna; Boo, Irene; Poumbourios, Pantelis; Drummer, Heidi E.

    2011-01-01

    The HCV envelope glycoproteins E1 and E2 contain eight and 18 highly conserved cysteine residues, respectively. Here, we examined the oxidation state of E1E2 heterodimers incorporated into retroviral pseudotyped particles (HCVpp) and investigated the significance of free sulfhydryl groups in cell culture-derived HCV (HCVcc) and HCVpp entry. Alkylation of free sulfhydryl groups on HCVcc/pp with a membrane-impermeable sulfhydryl-alkylating reagent 4-(N-maleimido)benzyl-α-trimethylammonium iodide (M135) prior to virus attachment to cells abolished infectivity in a dose-dependent manner. Labeling of HCVpp envelope proteins with EZ-Link maleimide-PEG2-biotin (maleimide-biotin) detected free thiol groups in both E1 and E2. Unlike retroviruses that employ disulfide reduction to facilitate virus entry, the infectivity of alkylated HCVcc could not be rescued by addition of exogenous reducing agents. Furthermore, the infectivity of HCVcc bound to target cells was not affected by addition of M135 indicative of a change in glycoprotein oxidation state from reduced to oxidized following virus attachment to cells. By contrast, HCVpp entry was reduced by 61% when treated with M135 immediately following attachment to cells, suggesting that the two model systems might demonstrate variations in oxidation kinetics. Glycoprotein oxidation was not altered following binding of HCVpp incorporated E1E2 to soluble heparin or recombinant CD81. These results suggest that HCV entry is dependent on the presence of free thiol groups in E1 and E2 prior to cellular attachment and reveals a new essential component of the HCV entry process. PMID:21768113

  7. Hepatitis C virus (HCV) envelope glycoproteins E1 and E2 contain reduced cysteine residues essential for virus entry.

    PubMed

    Fraser, Johanna; Boo, Irene; Poumbourios, Pantelis; Drummer, Heidi E

    2011-09-16

    The HCV envelope glycoproteins E1 and E2 contain eight and 18 highly conserved cysteine residues, respectively. Here, we examined the oxidation state of E1E2 heterodimers incorporated into retroviral pseudotyped particles (HCVpp) and investigated the significance of free sulfhydryl groups in cell culture-derived HCV (HCVcc) and HCVpp entry. Alkylation of free sulfhydryl groups on HCVcc/pp with a membrane-impermeable sulfhydryl-alkylating reagent 4-(N-maleimido)benzyl-α-trimethylammonium iodide (M135) prior to virus attachment to cells abolished infectivity in a dose-dependent manner. Labeling of HCVpp envelope proteins with EZ-Link maleimide-PEG2-biotin (maleimide-biotin) detected free thiol groups in both E1 and E2. Unlike retroviruses that employ disulfide reduction to facilitate virus entry, the infectivity of alkylated HCVcc could not be rescued by addition of exogenous reducing agents. Furthermore, the infectivity of HCVcc bound to target cells was not affected by addition of M135 indicative of a change in glycoprotein oxidation state from reduced to oxidized following virus attachment to cells. By contrast, HCVpp entry was reduced by 61% when treated with M135 immediately following attachment to cells, suggesting that the two model systems might demonstrate variations in oxidation kinetics. Glycoprotein oxidation was not altered following binding of HCVpp incorporated E1E2 to soluble heparin or recombinant CD81. These results suggest that HCV entry is dependent on the presence of free thiol groups in E1 and E2 prior to cellular attachment and reveals a new essential component of the HCV entry process. PMID:21768113

  8. Gene order for rubella virus structural proteins is NH/sub 2/-C-E2-E1-COOH

    SciTech Connect

    Oker-Blom, C.

    1984-08-01

    The order of translation in vivo of the genes coding for rubella virus structural proteins was studied in infected B-Vero cells. The proteins were sequentially pulse-chase labeled with (/sup 35/S)methionine after synchronization of translation initiation with hypertonic salt treatment. A sequential labeling procedure (window-labeling) to specifically label defined segments of the structural proteins was also used. The labeled proteins were identified by sodium dodecyl sulfate-gel electrophoresis after immunoprecipitation with specific antisera directed against the two virion glycoproteins (E1 and E2a/E2b) and the nucleocapsid (C) protein. The order of translation was found to be NH/sub 2/-C-E2-E1-COOH. We have previously shown that the structural proteins are synthesized in vitro from a cytoplasmic 24S subgenomic mRNA as a 110,000-dalton (p110) precursor. Here, it is shown that p110 is precipitated with anti-C, anti-E2, and anti-E1 sera, indicating that p110 is the precursor of all three structural proteins. Two major in vitro translation products (M/sub r/s, 66,000 and 62,000) that could represent preterminated polypeptide chains or proteolytic cleavage products were precipitated with anti-C and anti-Es sera, but not with anti-E1 serum, indicating, in conformity with the in vivo results, that the genes for the C and E2 proteins are adjacent to each other. Using these specific antisera, we have also confirmed the identity of the unglycosylated forms of E1 (M/sub r/, 53,000) and E2 (M/sub r/ 30,000) immunoprecipitated from tunicamycin-treated infected cells. 18 references, 6 figures.

  9. Bovine Collagen Peptides Compounds Promote the Proliferation and Differentiation of MC3T3-E1 Pre-Osteoblasts

    PubMed Central

    Liu, JunLi; Zhang, Bing; Song, ShuJun; Ma, Ming; Si, ShaoYan; Wang, YiHu; Xu, BingXin; Feng, Kai; Wu, JiGong; Guo, YanChuan

    2014-01-01

    Objective Collagen peptides (CP) compounds, as bone health supplements, are known to play a role in the treatment of osteoporosis. However, the molecular mechanisms of this process remain unclear. This study aimed to investigate the effects of bovine CP compounds on the proliferation and differentiation of MC3T3-E1 cells. Methods Mouse pre-osteoblast cell line MC3T3-E1 subclone 4 cells were treated with bovine CP compounds. Cell proliferation was analyzed by MTT assays and the cell cycle was evaluated by flow cytometry scanning. Furthermore, MC3T3-E1 cell differentiation was analyzed at the RNA level by real-time PCR and at the protein level by western blot analysis for runt-related transcription factor 2 (Runx2), a colorimetric p-nitrophenyl phosphate assay for alkaline phosphatase (ALP), and ELISA for osteocalcin (OC). Finally, alizarin red staining for mineralization was measured using Image Software Pro Plus 6.0. Results Cell proliferation was very efficient after treatment with different concentrations of bovine CP compounds, and the best concentration was 3 mg/mL. Bovine CP compounds significantly increased the percentage of MC3T3-E1 cells in G2/S phase. Runx2 expression, ALP activity, and OC production were significantly increased after treatment with bovine CP compounds for 7 or 14 days. Quantitative analyses with alizarin red staining showed significantly increased mineralization of MC3T3-E1 cells after treatment with bovine CP compounds for 14 or 21 days. Conclusions Bovine CP compounds increased osteoblast proliferation, and played positive roles in osteoblast differentiation and mineralized bone matrix formation. Taking all the experiments together, our study indicates a molecular mechanism for the potential treatment of osteoarthritis and osteoporosis. PMID:24926875

  10. Resolvin E1 analog RX-10045 0.1% reduces corneal stromal haze in rabbits when applied topically after PRK

    PubMed Central

    Torricelli, Andre A. M.; Santhanam, Abirami; Agrawal, Vandana

    2014-01-01

    Purpose To perform a masked study to determine whether resolvin E1 (RvE1), a lipid-derived immunomodulator, could regulate the development of corneal haze and opacity-related myofibroblasts after opacity-generating high correction photorefractive keratectomy (PRK) in rabbits. Methods Three groups of eight rabbits each were included in the study. Nine diopter (D) PRK for myopia was performed in each test cornea, and the eyes were treated with 30 µl of topical solution every 4 h (six times a day) for 5 days starting immediately after PRK. Group 1 was treated with 0.1% RX-10045, a prodrug of an RvE1 analog; group 2 was treated with 0.01% RX-10045; and group 3 was treated with vehicle control solution. At 1 month after PRK, haze was graded at the slit-lamp by a masked observer. Immunohistochemistry for α-smooth muscle actin (SMA) was performed on the central cornea of each test eye to determine the anterior stromal myofibroblast density. Results Corneal opacity was significantly lower in the 0.1% RX-10045 group, but not the 0.01% RX-10045 group, compared to the vehicle control group (p=0.029), at 1 month after −9.0D PRK. At 1 month after −9.0D PRK, SMA+ myofibroblast densities in the anterior stroma were not statistically significantly different among the three groups, although a trend toward lower myofibroblast generation was noted in the 0.1% RX-10045 group. Conclusions Topical 0.1% RX-10045, a prodrug of an RvE1 analog, reduces corneal opacity after haze-generating PRK in rabbits. Further studies are needed to determine the precise points at which RvE1 decreases corneal opacity after injury. PMID:25558174

  11. The Soybean-Specific Maturity Gene E1 Family of Floral Repressors Controls Night-Break Responses through Down-Regulation of FLOWERING LOCUS T Orthologs1[OPEN

    PubMed Central

    Xu, Meilan; Yamagishi, Noriko; Zhao, Chen; Takeshima, Ryoma; Kasai, Megumi; Watanabe, Satoshi; Kanazawa, Akira; Yoshikawa, Nobuyuki; Liu, Baohui; Yamada, Tetsuya; Abe, Jun

    2015-01-01

    Photoperiodism is a rhythmic change of sensitivity to light, which helps plants to adjust flowering time according to seasonal changes in daylength and to adapt to growing conditions at various latitudes. To reveal the molecular basis of photoperiodism in soybean (Glycine max), a facultative short-day plant, we analyzed the transcriptional profiles of the maturity gene E1 family and two FLOWERING LOCUS T (FT) orthologs (FT2a and FT5a). E1, a repressor for FT2a and FT5a, and its two homologs, E1-like-a (E1La) and E1Lb, exhibited two peaks of expression in long days. Using two different approaches (experiments with transition between light and dark phases and night-break experiments), we revealed that the E1 family genes were expressed only during light periods and that their induction after dawn in long days required a period of light before dusk the previous day. In the cultivar Toyomusume, which lacks the E1 gene, virus-induced silencing of E1La and E1Lb up-regulated the expression of FT2a and FT5a and led to early flowering. Therefore, E1, E1La, and E1Lb function similarly in flowering. Regulation of E1 and E1L expression by light was under the control of E3 and E4, which encode phytochrome A proteins. Our data suggest that phytochrome A-mediated transcriptional induction of E1 and its homologs by light plays a critical role in photoperiodic induction of flowering in soybean. PMID:26134161

  12. The Soybean-Specific Maturity Gene E1 Family of Floral Repressors Controls Night-Break Responses through Down-Regulation of FLOWERING LOCUS T Orthologs.

    PubMed

    Xu, Meilan; Yamagishi, Noriko; Zhao, Chen; Takeshima, Ryoma; Kasai, Megumi; Watanabe, Satoshi; Kanazawa, Akira; Yoshikawa, Nobuyuki; Liu, Baohui; Yamada, Tetsuya; Abe, Jun

    2015-08-01

    Photoperiodism is a rhythmic change of sensitivity to light, which helps plants to adjust flowering time according to seasonal changes in daylength and to adapt to growing conditions at various latitudes. To reveal the molecular basis of photoperiodism in soybean (Glycine max), a facultative short-day plant, we analyzed the transcriptional profiles of the maturity gene E1 family and two FLOWERING LOCUS T (FT) orthologs (FT2a and FT5a). E1, a repressor for FT2a and FT5a, and its two homologs, E1-like-a (E1La) and E1Lb, exhibited two peaks of expression in long days. Using two different approaches (experiments with transition between light and dark phases and night-break experiments), we revealed that the E1 family genes were expressed only during light periods and that their induction after dawn in long days required a period of light before dusk the previous day. In the cultivar Toyomusume, which lacks the E1 gene, virus-induced silencing of E1La and E1Lb up-regulated the expression of FT2a and FT5a and led to early flowering. Therefore, E1, E1La, and E1Lb function similarly in flowering. Regulation of E1 and E1L expression by light was under the control of E3 and E4, which encode phytochrome A proteins. Our data suggest that phytochrome A-mediated transcriptional induction of E1 and its homologs by light plays a critical role in photoperiodic induction of flowering in soybean. PMID:26134161

  13. Low-Level Expression of the E1B 20-Kilodalton Protein by Adenovirus 14p1 Enhances Viral Immunopathogenesis.

    PubMed

    Radke, Jay R; Yong, Sherri L; Cook, James L

    2016-01-01

    Adenovirus 14p1 (Ad14p1) is an emergent variant of Ad serotype 14 (Ad14) that has caused increased severity of respiratory illnesses during globally distributed outbreaks, including cases of acute respiratory distress syndrome and death. We found that human cell infection with Ad14p1 results in markedly decreased expression of the E1B 20-kilodalton (20K) protein compared to that with infection with wild-type (wt) Ad14. This reduced Ad14p1 E1B 20K expression caused a loss-of-function phenotype of Ad-infected cell corpses that, in contrast to cells infected with wt Ad14, either failed to repress or increased NF-κB-dependent, proinflammatory cytokine responses of responder human alveolar macrophages. A small-animal model of Ad14-induced lung infection was used to test the translational relevance of these in vitro observations. Intratracheal infection of Syrian hamsters with Ad14p1 caused a marked, patchy bronchopneumonia, whereas hamster infection with wt Ad14 caused minimal peribronchial inflammation. These results suggest that this difference in E1B 20K gene expression during Ad14p1 infection and its modulating effect on the interactions between Ad14-infected cells and the host innate immune response could explain the increased immunopathogenic potential and associated increase in clinical illness in some people infected with the Ad14p1 outbreak strain.IMPORTANCE We previously reported that Ad-infected human cells exhibit E1B 19K-dependent repression of virally induced, NF-κB-dependent macrophage cytokine responses (J. R. Radke, F. Grigera, D. S. Ucker, and J. L. Cook, J Virol 88:2658-2669, 2014, http://dx.doi.org/10.1128/JVI.02372-13). The more virulent, emergent strain of Ad14, Ad14p1, causes increased cytopathology in vitro, which suggested a possible E1B 20K defect. Whether there is a linkage between these observations was unknown. We show that there is markedly reduced expression of E1B 20K in Ad14p1-infected human cells and that this causes an increased

  14. Phosphorylation of bovine papillomavirus E1 by the protein kinase CK2 near the nuclear localization signal does not influence subcellular distribution of the protein in dividing cells.

    PubMed

    Lentz, Michael R; Shideler, Tess

    2016-01-01

    The bovine papillomavirus E1 helicase is essential for viral replication. In dividing cells, DNA replication maintains, but does not increase, the viral genome copy number. Replication is limited by low E1 expression and an E1 nucleocytoplasmic shuttling mechanism. Shuttling is controlled in part by phosphorylation of E1 by cellular kinases. Here we investigate conserved sites for phosphorylation by kinase CK2 within the E1 nuclear localization signal. When these CK2 sites are mutated to either alanine or aspartic acid, no change in replication phenotype is observed, and there is no effect on the subcellular distribution of E1, which remains primarily nuclear. This demonstrates that phosphorylation of E1 by CK2 at these sites is not a factor in regulating viral DNA replication in dividing cells. PMID:26467928

  15. Arg-Gly-Asp (RGD)-Modified E1A/E1B Double Mutant Adenovirus Enhances Antitumor Activity in Prostate Cancer Cells In Vitro and in Mice

    PubMed Central

    Wang, Hua; Cai, Zhi-Jian; Xu, Yi-Peng; Zhao, An; Su, Ying; Zhang, Gu; Zhu, Shao-Xing

    2016-01-01

    CAR is a transmembrane protein that is expressed in various epithelial and endothelial cells. CAR mediates adenoviral infection, as well as adenovirus-mediated oncolysis of AxdAdB-3, an E1A/E1B double-restricted oncolytic adenovirus, in prostate cancer cells. This study further assessed the therapeutic efficacy of AxdAdB-3 with Arg-Gly-Asp (RGD)-fiber modification (AxdAdB3-F/RGD), which enables integrin-dependent infection, in prostate cancer. Susceptibility of prostate cancer cells LNCaP, PC3, and DU145 to adenovirus infection was associated with CAR expression. All of the prostate cancer cell lines expressed integrin αvβ3 and αvβ5. AxdAdB-3 was more cytopathic in CAR-positive prostate cancer cells than in CAR-negative cells, whereas AxdAdB3-F/RGD caused potent oncolysis in both CAR-positive and CAR-negative prostate cancer cells. In contrast, AxdAdB3-F/RGD was not cytopathic against normal prostate epithelial cells, RWPE-1. Intratumoral injection of AxdAdB3-F/RGD into CAR-negative prostate cancer cell xenografts in nude mice inhibited tumor growth. The current study demonstrates that E1A/E1B double-restricted oncolytic adenovirus with an RGD-fiber modification enhances infection efficiency and anti-tumor activity in CAR-deficient prostate cancer cells, while sparing normal cells. Future studies will evaluate the therapeutic potential of AxdAdB3-F/RGD in prostate cancer. PMID:26799485

  16. Inhibition of human Cytochrome P450 2E1 and 2A6 by aldehydes: Structure and activity relationships

    PubMed Central

    Kandagatla, Suneel K.; Mack, Todd; Simpson, Sean; Sollenberger, Jill; Helton, Eric; Raner, Gregory M.

    2014-01-01

    The purpose of this study was to probe active site structure and dynamics of human cytochrome P4502E1 and P4502A6 using a series of related short chain fatty aldehydes. Binding efficiency of the aldehydes was monitored via their ability to inhibit the binding and activation of the probe substrates p-nitrophenol (2E1) and coumarin (2A6). Oxidation of the aldehydes was observed in reactions with individually expressed 2E1, but not 2A6, suggesting alternate binding modes. For saturated aldehydes the optimum chain length for inhibition of 2E1 was 9 carbons (KI=7.8 ±0.3 μM), whereas for 2A6 heptanal was most potent (KI=15.8 ±1.1 μM). A double bond in the 2-position of the aldehyde significantly decreased the observed KI relative to the corresponding saturated compound in most cases. A clear difference in the effect of the double bond was observed between the two isoforms. With 2E1, the double bond appeared to remove steric constraints on aldehyde binding with KI values for the 5–12 carbon compounds ranging between 2.6 ± 0.1 μM and 12.8± 0.5 μM, whereas steric effects remained the dominant factor in the binding of the unsaturated aldehydes to 2A6 (observed KI values between 7.0± 0.5 μM and >1000 μM). The aldehyde function was essential for effective inhibition, as the corresponding carboxylic acids had very little effect on enzyme activity over the same range of concentrations, and branching at the 3-position of the aldehydes increased the corresponding KI value in all cases examined. The results suggest that a conjugated π-system may be a key structural determinant in the binding of these compounds to both enzymes, and may also be an important feature for the expansion of the active site volume in 2E1. PMID:24924949

  17. Control of adenovirus E1B mRNA synthesis by a shift in the activities of RNA splice sites.

    PubMed Central

    Montell, C; Fisher, E F; Caruthers, M H; Berk, A J

    1984-01-01

    The primary transcript from adenovirus 2 early region 1B (E1B) is processed by differential RNA splicing into two overlapping mRNAs, 13S and 22S. The 22S mRNA is the major E1B mRNA during the early phase of infection, whereas the 13S mRNA predominates during the late phase. In previous work, it has been shown that this shift in proportions of the E1B mRNAs is influenced by increased cytoplasmic stability of the 13S mRNA at late times in infection. Two observations presented here demonstrate that the increase in proportion of the 13S mRNA at late times is also regulated by a change in the specificity of RNA splicing. First, the relative concentrations of the 13S to 22S nuclear RNAs were not constant throughout infection but increased at late times. Secondly, studies with the mutant, adenovirus 2 pm2250 , provided evidence that there was an increased propensity to utilize a 5' splice in the region of the 13S 5' splice site at late times in infection. Adenovirus 2 pm2250 has a G----C transversion in the first base of E1B 13S mRNA intron preventing splicing of the 13S mRNA but not of the 22S mRNA. During the early phase of a pm2250 infection, the E1B primary transcripts were processed into the 22S mRNA only. However, during the late phase, when the 13S mRNA normally predominates, E1B primary transcripts were also processed by RNA splicing at two formerly unused or cryptic 5' splice sites. Both cryptic splice sites were located much closer to the disrupted 13S 5' splice site than to the 22S 5' splice site. Thus, the temporal increase in proportion of the 13S mRNA to the 22S mRNA is regulated by two processes, an increase in cytoplasmic stability of the 13S mRNA and an increased propensity to utilize the 13S 5' splice site during the late phase of infection. Adenovirus 2 pm2250 was not defective for productive infection of HeLa cells or for transformation of rat cells. Images PMID:6727875

  18. Dissection of the C-terminal region of E1A redefines the roles of CtBP and other cellular targets in oncogenic transformation.

    PubMed

    Cohen, M J; Yousef, A F; Massimi, P; Fonseca, G J; Todorovic, B; Pelka, P; Turnell, A S; Banks, L; Mymryk, J S

    2013-09-01

    Human adenovirus E1A makes extensive connections with the cellular protein interaction network. By doing so, E1A can manipulate many cellular programs, including cell cycle progression. Through these reprogramming events, E1A functions as a growth-promoting oncogene and has been used extensively to investigate mechanisms contributing to oncogenesis. Nevertheless, it remains unclear how the C-terminal region of E1A contributes to oncogenic transformation. Although this region is required for transformation in cooperation with E1B, it paradoxically suppresses transformation in cooperation with activated Ras. Previous analysis has suggested that the interaction of E1A with CtBP plays a pivotal role in both activities. However, some C-terminal mutants of E1A retain CtBP binding and yet exhibit defects in transformation, suggesting that other targets of this region are also necessary. To explore the roles of these additional factors, we performed an extensive mutational analysis of the C terminus of E1A. We identified key residues that are specifically required for binding all known targets of the C terminus of E1A. We further tested each mutant for the ability to both localize to the nucleus and transform primary rat cells in cooperation with E1B-55K or Ras. Interaction of E1A with importin α3/Qip1, dual-specificity tyrosine-regulated kinase 1A (DYRK1A), HAN11, and CtBP influenced transformation with E1B-55K. Interestingly, the interaction of E1A with DYRK1A and HAN11 appeared to play a role in suppression of transformation by activated Ras whereas interaction with CtBP was not necessary. This unexpected result suggests a need for revision of current models and provides new insight into transformation by the C terminus of E1A. PMID:23864635

  19. Fusion of adenovirus E1A to the glucocorticoid receptor by high-resolution deletion cloning creates a hormonally inducible viral transactivator.

    PubMed Central

    Becker, D M; Hollenberg, S M; Ricciardi, R P

    1989-01-01

    The 289-amino-acid E1A protein of adenovirus type 2 stimulates transcription from early viral and certain cellular promoters. Its mechanism is not known, and there exist no temperature-sensitive mutants of E1A that could help to elucidate the details of E1A transcriptional activation. To create for E1A such a conditional phenotype, we fused portions of E1A to the human glucocorticoid receptor (GR) to make transactivation by E1A dependent on the presence of dexamethasone. Nested subsets of the E1A coding region, centered around the 46-amino-acid transactivating domain, were substituted for the DNA-binding domain of the GR. One of the resulting chimeric proteins (GR/E1A-99), which included the entire E1A transactivating domain, stimulated expression from a viral early promoter (E3) exclusively in the presence of hormone. GR/E1A-99 did not transactivate a GR-responsive promoter. It therefore exhibited the promoter specificity of E1A while possessing the hormone inducibility of the GR. Two smaller chimeras that contained only portions of the E1A transactivating domain failed to transactivate E3. These three chimeras were constructed by a novel strategy, high-resolution deletion cloning. In this procedure, series of unidirectional deletions were made with exonuclease III on each side of the E1A coding region at a resolution of 1 to 2 nucleotides. The large number of in-frame fragments present in the collection of deleted clones facilitated the construction of the GR/E1A chimeras and can be used to create many additional fusions. Images PMID:2550806

  20. Diurnal Expression Pattern, Allelic Variation, and Association Analysis Reveal Functional Features of the E1 Gene in Control of Photoperiodic Flowering in Soybean

    PubMed Central

    Wu, Hongyan; Zhang, Yupeng; Zhang, Xingzheng; Yang, Jiayin; Wang, Yaying; Yang, Guang; Qiu, Hongmei; Cui, Tingting; Xia, Zhengjun

    2015-01-01

    Although four maturity genes, E1 to E4, in soybean have been successfully cloned, their functional mechanisms and the regulatory network of photoperiodic flowering remain to be elucidated. In this study, we investigated how the diurnal expression pattern of the E1 gene is related to photoperiodic length; and to what extent allelic variation in the B3-like domain of the E1 gene is associated with flowering time phenotype. The bimodal expression of the E1 gene peaked first at around 2 hours after dawn in long-day condition. The basal expression level of E1 was enhanced by the long light phase, and decreased by duration of dark. We identified a 5bp (3 SNP and 2-bp deletion) mutation, referred to an e1-b3a, which occurs in the middle of B3 domain of the E1 gene in the early flowering cultivar Yanhuang 3. Subcellular localization analysis showed that the putative truncated e1-b3a protein was predominately distributed in nuclei, indicating the distribution pattern of e1-b3a was similar to that of E1, but not to that of e1-as. Furthermore, genetic analysis demonstrated allelic variations at the E1 locus significantly underlay flowering time in three F2 populations. Taken together, we can conclude the legume specific E1 gene confers some special features in photoperiodic control of flowering in soybean. Further characterization of the E1 gene will extend our understanding of the soybean flowering pathway in soybean. PMID:26275311

  1. Sino-VirtualMoon: A 3D web platform using Chang’E-1 data for collaborative research

    NASA Astrophysics Data System (ADS)

    Chen, Min; Lin, Hui; Wen, Yongning; He, Li; Hu, Mingyuan

    2012-05-01

    The successful launch of the Chinese Chang’E-1 satellite created a valuable opportunity for lunar research, and represented China’s remarkable leap in deep space exploration. With the observed data acquired by Chang’E-1 satellite, a web platform was developed aims at providing an open research workspace for experts to conduct collaborative scientific research on the Moon. Excepting for supporting 3D visualization, the platform also provides collaborative tools for the basic geospatial analysis of the Moon, and supports collaborative simulation about the dynamic formation of lunar impact craters caused by the collision of meteors (or small asteroids). Based on this platform, related multidisciplinary experts can contribute their domain knowledge conveniently for collaborative scientific research of the Moon.

  2. FGF-2 signaling induces downregulation of TAZ protein in osteoblastic MC3T3-E1 cells

    SciTech Connect

    Eda, Homare; Aoki, Katsuhiko; Marumo, Keishi; Fujii, Katsuyuki; Ohkawa, Kiyoshi

    2008-02-08

    Transcriptional coactivator with PDZ-binding motif (TAZ) protein is a coactivator of Runx2 and corepressor of PPAR{gamma}. It also induces differentiation of mesenchymal cells into osteoblasts. In this study, we found that FGF-2, which inhibits bone mineralization and stimulates cell proliferation, reduced the TAZ protein expression level in osteoblast-like cells, MC3T3-E1. This reduction was recovered by removing FGF-2 from the culture medium, which also restored the osteoblastic features of MC3T3-E1 cells. Furthermore, FGF-2-induced reduction of TAZ is blocked by a SAPK/JNK-specific inhibitor. These findings suggest that the expression of TAZ protein is involved in osteoblast proliferation and differentiation. This may help elucidate the discrepancies in the effect of FGF-2 and contribute to the understanding of FGF/FGFR-associated craniosynostosis syndrome etiology and treatment.

  3. Ectopic osteogenic tissue formation by MC3T3-E1 cell-laden chitosan/hydroxyapatite composite scaffold.

    PubMed

    Koç, Aysel; Elçin, Ayşe Eser; Elçin, Yaşar Murat

    2016-09-01

    This study evaluates the suitability of a macroporous three-dimensional chitosan/hydroxyapatite (CS/HA) composite as a bone tissue engineering scaffold using MC3T3-E1 cells. The CS/HA scaffold was produced by freeze-drying, and characterized by means of SEM and FTIR. In vitro findings demonstrated that CS/HA supported attachment and proliferation of cells, and stimulated extracellular matrix (ECM) production. Tissue biocompatibility and osteogenic capacity of the cell-laden constructs were evaluated in an ectopic Wistar rat model. In vivo results showed that the MC3T3-E1 cell-laden CS/HA was essentially histocompatible, promoted neovascularization and calcified matrix formation, and secreted osteoblast-specific protein. We conclude that the composite scaffold evaluated has potential for applications in bone regeneration. PMID:25968048

  4. Deoxyactein Isolated from Cimicifuga racemosa protects osteoblastic MC3T3-E1 cells against antimycin A-induced cytotoxicity.

    PubMed

    Choi, Eun Mi

    2013-06-01

    Deoxyactein is one of the major constituents isolated from Cimicifuga racemosa. In the present study, we investigated the protective effects of deoxyactein on antimycin A (mitochondrial electron transport inhibitor)-induced toxicity in osteoblastic MC3T3-E1 cells. Exposure of MC3T3-E1 cells to antimycin A caused significant cell viability loss, as well as mitochondrial membrane potential dissipation, complex IV inactivation, ATP loss, intracellular calcium ([Ca(2+) ]i ) elevation and oxidative stress. Pretreatment with deoxyactein prior to antimycin A exposure significantly reduced antimycin A-induced cell damage by preventing mitochondrial membrane potential dissipation, complex IV inactivation, ATP loss, [Ca(2+) ]i elevation and oxidative stress. Moreover, deoxyactein increased the activation of PI3K (phosphoinositide 3-kinase), Akt (protein kinase B) and CREB (cAMP-response element-binding protein) inhibited by antimycin A. All these data indicate that deoxyactein may reduce or prevent osteoblasts degeneration in osteoporosis or other degenerative disorders. PMID:22180388

  5. The PI3K/Akt Pathway Regulates Oxygen Metabolism via Pyruvate Dehydrogenase (PDH)-E1α Phosphorylation.

    PubMed

    Cerniglia, George J; Dey, Souvik; Gallagher-Colombo, Shannon M; Daurio, Natalie A; Tuttle, Stephen; Busch, Theresa M; Lin, Alexander; Sun, Ramon; Esipova, Tatiana V; Vinogradov, Sergei A; Denko, Nicholas; Koumenis, Constantinos; Maity, Amit

    2015-08-01

    Inhibition of the PI3K/Akt pathway decreases hypoxia within SQ20B human head and neck cancer xenografts. We set out to understand the molecular mechanism underlying this observation. We measured oxygen consumption using both a Clark electrode and an extracellular flux analyzer. We made these measurements after various pharmacologic and genetic manipulations. Pharmacologic inhibition of the PI3K/mTOR pathway or genetic inhibition of Akt/PI3K decreased the oxygen consumption rate (OCR) in vitro in SQ20B and other cell lines by 30% to 40%. Pharmacologic inhibition of this pathway increased phosphorylation of the E1α subunit of the pyruvate dehydrogenase (PDH) complex on Ser293, which inhibits activity of this critical gatekeeper of mitochondrial respiration. Expressing wild-type PTEN in a doxycycline-inducible manner in a cell line with mutant PTEN led to an increase in PDH-E1α phosphorylation and a decrease in OCR. Pretreatment of SQ20B cells with dichloroacetate (DCA), which inhibits PDH-E1α phosphorylation by inhibiting dehydrogenase kinases (PDK), reversed the decrease in OCR in response to PI3K/Akt/mTOR inhibition. Likewise, introduction of exogenous PDH-E1α that contains serine to alanine mutations, which can no longer be regulated by phosphorylation, also blunted the decrease in OCR seen with PI3K/mTOR inhibition. Our findings highlight an association between the PI3K/mTOR pathway and tumor cell oxygen consumption that is regulated in part by PDH phosphorylation. These results have important implications for understanding the effects of PI3K pathway activation in tumor metabolism and also in designing cancer therapy trials that use inhibitors of this pathway. PMID:25995437

  6. Cytochrome P450 2E1 is responsible for the initiation of 1,2-dichloropropane-induced liver damage.

    PubMed

    Yanagiba, Yukie; Suzuki, Tetsuya; Suda, Megumi; Hojo, Rieko; Gonzalez, Frank J; Nakajima, Tamie; Wang, Rui-Sheng

    2016-09-01

    1,2-Dichloropropane (1,2-DCP), a solvent, which is the main component of the cleaner used in the offset printing companies in Japan, is suspected to be the causative agent of bile duct cancer, which has been recently reported at high incidence in those offset printing workplaces. While there are some reports about the acute toxicity of 1,2-DCP, no information about its metabolism related to toxicity in animals is available. As part of our efforts toward clarifying the role of 1,2-DCP in the development of cancer, we studied the metabolic pathways and the hepatotoxic effect of 1,2-DCP in mice with or without cytochrome P450 2E1 (CYP2E1) activity. In an in vitro reaction system containing liver homogenate, 1,2-DCP was only metabolized by liver tissue of wild-type mice but not by that of cyp2e1-null mice. Furthermore, the kinetics of the solvent in mice revealed a great difference between the two genotypes; 1,2-DCP administration resulted in dose-dependent hepatic damage, as shown biochemically and pathologically, but this effect was only observed in wild-type mice. The nuclear factor κB p52 pathway was involved in the liver response to 1,2-DCP. Our results clearly indicate that the oxidative metabolism of 1,2-DCP in mice is exclusively catalyzed by CYP2E1, and this step is indispensable for the manifestation of the hepatotoxic effect of the solvent. PMID:25681370

  7. Role of CYP2E1 and saturation kinetics in the bioactivation of thioacetamide: Effects of diet restriction and phenobarbital

    SciTech Connect

    Chilakapati, Jaya; Korrapati, Midhun C.; Shankar, Kartik; Hill, Ronald A.; Warbritton, Alan; Latendresse, John R.; Mehendale, Harihara M. . E-mail: mehendale@ulm.edu

    2007-02-15

    Thioacetamide (TA) undergoes saturation toxicokinetics in ad libitum (AL) fed rats. Diet restriction (DR) protects rats from lethal dose of TA despite increased bioactivation-mediated liver injury via CYP2E1 induction. While a low dose (50 mg TA/kg) produces 6-fold higher initial injury, a 12-fold higher dose produces delayed and mere 2.5-fold higher injury. The primary objective was to determine if this less-than-expected increase in injury is due to saturation toxicokinetics. Rats on AL and DR for 21 days received either 50 or 600 mg TA/kg i.p. T {sub 1/2} and AUCs for TA and TA-S-oxide were consistent with saturable kinetics. Covalent binding of {sup 14}C-TA-derived-radiolabel to liver macromolecules after low dose was 2-fold higher in DR than AL rats. However, following lethal dose, no differences were found between AL and DR. This lack of dose-dependent response appears to be due to saturation of bioactivation at the higher dose. The second objective was to investigate the effect of phenobarbital pretreatment (PB) on TA-initiated injury following a sub-lethal dose (500 mg/kg). PB induced CYP2B1/2 {approx} 350-fold, but did not increase covalent binding of {sup 14}C-TA, TA-induced liver injury and mortality, suggesting that CYP2B1/2 has no major role in TA bioactivation. The third objective was to investigate the role of CYP2E1 using cyp2e1 knockout mice (KO). Injury was assessed over time (0-48 h) in wild type (WT) and KO mice after LD{sub 100} dose (500 mg/kg) in WT. While WT mice exhibited robust injury which progressed to death, KO mice exhibited neither initiation nor progression of injury. These findings confirm that CYP2E1 is responsible for TA bioactivation.

  8. E1, E2 and M1 transition parameters for some levels over ionization limit of Ne III

    NASA Astrophysics Data System (ADS)

    Eser, Selda; Özdemir, Leyla

    2016-07-01

    We have reported the level energies and radiative transition ( E1 , E2 and M1 parameters, such as wavelengths, transition rates, oscillator strengths and line strengths for some levels over the ionization limit of Ne III (oxygen-like). The calculations have been performed using the general-purpose relativistic atomic structure package (GRASP) based on the fully relativistic multiconfiguration Dirac-Fock (MCDF) method. The results obtained have been compared with the available theoretical and experimental values in the literature.

  9. 11 CFR 102.13 - Authorization of political committees (2 U.S.C. 432(e) (1) and (3)).

    Code of Federal Regulations, 2011 CFR

    2011-01-01

    ... 11 Federal Elections 1 2011-01-01 2011-01-01 false Authorization of political committees (2 U.S.C. 432(e) (1) and (3)). 102.13 Section 102.13 Federal Elections FEDERAL ELECTION COMMISSION GENERAL REGISTRATION, ORGANIZATION, AND RECORDKEEPING BY POLITICAL COMMITTEES (2 U.S.C. 433) § 102.13 Authorization of political committees (2 U.S.C. 432(e)...

  10. Association between Rare Variants in AP4E1, a Component of Intracellular Trafficking, and Persistent Stuttering

    PubMed Central

    Raza, M. Hashim; Mattera, Rafael; Morell, Robert; Sainz, Eduardo; Rahn, Rachel; Gutierrez, Joanne; Paris, Emily; Root, Jessica; Solomon, Beth; Brewer, Carmen; Basra, M. Asim Raza; Khan, Shaheen; Riazuddin, Sheikh; Braun, Allen; Bonifacino, Juan S.; Drayna, Dennis

    2015-01-01

    Stuttering is a common, highly heritable neurodevelopmental disorder characterized by deficits in the volitional control of speech. Whole-exome sequencing identified two heterozygous AP4E1 coding variants, c.1549G>A (p.Val517Ile) and c.2401G>A (p.Glu801Lys), that co-segregate with persistent developmental stuttering in a large Cameroonian family, and we observed the same two variants in unrelated Cameroonians with persistent stuttering. We found 23 other rare variants, including predicted loss-of-function variants, in AP4E1 in unrelated stuttering individuals in Cameroon, Pakistan, and North America. The rate of rare variants in AP4E1 was significantly higher in unrelated Pakistani and Cameroonian stuttering individuals than in population-matched control individuals, and coding variants in this gene are exceptionally rare in the general sub-Saharan West African, South Asian, and North American populations. Clinical examination of the Cameroonian family members failed to identify any symptoms previously reported in rare individuals carrying homozygous loss-of-function mutations in this gene. AP4E1 encodes the ε subunit of the heterotetrameric (ε-β4-μ4-σ4) AP-4 complex, involved in protein sorting at the trans-Golgi network. We found that the μ4 subunit of AP-4 interacts with NAGPA, an enzyme involved in the synthesis of the mannose 6-phosphate signal that targets acid hydrolases to the lysosome and the product of a gene previously associated with stuttering. These findings implicate deficits in intracellular trafficking in persistent stuttering. PMID:26544806

  11. Study of M1 and E1 excitations by high-resolution proton inelastic scattering measurement at forward angles

    SciTech Connect

    Tamii, A.; Adachi, T.; Hatanaka, K.; Hashimoto, H.; Kaneda, T.; Matsubara, H.; Okamura, H.; Sakemi, Y.; Shimizu, Y.; Tameshige, Y.; Yosoi, M.; Carter, J.; Dozono, M.; Fujita, H.; Fujita, Y.; Itoh, M.; Kawabata, T.; Nakanishi, K.; Sasamoto, Y.; Neumann-Cosel, P. von

    2007-06-13

    Experimental technique for measuring proton inelastic scattering with high-resolution at 295 MeV and at forward angles including zero degrees is described. The method is useful for extracting spin part of the M1 strength via nuclear excitation as well as E1 strength via Coulomb excitation. An excitation energy resolution of 20 keV, good scattering angle resolution, and low background condition have been achieved. The experimental technique was applied for several sd and pf shell nuclei.

  12. Identification, molecular characterization, and cellular studies of an apolipoprotein E mutant (E1) in three unrelated families with hyperlipidemia.

    PubMed

    Miller, D B; Hegele, R A; Wolfe, B M; Huff, M W

    1995-03-01

    Remnants of triglyceride-rich lipoproteins accumulate in plasma of subjects with type III hyperlipoproteinemia (HLP) due to defective clearance by hepatic receptors. Although most subjects with type III HLP are homozygous for apolipoprotein (apo) E2 (arg158-->cys, R158C), a variant that binds defectively to cell surface receptors, some individuals with type III HLP have rare mutations of apo E. We identified six subjects from three families with type III HLP who had either an apo E3/1 or E4/1 phenotype by isoelectric focusing. Using DNA restriction isotyping with HhaI, all six subjects were determined to have only one apo E allele encoding cys158 and the other encoding arg158. Subsequently, digestion of polymerase chain reaction-amplified portions of exon 4 of the apo E gene with endonucleases HaeIII, TaqI, and Sau3AI demonstrated a second DNA variant that encoded a single amino acid substitution (gly127-->asp, G127D) due to a guanosine-to-adenosine nucleotide change resulting in the apo E1 isoform (G127D, R158C), which had arisen from a parent apo E2 allele. This mutation was confirmed with direct DNA sequencing. Incubation of very low density lipoprotein (VLDL) isolated from hyperlipidemic apo E1 subjects with J774 macrophages resulted in a 7- to 12-fold increase in cellular cholesterol ester compared with VLDL from apo E2/2 subjects. Although heterozygosity for apo E1 alone did not impair the interaction of VLDL with cellular receptors in vitro, its presence in subjects with type III HLP suggests that apo E1, perhaps in combination with secondary factors, may be causative for the dyslipidemia. PMID:7883834

  13. Interactions between cell growth-regulating domains in the products of the adenovirus E1A oncogene

    SciTech Connect

    Moran, B.; Zerler, B.

    1988-04-01

    Among the various biological activities expressed by the products of the adenovirus E1A gene are the abilities to induce cellular DNA synthesis and proliferation in quiescent primary baby rat kidney cells. The functional sites for these activities lie principally within two regions of the E1A proteins: an N-terminal region and a small second region of approximately 20 amino acids further downstream. To study the biological functions of the first domain, the authors constructed an in-frame deletion of amino acid positions 23 through 107 of the E1A products. This deletion did not impede the ability of the E1A products to transactivate the adenovirus early region 3 promoter in a transient-expression assay in HeLa cells. The ability to induce DNA synthesis in quiescent baby rat kidney cells was, however, lost in the absence of these sequences. Deletion of the small second region induced a form of S phase in which DNA synthesis occurred in the apparent absence of controls required for the cessation of DNA synthesis and progression through the remainder of the cell cycle. These cells did not appear to accumulate in or before G2, and many appeared to have a DNA content greater than that in G2. The functions of both domains are required for production of transformed foci in a ras cooperation assay. Focus formation occurred, however, even when the two domains were introduced on two separate plasmids. This complementation effect appeared to require expression of both of the mutant proteins and did not appear to result merely from recombination at the DNA level.

  14. Functional expression of 5-HT{sub 2A} receptor in osteoblastic MC3T3-E1 cells

    SciTech Connect

    Hirai, Takao; Kaneshige, Kota; Kurosaki, Teruko; Nishio, Hiroaki

    2010-05-28

    In the previous study, we reported the gene expression for proteins related to the function of 5-hydroxytryptamine (5-HT, serotonin) and elucidated the expression patterns of 5-HT{sub 2} receptor subtypes in mouse osteoblasts. In the present study, we evaluated the possible involvement of 5-HT receptor subtypes and its inactivation system in MC3T3-E1 cells, an osteoblast cell line. DOI, a 5-HT{sub 2A} and 5-HT{sub 2C} receptor selective agonist, as well as 5-HT concentration-dependently increased proliferative activities of MC3T3-E1 cells in their premature period. This effect of 5-HT on cell proliferation were inhibited by ketanserin, a 5-HT{sub 2A} receptor specific antagonist. Moreover, both DOI-induced cell proliferation and phosphorylation of ERK1 and 2 proteins were inhibited by PD98059 and U0126, selective inhibitors of MEK in a concentration-dependent manner. Furthermore, treatment with fluoxetine, a 5-HT specific re-uptake inhibitor which inactivate the function of extracellular 5-HT, significantly increased the proliferative activities of MC3T3-E1 cells in a concentration-dependent manner. Our data indicate that 5-HT fill the role for proliferation of osteoblast cells in their premature period. Notably, 5-HT{sub 2A} receptor may be functionally expressed to regulate mechanisms underlying osteoblast cell proliferation, at least in part, through activation of ERK/MAPK pathways in MC3T3-E1 cells.

  15. Association between Rare Variants in AP4E1, a Component of Intracellular Trafficking, and Persistent Stuttering.

    PubMed

    Raza, M Hashim; Mattera, Rafael; Morell, Robert; Sainz, Eduardo; Rahn, Rachel; Gutierrez, Joanne; Paris, Emily; Root, Jessica; Solomon, Beth; Brewer, Carmen; Basra, M Asim Raza; Khan, Shaheen; Riazuddin, Sheikh; Braun, Allen; Bonifacino, Juan S; Drayna, Dennis

    2015-11-01

    Stuttering is a common, highly heritable neurodevelopmental disorder characterized by deficits in the volitional control of speech. Whole-exome sequencing identified two heterozygous AP4E1 coding variants, c.1549G>A (p.Val517Ile) and c.2401G>A (p.Glu801Lys), that co-segregate with persistent developmental stuttering in a large Cameroonian family, and we observed the same two variants in unrelated Cameroonians with persistent stuttering. We found 23 other rare variants, including predicted loss-of-function variants, in AP4E1 in unrelated stuttering individuals in Cameroon, Pakistan, and North America. The rate of rare variants in AP4E1 was significantly higher in unrelated Pakistani and Cameroonian stuttering individuals than in population-matched control individuals, and coding variants in this gene are exceptionally rare in the general sub-Saharan West African, South Asian, and North American populations. Clinical examination of the Cameroonian family members failed to identify any symptoms previously reported in rare individuals carrying homozygous loss-of-function mutations in this gene. AP4E1 encodes the ε subunit of the heterotetrameric (ε-β4-μ4-σ4) AP-4 complex, involved in protein sorting at the trans-Golgi network. We found that the μ4 subunit of AP-4 interacts with NAGPA, an enzyme involved in the synthesis of the mannose 6-phosphate signal that targets acid hydrolases to the lysosome and the product of a gene previously associated with stuttering. These findings implicate deficits in intracellular trafficking in persistent stuttering. PMID:26544806

  16. Therapeutic targets for olive pollen allergy defined by gene markers modulated by Ole e 1-derived peptides.

    PubMed

    Calzada, David; Aguerri, Miriam; Baos, Selene; Montaner, David; Mata, Manuel; Dopazo, Joaquín; Quiralte, Joaquín; Florido, Fernando; Lahoz, Carlos; Cárdaba, Blanca

    2015-04-01

    Two regions of Ole e 1, the major olive-pollen allergen, have been characterized as T-cell epitopes, one as immunodominant region (aa91-130) and the other, as mainly recognized by non-allergic subjects (aa10-31). This report tries to characterize the specific relevance of these epitopes in the allergic response to olive pollen by analyzing the secreted cytokines and the gene expression profiles induced after specific stimulation of peripheral blood mononuclear cells (PBMCs). PBMCs from olive pollen-allergic and non-allergic control subjects were stimulated with olive-pollen extract and Ole e 1 dodecapeptides containing relevant T-cell epitopes. Levels of cytokines were measured in cellular supernatants and gene expression was determined by microarrays, on the RNAs extracted from PBMCs. One hundred eighty-nine differential genes (fold change >2 or <-2, P<0.05) were validated by qRT-PCR in a large population. It was not possible to define a pattern of response according the overall cytokine results but interesting differences were observed, mainly in the regulatory cytokines. Principal component (PCA) gene-expression analysis defined clusters that correlated with the experimental conditions in the group of allergic subjects. Gene expression and functional analyses revealed differential genes and pathways among the experimental conditions. A set of 51 genes (many essential to T-cell tolerance and homeostasis) correlated with the response to aa10-31 of Ole e 1. In conclusion, two peptides derived from Ole e 1 could regulate the immune response in allergic patients, by gene-expression modification of several regulation-related genes. These results open new research ways to the regulation of allergy by Oleaceae family members. PMID:25553522

  17. R237 and h238 are key amino acids in the rubella virus E1 neutralization epitope.

    PubMed

    Li, Zhen-Mei; Chu, Fu-Lu; Wen, Hong-Ling; Sun, Zi-Hao; Li, Guo-Hong; Xie, Wen-Yan; Wang, Zhi-Yu

    2014-11-01

    Residues 221-239 of rubella virus E1 glycoprotein contain antibody neutralization domains, and the solvent-exposed charged amino acids at the binding interface may be crucial for binding ability. However, the role of charged amino acid residues on the E1 epitope in peptide-antibody binding is unknown. To investigate the role of single amino acid substitutions on the important neutralizing epitope, biolayer interferometry and serological tests were performed. There are three charged residues in the neutralizing epitope: D229, R237, and H238. Substitution of D229 for amino acid A had no influence on the binding activity of the antibody to the peptide. However, substitutions of R237 or H238 for charged amino acid H or R were found to abolish the binding activity. Furthermore, substitution of an uncharged amino acid Q236 for a charged amino acid D was found to reduce the binding activity significantly. Thus, R237 and H238 are key amino acids in the rubella virus E1 neutralization epitope. PMID:25226223

  18. DNA Damage Reduces the Quality, but Not the Quantity of Human Papillomavirus 16 E1 and E2 DNA Replication

    PubMed Central

    Bristol, Molly L.; Wang, Xu; Smith, Nathan W.; Son, Minkyeong P.; Evans, Michael R.; Morgan, Iain M.

    2016-01-01

    Human papillomaviruses (HPVs) are causative agents in almost all cervical carcinomas. HPVs are also causative agents in head and neck cancer, the cases of which are increasing rapidly. Viral replication activates the DNA damage response (DDR) pathway; associated proteins are recruited to replication foci, and this pathway may serve to allow for viral genome amplification. Likewise, HPV genome double-strand breaks (DSBs) could be produced during replication and could lead to linearization and viral integration. Many studies have shown that viral integration into the host genome results in unregulated expression of the viral oncogenes, E6 and E7, promoting HPV-induced carcinogenesis. Previously, we have demonstrated that DNA-damaging agents, such as etoposide, or knocking down viral replication partner proteins, such as topoisomerase II β binding protein I (TopBP1), does not reduce the level of DNA replication. Here, we investigated whether these treatments alter the quality of DNA replication by HPV16 E1 and E2. We confirm that knockdown of TopBP1 or treatment with etoposide does not reduce total levels of E1/E2-mediated DNA replication; however, the quality of replication is significantly reduced. The results demonstrate that E1 and E2 continue to replicate under genomically-stressed conditions and that this replication is mutagenic. This mutagenesis would promote the formation of substrates for integration of the viral genome into that of the host, a hallmark of cervical cancer. PMID:27338449

  19. DNA Damage Reduces the Quality, but Not the Quantity of Human Papillomavirus 16 E1 and E2 DNA Replication.

    PubMed

    Bristol, Molly L; Wang, Xu; Smith, Nathan W; Son, Minkyeong P; Evans, Michael R; Morgan, Iain M

    2016-01-01

    Human papillomaviruses (HPVs) are causative agents in almost all cervical carcinomas. HPVs are also causative agents in head and neck cancer, the cases of which are increasing rapidly. Viral replication activates the DNA damage response (DDR) pathway; associated proteins are recruited to replication foci, and this pathway may serve to allow for viral genome amplification. Likewise, HPV genome double-strand breaks (DSBs) could be produced during replication and could lead to linearization and viral integration. Many studies have shown that viral integration into the host genome results in unregulated expression of the viral oncogenes, E6 and E7, promoting HPV-induced carcinogenesis. Previously, we have demonstrated that DNA-damaging agents, such as etoposide, or knocking down viral replication partner proteins, such as topoisomerase II β binding protein I (TopBP1), does not reduce the level of DNA replication. Here, we investigated whether these treatments alter the quality of DNA replication by HPV16 E1 and E2. We confirm that knockdown of TopBP1 or treatment with etoposide does not reduce total levels of E1/E2-mediated DNA replication; however, the quality of replication is significantly reduced. The results demonstrate that E1 and E2 continue to replicate under genomically-stressed conditions and that this replication is mutagenic. This mutagenesis would promote the formation of substrates for integration of the viral genome into that of the host, a hallmark of cervical cancer. PMID:27338449

  20. Collagen-derived dipeptide prolyl-hydroxyproline promotes differentiation of MC3T3-E1 osteoblastic cells

    SciTech Connect

    Kimira, Yoshifumi; Ogura, Kana; Taniuchi, Yuri; Kataoka, Aya; Inoue, Naoki; Sugihara, Fumihito; Nakatani, Sachie; Shimizu, Jun; Wada, Masahiro; Mano, Hiroshi

    2014-10-24

    Highlights: • Pro-Hyp did not affect MC3T3-E1 cell proliferation and matrix mineralization. • Pro-Hyp significantly increased alkaline phosphatase activity. • Pro-Hyp significantly upregulated gene expression of Runx2, Osterix, and Col1α1. - Abstract: Prolyl-hydroxyproline (Pro-Hyp) is one of the major constituents of collagen-derived dipeptides. The objective of this study was to investigate the effects of Pro-Hyp on the proliferation and differentiation of MC3T3-E1 osteoblastic cells. Addition of Pro-Hyp did not affect MC3T3-E1 cell proliferation and matrix mineralization but alkaline phosphatase activity was significantly increased. Furthermore, cells treated with Pro-Hyp significantly upregulated gene expression of Runx2, Osterix, and Col1α1. These results indicate that Pro-Hyp promotes osteoblast differentiation. This study demonstrates for the first time that Pro-Hyp has a positive effect on osteoblast differentiation with upregulation of Runx2, Osterix, and Collα1 gene expression.