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

DOE Office of Scientific and Technical Information (OSTI.GOV)

Xie, Gary; Detter, Chris; Bruce, David

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 DNAmore » 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.« less

In vivo synergistic activity of a CAZyme cassette from Acidothermus cellulolyticus significantly improves the cellulolytic activity of the C. bescii exoproteome

DOE PAGES

Kim, Sun -Ki; Chung, Daehwan; Himmel, Michael E.; ...

2017-06-26

The use of microbial cells to convert plant biomass directly to fuels and chemicals is referred to as consolidated bioprocessing (CBP). Members of the bacterial genus, Caldicellulosiruptor (Gram-positive, anaerobic hyperthermophiles) are capable of deconstructing plant biomass without enzymatic or chemical pretreatment. This is accomplished by the production and secretion of free, multi-domain enzymes that outperform commercial enzyme cocktails on some substrates. Here, we show that the exoproteome of C. bescii may be enhanced by the heterologous expression of enzymes from Acidothermus cellulolyticus that act synergistically to improve sugar release from complex substrates; as well as improve cell growth. In thismore » work, co-expression of the A. cellulolyticus Acel_0615 ..beta..-glucanase (GH6 and GH12) and E1 endoglucanase (GH5) enzymes resulted in an increase in the activity of the exoproteome on Avicel; as well as an increase in growth of C. bescii on Avicel compared to the parental strain or the strain expressing the ..beta..-glucanase alone. As a result, our ability to engineer the composition and effectiveness of the exoproteome of these bacteria provides insight into the natural mechanism of plant cell wall deconstruction, as well as future directions for improving CBP.« less

In vivo synergistic activity of a CAZyme cassette from Acidothermus cellulolyticus significantly improves the cellulolytic activity of the C. bescii exoproteome

DOE Office of Scientific and Technical Information (OSTI.GOV)

Kim, Sun -Ki; Chung, Daehwan; Himmel, Michael E.

The use of microbial cells to convert plant biomass directly to fuels and chemicals is referred to as consolidated bioprocessing (CBP). Members of the bacterial genus, Caldicellulosiruptor (Gram-positive, anaerobic hyperthermophiles) are capable of deconstructing plant biomass without enzymatic or chemical pretreatment. This is accomplished by the production and secretion of free, multi-domain enzymes that outperform commercial enzyme cocktails on some substrates. Here, we show that the exoproteome of C. bescii may be enhanced by the heterologous expression of enzymes from Acidothermus cellulolyticus that act synergistically to improve sugar release from complex substrates; as well as improve cell growth. In thismore » work, co-expression of the A. cellulolyticus Acel_0615 ..beta..-glucanase (GH6 and GH12) and E1 endoglucanase (GH5) enzymes resulted in an increase in the activity of the exoproteome on Avicel; as well as an increase in growth of C. bescii on Avicel compared to the parental strain or the strain expressing the ..beta..-glucanase alone. As a result, our ability to engineer the composition and effectiveness of the exoproteome of these bacteria provides insight into the natural mechanism of plant cell wall deconstruction, as well as future directions for improving CBP.« less

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

DOE Office of Scientific and Technical Information (OSTI.GOV)

Barabote, Ravi D.; Xie, Gary; Leu, David H.

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 enzymesmore » 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

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.

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.

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

DOE Office of Scientific and Technical Information (OSTI.GOV)

Sun, Y.; Cheng, J. J.; Himmel, M. E.

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.more » 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.« less

Heterologous expression of family 10 xylanases from Acidothermus cellulolyticus enhances the exoproteome of Caldicellulosiruptor bescii and growth on xylan substrates

DOE PAGES

Kim, Sun -Ki; Chung, Daehwan; Himmel, Michael E.; ...

2016-08-22

The ability to deconstruct plant biomass without conventional pretreatment has made members of the genus Caldicellulosiruptor the target of investigation for the consolidated processing of lignocellulosic biomass to biofuels and bioproducts. These Gram-positive bacteria are hyperthermophilic anaerobes and the most thermophilic cellulolytic organisms so far described. They use both C5 and C6 sugars simultaneously and have the ability to grow well on xylan, a major component of plant cell walls. This is an important advantage for their use to efficiently convert biomass at yields sufficient for an industrial process. For commodity chemicals, yield from substrate is perhaps the most importantmore » economic factor. In an attempt to improve even further the ability of C. bescii to use xylan, we introduced two xylanases from Acidothermus cellulolyticus. Acel_0180 includes tandem carbohydrate-binding modules (CBM2 and CBM3) located at the C-terminus, one of which, CBM2, is not present in C. bescii. Also, the sequences of Xyn10A and Acel_0180 have very little homology with the GH10 domains present in C. bescii. For these reasons, we selected these xylanases as potential candidates for synergistic interaction with those in the C. bescii exoproteome. As a result, heterologous expression of two xylanases from Acidothermus cellulolyticus in Caldicellulosiruptor bescii resulted in a modest, but significant increase in the activity of the exoproteome of C. bescii on xylan substrates. Even though the increase in extracellular activity was modest, the ability of C. bescii to grow on these substrates was dramatically improved suggesting that the xylan substrate/microbe interaction substantially increased deconstruction over the secreted free enzymes alone. In conclusion, we anticipate that the ability to efficiently use xylan, a major component of plant cell walls for conversion of plant biomass to products of interest, will allow the conversion of renewable, sustainable, and

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

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

DOE Office of Scientific and Technical Information (OSTI.GOV)

Chou, Hong L.; Dai, Ziyu; Hsieh, Chia W.

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 hybridmore » 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

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

DOE Office of Scientific and Technical Information (OSTI.GOV)

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

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 E1more » 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.« less

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.

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.

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

DOE PAGES

Inoue, Hiroyuki; Decker, Stephen R.; Taylor, Larry E.; ...

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

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

DOE Office of Scientific and Technical Information (OSTI.GOV)

Inoue, Hiroyuki; Decker, Stephen R.; Taylor, Larry E.

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

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

DOE PAGES

Chung, Daehwan; Young, Jenna; Cha, Minseok; ...

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

Enhancement of β-xylosidase productivity in cellulase producing fungus Acremonium cellulolyticus

PubMed Central

2011-01-01

Enzymatic hydrolysis is one of the most important processes in bioethanol production from lignocellulosic biomass. Acremonium cellulolyticus is a filamentous fungus with high cellulase production but productivity of hemicellulase, especially β-xylosidase, is lower than other filamentous fungi. We identified 2.4 Kb β-xylosidase gene in the A. cellulolyticus genome sequence information and it encoded 798 amino acids without introns. To enhance hemicellulase productivity in A. cellulolyticus, we transformed this fungus with the identified β-xylosidase gene driven by the cellobiohydrolase Ι (cbh1) promoter, using the protoplast-polyethyleneglycol (PEG) method, and obtained a transformant, YKX1. Hydrolysis rate of xylooligosaccharides was more than 50-fold higher using culture supernatant from YKX1 than that from the parental strain, Y-94. Total cellulase activity (measured by filter paper assay) in YKX1 was not affected by the cbh1 promoter used for expression of β-xylosidase, and induced by cellulose. Since YKX1 can produce larger amount of β-xylosidase without affecting cellulase productivity, it is considered to be beneficial for practical monosaccharide recoveries from lignocellulosic biomass. PMID:21906369

Enhancement of β-xylosidase productivity in cellulase producing fungus Acremonium cellulolyticus.

PubMed

Kanna, Machi; Yano, Shinichi; Inoue, Hiroyuki; Fujii, Tatsuya; Sawayama, Shigeki

2011-06-30

Enzymatic hydrolysis is one of the most important processes in bioethanol production from lignocellulosic biomass. Acremonium cellulolyticus is a filamentous fungus with high cellulase production but productivity of hemicellulase, especially β-xylosidase, is lower than other filamentous fungi. We identified 2.4 Kb β-xylosidase gene in the A. cellulolyticus genome sequence information and it encoded 798 amino acids without introns. To enhance hemicellulase productivity in A. cellulolyticus, we transformed this fungus with the identified β-xylosidase gene driven by the cellobiohydrolase Ι (cbh1) promoter, using the protoplast-polyethyleneglycol (PEG) method, and obtained a transformant, YKX1. Hydrolysis rate of xylooligosaccharides was more than 50-fold higher using culture supernatant from YKX1 than that from the parental strain, Y-94. Total cellulase activity (measured by filter paper assay) in YKX1 was not affected by the cbh1 promoter used for expression of β-xylosidase, and induced by cellulose. Since YKX1 can produce larger amount of β-xylosidase without affecting cellulase productivity, it is considered to be beneficial for practical monosaccharide recoveries from lignocellulosic biomass.

Quantitative Evaluation of E1 Endoglucanase Recovery from Tobacco Leaves Using the Vacuum Infiltration-Centrifugation Method

PubMed Central

Kingsbury, Nathaniel J.; McDonald, Karen A.

2014-01-01

As a production platform for recombinant proteins, plant leaf tissue has many advantages, but commercialization of this technology has been hindered by high recovery and purification costs. Vacuum infiltration-centrifugation (VI-C) is a technique to obtain extracellularly-targeted products from the apoplast wash fluid (AWF). Because of its selective recovery of secreted proteins without homogenizing the whole tissue, VI-C can potentially reduce downstream production costs. Lab scale experiments were conducted to quantitatively evaluate the VI-C method and compared to homogenization techniques in terms of product purity, concentration, and other desirable characteristics. From agroinfiltrated Nicotiana benthamiana leaves, up to 81% of a truncated version of E1 endoglucanase from Acidothermus cellulolyticus was recovered with VI-C versus homogenate extraction, and average purity and concentration increases of 4.2-fold and 3.1-fold, respectively, were observed. Formulas were developed to predict recovery yields of secreted protein obtained by performing multiple rounds of VI-C on the same leaf tissue. From this, it was determined that three rounds of VI-C recovered 97% of the total active recombinant protein accessible to the VI-C procedure. The results suggest that AWF recovery is an efficient process that could reduce downstream processing steps and costs for plant-made recombinant proteins. PMID:24971334

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

DOE Office of Scientific and Technical Information (OSTI.GOV)

Gao, Johnway; Hooker, Brian S.; Skeen, R S.

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.

Enhanced conversion of plant biomass into glucose using transgenic rice-produced endoglucanase for cellulosic ethanol.

PubMed

Oraby, Hesham; Venkatesh, Balan; Dale, Bruce; Ahmad, Rashid; Ransom, Callista; Oehmke, James; Sticklen, Mariam

2007-12-01

The catalytic domain of Acidothermus cellulolyticus thermostable endoglucanase gene (encoding for endo-1,4-beta-glucanase enzyme or E1) was constitutively expressed in rice. Molecular analyses of T1 plants confirmed presence and expression of the transgene. The amount of E1 enzyme accounted for up to 4.9% of the plant total soluble proteins, and its accumulation had no apparent deleterious effects on plant growth and development. Approximately 22 and 30% of the cellulose of the Ammonia Fiber Explosion (AFEX)-pretreated rice and maize biomass respectively was converted into glucose using rice E1 heterologous enzyme. As rice is the major food crop of the world with minimal use for its straw, our results suggest a successful strategy for producing biologically active hydrolysis enzymes in rice to help generate alcohol fuel, by substituting the wasteful and polluting practice of rice straw burning with an environmentally friendly technology.

Crystallization and preliminary X-ray crystallographic analysis of a putative feruloyl esterase from Talaromyces cellulolyticus

PubMed Central

Watanabe, Masahiro; Ishikawa, Kazuhiko

2014-01-01

Feruloyl esterase (FAE; EC 3.1.1.73) catalyzes the cleavage of the ester bond between ferulic acid and polysaccharides in plant cell walls, and thus holds significant potential for the industrial utilization of biomass saccharification. A feruloyl esterase was identified from the genome database of Talaromyces cellulolyticus (formerly known as Acremonium cellulolyticus). The gene consists of the catalytic domain and a carbohydrate-binding module connected through a serine/threonine-rich linker region. The recombinant enzyme was prepared, purified and crystallized at 293 K using 0.1 M imidazole pH 8.0, 0.2 M calcium acetate, 14% PEG 8000 as the precipitant. The crystal diffracted to 2.6 Å resolution and the crystal system is primitive orthorhombic, with unit-cell parameters a = 90.9, b = 123.4, c = 135.4 Å. Four molecules are assumed to be present per asymmetric unit, corresponding to a Matthews coefficient of 2.50 Å3 Da−1 and a solvent content of 50.88%(v/v). PMID:25484222

Crystallization and preliminary X-ray crystallographic analysis of a putative feruloyl esterase from Talaromyces cellulolyticus.

PubMed

Watanabe, Masahiro; Ishikawa, Kazuhiko

2014-12-01

Feruloyl esterase (FAE; EC 3.1.1.73) catalyzes the cleavage of the ester bond between ferulic acid and polysaccharides in plant cell walls, and thus holds significant potential for the industrial utilization of biomass saccharification. A feruloyl esterase was identified from the genome database of Talaromyces cellulolyticus (formerly known as Acremonium cellulolyticus). The gene consists of the catalytic domain and a carbohydrate-binding module connected through a serine/threonine-rich linker region. The recombinant enzyme was prepared, purified and crystallized at 293 K using 0.1 M imidazole pH 8.0, 0.2 M calcium acetate, 14% PEG 8000 as the precipitant. The crystal diffracted to 2.6 Å resolution and the crystal system is primitive orthorhombic, with unit-cell parameters a = 90.9, b = 123.4, c = 135.4 Å. Four molecules are assumed to be present per asymmetric unit, corresponding to a Matthews coefficient of 2.50 Å(3) Da(-1) and a solvent content of 50.88%(v/v).

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

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

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.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Dai, Ziyu; Hooker, Brian S.; Anderson, Daniel B.

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 tomore » 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.« less

Elaborate cellulosome architecture of Acetivibrio cellulolyticus revealed by selective screening of cohesin-dockerin interactions.

PubMed

Hamberg, Yuval; Ruimy-Israeli, Vered; Dassa, Bareket; Barak, Yoav; Lamed, Raphael; Cameron, Kate; Fontes, Carlos M G A; Bayer, Edward A; Fried, Daniel B

2014-01-01

Cellulosic waste represents a significant and underutilized carbon source for the biofuel industry. Owing to the recalcitrance of crystalline cellulose to enzymatic degradation, it is necessary to design economical methods of liberating the fermentable sugars required for bioethanol production. One route towards unlocking the potential of cellulosic waste lies in a highly complex class of molecular machines, the cellulosomes. Secreted mainly by anaerobic bacteria, cellulosomes are structurally diverse, cell surface-bound protein assemblies that can contain dozens of catalytic components. The key feature of the cellulosome is its modularity, facilitated by the ultra-high affinity cohesin-dockerin interaction. Due to the enormous number of cohesin and dockerin modules found in a typical cellulolytic organism, a major bottleneck in understanding the biology of cellulosomics is the purification of each cohesin- and dockerin-containing component, prior to analyses of their interaction. As opposed to previous approaches, the present study utilized proteins contained in unpurified whole-cell extracts. This strategy was made possible due to an experimental design that allowed for the relevant proteins to be "purified" via targeted affinity interactions as a function of the binding assay. The approach thus represents a new strategy, appropriate for future medium- to high-throughput screening of whole genomes, to determine the interactions between cohesins and dockerins. We have selected the cellulosome of Acetivibrio cellulolyticus for this work due to its exceptionally complex cellulosome systems and intriguing diversity of its cellulosomal modular components. Containing 41 cohesins and 143 dockerins, A. cellulolyticus has one of the largest number of potential cohesin-dockerin interactions of any organism, and contains unusual and novel cellulosomal features. We have surveyed a representative library of cohesin and dockerin modules spanning the cellulosome's total cohesin

Discriminating between stabilizing and destabilizing protein design mutations via recombination and simulation.

PubMed

Johnson, Lucas B; Gintner, Lucas P; Park, Sehoo; Snow, Christopher D

2015-08-01

Accuracy of current computational protein design (CPD) methods is limited by inherent approximations in energy potentials and sampling. These limitations are often used to qualitatively explain design failures; however, relatively few studies provide specific examples or quantitative details that can be used to improve future CPD methods. Expanding the design method to include a library of sequences provides data that is well suited for discriminating between stabilizing and destabilizing design elements. Using thermophilic endoglucanase E1 from Acidothermus cellulolyticus as a model enzyme, we computationally designed a sequence with 60 mutations. The design sequence was rationally divided into structural blocks and recombined with the wild-type sequence. Resulting chimeras were assessed for activity and thermostability. Surprisingly, unlike previous chimera libraries, regression analysis based on one- and two-body effects was not sufficient for predicting chimera stability. Analysis of molecular dynamics simulations proved helpful in distinguishing stabilizing and destabilizing mutations. Reverting to the wild-type amino acid at destabilized sites partially regained design stability, and introducing predicted stabilizing mutations in wild-type E1 significantly enhanced thermostability. The ability to isolate stabilizing and destabilizing elements in computational design offers an opportunity to interpret previous design failures and improve future CPD methods. © The Author 2015. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.

The Impact of DNA Topology and Guide Length on Target Selection by a Cytosine-Specific Cas9.

PubMed

Tsui, Tsz Kin Martin; Hand, Travis H; Duboy, Emily C; Li, Hong

2017-06-16

Cas9 is an RNA-guided DNA cleavage enzyme being actively developed for genome editing and gene regulation. To be cleaved by Cas9, a double stranded DNA, or the protospacer, must be complementary to the guide region, typically 20-nucleotides in length, of the Cas9-bound guide RNA, and adjacent to a short Cas9-specific element called Protospacer Adjacent Motif (PAM). Understanding the correct juxtaposition of the protospacer- and PAM-interaction with Cas9 will enable development of versatile and safe Cas9-based technology. We report identification and biochemical characterization of Cas9 from Acidothermus cellulolyticus (AceCas9). AceCas9 depends on a 5'-NNNCC-3' PAM and is more efficient in cleaving negative supercoils than relaxed DNA. Kinetic as well as in vivo activity assays reveal that AceCas9 achieves optimal activity when combined with a guide RNA containing a 24-nucleotide complementarity region. The cytosine-specific, DNA topology-sensitive, and extended guide-dependent properties of AceCas9 may be explored for specific genome editing applications.

High temperature pre-digestion of corn stover biomass for improved product yields

DOE PAGES

Brunecky, Roman; Hobdey, Sarah E.; Taylor, Larry E.; ...

2014-12-03

Introduction: The efficient conversion of lignocellulosic feedstocks remains a key step in the commercialization of biofuels. One of the barriers to cost-effective conversion of lignocellulosic biomass to sugars remains the enzymatic saccharification process step. Here, we describe a novel hybrid processing approach comprising enzymatic pre-digestion with newly characterized hyperthermophilic enzyme cocktails followed by conventional saccharification with commercial enzyme preparations. Dilute acid pretreated corn stover was subjected to this new procedure to test its efficacy. Thermal tolerant enzymes from Acidothermus cellulolyticus and Caldicellulosiruptor bescii were used to pre-digest pretreated biomass at elevated temperatures prior to saccharification by the commercial cellulase formulation.more » Results: We report that pre-digestion of biomass with these enzymes at elevated temperatures prior to addition of the commercial cellulase formulation increased conversion rates and yields when compared to commercial cellulase formulation alone under low solids conditions. In conclusion, Our results demonstrating improvements in rates and yields of conversion point the way forward for hybrid biomass conversion schemes utilizing catalytic amounts of hyperthermophilic enzymes.« less

Heterologous expression of a β-d-glucosidase in Caldicellulosiruptor bescii has a surprisingly modest effect on the activity of the exoproteome and growth on crystalline cellulose

DOE Office of Scientific and Technical Information (OSTI.GOV)

Kim, Sun-Ki; Chung, Daehwan; Himmel, Michael E.

Members of the genus Caldicellulosiruptor are the most thermophilic cellulolytic bacteria so far described and are capable of efficiently utilizing complex lignocellulosic biomass without conventional pretreatment. Previous studies have shown that accumulation of high concentrations of cellobiose and, to a lesser extent, cellotriose, inhibits cellulase activity both in vivo and in vitro and high concentrations of cellobiose are present in C. bescii fermentations after 90 h of incubation. For some cellulolytic microorganisms, β-d-glucosidase is essential for the efficient utilization of cellobiose as a carbon source and is an essential enzyme in commercial preparations for efficient deconstruction of plant biomass. Inmore » spite of its ability to grow efficiently on crystalline cellulose, no extracellular β-d-glucosidase or its GH1 catalytic domain could be identified in the C. bescii genome. In order to investigate whether the addition of a secreted β-d-glucosidase would improve growth and cellulose utilization by C. bescii, we also cloned and expressed a thermostable β-d-glucosidase from Acidothermus cellulolyticus (Acel_0133) in C. bescii using the CelA signal sequence for protein export. The effect of this addition was modest, suggesting that ..beta..-d-glucosidase is not rate limiting for cellulose deconstruction and utilization by C. bescii.« less

Heterologous expression of a β-d-glucosidase in Caldicellulosiruptor bescii has a surprisingly modest effect on the activity of the exoproteome and growth on crystalline cellulose

DOE PAGES

Kim, Sun-Ki; Chung, Daehwan; Himmel, Michael E.; ...

2017-09-23

Members of the genus Caldicellulosiruptor are the most thermophilic cellulolytic bacteria so far described and are capable of efficiently utilizing complex lignocellulosic biomass without conventional pretreatment. Previous studies have shown that accumulation of high concentrations of cellobiose and, to a lesser extent, cellotriose, inhibits cellulase activity both in vivo and in vitro and high concentrations of cellobiose are present in C. bescii fermentations after 90 h of incubation. For some cellulolytic microorganisms, β-d-glucosidase is essential for the efficient utilization of cellobiose as a carbon source and is an essential enzyme in commercial preparations for efficient deconstruction of plant biomass. Inmore » spite of its ability to grow efficiently on crystalline cellulose, no extracellular β-d-glucosidase or its GH1 catalytic domain could be identified in the C. bescii genome. In order to investigate whether the addition of a secreted β-d-glucosidase would improve growth and cellulose utilization by C. bescii, we also cloned and expressed a thermostable β-d-glucosidase from Acidothermus cellulolyticus (Acel_0133) in C. bescii using the CelA signal sequence for protein export. The effect of this addition was modest, suggesting that ..beta..-d-glucosidase is not rate limiting for cellulose deconstruction and utilization by C. bescii.« less

Purification and characterization of a GH43 β-xylosidase from Enterobacter sp. identified and cloned from forest soil bacteria.

PubMed

Campos, Eleonora; Negro Alvarez, María José; Sabarís di Lorenzo, Gonzalo; Gonzalez, Sergio; Rorig, Marcela; Talia, Paola; Grasso, Daniel H; Sáez, Felicia; Manzanares Secades, Paloma; Ballesteros Perdices, Mercedes; Cataldi, Angel A

2014-01-01

The use of lignocellulosic biomass for second generation biofuels requires optimization of enzymatic breakdown of plant cell walls. In this work, cellulolytic bacteria were isolated from a native and two cultivated forest soil samples. Amplification of glycosyl hydrolases was attempted by using a low stringency-degenerate primer PCR strategy, using total soil DNA and bulk DNA pooled from positive colonies as template. A set of primers was designed based on Acidothermus cellulolyticus genome, by search of conserved domains of glycosyl hydrolases (GH) families of interest. Using this approach, a fragment containing an open reading frame (ORF) with 98% identity to a putative GH43 beta-xylosidase coding gene from Enterobacter cloacae was amplified and cloned. The full protein was expressed in Escherichia coli as N-terminal or C-terminal His-tagged fusions and purified under native conditions. Only N-terminal fusion protein, His-Xyl43, presented beta-xylosidase activity. On pNPX, optimal activity was achieved at pH 6 and 40 °C and Km and Kcat values were 2.92 mM and 1.32 seg(-1), respectively. Activity was also demonstrated on xylobiose (X2), with Km 17.8 mM and Kcat 380 s(-1). These results demonstrated that Xyl43 is a functional beta-xylosidase and it is the first evidence of this activity for Enterobacter sp. Copyright © 2013 Elsevier GmbH. All rights reserved.

The role of disulfide bond in hyperthermophilic endocellulase.

PubMed

Kim, Han-Woo; Ishikawa, Kazuhiko

2013-07-01

The hyperthermophilic endocellulase, EGPh (glycosyl hydrolase family 5) from Pyrococcus horikoshii possesses 4 cysteine residues forming 2 disulfide bonds, as identified by structural analysis. One of the disulfide bonds is located at the proximal region of the active site in EGPh, which exhibits a distinct pattern from that of the thermophilic endocellulase EGAc (glycosyl hydrolase family 5) of Acidothermus cellulolyticus despite the structural similarity between the two endocellulases. The structural similarity between EGPh and EGAc suggests that EGPh possesses a structure suitable for changing the position of the disulfide bond corresponding to that in EGAc. Introduction of this alternative disulfide bond in EGPh, while removing the original disulfide bond, did not result in a loss of enzymatic activity but the EGPh was no longer hyperthermostable. These results suggest that the contribution of disulfide bond to hyperthermostability at temperature higher than 100 °C is restrictive, and that its impact is dependent on the specific structural environment of the hyperthermophilic proteins. The data suggest that the structural position and environment of the disulfide bond has a greater effect on high-temperature thermostability of the enzyme than on the potential energy of the dihedral angle that contributes to disulfide bond cleavage.

Production of d-allulose from d-glucose by Escherichia coli transformant cells co-expressing d-glucose isomerase and d-psicose 3-epimerase genes.

PubMed

Zhang, Wenli; Li, Hao; Jiang, Bo; Zhang, Tao; Mu, Wanmeng

2017-08-01

d-Allulose is a novel and low-calorie rare monosaccharide that is a C-3 epimer of d-fructose. Because of its excellent physiological properties and commercial potential, d-allulose has attracted researchers' interests. Based on the Izumoring strategy, d-allulose is converted from d-fructose by d-psicose 3-epimerase (DPEase), while d-fructose is converted from d-glucose by d-glucose isomerase (GIase). In this study, we created a cellular system capable of converting d-glucose to d-allulose in a one-step process that co-expressed the GIase from Acidothermus cellulolyticus and the DPEase from Dorea sp. CAG. The co-expression plasmid pETDuet-Dosp-DPE/Acce-GI was generated and transformed into Escherichia coli BL21(DE3) cells. The recombinant co-expression cells exhibited maximum catalytic activity at pH 6.5 and 75 °C. These cells were thermostable at less than 60 °C. The addition of Co 2+ significantly increased the catalytic activity by 10.8-fold. When the reaction equilibrium was reached, the ratio of d-glucose, d-fructose and d-allulose was approximately 6.5:7:3, respectively. A recombinant co-expression strain that catalysed the bioconversion of d-allulose from d-glucose in a one-step process was created and characterised. When adding 500 g L -1 d-glucose as a substrate, 204.3 g L -1 d-fructose and 89.1 g L -1 d-allulose were produced. © 2016 Society of Chemical Industry. © 2016 Society of Chemical Industry.

Screening Currency Notes for Microbial Pathogens and Antibiotic Resistance Genes Using a Shotgun Metagenomic Approach

PubMed Central

Jalali, Saakshi; Kohli, Samantha; Latka, Chitra; Bhatia, Sugandha; Vellarikal, Shamsudheen Karuthedath; Sivasubbu, Sridhar; Scaria, Vinod; Ramachandran, Srinivasan

2015-01-01

Fomites are a well-known source of microbial infections and previous studies have provided insights into the sojourning microbiome of fomites from various sources. Paper currency notes are one of the most commonly exchanged objects and its potential to transmit pathogenic organisms has been well recognized. Approaches to identify the microbiome associated with paper currency notes have been largely limited to culture dependent approaches. Subsequent studies portrayed the use of 16S ribosomal RNA based approaches which provided insights into the taxonomical distribution of the microbiome. However, recent techniques including shotgun sequencing provides resolution at gene level and enable estimation of their copy numbers in the metagenome. We investigated the microbiome of Indian paper currency notes using a shotgun metagenome sequencing approach. Metagenomic DNA isolated from samples of frequently circulated denominations of Indian currency notes were sequenced using Illumina Hiseq sequencer. Analysis of the data revealed presence of species belonging to both eukaryotic and prokaryotic genera. The taxonomic distribution at kingdom level revealed contigs mapping to eukaryota (70%), bacteria (9%), viruses and archae (~1%). We identified 78 pathogens including Staphylococcus aureus, Corynebacterium glutamicum, Enterococcus faecalis, and 75 cellulose degrading organisms including Acidothermus cellulolyticus, Cellulomonas flavigena and Ruminococcus albus. Additionally, 78 antibiotic resistance genes were identified and 18 of these were found in all the samples. Furthermore, six out of 78 pathogens harbored at least one of the 18 common antibiotic resistance genes. To the best of our knowledge, this is the first report of shotgun metagenome sequence dataset of paper currency notes, which can be useful for future applications including as bio-surveillance of exchangeable fomites for infectious agents. PMID:26035208

26 CFR 1.367(e)-0 - Outline of §§ 1.367(e)-1 and 1.367(e)-2.

Code of Federal Regulations, 2011 CFR

2011-04-01

... 26 Internal Revenue 4 2011-04-01 2011-04-01 false Outline of §§ 1.367(e)-1 and 1.367(e)-2. 1.367(e)-0 Section 1.367(e)-0 Internal Revenue INTERNAL REVENUE SERVICE, DEPARTMENT OF THE TREASURY (CONTINUED) INCOME TAX (CONTINUED) INCOME TAXES Effects on Corporation § 1.367(e)-0 Outline of §§ 1.367(e)-1...

Superactive cellulase formulation using cellobiohydrolase-1 from Penicillium funiculosum

DOE Office of Scientific and Technical Information (OSTI.GOV)

Adney, William S.; Baker, John O.; Decker, Stephen R.

2008-11-11

Purified cellobiohydrolase I (glycosyl hydrolase family 7 (Cel7A) enzymes from Penicillium funiculosum demonstrate a high level of specific performance in comparison to other Cel7 family member enzymes when formulated with purified EIcd endoglucanase from A. cellulolyticus and tested on pretreated corn stover. This result is true of the purified native enzyme, as well as recombinantly expressed enzyme, for example, that enzyme expressed in a non-native Aspergillus host. In a specific example, the specific performance of the formulation using purified recombinant Cel7A from Penicillium funiculosum expressed in A. awamori is increased by more than 200% when compared to a formulation usingmore » purified Cel7A from Trichoderma reesei.« less

Superactive cellulase formulation using cellobiohydrolase-1 from Penicillium funiculosum

DOEpatents

Adney, William S.; Baker, John O.; Decker, Stephen R.; Chou, Yat-Chen; Himmel, Michael E.; Ding, Shi-You

2012-10-09

Purified cellobiohydrolase I (glycosyl hydrolase family 7 (Cel7A)) enzymes from Penicillium funiculosum demonstrate a high level of specific performance in comparison to other Cel7 family member enzymes when formulated with purified EIcd endoglucanase from A. cellulolyticus and tested on pretreated corn stover. This result is true of the purified native enzyme, as well as recombinantly expressed enzyme, for example, that enzyme expressed in a non-native Aspergillus host. In a specific example, the specific performance of the formulation using purified recombinant Cel7A from Penicillium funiculosum expressed in A. awamori is increased by more than 200% when compared to a formulation using purified Cel7A from Trichoderma reesei.

Superactive cellulase formulation using cellobiohydrolase-1 from Penicillium funiculosum

DOEpatents

Adney, William S.; Baker, John O.; Decker, Stephen R.; Chou, Yat-Chen; Himmel, Michael E.; Ding, Shi-You

2008-11-11

Purified cellobiohydrolase I (glycosyl hydrolase family 7 (Cel7A) enzymes from Penicillium funiculosum demonstrate a high level of specific performance in comparison to other Cel7 family member enzymes when formulated with purified EIcd endoglucanase from A. cellulolyticus and tested on pretreated corn stover. This result is true of the purified native enzyme, as well as recombinantly expressed enzyme, for example, that enzyme expressed in a non-native Aspergillus host. In a specific example, the specific performance of the formulation using purified recombinant Cel7A from Penicillium funiculosum expressed in A. awamori is increased by more than 200% when compared to a formulation using purified Cel7A from Trichoderma reesei.

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

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 § 1.514(e)-1...

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

Code of Federal Regulations, 2011 CFR

2011-04-01

... 26 Internal Revenue 7 2011-04-01 2009-04-01 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 § 1.514(e)-1...

mTORC1 signalling and eIF4E/4E-BP1 translation initiation factor stoichiometry influence recombinant protein productivity from GS-CHOK1 cells.

PubMed

Jossé, Lyne; Xie, Jianling; Proud, Christopher G; Smales, C Mark

2016-12-15

Many protein-based biotherapeutics are produced in cultured Chinese hamster ovary (CHO) cell lines. Recent reports have demonstrated that translation of recombinant mRNAs and global control of the translation machinery via mammalian target of rapamycin (mTOR) signalling are important determinants of the amount and quality of recombinant protein such cells can produce. mTOR complex 1 (mTORC1) is a master regulator of cell growth/division, ribosome biogenesis and protein synthesis, but the relationship between mTORC1 signalling, cell growth and proliferation and recombinant protein yields from mammalian cells, and whether this master regulating signalling pathway can be manipulated to enhance cell biomass and recombinant protein production (rPP) are not well explored. We have investigated mTORC1 signalling and activity throughout batch culture of a panel of sister recombinant glutamine synthetase-CHO cell lines expressing different amounts of a model monoclonal IgG4, to evaluate the links between mTORC1 signalling and cell proliferation, autophagy, recombinant protein expression, global protein synthesis and mRNA translation initiation. We find that the expression of the mTORC1 substrate 4E-binding protein 1 (4E-BP1) fluctuates throughout the course of cell culture and, as expected, that the 4E-BP1 phosphorylation profiles change across the culture. Importantly, we find that the eIF4E/4E-BP1 stoichiometry positively correlates with cell productivity. Furthermore, eIF4E amounts appear to be co-regulated with 4E-BP1 amounts. This may reflect a sensing of either change at the mRNA level as opposed to the protein level or the fact that the phosphorylation status, as well as the amount of 4E-BP1 present, is important in the co-regulation of eIF4E and 4E-BP1. © 2016 The Author(s).

Protein phosphatase PPM1G regulates protein translation and cell growth by dephosphorylating 4E binding protein 1 (4E-BP1).

PubMed

Liu, Jianyu; Stevens, Payton D; Eshleman, Nichole E; Gao, Tianyan

2013-08-09

Protein translation initiation is a tightly controlled process responding to nutrient availability and mitogen stimulation. Serving as one of the most important negative regulators of protein translation, 4E binding protein 1 (4E-BP1) binds to translation initiation factor 4E and inhibits cap-dependent translation in a phosphorylation-dependent manner. Although it has been demonstrated previously that the phosphorylation of 4E-BP1 is controlled by mammalian target of rapamycin in the mammalian target of rapamycin complex 1, the mechanism underlying the dephosphorylation of 4E-BP1 remains elusive. Here, we report the identification of PPM1G as the phosphatase of 4E-BP1. A coimmunoprecipitation experiment reveals that PPM1G binds to 4E-BP1 in cells and that purified PPM1G dephosphorylates 4E-BP1 in vitro. Knockdown of PPM1G in 293E and colon cancer HCT116 cells results in an increase in the phosphorylation of 4E-BP1 at both the Thr-37/46 and Ser-65 sites. Furthermore, the time course of 4E-BP1 dephosphorylation induced by amino acid starvation or mammalian target of rapamycin inhibition is slowed down significantly in PPM1G knockdown cells. Functionally, the amount of 4E-BP1 bound to the cap-dependent translation initiation complex is decreased when the expression of PPM1G is depleted. As a result, the rate of cap-dependent translation, cell size, and protein content are increased in PPM1G knockdown cells. Taken together, our study has identified protein phosphatase PPM1G as a novel regulator of cap-dependent protein translation by negatively controlling the phosphorylation of 4E-BP1.

Molecular mechanism of the dual activity of 4EGI-1: Dissociating eIF4G from eIF4E but stabilizing the binding of unphosphorylated 4E-BP1

DOE PAGES

Sekiyama, Naotaka; Arthanari, Haribabu; Papadopoulos, Evangelos; ...

2015-07-13

The eIF4E-binding protein (4E-BP) is a phosphorylation-dependent regulator of protein synthesis. The nonphosphorylated or minimally phosphorylated form binds translation initiation factor 4E (eIF4E), preventing binding of eIF4G and the recruitment of the small ribosomal subunit. Signaling events stimulate serial phosphorylation of 4E-BP, primarily by mammalian target of rapamycin complex 1 (mTORC1) at residues T 37/T 46, followed by T 70 and S 65. Hyperphosphorylated 4E-BP dissociates from eIF4E, allowing eIF4E to interact with eIF4G and translation initiation to resume. Because overexpression of eIF4E is linked to cellular transformation, 4E-BP is a tumor suppressor, and up-regulation of its activity is amore » goal of interest for cancer therapy. A recently discovered small molecule, eIF4E/eIF4G interaction inhibitor 1 (4EGI-1), disrupts the eIF4E/eIF4G interaction and promotes binding of 4E-BP1 to eIF4E. Structures of 14- to 16-residue 4E-BP fragments bound to eIF4E contain the eIF4E consensus binding motif, 54YXXXXLΦ 60 (motif 1) but lack known phosphorylation sites. We report in this paper a 2.1-Å crystal structure of mouse eIF4E in complex with m 7GTP and with a fragment of human 4E-BP1, extended C-terminally from the consensus-binding motif (4E-BP1 50–84). The extension, which includes a proline-turn-helix segment (motif 2) followed by a loop of irregular structure, reveals the location of two phosphorylation sites (S 65 and T 70). Our major finding is that the C-terminal extension (motif 3) is critical to 4E-BP1–mediated cell cycle arrest and that it partially overlaps with the binding site of 4EGI-1. Finally, the binding of 4E-BP1 and 4EGI-1 to eIF4E is therefore not mutually exclusive, and both ligands contribute to shift the equilibrium toward the inhibition of translation initiation.« less

Distinct transcriptomic changes in E14.5 mouse skeletal muscle lacking RYR1 or Cav1.1 converge at E18.5

PubMed Central

Henry, Margit; Rotshteyn, Tamara; Brunn, Anna; Carstov, Mariana; Deckert, Martina; Hescheler, Jürgen; Sachinidis, Agapios; Pfitzer, Gabriele

2018-01-01

In skeletal muscle the coordinated actions of two mechanically coupled Ca2+ channels—the 1,4-dihydropyridine receptor (Cav1.1) and the type 1 ryanodine receptor (RYR1)–underlie the molecular mechanism of rapid cytosolic [Ca2+] increase leading to contraction. While both [Ca2+]i and contractile activity have been implicated in the regulation of myogenesis, less is known about potential specific roles of Cav1.1 and RYR1 in skeletal muscle development. In this study, we analyzed the histology and the transcriptomic changes occurring at E14.5 –the end of primary myogenesis and around the onset of intrauterine limb movement, and at E18.5 –the end of secondary myogenesis, in WT, RYR1-/-, and Cav1.1-/- murine limb skeletal muscle. At E14.5 the muscle histology of both mutants exhibited initial alterations, which became much more severe at E18.5. Immunohistological analysis also revealed higher levels of activated caspase-3 in the Cav1.1-/- muscles at E14.5, indicating an increase in apoptosis. With WT littermates as controls, microarray analyses identified 61 and 97 differentially regulated genes (DEGs) at E14.5, and 493 and 1047 DEGs at E18.5, in RYR1-/- and Cav1.1-/- samples, respectively. Gene enrichment analysis detected no overlap in the affected biological processes and pathways in the two mutants at E14.5, whereas at E18.5 there was a significant overlap of DEGs in both mutants, affecting predominantly processes linked to muscle contraction. Moreover, the E18.5 vs. E14.5 comparison revealed multiple genotype-specific DEGs involved in contraction, cell cycle and miRNA-mediated signaling in WT, neuronal and bone development in RYR1-/-, and lipid metabolism in Cav1.1-/- samples. Taken together, our study reveals discrete changes in the global transcriptome occurring in limb skeletal muscle from E14.5 to E18.5 in WT, RYR1-/- and Cav1.1-/- mice. Our results suggest distinct functional roles for RYR1 and Cav1.1 in skeletal primary and secondary myogenesis. PMID

Molecular docking studies of (1E,3E,5E)-1,6-Bis(substituted phenyl)hexa-1,3,5-triene and 1,4-Bis(substituted trans-styryl)benzene analogs as novel tyrosinase inhibitors.

PubMed

Ha, Young Mi; Lee, Hye Jin; Park, Daeui; Jeong, Hyoung Oh; Park, Ji Young; Park, Yun Jung; Lee, Kyung Jin; Lee, Ji Yeon; Moon, Hyung Ryong; Chung, Hae Young

2013-01-01

We simulated the docking of the tertiary structure of mushroom tyrosinase with our compounds. From the structure-tyrosinase inhibitory activity relationship, it is notable that compounds 4, 8 and 11 showed similar or better activity rates than kojic acid which was used as a positive control. Compounds 17, 21, and 23 among benzene analogs that possess the same substituent showed significantly lower tyrosinase inhibitory effects. Therefore, we have confirmed that among the compounds showing better tyrosinase inhibitory effects than kojic acid, the compounds with triene analogs have better tyrosinase inhibitory effect than the compounds with benzene analogs. Docking simulation suggested the mechanism of compounds by several key residues which had possible hydrogen bonding interactions. The pharmacophore model underlined the features of active compounds, 4,4'-((1E,3E,5E)-hexa-1,3,5-triene-1,6-diyl)diphenol, 5,5'-((1E,3E,5E)-hexa-1,3,5-triene-1,6-diyl)bis(2-methoxy-phenol), and 5,5'-((1E,3E,5E)-hexa-1,3,5-triene-1,6-diyl)dibenzene-1,3-diol among triene derivatives which had several hydrogen bond groups on both terminal rings. The soundness of the docking results and the agreement with the pharmacophores suggest that it can be conveniently exploited to design inhibitors with an improved affinity for tyrosinase.

Functional synergy between DP-1 and E2F-1 in the cell cycle-regulating transcription factor DRTF1/E2F.

PubMed Central

Bandara, L R; Buck, V M; Zamanian, M; Johnston, L H; La Thangue, N B

1993-01-01

It is widely believed that the cellular transcription factor DRTF1/E2F integrates cell cycle events with the transcription apparatus because during cell cycle progression in mammalian cells it interacts with molecules that are important regulators of cellular proliferation, such as the retinoblastoma tumour suppressor gene product (pRb), p107, cyclins and cyclin-dependent kinases. Thus, pRb, which negatively regulates early cell cycle progression and is frequently mutated in tumour cells, and the Rb-related protein p107, bind to and repress the transcriptional activity of DRTF1/E2F. Viral oncoproteins, such as adenovirus E1a and SV40 large T antigen, overcome such repression by sequestering pRb and p107 and in so doing are likely to activate genes regulated by DRTF1/E2F, such as cdc2, c-myc and DHFR. Two sequence-specific DNA binding proteins, E2F-1 and DP-1, which bind to the E2F site, contain a small region of similarity. The functional relationship between them has, however, been unclear. We report here that DP-1 and E2F-1 exist in a DNA binding complex in vivo and that they bind efficiently and preferentially as a heterodimer to the E2F site. Moreover, studies in yeast and Drosophila cells indicate that DP-1 and E2F-1 interact synergistically in E2F site-dependent transcriptional activation. Images PMID:8223441

The Aryl Hydrocarbon Receptor Binds to E2F1 and Inhibits E2F1-induced Apoptosis

PubMed Central

Marlowe, Jennifer L.; Fan, Yunxia; Chang, Xiaoqing; Peng, Li; Knudsen, Erik S.; Xia, Ying

2008-01-01

Cellular stress by DNA damage induces checkpoint kinase-2 (CHK2)-mediated phosphorylation and stabilization of the E2F1 transcription factor, leading to induction of apoptosis by activation of a subset of proapoptotic E2F1 target genes, including Apaf1 and p73. This report characterizes an interaction between the aryl hydrocarbon (Ah) receptor (AHR), a ligand-activated transcription factor, and E2F1 that results in the attenuation of E2F1-mediated apoptosis. In Ahr−/− fibroblasts stably transfected with a doxycycline-regulated AHR expression vector, inhibition of AHR expression causes a significant elevation of oxidative stress, γH2A.X histone phosphorylation, and E2F1-dependent apoptosis, which can be blocked by small interfering RNA-mediated knockdown of E2F1 expression. In contrast, ligand-dependent AHR activation protects these cells from etoposide-induced cell death. In cells expressing both proteins, AHR and E2F1 interact independently of the retinoblastoma protein (RB), because AHR and E2F1 coimmunoprecipitate from extracts of RB-negative cells. Additionally, chromatin immunoprecipitation assays indicate that AHR and E2F1 bind to the Apaf1 promoter at a region containing a consensus E2F1 binding site but no AHR binding sites. AHR activation represses Apaf1 and TAp73 mRNA induction by a constitutively active CHK2 expression vector. Furthermore, AHR overexpression blocks the transcriptional induction of Apaf1 and p73 and the accumulation of sub-G0/G1 cells resulting from ectopic overexpression of E2F1. These results point to a proproliferative, antiapoptotic function of the Ah receptor that likely plays a role in tumor progression. PMID:18524851

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 the...

Probability 1/e

ERIC Educational Resources Information Center

Koo, Reginald; Jones, Martin L.

2011-01-01

Quite a number of interesting problems in probability feature an event with probability equal to 1/e. This article discusses three such problems and attempts to explain why this probability occurs with such frequency.

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

Code of Federal Regulations, 2014 CFR

2014-04-01

... 26 Internal Revenue 2 2014-04-01 2014-04-01 false Change in method. 1.167(e)-1 Section 1.167(e)-1... method. (a) In general. (1) Any change in the method of computing the depreciation allowances with respect to a particular account (other than a change in method permitted or required by reason of the...

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

Code of Federal Regulations, 2010 CFR

2010-04-01

... 26 Internal Revenue 10 2010-04-01 2010-04-01 false Special sourcing rule. 1.927(e)-1 Section 1.927(e)-1 Internal Revenue INTERNAL REVENUE SERVICE, DEPARTMENT OF THE TREASURY (CONTINUED) INCOME TAX... sale of export property giving rise to foreign trading gross receipts of a FSC that is treated as from...

E&V Guidebook, Version 1.1

DTIC Science & Technology

1989-08-15

checklist 14.1] Chapter 3, Language-related issues, extracts from the Ada language reference manua! [,_D 1982] those features exp!icitly a!owcd to vay...welcome. Please send comments electronically (preferred) to szymansk~aja;po.sei.cmu.edu, or by regular mail to Mr. Raymond Szymanski , AFWAL/AAAF, Wright...of Tool Features for the Ada Programming Support Environment (APSE) 4-3 4.3 E&V Report: DoD APSE Analysis 4-4 4.4 Classification Schema/E&V Taxonomy

E4orf1 Limits the Oncolytic Potential of the E1B-55K Deletion Mutant Adenovirus▿

PubMed Central

Thomas, Michael A.; Broughton, Robin S.; Goodrum, Felicia D.; Ornelles, David A.

2009-01-01

Clinical trials have shown oncolytic adenoviruses to be tumor selective with minimal toxicity toward normal tissue. The virus ONYX-015, in which the gene encoding the early region 1B 55-kDa (E1B-55K) protein is deleted, has been most effective when used in combination with either chemotherapy or radiation therapy. Therefore, improving the oncolytic nature of tumor-selective adenoviruses remains an important objective for improving this form of cancer therapy. Cells infected during the G1 phase of the cell cycle with the E1B-55K deletion mutant virus exhibit a reduced rate of viral late protein synthesis, produce fewer viral progeny, and are less efficiently killed than cells infected during the S phase. Here we demonstrate that the G1 restriction imposed on the E1B-55K deletion mutant virus is due to the viral oncogene encoded by open reading frame 1 of early region 4 (E4orf1). E4orf1 has been reported to signal through the phosphatidylinositol 3′-kinase pathway leading to the activation of Akt, mTOR, and p70 S6K. Evidence presented here shows that E4orf1 may also induce the phosphorylation of Akt and p70 S6K in a manner that depends on Rac1 and its guanine nucleotide exchange factor Tiam1. Accordingly, agents that have been reported to disrupt the Tiam1-Rac1 interaction or to prevent phosphorylation of the ribosomal S6 kinase partially alleviated the E4orf1 restriction to late viral protein synthesis and enhanced tumor cell killing by the E1B-55K mutant virus. These results demonstrate that E4orf1 limits the oncolytic nature of a conditionally replicating adenovirus such as ONYX-015. The therapeutic value of similar oncolytic adenoviruses may be improved by abrogating E4orf1 function. PMID:19129452

E4orf1 limits the oncolytic potential of the E1B-55K deletion mutant adenovirus.

PubMed

Thomas, Michael A; Broughton, Robin S; Goodrum, Felicia D; Ornelles, David A

2009-03-01

Clinical trials have shown oncolytic adenoviruses to be tumor selective with minimal toxicity toward normal tissue. The virus ONYX-015, in which the gene encoding the early region 1B 55-kDa (E1B-55K) protein is deleted, has been most effective when used in combination with either chemotherapy or radiation therapy. Therefore, improving the oncolytic nature of tumor-selective adenoviruses remains an important objective for improving this form of cancer therapy. Cells infected during the G(1) phase of the cell cycle with the E1B-55K deletion mutant virus exhibit a reduced rate of viral late protein synthesis, produce fewer viral progeny, and are less efficiently killed than cells infected during the S phase. Here we demonstrate that the G(1) restriction imposed on the E1B-55K deletion mutant virus is due to the viral oncogene encoded by open reading frame 1 of early region 4 (E4orf1). E4orf1 has been reported to signal through the phosphatidylinositol 3'-kinase pathway leading to the activation of Akt, mTOR, and p70 S6K. Evidence presented here shows that E4orf1 may also induce the phosphorylation of Akt and p70 S6K in a manner that depends on Rac1 and its guanine nucleotide exchange factor Tiam1. Accordingly, agents that have been reported to disrupt the Tiam1-Rac1 interaction or to prevent phosphorylation of the ribosomal S6 kinase partially alleviated the E4orf1 restriction to late viral protein synthesis and enhanced tumor cell killing by the E1B-55K mutant virus. These results demonstrate that E4orf1 limits the oncolytic nature of a conditionally replicating adenovirus such as ONYX-015. The therapeutic value of similar oncolytic adenoviruses may be improved by abrogating E4orf1 function.

Diagnosis of 25 genotypes of human papillomaviruses for their physical statuses in cervical precancerous/cancerous lesions: a comparison of E2/E6E7 ratio-based vs. multiple E1-L1/E6E7 ratio-based detection techniques.

PubMed

Zhang, Rong; He, Yi-feng; Chen, Mo; Chen, Chun-mei; Zhu, Qiu-jing; Lu, Huan; Wei, Zhen-hong; Li, Fang; Zhang, Xiao-xin; Xu, Cong-jian; Yu, Long

2014-10-02

Cervical lesions caused by integrated human papillomavirus (HPV) infection are highly dangerous because they can quickly develop into invasive cancers. However, clinicians are currently hampered by the lack of a quick, convenient and precise technique to detect integrated/mixed infections of various genotypes of HPVs in the cervix. This study aimed to develop a practical tool to determine the physical status of different HPVs and evaluate its clinical significance. The target population comprised 1162 women with an HPV infection history of > six months and an abnormal cervical cytological finding. The multiple E1-L1/E6E7 ratio analysis, a novel technique, was developed based on determining the ratios of E1/E6E7, E2/E6E7, E4E5/E6E7, L2/E6E7 and L1/E6E7 within the viral genome. Any imbalanced ratios indicate integration. Its diagnostic and predictive performances were compared with those of E2/E6E7 ratio analysis. The detection accuracy of both techniques was evaluated using the gold-standard technique "detection of integrated papillomavirus sequences" (DIPS). To realize a multigenotypic detection goal, a primer and probe library was established. The integration rate of a particular genotype of HPV was correlated with its tumorigenic potential and women with higher lesion grades often carried lower viral loads. The E1-L1/E6E7 ratio analysis achieved 92.7% sensitivity and 99.0% specificity in detecting HPV integration, while the E2/E6E7 ratio analysis showed a much lower sensitivity (75.6%) and a similar specificity (99.3%). Interference due to episomal copies was observed in both techniques, leading to false-negative results. However, some positive results of E1-L1/E6E7 ratio analysis were missed by DIPS due to its stochastic detection nature. The E1-L1/E6E7 ratio analysis is more efficient than E2/E6E7 ratio analysis and DIPS in predicting precancerous/cancerous lesions, in which both positive predictive values (36.7%-82.3%) and negative predictive values (75

Genetic polymorphism analysis of cytochrome P4502E1 (CYP2E1) in a Chinese Tibetan population

PubMed Central

Wang, Li; Ren, Guoxia; Li, Jingjie; Zhu, Linhao; Niu, Fanglin; Yan, Mengdan; Li, Jing; Yuan, Dongya; Jin, Tianbo

2017-01-01

Abstract Cytochrome P4502E1 (CYP2E1) gene genetic polymorphisms vary markedly in frequency among different ethnic and racial groups. We studied the genotype distributions and allele frequencies of 3 CYP2E1 polymorphisms: CYP2E1∗1A, CYP2E1∗7A, and CYP2E1∗7C by polymerase chain reaction technique in a sample of 100 healthy subjects representing Tibetan population. The frequencies of CYP2E1∗1A, ∗7A, and ∗7C alleles were 0.705, 0.125, and 0.170, respectively. Compared with other populations, we found that the allele frequencies of the variants −352A>G (rs2070672) and −333A>T (rs2070673) in this Tibetan population have significant differences compared with European-American, African-American, Japanese, Korean, and other different geographic areas in Chinese Han population. Furthermore, the results of protein prediction revealed that the variant 6397G>A (rs61710826) could influence the protein structure and function. These findings in this study would be valuable for pharmacogenetics for drug therapy and drug discovery. However, further studies in larger samples are warranted to confirm our results. PMID:29381998

The B(E2;4^+1->2^+1) / B(E2;2^+1->0^+1) Ratio in Even-Even Nuclei

NASA Astrophysics Data System (ADS)

Loelius, C.; Sharon, Y. Y.; Zamick, L.; G"Urdal, G.

2009-10-01

We considered 207 even-even nuclei throughout the chart of nuclides for which the NNDC Tables had data on the energies and lifetimes of the 2^+1 and 4^+1 states. Using these data we calculated for each nucleus the electric quadrupole transition strengths B(E2;4^+1->2^+1) and B(E2;2^+1->0^+1), as well as their ratio. The internal conversion coefficients were obtained by using the NNDC HSICC calculator. For each nucleus we plotted the B(E2) ratio against A, N, and Z. We found that for close to 90% of the nuclei considered the ratio had values between 0.5 and 2.5. Most of the outliers had magic numbers of protons or neutrons. Our ratio results were compared with the theoretical predictions for this ratio by different models--10/7 in the rotational model and 2 in the simplest vibrational model. In the rotational regions (for 150 < A < 180 and A > 220) the ratios were indeed close to 10/7. For the few nuclei thought to be vibrational the ratios were usually less than 2. Otherwise, we got a wide scatter of ratio values. Hence other models, including the NpNn scheme, must be considered in interpreting these results.

Structural insights into eRF3 and stop codon recognition by eRF1

PubMed Central

Cheng, Zhihong; Saito, Kazuki; Pisarev, Andrey V.; Wada, Miki; Pisareva, Vera P.; Pestova, Tatyana V.; Gajda, Michal; Round, Adam; Kong, Chunguang; Lim, Mengkiat; Nakamura, Yoshikazu; Svergun, Dmitri I.; Ito, Koichi; Song, Haiwei

2009-01-01

Eukaryotic translation termination is mediated by two interacting release factors, eRF1 and eRF3, which act cooperatively to ensure efficient stop codon recognition and fast polypeptide release. The crystal structures of human and Schizosaccharomyces pombe full-length eRF1 in complex with eRF3 lacking the GTPase domain revealed details of the interaction between these two factors and marked conformational changes in eRF1 that occur upon binding to eRF3, leading eRF1 to resemble a tRNA molecule. Small-angle X-ray scattering analysis of the eRF1/eRF3/GTP complex suggested that eRF1's M domain contacts eRF3's GTPase domain. Consistently, mutation of Arg192, which is predicted to come in close contact with the switch regions of eRF3, revealed its important role for eRF1's stimulatory effect on eRF3's GTPase activity. An ATP molecule used as a crystallization additive was bound in eRF1's putative decoding area. Mutational analysis of the ATP-binding site shed light on the mechanism of stop codon recognition by eRF1. PMID:19417105

Effects of different cellulases on the release of phenolic acids from rice straw during saccharification.

PubMed

Xue, Yiyun; Wang, Xiahui; Chen, Xingxuan; Hu, Jiajun; Gao, Min-Tian; Li, Jixiang

2017-06-01

Effects of different cellulases on the release of phenolic acids from rice straw during saccharification were investigated in this study. All cellulases tested increased the contents of phenolic acids during saccharification. However, few free phenolic acids were detected, as they were present in conjugated form after saccharification when the cellulases from Trichoderma reesei, Trichoderma viride and Aspergillus niger were used. On the other hand, phenolic acids were present in free form when the Acremonium cellulolyticus cellulase was used. Assays of enzyme activity showed that, besides high cellulase activity, the A. cellulolyticus cellulase exhibited high feruloyl esterase (FAE) activity. A synergistic interaction between FAE and cellulase led to the increase in free phenolic acids, and thus an increase in antioxidative and antiradical activities of the phenolic acids. Moreover, a cost estimation demonstrated the feasibility of phenolic acids as value-added products to reduce the total production cost of ethanol. Copyright © 2017 Elsevier Ltd. All rights reserved.

40 CFR 721.10171 - 1H-benz(e)indolium, 1,1,2,3-tetramethyl-, 4-methylbenzenesulfonic acid (1:1).

Code of Federal Regulations, 2010 CFR

2010-07-01

... 40 Protection of Environment 30 2010-07-01 2010-07-01 false 1H-benz(e)indolium, 1,1,2,3... SUBSTANCES Significant New Uses for Specific Chemical Substances § 721.10171 1H-benz(e)indolium, 1,1,2,3... reporting. (1) The chemical substance identified as 1H-benz(e)indolium, 1,1,2,3-tetramethyl-, 4...

26 CFR 1.503(e)-2 - Requirements.

Code of Federal Regulations, 2014 CFR

2014-04-01

... 26 Internal Revenue 7 2014-04-01 2013-04-01 true Requirements. 1.503(e)-2 Section 1.503(e)-2...) INCOME TAXES (CONTINUED) Exempt Organizations § 1.503(e)-2 Requirements. (a) In general. The requirements... price may not be a valid price for 1,000 bonds and the purchase may therefore not meet the requirements...

26 CFR 1.503(e)-2 - Requirements.

Code of Federal Regulations, 2011 CFR

2011-04-01

... 26 Internal Revenue 7 2011-04-01 2009-04-01 true Requirements. 1.503(e)-2 Section 1.503(e)-2...) INCOME TAXES (CONTINUED) Exempt Organizations § 1.503(e)-2 Requirements. (a) In general. The requirements... price may not be a valid price for 1,000 bonds and the purchase may therefore not meet the requirements...

26 CFR 1.503(e)-2 - Requirements.

Code of Federal Regulations, 2013 CFR

2013-04-01

... 26 Internal Revenue 7 2013-04-01 2013-04-01 false Requirements. 1.503(e)-2 Section 1.503(e)-2...) INCOME TAXES (CONTINUED) Exempt Organizations § 1.503(e)-2 Requirements. (a) In general. The requirements... price may not be a valid price for 1,000 bonds and the purchase may therefore not meet the requirements...

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

Code of Federal Regulations, 2010 CFR

2010-04-01

... relate to section 1402(e) as amended by section 115(b)(2) of the Social Security Amendments of 1967 (81 Stat. 839) and apply to taxable years ending after 1967. Section 1.1402(e)-5A reflects changes made by... effect prior to amendment by the Social Security Amendments of 1967) applicable to taxable years ending...

The Maize Gene terpene synthase 1 Encodes a Sesquiterpene Synthase Catalyzing the Formation of (E)-β-Farnesene, (E)-Nerolidol, and (E,E)-Farnesol after Herbivore Damage1

PubMed Central

Schnee, Christiane; Köllner, Tobias G.; Gershenzon, Jonathan; Degenhardt, Jörg

2002-01-01

Maize (Zea mays) emits a mixture of volatile compounds upon attack by the Egyptian cotton leafworm (Spodoptera littoralis). These substances, primarily mono- and sesquiterpenes, are used by parasitic wasps to locate the lepidopteran larvae, which are their natural hosts. This interaction among plant, lepidopteran larvae, and hymenopteran parasitoids benefits the plant and has been termed indirect defense. The committed step in the biosynthesis of the different skeletal types of mono- and sesquiterpenes is catalyzed by terpene synthases, a class of enzymes that forms a large variety of mono- and sesquiterpene products from prenyl diphosphate precursors. We isolated a terpene synthase gene, terpene synthase 1 (tps1), from maize that exhibits only a low degree of sequence identity to previously identified terpene synthases. Upon expression in a bacterial system, the encoded enzyme produced the acyclic sesquiterpenes, (E)-β-farnesene, (E,E)-farnesol, and (3R)-(E)-nerolidol, the last an intermediate in the formation of (3E)-4,8-dimethyl-1,3,7-nonatriene. Both (E)-β-farnesene and (3E)-4,8-dimethyl-1,3,7-nonatriene are prominent compounds of the maize volatile blend that is emitted after herbivore damage. The biochemical characteristics of the encoded enzyme are similar to those of terpene synthases from both gymnosperms and dicotyledonous angiosperms, suggesting that catalysis involves a similar electrophilic reaction mechanism. The transcript level of tps1 in the maize cv B73 was elevated after herbivory, mechanical damage, and treatment with elicitors. In contrast, the increase in the transcript level of the tps1 gene or gene homolog in the maize cv Delprim after herbivory was less pronounced, suggesting that the regulation of terpene synthase expression may vary among maize varieties. PMID:12481088

26 CFR 1.503(e)-1 - Special rules.

Code of Federal Regulations, 2010 CFR

2010-04-01

... adequate security and a reasonable rate of interest) the acquisition of a bond, debenture, note, or...) Section 503(e) does not affect the requirement in section 503(b)(1) of a reasonable rate of interest. Thus... indebtedness does not bear a reasonable rate of interest. (3) The provisions of section 503(e) do not limit the...

Evolution of the ubiquitin-activating enzyme Uba1 (E1)

NASA Astrophysics Data System (ADS)

Allan, Douglas C.; Phillips, J. C.

2017-10-01

Ubiquitin tags diseased proteins and initiates an enzyme conjugation cascade, which has three stages. The first-stage enzyme Uba1 (E1) has evolved only modestly from slime mold to humans, and is > 14 times larger than Ub. Here we use critical point thermodynamic scaling theory to connect Uba1 (E1) evolution from yeast and slime mold to fruit flies and humans to subtle changes in its amino acid sequences.

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 are...

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 are...

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 are...

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 are...

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 of...

Plasmalogen modulation attenuates atherosclerosis in ApoE- and ApoE/GPx1-deficient mice.

PubMed

Rasmiena, Aliki A; Barlow, Christopher K; Stefanovic, Nada; Huynh, Kevin; Tan, Ricardo; Sharma, Arpeeta; Tull, Dedreia; de Haan, Judy B; Meikle, Peter J

2015-12-01

We previously reported a negative association of circulating plasmalogens (phospholipids with proposed atheroprotective properties) with coronary artery disease. Plasmalogen modulation was previously demonstrated in animals but its effect on atherosclerosis was unknown. We assessed the effect of plasmalogen enrichment on atherosclerosis of murine models with differing levels of oxidative stress. Six-week old ApoE- and ApoE/glutathione peroxidase-1 (GPx1)-deficient mice were fed a high-fat diet with/without 2% batyl alcohol (precursor to plasmalogen synthesis) for 12 weeks. Mass spectrometry analysis of lipids showed that batyl alcohol supplementation to ApoE- and ApoE/GPx1-deficient mice increased the total plasmalogen levels in both plasma and heart. Oxidation of plasmalogen in the treated mice was evident from increased level of plasmalogen oxidative by-product, sn-2 lysophospholipids. Atherosclerotic plaque in the aorta was reduced by 70% (P = 5.69E-07) and 69% (P = 2.00E-04) in treated ApoE- and ApoE/GPx1-deficient mice, respectively. A 40% reduction in plaque (P = 7.74E-03) was also seen in the aortic sinus of only the treated ApoE/GPx1-deficient mice. Only the treated ApoE/GPx1-deficient mice showed a decrease in VCAM-1 staining (-28%, P = 2.43E-02) in the aortic sinus and nitrotyrosine staining (-78%, P = 5.11E-06) in the aorta. Plasmalogen enrichment via batyl alcohol supplementation attenuated atherosclerosis in ApoE- and ApoE/GPx1-deficient mice, with a greater effect in the latter group. Plasmalogen enrichment may represent a viable therapeutic strategy to prevent atherosclerosis and reduce cardiovascular disease risk, particularly under conditions of elevated oxidative stress and inflammation. Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.

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

USDA-ARS?s Scientific Manuscript database

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,...

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

Anti-E1E2 antibodies status prior therapy favors direct-acting antiviral treatment efficacy.

PubMed

Virlogeux, Victor; Berthillon, Pascale; Bordes, Isabelle; Larrat, Sylvie; Crouy, Stéphanie; Scholtès, Caroline; Pradat, Pierre; Maynard, Marianne; Zoulim, Fabien; Leroy, Vincent; Chemin, Isabelle; Trépo, Christian; Petit, Marie-Anne

2018-03-15

Presence of anti-E1E2 antibodies was previously associated with spontaneous cure of hepatitis C virus (HCV) and predictive before treatment of a sustained virological response (SVR) to bi- or tri-therapy in naïve or experienced patients, regardless of HCV genotype. We investigated the impact of anti-E1E2 seroprevalence at baseline on treatment response in patients receiving direct-acting antiviral (DAA) therapy. We screened anti-E1E2 antibodies by ELISA in serum samples collected at treatment initiation for two groups of patients: 59 with SVR at the end of DAA treatment and 44 relapsers after DAA treatment. Nineteen patients received a combination of ribavirin (RBV) or PEG-interferon/ribavirin with sofosbuvir or daclatasvir and others received interferon-free treatment with DAA±RBV. HCV viral load was measured at different time points during treatment in a subgroup of patients. A significant association was observed between presence of anti-E1E2 and HCV viral load<6log10 prior treatment. Among patients with anti-E1E2 at baseline, 70% achieved SVR whereas among patients without anti-E1E2, only 45% achieved SVR. Conversely, 66% of patients experiencing DAA-failure were anti-E1E2 negative at baseline. In the multivariate analysis, presence of anti-E1E2 was significantly associated with SVR after adjustment on potential cofounders such as age, sex, fibrosis stage, prior HCV treatment and alanine aminotransferase (ALT) level. The presence of anti-E1E2 at treatment initiation is a predictive factor of SVR among patients treated with DAA and more likely among patients with low initial HCV viral load (<6log10). Absence of anti-E1E2 at baseline could predict DAA-treatment failure. Copyright © 2018 Elsevier Masson SAS. All rights reserved.

E&V (Evaluation and Validation) Reference Manual, Version 1.1

DTIC Science & Technology

1988-10-20

E&V. This model will allow the user to arrive at E&V techniques through many different paths, and provides a means to extract useful information...electronically (preferred) to szymansk@ajpo.sei.cmu.edu or by regular mail to Mr. Raymond Szymanski , AFWAL/AAAF, Wright Patterson AFB, OH 45433-6543. ES-2 E&V...1, 1-3 illustrate the types of infor- mation to be extracted from each document. Chapter 2 provides a more detailed description of the structure and

E6/E7-P53-POU2F1-CTHRC1 axis promotes cervical cancer metastasis and activates Wnt/PCP pathway

PubMed Central

Zhang, Rong; Lu, Huan; Lyu, Yuan-yuan; Yang, Xiao-mei; Zhu, Lin-yan; Yang, Guang-dong; Jiang, Peng-cheng; Re, Yuan; Song, Wei-wei; Wang, Jin-hao; Zhang, Can-can; Gu, Fei; Luo, Tian-jiao; Wu, Zhi-yong; Xu, Cong-jian

2017-01-01

Cervical cancer is an infectious cancer and the most common gynecologic cancer worldwide. E6/E7, the early genes of the high-risk mucosal human papillomavirus type, play key roles in the carcinogenic process of cervical cancer. However, little was known about its roles in modulating tumor microenvironment, particular extracellular matrix (ECM). In this study, we found that E6/E7 could regulate multiple ECM proteins, especially collagen triple helix repeat containing 1 (CTHRC1). CTHRC1 is highly expressed in cervical cancer tissue and serum and closely correlated with clinicopathological parameters. CTHRC1 promotes cervical cancer cell migration and invasion in vitro and metastasis in vivo. E6/E7 regulates the expression of CTHRC1 in cervical cancer by E6/E7-p53-POU2F1 (POU class 2 homeobox 1) axis. Futhermore, CTHRC1 activates Wnt/PCP signaling pathway. Take together, E6/E7-p53-POU2F1-CTHRC1 axis promotes cervical cancer cell invasion and metastasis and may act as a potential therapeutic target for interventions against cervical cancer invasion and metastasis. PMID:28303973

E-Peptides Control Bioavailability of IGF-1

PubMed Central

Piszczek, Agnieszka; Perlas, Emarald; Winn, Nadine; Nastasi, Tommaso; Rosenthal, Nadia

2012-01-01

Insulin-like growth factor 1 (IGF-1) is a potent cytoprotective growth factor that has attracted considerable attention as a promising therapeutic agent. Transgenic over-expression of IGF-1 propeptides facilitates protection and repair in a broad range of tissues, although transgenic mice over-expressing IGF-1 propeptides display little or no increase in IGF-1 serum levels, even with high levels of transgene expression. IGF-1 propeptides are encoded by multiple alternatively spliced transcripts including C-terminal extension (E) peptides, which are highly positively charged. In the present study, we use decellularized mouse tissue to show that the E-peptides facilitate in vitro binding of murine IGF-1 to the extracellular matrix (ECM) with varying affinities. This property is independent of IGF-1, since proteins consisting of the E-peptides fused to relaxin, a related member of the insulin superfamily, bound equally avidly to decellularized ECM. Thus, the E-peptides control IGF-1 bioavailability by preventing systemic circulation, offering a potentially powerful way to tether IGF-1 and other therapeutic proteins to the site of synthesis and/or administration. PMID:23251442

Cooperative transformation and coexpression of bovine papillomavirus type 1 E5 and E7 proteins.

PubMed

Bohl, J; Hull, B; Vande Pol, S B

2001-01-01

Productively infected bovine fibropapillomas were examined for bovine papillomavirus type 1 (BPV-1) E7 localization. BPV-1 E7 was observed in the cytoplasm of basal and lower spinous epithelial cells, coexpressed in the cytoplasm of basal cells with the E5 oncoprotein. E7 was also observed in nucleoli throughout the basal and spinous layers but not in the granular cell layer. Ectopic expression of E7 in cultured epithelial cells gave rise to localization similar to that seen in productive fibropapillomas, with cytoplasmic and nucleolar expression observed. Consistent with the coexpression of E7 and E5 in basal keratinocytes, BPV-1 E7 cooperated with E5 as well as E6 in an anchorage independence transformation assay. While E5 is expressed in both basal and superficial differentiating keratinocytes, BPV-1 E7 is only observed in basal and lower spinous epithelial cells. Therefore, BPV-1 E7 may serve to modulate the cellular response of basal epithelial cells to E5 expression.

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

Code of Federal Regulations, 2013 CFR

2013-04-01

... TAX (CONTINUED) INCOME TAXES (CONTINUED) Empowerment Zone Employment Credit § 1.1397E-1 Qualified zone... section 1397E(d)(2)(B)(i), (ii), (iii), (iv) or (v). Services of employees of the eligible local education... empowerment zone or enterprise community (as defined in section 1393), or there is a reasonable expectation...

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

Code of Federal Regulations, 2014 CFR

2014-04-01

... TAX (CONTINUED) INCOME TAXES (CONTINUED) Empowerment Zone Employment Credit § 1.1397E-1 Qualified zone... section 1397E(d)(2)(B)(i), (ii), (iii), (iv) or (v). Services of employees of the eligible local education... empowerment zone or enterprise community (as defined in section 1393), or there is a reasonable expectation...

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

Code of Federal Regulations, 2012 CFR

2012-04-01

... TAX (CONTINUED) INCOME TAXES (CONTINUED) Empowerment Zone Employment Credit § 1.1397E-1 Qualified zone... section 1397E(d)(2)(B)(i), (ii), (iii), (iv) or (v). Services of employees of the eligible local education... empowerment zone or enterprise community (as defined in section 1393), or there is a reasonable expectation...

Ubiquitination independent of E1 and E2 enzymes by bacterial effectors

DOE Office of Scientific and Technical Information (OSTI.GOV)

Qiu, Jiazhang; Sheedlo, Michael J.; Yu, Kaiwen

Signaling by ubiquitination regulates virtually every cellular process in eukaryotes. Covalent attachment of ubiquitin to a substrate is catalyzed by the E1, E2 and E3 three-enzyme cascade 1, which links the C terminus of ubiquitin via an isopeptide bond mostly to the ε-amino group of a lysine of the substrate. 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 2. For example, many bacterial pathogens exploit ubiquitin signaling using virulence factors that function as E3 ligases, deubiquitinases 3 or asmore » enzymes that directly attack ubiquitin 4. The bacterial pathogen Legionella pneumophila utilizes approximately 300 effectors that modulate diverse host processes to create a niche permissive for its replication in phagocytes 5. Here we demonstrate that members of the SidE effector family (SidEs) of L. pneumophila ubiquitinate multiple Rab small GTPases associated with the endoplasmic reticulum (ER). Moreover, we show that these proteins are capable of catalyzing ubiquitination without the need for the E1 and E2 enzymes. The E1/E2-independent ubiquitination catalyzed by these enzymes requires NAD but not ATP and Mg2+. A putative mono ADP-ribosyltransferase (mART) motif critical for the ubiquitination activity is also essential for the role of SidEs in intracellular bacterial replication in a protozoan host. These results establish that ubiquitination can be catalyzed by a single enzyme.« less

Resolvin E1 (Rv E1 ) attenuates LPS induced inflammation and subsequent atrophy in C2C12 myotubes.

PubMed

Baker, Luke A; Martin, Neil R W; Kimber, Marc C; Pritchard, Gareth J; Lindley, Martin R; Lewis, Mark P

2018-03-25

Resolution of inflammation is now known to be an active process which in part is instigated and controlled by specialized pro-resolving lipid mediators (SPM's) derived from dietary omega-3 fatty acids. Resolvin E1 (R v E 1 ) is one of these SPM's derived from the omega-3 fatty acid eicosapentaenoic acid. Using both molecular and phenotypic functional measures we report that in a model of Lipopolysaccharide (LPS) induced inflammation, R v E 1 attenuated mRNA levels of both interlukin-6 and monocyte chemoattractant protein-1 whilst having no effect on tumor necrosis factor-α or interlukin-1β in C2C12 skeletal muscle myotubes. Findings at the molecular level were transferred into similar changes in extracellular protein levels of the corresponding genes with the greatest attenuation being noted in IL-6 protein concentrations. R v E 1 instigated beneficial morphological changes through the prevention of LPS induced skeletal muscle atrophy, in tandem with attenuation of the LPS induced reduction in contractile force in tissue engineered skeletal muscle. These findings demonstrate, in our model of endotoxin induced inflammation in skeletal muscle, that R v E 1 has pro-resolving properties in this cell type. Our data provides rationale for further investigation into the mechanistic action of R v E 1 in skeletal muscle, with the vision of having potential benefits for the prevention/resolution of in-vivo skeletal muscle atrophy. © 2018 Wiley Periodicals, Inc.

E1 and M1 γ-strength functions in 144Nd

DOE PAGES

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 experimentalmore » data best.« less

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

Code of Federal Regulations, 2010 CFR

2010-04-01

... 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 in connection with certain railroad rolling stock. (a) Allowance of deduction—(1) Election. Under section 263(e...

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

Code of Federal Regulations, 2010 CFR

2010-04-01

...)(6) (exception where stock held during entire existence of corporation) and section 1059(e)(2... dividend is a qualifying dividend. (b) Reorganizations. For purposes of section 1059(e)(1), any exchange...

26 CFR 49.4264(e)-1 - Round trips.

Code of Federal Regulations, 2011 CFR

2011-04-01

... 26 Internal Revenue 16 2011-04-01 2011-04-01 false Round trips. 49.4264(e)-1 Section 49.4264(e)-1... TAXES FACILITIES AND SERVICES EXCISE TAXES Transportation of Persons § 49.4264(e)-1 Round trips. (a) In general. For purposes of the regulations in this subpart, a round trip shall be considered to consist of...

26 CFR 49.4264(e)-1 - Round trips.

Code of Federal Regulations, 2013 CFR

2013-04-01

... 26 Internal Revenue 16 2013-04-01 2013-04-01 false Round trips. 49.4264(e)-1 Section 49.4264(e)-1... TAXES FACILITIES AND SERVICES EXCISE TAXES Transportation of Persons § 49.4264(e)-1 Round trips. (a) In general. For purposes of the regulations in this subpart, a round trip shall be considered to consist of...

26 CFR 49.4264(e)-1 - Round trips.

Code of Federal Regulations, 2012 CFR

2012-04-01

... 26 Internal Revenue 16 2012-04-01 2012-04-01 false Round trips. 49.4264(e)-1 Section 49.4264(e)-1... TAXES FACILITIES AND SERVICES EXCISE TAXES Transportation of Persons § 49.4264(e)-1 Round trips. (a) In general. For purposes of the regulations in this subpart, a round trip shall be considered to consist of...

E1 transitions from octupole vibration states

NASA Astrophysics Data System (ADS)

Cottle, P. D.

1993-04-01

Electric dipole moments are extracted from data for E1 transitions deexciting octupole vibration states in nineteen nuclei. The moments are then compared to values calculated using the droplet model prescription of Dorso, Myers, and Swiatecki. It is found that the E1 moments in quadrupole deformed nuclei can be reproduced with the droplet model using the same model parameters that reproduce atomic masses and fission barriers. This result supports the suggestion of Butler and Nazarewicz that single particle effects are usually much smaller than macroscopic effects in E1 transitions associated with octupole vibrations in reflection symmetric deformed nuclei.

Characterisation of Translation Elongation Factor eEF1B Subunit Expression in Mammalian Cells and Tissues and Co-Localisation with eEF1A2

PubMed Central

Janikiewicz, Justyna; Doig, Jennifer; Abbott, Catherine M.

2014-01-01

Translation elongation is the stage of protein synthesis in which the translation factor eEF1A plays a pivotal role that is dependent on GTP exchange. In vertebrates, eEF1A can exist as two separately encoded tissue-specific isoforms, eEF1A1, which is almost ubiquitously expressed, and eEF1A2, which is confined to neurons and muscle. The GTP exchange factor for eEF1A1 is a complex called eEF1B made up of subunits eEF1Bα, eEF1Bδ and eEF1Bγ. Previous studies have cast doubt on the ability of eEF1B to interact with eEF1A2, suggesting that this isoform might use a different GTP exchange factor. We show that eEF1B subunits are all widely expressed to varying degrees in different cell lines and tissues, and at different stages of development. We show that ablation of any of the subunits in human cell lines has a small but significant impact on cell viability and cycling. Finally, we show that both eEF1A1 and eEF1A2 colocalise with all eEF1B subunits, in such close proximity that they are highly likely to be in a complex. PMID:25436608

Synthesis and structural study on (1E,2E,1'E,2'E)-3,3'-bis[(4-bromophenyl)-3,3'-(4-methy-1,2-phenylene diimine)] acetaldehyde dioxime: A combined experimental and theoretical study

NASA Astrophysics Data System (ADS)

Topal, T.; Kart, H. H.; Tunay Taşlı, P.; Karapınar, E.

2015-06-01

Tetradentate (1E,2E,1'E,2'E)-3,3'-bis[(4-bromophenyl)-3,3'-(4-methy-1,2-phenylene diimine)] acetaldehyde dioxime which possess N4 donor sets derived from the condensation of isonitroso- p-bromoacetophenone and 3,4-diaminotoluene are synthesized and characterized. The characterization of tetradentate Schiff base ligand has been deduced from LC-MS, FTIR, 13C and 1H NMR spectra and elemental analysis. Furthermore, the molecular geometry, infrared and NMR spectra of the title molecule in the ground state have been calculated by using the quantum chemical computational methods such as density functional theory (DFT) and ab initio Hartree-Fock (HF) methods with the 6-31G(d) and 6-311G(d) basis sets. The computed bond lengths and bond angles by using the both methods show the good agreement with each other. Moreover, the vibrational frequencies have been calculated and the scaled values have been compared with the experimental FTIR spectroscopic data. Assignments of the vibrational modes are made on the basis of potential energy distribution (PED) calculated from by using VEDA program. The correlations between the observed and calculated frequencies are in good agreement with each other as well as the correlation of the NMR data.

E1A promoter of bovine adenovirus type 3.

PubMed

Xing, Li; Tikoo, Suresh Kumar

2006-12-01

Conserved motifs of eukaryotic gene promoters, such as TATA box and CAAT box sequences, of E1A of human adenoviruses (e.g human adenovirus 5) lie between the left inverted terminal repeat (ITR) and the ATG of E1A. However, analysis of the left end of the bovine adenovirus 3 (BAdV-3) genome revealed that the conserved sequences of the E1A promoter are present only in the ITR. As such, the promoter activity of ITR was tested in the context of a BAdV-3 vector or a plasmid-based system. Different regions of the left end of the BAdV-3 genome initiated transcription of the red fluorescent protein gene in a plasmid-based system. Moreover, BAdV-3 mutants in which the open reading frame of E1A was placed immediately downstream of the ITR produced E1A transcript and could be propagated in non-E1A-complementing Madin-Darby bovine kidney cells. These results suggest that the left ITR contains the sole BAdV-3 E1A promoter.

CYP2E1 Metabolism of Styrene Involves Allostery

PubMed Central

Hartman, Jessica H.; Boysen, Gunnar

2012-01-01

We are the first to report allosterism during styrene oxidation by recombinant CYP2E1 and human liver microsomes. At low styrene concentrations, oxidation is inefficient because of weak binding to CYP2E1 (Ks = 830 μM). A second styrene molecule then binds CYP2E1 with higher affinity (Kss = 110 μM) and significantly improves oxidation to achieve a kcat of 6.3 nmol · min−1 · nmol CYP2E1−1. The transition between these metabolic cycles coincides with reported styrene concentrations in blood from exposed workers; thus, this CYP2E1 mechanism may be relevant in vivo. Scaled modeling of the in vitro-positive allosteric mechanism for styrene metabolism to its in vivo clearance led to significant deviations from the traditional model based on Michaelis-Menten kinetics. Low styrene levels were notably much less toxic than generally assumed. We interrogated the allosteric mechanism using the CYP2E1-specific inhibitor and drug 4-methylpyrazole, which we have shown binds two CYP2E1 sites. From the current studies, styrene was a positive allosteric effector on 4-methylpyrazole binding, based on a 10-fold increase in 4-methylpyrazole binding affinity from Ki 0.51 to Ksi 0.043 μM. The inhibitor was a negative allosteric effector on styrene oxidation, because kcat decreased 6-fold to 0.98 nmol · min−1 · nmol CYP2E1−1. Consequently, mixtures of styrene and other molecules can induce allosteric effects on binding and metabolism by CYP2E1 and thus mitigate the efficiency of their metabolism and corresponding effects on human health. Taken together, our elucidation of mechanisms for these allosteric reactions provides a powerful tool for further investigating the complexities of CYP2E1 metabolism of drugs and pollutants. PMID:22807108

Analysis of eIF4E and 4EBP1 mRNAs in head and neck cancer.

PubMed

Sunavala-Dossabhoy, Gulshan; Palaniyandi, Senthilnathan; Clark, Cheryl; Nathan, Cherie-Ann O; Abreo, Fleurette W; Caldito, Gloria

2011-10-01

The eukaryotic translation initiation factor 4E (eIF4E) in conjunction with its binding protein, 4EBP1, regulates the translation of cap-dependent mRNAs. An aberrant increase in eIF4E shifts the balance in favor of translation of transcripts that promote cell proliferation and malignancy. eIF4E protein is commonly elevated in head and neck squamous cell carcinomas (HNSCC), and its overexpression is associated with increased recurrence. An underlying mechanism for eIF4E overexpression is gene amplification, and we wanted to determine whether eIF4E mRNA could serve as a prognostic maker of HNSCC. Tumor specimens from 26 HNSCC patients and oral tissues from 17 control subjects were examined for eIF4E and 4EBP1 by semiquantitative RT-PCR and correlated with clinical and pathologic findings. Unlike eIF4E mRNA alone, expression of eIF4E relative to 4EBP1 was a more precise predictor of HNSCC and its progression (P < .01, Wilcoxon rank sum test). Eight of 26 patients (31%) had elevated eIF4E:4EBP1 (4E:4EBP1; >25), and 7 of these (87.5%) had recurrence. Alternately, from 18 patients with low 4E:4EBP1 (<25; 69%), only 5 patients had recurrence (30.1%). To determine the probability of no recurrence, Kaplan-Meier analysis showed significantly poor disease-free survival in patients with elevated 4E:4EBP1 than those with low ratios (P < .01, log rank test). Elevated 4E:4EBP1 significantly correlated with increased disease recurrence. Because 4EBP1 modulates eIF4E activity, our results highlight the importance of incorporating a joint analysis of eIF4E and 4EBP1 mRNAs in HNSCC patient care decisions. Copyright © 2011 The American Laryngological, Rhinological, and Otological Society, Inc.

CDH1 regulates E2F1 degradation in response to differentiation signals in keratinocytes

PubMed Central

Singh, Randeep K.; Dagnino, Lina

2017-01-01

The E2F1 transcription factor plays key roles in skin homeostasis. In the epidermis, E2F1 expression is essential for normal proliferation of undifferentiated keratinocytes, regeneration after injury and DNA repair following UV radiation-induced photodamage. Abnormal E2F1 expression promotes nonmelanoma skin carcinoma. In addition, E2F1 must be downregulated for proper keratinocyte differentiation, but the relevant mechanisms involved remain poorly understood. We show that differentiation signals induce a series of post-translational modifications in E2F1 that are jointly required for its downregulation. Analysis of the structural determinants that govern these processes revealed a central role for S403 and T433. In particular, substitution of these two amino acid residues with non-phosphorylatable alanine (E2F1 ST/A) interferes with E2F1 nuclear export, K11- and K48-linked polyubiquitylation and degradation in differentiated keratinocytes. In contrast, replacement of S403 and T433 with phosphomimetic aspartic acid to generate a pseudophosphorylated E2F1 mutant protein (E2F1 ST/D) generates a protein that is regulated in a manner indistinguishable from that of wild type E2F1. Cdh1 is an activating cofactor that interacts with the anaphase-promoting complex/cyclosome (APC/C) ubiquitin E3 ligase, promoting proteasomal degradation of various substrates. We found that Cdh1 associates with E2F1 in keratinocytes. Inhibition or RNAi-mediated silencing of Cdh1 prevents E2F1 degradation in response to differentiation signals. Our results reveal novel regulatory mechanisms that jointly modulate post-translational modifications and downregulation of E2F1, which are necessary for proper epidermal keratinocyte differentiation. PMID:27903963

CDH1 regulates E2F1 degradation in response to differentiation signals in keratinocytes.

PubMed

Singh, Randeep K; Dagnino, Lina

2017-01-17

The E2F1 transcription factor plays key roles in skin homeostasis. In the epidermis, E2F1 expression is essential for normal proliferation of undifferentiated keratinocytes, regeneration after injury and DNA repair following UV radiation-induced photodamage. Abnormal E2F1 expression promotes nonmelanoma skin carcinoma. In addition, E2F1 must be downregulated for proper keratinocyte differentiation, but the relevant mechanisms involved remain poorly understood. We show that differentiation signals induce a series of post-translational modifications in E2F1 that are jointly required for its downregulation. Analysis of the structural determinants that govern these processes revealed a central role for S403 and T433. In particular, substitution of these two amino acid residues with non-phosphorylatable alanine (E2F1 ST/A) interferes with E2F1 nuclear export, K11- and K48-linked polyubiquitylation and degradation in differentiated keratinocytes. In contrast, replacement of S403 and T433 with phosphomimetic aspartic acid to generate a pseudophosphorylated E2F1 mutant protein (E2F1 ST/D) generates a protein that is regulated in a manner indistinguishable from that of wild type E2F1. Cdh1 is an activating cofactor that interacts with the anaphase-promoting complex/cyclosome (APC/C) ubiquitin E3 ligase, promoting proteasomal degradation of various substrates. We found that Cdh1 associates with E2F1 in keratinocytes. Inhibition or RNAi-mediated silencing of Cdh1 prevents E2F1 degradation in response to differentiation signals. Our results reveal novel regulatory mechanisms that jointly modulate post-translational modifications and downregulation of E2F1, which are necessary for proper epidermal keratinocyte differentiation.

A MUB E2 structure reveals E1 selectivity between cognate ubiquitin E2s in eukaryotes

NASA Astrophysics Data System (ADS)

Lu, Xiaolong; Malley, Konstantin R.; Brenner, Caitlin C.; Koroleva, Olga; Korolev, Sergey; Downes, Brian P.

2016-08-01

Ubiquitin (Ub) is a protein modifier that controls processes ranging from protein degradation to endocytosis, but early-acting regulators of the three-enzyme ubiquitylation cascade are unknown. Here we report that the prenylated membrane-anchored ubiquitin-fold protein (MUB) is an early-acting regulator of subfamily-specific E2 activation. An AtMUB3:AtUBC8 co-crystal structure defines how MUBs inhibit E2~Ub formation using a combination of E2 backside binding and a MUB-unique lap-bar loop to block E1 access. Since MUBs tether Arabidopsis group VI E2 enzymes (related to HsUbe2D and ScUbc4/5) to the plasma membrane, and inhibit E2 activation at physiological concentrations, they should function as potent plasma membrane localized regulators of Ub chain synthesis in eukaryotes. Our findings define a biochemical function for MUB, a family of highly conserved Ub-fold proteins, and provide an example of selective activation between cognate Ub E2s, previously thought to be constitutively activated by E1s.

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

DOE Office of Scientific and Technical Information (OSTI.GOV)

Turner, Roberta L.; Wilkinson, John C., E-mail: john.wilkinson@ndsu.edu; Ornelles, David A., E-mail: ornelles@wakehealth.edu

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)more » 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.« less

Therapeutic Strategies Against Cyclin E1 Amplified Ovarian Cancers

DTIC Science & Technology

2017-10-01

interaction will lead to enhancement of RB/E2F interaction and suppression of E2F- dependent oncogenic activity resulting in activity against CCNE1-amplified...relevant for CCNE1-amplified ovarian tumors which are dependent on hyperactive HR and are sensitive to suppression of BRCA1. 15. SUBJECT TERMS Ovarian...enhancement of RB/E2F interaction and suppression of E2F- dependent oncogenic activity resulting in activity against CCNE1-amplified cells. In the third

Structure of a Glomulin-RBX1-CUL1 Complex: Inhibition of a RING E3 Ligase through Masking of Its E2-Binding Surface

DOE Office of Scientific and Technical Information (OSTI.GOV)

Duda, David M.; Olszewski, Jennifer L.; Tron, Adriana E.

2012-11-01

The approximately 300 human cullin-RING ligases (CRLs) are multisubunit E3s in which a RING protein, either RBX1 or RBX2, recruits an E2 to catalyze ubiquitination. RBX1-containing CRLs also can bind Glomulin (GLMN), which binds RBX1's RING domain, regulates the RBX1-CUL1-containing SCF{sup FBW7} complex, and is disrupted in the disease Glomuvenous Malformation. Here we report the crystal structure of a complex between GLMN, RBX1, and a fragment of CUL1. Structural and biochemical analyses reveal that GLMN adopts a HEAT-like repeat fold that tightly binds the E2-interacting surface of RBX1, inhibiting CRL-mediated chain formation by the E2 CDC34. The structure explains themore » basis for GLMN's selectivity toward RBX1 over RBX2, and how disease-associated mutations disrupt GLMN-RBX1 interactions. Our study reveals a mechanism for RING E3 ligase regulation, whereby an inhibitor blocks E2 access, and raises the possibility that other E3s are likewise controlled by cellular proteins that mask E2-binding surfaces to mediate inhibition.« less

Structure of a Glomulin-RBX1-CUL1 complex: inhibition of a RING E3 ligase through masking of its E2-binding surface

PubMed Central

Duda, David M.; Olszewski, Jennifer L.; Tron, Adriana E.; Hammel, Michal; Lambert, Lester J.; Waddell, M. Brett; Mittag, Tanja; DeCaprio, James A.; Schulman, Brenda A.

2012-01-01

Summary The ~300 human Cullin-RING ligases (CRLs) are multisubunit E3s in which a RING protein, either RBX1 or RBX2, recruits an E2 to catalyze ubiquitination. RBX1-containing CRLs also can bind Glomulin (GLMN), which binds RBX1’s RING domain, regulates the RBX1-CUL1-containing SCFFBW7 complex, and is disrupted in the disease Glomuvenous Malformation. Here we report the crystal structure of a complex between GLMN, RBX1, and a fragment of CUL1. Structural and biochemical analyses reveal that GLMN adopts a HEAT-like repeat fold that tightly binds the E2-interacting surface of RBX1, inhibiting CRL-mediated chain formation by the E2 CDC34. The structure explains the basis for GLMN’s selectivity toward RBX1 over RBX2, and how disease-associated mutations disrupt GLMN-RBX1 interactions. Our study reveals a mechanism for RING E3 ligase regulation whereby an inhibitor blocks E2 access, and raises the possibility that other E3s are likewise controlled by cellular proteins that mask E2-binding surfaces to mediate inhibition. PMID:22748924

CYP2E1 Potentiates Ethanol-induction of Hypoxia and HIF-1α in vivo

PubMed Central

Wang, Xiaodong; Wu, Defeng; Yang, Lili; Gan, Lixia; Cederbaum, Arthur I

2013-01-01

Ethanol induces hypoxia and elevates HIF-1α in the liver. CYP2E1 plays a role in the mechanisms by which ethanol generates oxidative stress, fatty liver and liver injury. The current study evaluated whether CYP2E1 contributes to ethanol-induced hypoxia and activation of HIF-1α in vivo and whether HIF-1α protects against or promotes CYP2E1-dependent toxicity in vitro. Wild type (WT), CYP2E1-knockin (KI) and CYP2E1 knockout (KO) mice were fed ethanol chronically; pair fed controls received isocaloric dextrose. Ethanol produced liver injury in the KI mice to a much greater extent than in the WT and KO mice. Protein levels of HIF-1α and downstream targets of HIF-1α activation were elevated in the ethanol-fed KI mice compared to the WT and KO mice. Levels of HIF prolylhydroxlase 2 which promotes HIF-1α degradation were decreased in the ethanol-fed KI mice in association with the increases in HIF-1α. Hypoxia occurred in the ethanol-fed CYP2E1 KI mice as shown by an increased area of staining using the hypoxia-specific marker pimonidazole. Hypoxia was lower in the ethanol-fed WT mice and lowest in the ethanol fed KO mice and all the dextrose-fed mice. In situ double staining showed that pimonidazole and CYP2E1 were co-localized to the same area of injury in the hepatic centrilobule. Increased protein levels of HIF-1α were also found after acute ethanol treatment of KI mice. Treatment of HepG2 E47 cells which express CYP2E1 with ethanol plus arachidonic (AA) acid or ethanol plus buthionine sulfoximine (BSO) which depletes GSH caused loss of cell viability to greater extent than in HepG2 C34 cells which do not express CYP2E1. These treatments elevated protein levels of HIF-1α to a greater extent in E47 cells than C34 cells. 2-Methoxyestradiol, an inhibitor of HIF-1α, blunted the toxic effects of ethanol plus AA and ethanol plus BSO in the E47 cells in association with inhibition of HIF-1α. The HIF-1α inhibitor also blocked the elevated oxidative stress produced

HPV16 E6 regulates annexin 1 (ANXA1) protein expression in cervical carcinoma cell lines

DOE Office of Scientific and Technical Information (OSTI.GOV)

Calmon, Marilia Freitas; Sichero, Laura; Boccardo, Enrique

Annexin 1 (ANXA1) is a substrate for E6AP mediated ubiquitylation. It has been hypothesized that HPV 16 E6 protein redirects E6AP away from ANXA1, increasing its stability and possibly contributing to viral pathogenesis. We analyzed ANXA1 expression in HPV-positive and negative cervical carcinoma-derived cells, in cells expressing HPV-16 oncogenes and in cells transduced with shRNA targeting E6AP. We observed that ANXA1 protein expression increased in HPV-16-positive tumor cells, in keratinocytes expressing HPV-16 E6wt (wild-type) or E6/E7 and C33 cells expressing HPV-16 E6wt. ANXA1 protein expression decreased in cells transfected with E6 Dicer-substrate RNAs (DsiRNA) and C33 cells cotransduced with HPV-16more » E6wt and E6AP shRNA. Moreover, colony number and proliferation rate decreased in HPV16-positive cells transduced with ANXA1 shRNA. We observed that in cells infected with HPV16, the E6 binds to E6AP to degrade p53 and upregulate ANXA1. We suggest that ANXA1 may play a role in HPV-mediated carcinogenesis. - Highlights: • ANXA1 upregulation requires the presence of E6 and E6AP and is dependent on E6 integrity. • E6 binds to E6AP to degrade p53 and upregulate ANXA1 in cells infected with HPV16. • ANXA1 plays a role in cell proliferation in HPV-positive cervical cells.« less

The nuclear receptor NR2E1/TLX controls senescence.

PubMed

O'Loghlen, Ana; Martin, Nadine; Krusche, Benjamin; Pemberton, Helen; Alonso, Marta M; Chandler, Hollie; Brookes, Sharon; Parrinello, Simona; Peters, Gordon; Gil, Jesús

2015-07-30

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 tumors including glioblastomas. Despite NR2E1 regulating targets like p21(CIP1) or PTEN we still lack a full explanation for its role in NSC self-renewal and tumorigenesis. We know that polycomb repressive complexes 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 p16(INK4a) and direct repression of p21(CIP1). In addition NR2E1 expression also counteracts oncogene-induced senescence. 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.

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

PubMed Central

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; Oualid, Farid El; Ovaa, Huib

2016-01-01

Post-translational modifications of proteins with ubiquitin (Ub) and ubiquitin-like (Ubl) modifiers, orchestrated by a cascade of specialized E1, E2 and E3 enzymes, control a staggering breadth of cellular processes. To monitor catalysis along these complex reaction pathways, we developed a cascading activity-based probe, UbDha. Akin 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 ubiquitin-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 activities in living cells, presents novel and versatile tools to interrogate the Ub/Ubl cascades. PMID:27182664

[Changes of biological behavioral of E. coli K1 after ppk1 gene deletion].

PubMed

Peng, Liang; Pan, Jiayun; Luo, Su; Yang, Zhenghui; Huang, Mufang; Cao, Hong

2014-06-01

To study the changes in biological behaviors of meningitis E. coli K1 strain E44 after deletion of polyphosphate kinase 1 (ppk1) gene and explore the role of ppk1 in the pathogenesis of E. coli K1-induced meningitis. The wild-type strain E. coli K1 and ppk1 deletion mutant were exposed to heat at 56 degrees celsius; for 6 min, and their survival rates were determined. The adhesion and invasion of the bacteria to human brain microvascular endothelial cells (HBMECs) were observed using electron microscopy and quantitative tests. HBMECs were co-incubated with wild-type strain or ppk1 deletion mutant, and the cytoskeleton rearrangement was observed under laser scanning confocal microscope. The survival rate of the ppk1 deletion mutant was significantly lower than that of the wild-type strain after heat exposure. The ppk1 deletion mutant also showed lowered cell adhesion and invasion abilities and weakened ability to induce cytoskeleton rearrangement in HBMECs. ppk1 gene is important for E.coli K1 for heat resistance, cell adhesion and invasion, and for inducing cytoskeletal rearrangement in HBMECs.

Artificial Recruitment of UAF1-USP Complexes by a PHLPP1-E1 Chimeric Helicase Enhances Human Papillomavirus DNA Replication

PubMed Central

Gagnon, David; Lehoux, Michaël

2015-01-01

ABSTRACT The E1 helicase from anogenital human papillomavirus (HPV) types interacts with the cellular WD repeat-containing protein UAF1 in complex with the deubiquitinating enzyme USP1, USP12, or USP46. This interaction stimulates viral DNA replication and is required for maintenance of the viral episome in keratinocytes. E1 associates with UAF1 through a short UAF1-binding site (UBS) located within the N-terminal 40 residues of the protein. Here, we investigated if the E1 UBS could be replaced by the analogous domain from an unrelated protein, the pleckstrin homology domain and leucine-rich repeat protein phosphatase 1 (PHLPP1). We found that PHLPP1 and E1 interact with UAF1 in a mutually exclusive manner and mapped the minimal PHLPP1 UBS (PUBS) to a 100-amino-acid region sufficient for assembly into UAF1-USP complexes. Similarly to the E1 UBS, overexpression of PUBS in trans inhibited HPV DNA replication, albeit less efficiently. Characterization of a PHLPP1-E1 chimeric helicase revealed that PUBS could partially substitute for the E1 UBS in enhancing viral DNA replication and that the stimulatory effect of PUBS likely involves recruitment of UAF1-USP complexes, as it was abolished by mutations that weaken UAF1-binding and by overexpression of catalytically inactive USPs. Although functionally similar to the E1 UBS, PUBS is larger in size and requires both the WD repeat region and C-terminal ubiquitin-like domain of UAF1 for interaction, in contrast to E1, which does not contact the latter. Overall, this comparison of two heterologous UBSs indicates that these domains function as transferable protein interaction modules and provide further evidence that the association of E1 with UAF1-containing deubiquitinating complexes stimulates HPV DNA replication. IMPORTANCE The E1 protein from anogenital HPV types interacts with the UAF1-associated deubiquitinating enzymes USP1, USP12, and USP46 to stimulate replication of the viral genome. Little is known about the

Artificial Recruitment of UAF1-USP Complexes by a PHLPP1-E1 Chimeric Helicase Enhances Human Papillomavirus DNA Replication.

PubMed

Gagnon, David; Lehoux, Michaël; Archambault, Jacques

2015-06-01

The E1 helicase from anogenital human papillomavirus (HPV) types interacts with the cellular WD repeat-containing protein UAF1 in complex with the deubiquitinating enzyme USP1, USP12, or USP46. This interaction stimulates viral DNA replication and is required for maintenance of the viral episome in keratinocytes. E1 associates with UAF1 through a short UAF1-binding site (UBS) located within the N-terminal 40 residues of the protein. Here, we investigated if the E1 UBS could be replaced by the analogous domain from an unrelated protein, the pleckstrin homology domain and leucine-rich repeat protein phosphatase 1 (PHLPP1). We found that PHLPP1 and E1 interact with UAF1 in a mutually exclusive manner and mapped the minimal PHLPP1 UBS (PUBS) to a 100-amino-acid region sufficient for assembly into UAF1-USP complexes. Similarly to the E1 UBS, overexpression of PUBS in trans inhibited HPV DNA replication, albeit less efficiently. Characterization of a PHLPP1-E1 chimeric helicase revealed that PUBS could partially substitute for the E1 UBS in enhancing viral DNA replication and that the stimulatory effect of PUBS likely involves recruitment of UAF1-USP complexes, as it was abolished by mutations that weaken UAF1-binding and by overexpression of catalytically inactive USPs. Although functionally similar to the E1 UBS, PUBS is larger in size and requires both the WD repeat region and C-terminal ubiquitin-like domain of UAF1 for interaction, in contrast to E1, which does not contact the latter. Overall, this comparison of two heterologous UBSs indicates that these domains function as transferable protein interaction modules and provide further evidence that the association of E1 with UAF1-containing deubiquitinating complexes stimulates HPV DNA replication. The E1 protein from anogenital HPV types interacts with the UAF1-associated deubiquitinating enzymes USP1, USP12, and USP46 to stimulate replication of the viral genome. Little is known about the molecular nature of the E1

Production of β-xylosidase from Trichoderma asperellum KIF125 and its application in efficient hydrolysis of pretreated rice straw with fungal cellulase.

PubMed

Inoue, Hiroyuki; Kitao, Chiaki; Yano, Shinichi; Sawayama, Shigeki

2016-11-01

On-site cellulase and hemicellulase production is a promising way to reduce enzyme cost in the commercialization of the lignocellulose-to-ethanol process. A hemicellulase-producing fungal strain suitable for on-site enzyme production was selected from cultures prepared using wet disc-milling rice straw (WDM-RS) and identified as Trichoderma asperellum KIF125. KIF125 hemicellulase showed uniquely high abundance of β-xylosidase in the xylanolytic enzyme system compared to other fungal hemicellulase preparations. Supplementation of Talaromyces cellulolyticus cellulase with KIF125 hemicellulase was more effective than that with the hemicellulases from other fungal sources in reducing the total enzyme loading for the improvement of xylose yield in the hydrolysis of ball-milling RS, due to its high β-xylosidase dominance. β-Xylosidase in KIF125 hemicellulase was purified and classified as a glycosyl hydrolase family 3 enzyme with relatively high specificity for xylobiose. The production of KIF125 β-xylosidase in the fermentor was estimated as 118 U/g-WDM-RS (2350 U/L culture) at 48 h. These results demonstrate that KIF125 is promising as a practical hemicellulase source to combine with on-site cellulase production using T. cellulolyticus.

TFDP3 was expressed in coordination with E2F1 to inhibit E2F1-mediated apoptosis in prostate cancer.

PubMed

Ma, Yueyun; Xin, Yijuan; Li, Rui; Wang, Zhe; Yue, Qiaohong; Xiao, Fengjing; Hao, Xiaoke

2014-03-10

TFDP3 has been previously identified as an inhibitor of E2F molecules. It has been shown to suppress E2F1-induced apoptosis dependent P53 and to play a potential role in carcinogenesis. However, whether it indeed helps cancer cells tolerate apoptosis stress in cancer tissues remains unknown. TFDP3 expression was assessed by RT-PCR, in situ hybridization and immunohistochemistry in normal human tissues, cancer tissues and prostate cancer tissues. The association between TFDP3 and E2F1 in prostate cancer development was analyzed in various stages. Apoptosis was evaluated with annexin-V and propidium iodide staining and flow-cytometry. The results show that, in 96 samples of normal human tissues, TFDP3 could be detected in the cerebrum, esophagus, stomach, small intestine, bronchus, breast, ovary, uterus, and skin, but seldom in the lung, muscles, prostate, and liver. In addition, TFDP3 was highly expressed in numerous cancer tissues, such as brain-keratinous, lung squamous cell carcinoma, testicular seminoma, cervical carcinoma, skin squamous cell carcinoma, gastric adenocarcinoma, liver cancer, and prostate cancer. Moreover, TFDP3 was positive in 23 (62.2%) of 37 prostate cancer samples regardless of stage. Furthermore, immunohistochemistry results show that TFDP3 was always expressed in coordination with E2F1 at equivalent expression levels in prostate cancer tissues, and was highly expressed particularly in samples of high stage. When E2F1 was extrogenously expressed in LNCap cells, TFDP3 could be induced, and the apoptosis induced by E2F1 was significantly decreased. It was demonstrated that TFDP3 was a broadly expressed protein corresponding to E2F1 in human tissues, and suggested that TFDP3 is involved in prostate cancer cell survival by suppressing apoptosis induced by E2F1. Copyright © 2013 Elsevier B.V. All rights reserved.

Active sites prediction and binding analysis E1-E2 protein human papillomavirus with biphenylsulfonacetic acid

NASA Astrophysics Data System (ADS)

Iryani, I.; Amelia, F.; Iswendi, I.

2018-04-01

Cervix cancer triggered by Human papillomavirus infection is the second cause to woman death in worldwide. The binding site of E1-E2 protein of HPV 16 is not known from a 3-D structure yet, so in this study we address this issue to study the structure of E1-E2 protein from Human papillomavirus type 16 and to find its potential binding sites using biphenylsulfonacetic acid as inhibitor. Swiss model was used for 3D structure prediction and PDB: 2V9P (E1 protein) and 2NNU (E2 protein) having 52.32% and 100% identity respectively was selected as a template. The 3D model structure developed of E1 and E2 in the core and allowed regions were 99.2% and 99.5%. The ligand binding sites were predicted using online server meta pocket 2.0 and MOE 2009.10 was used for docking. E1-and E2 protein of HPV-16 has three potential binding site that can interact with the inhibitors. The Docking biphenylsulfonacetic acid using these binding sites shows that ligand interact with the protein through hydrogen bonds on Lys 403, Arg 410, His 551 in the first pocket, on Tyr 32, Leu 99 in the second pocket, and Lys 558m Lys 517 in the third pocket.

Novel mechanism of JNK pathway activation by adenoviral E1A

PubMed Central

Morrison, Helen; Pospelova, Tatiana V.; Pospelov, Valery A.; Herrlich, Peter

2014-01-01

The adenoviral oncoprotein E1A influences cellular regulation by interacting with a number of cellular proteins. In collaboration with complementary oncogenes, E1A fully transforms primary cells. As part of this action, E1A inhibits transcription of c-Jun:Fos target genes while promoting that of c-Jun:ATF2-dependent genes including jun. Both c-Jun and ATF2 are hyperphosphorylated in response to E1A. In the current study, E1A was fused with the ligand binding domain of the estrogen receptor (E1A-ER) to monitor the immediate effect of E1A activation. With this approach we now show that E1A activates c-Jun N-terminal kinase (JNK), the upstream kinases MKK4 and MKK7, as well as the small GTPase Rac1. Activation of the JNK pathway requires the N-terminal domain of E1A, and, importantly, is independent of transcription. In addition, it requires the presence of ERM proteins. Downregulation of signaling components upstream of JNK inhibits E1A-dependent JNK/c-Jun activation. Taking these findings together, we show that E1A activates the JNK/c-Jun signaling pathway upstream of Rac1 in a transcription-independent manner, demonstrating a novel mechanism of E1A action. PMID:24742962

Transient replication of BPV-1 requires two viral polypeptides encoded by the E1 and E2 open reading frames.

PubMed Central

Ustav, M; Stenlund, A

1991-01-01

Bovine papillomavirus (BPV) DNA is maintained as an episome with a constant copy number in transformed cells and is stably inherited. To study BPV replication we have developed a transient replication assay based on a highly efficient electroporation procedure. Using this assay we have determined that in the context of the viral genome two of the viral open reading frames, E1 and E2, are required for replication. Furthermore we show that when produced from expression vectors in the absence of other viral gene products, the full length E2 transactivator polypeptide and a 72 kd polypeptide encoded by the E1 open reading frame in its entirety, are both necessary and sufficient for replication BPV in C127 cells. Images PMID:1846806

Transient replication of BPV-1 requires two viral polypeptides encoded by the E1 and E2 open reading frames.

PubMed

Ustav, M; Stenlund, A

1991-02-01

Bovine papillomavirus (BPV) DNA is maintained as an episome with a constant copy number in transformed cells and is stably inherited. To study BPV replication we have developed a transient replication assay based on a highly efficient electroporation procedure. Using this assay we have determined that in the context of the viral genome two of the viral open reading frames, E1 and E2, are required for replication. Furthermore we show that when produced from expression vectors in the absence of other viral gene products, the full length E2 transactivator polypeptide and a 72 kd polypeptide encoded by the E1 open reading frame in its entirety, are both necessary and sufficient for replication BPV in C127 cells.

E-ELT M1 test facility

NASA Astrophysics Data System (ADS)

Dimmler, M.; Marrero, J.; Leveque, S.; Barriga, P.; Sedghi, B.; Mueller, M.

2012-09-01

During the advanced design phase of the European Extremely Large Telescope (E-ELT) several critical components have been prototyped. During the last year some of them have been tested in dedicated test stands. In particular, a representative section of the E-ELT primary mirror has been assembled with 2 active and 2 passive segments. This test stand is equipped with complete prototype segment subunits, i.e. including support mechanisms, glass segments, edge sensors, position actuators as well as additional metrology for monitoring. The purpose is to test various procedures such as calibration, alignment and handling and to study control strategies. In addition the achievable component and subsystem performances are evaluated, and interface issues are identified. In this paper an overview of the activities related to the E-ELT M1 Test Facility will be given. Experiences and test results are presented.

Control of SIV infection and subsequent induction of pandemic H1N1 immunity in rhesus macaques using an Ad5 [E1-, E2b-] vector platform.

PubMed

Gabitzsch, Elizabeth S; Balint-Junior, Joseph P; Xu, Younong; Balcaitis, Stephanie; Sanders-Beer, Brigitte; Karl, Julie; Weinhold, Kent J; Paessler, Slobodan; Jones, Frank R

2012-11-26

Anti-vector immunity mitigates immune responses induced by recombinant adenovirus vector vaccines, limiting their prime-boost capabilities. We have developed a novel gene delivery and expression platform (Ad5 [E1-, E2b-]) that induces immune responses despite pre-existing and/or developed concomitant Ad5 immunity. In the present study, we evaluated if this new Ad5 platform could overcome the adverse condition of pre-existing Ad5 immunity to induce effective immune responses in prime-boost immunization regimens against two different infectious diseases in the same animal. Ad5 immune rhesus macaques (RM) were immunized multiple times with the Ad5 [E1-, E2b-] platform expressing antigens from simian immunodeficiency virus (SIV). Immunized RM developed cell-mediated immunity against SIV antigens Gag, Pol, Nef and Env as well as antibody against Env. Vaccinated and vector control RMs were challenged intra-rectally with homologous SIVmac239. During a 7-week follow-up, there was perturbation of SIV load in some immunized RM. At 7 weeks post-challenge, eight immunized animals (53%) did not have detectable SIV, compared to two RM controls (13%) (P<0.02; log-rank Mantel-Cox test). There was no correlation of protective MHC contributing to infection control. The RM without detectable circulating SIV, now hyper immune to Ad5, were then vaccinated with the same Ad5 [E1-, E2b-] platform expressing H1N1 influenza hemagglutinin (HA). Thirty days post Ad5 [E1-, E2b-]-HA vaccination, significant levels of influenza neutralizing antibody were induced in all animals that increased after an Ad5 [E1-, E2b-]-HA homologous boost. These data demonstrate the versatility of this new vector platform to immunize against two separate disease targets in the same animal despite the presence of immunity against the delivery platform, permitting homologous repeat immunizations with an Ad5 gene delivery platform. Copyright © 2012 Elsevier Ltd. All rights reserved.

Complete replication-competent adenovirus 11p vectors with E1 or E3 insertions show improved heat stability

DOE Office of Scientific and Technical Information (OSTI.GOV)

Mei, Ya-Fang, E-mail: ya-fang.mei@umu.se

2016-10-15

Conventional adenovirus vectors harboring E1 or E3 deletions followed by the insertion of an exogenous gene show considerably reduced virion stability. Here, we report strategies to generate complete replication-competent Ad11p(RCAd11p) vectors that overcome the above disadvantage. A GFP cassette was successfully introduced either upstream of E1A or in the E3A region. The resulting vectors showed high expression levels of the hexon and E1genes and also strongly induced the cytopathic effect in targeted cells. When harboring oversized genomes, the RCAd11pE1 and RCAd11pE3 vectors showed significantly improved heat stability in comparison to Ad11pwt;of the three, RCAd11pE3 was the most tolerant to heatmore » treatment. Electron microscopy showed that RCAd11pE3, RCAd11pE1, Ad11pwt, and Ad11pE1 Delmanifested dominant, moderate, minimum, or no full virus particles after heat treatment at 47 °C for 5 h. Our results demonstrated that both genome size and the insertion site in the viral genome affect virion stability. -- Highlights: •Replicating adenovirus 11p GFP vectors at the E1 or E3 region were generated. •RCAd11pE3 and RCAd11pE1 vectors manifested significantly improved heat stability. •RCAd11pE3 and RCAd11pE1 showed more full viral particles than Ad11pwt after heating. •We demonstrated that both genome size and the insertion site affect virion stability.« less

Bioactive constituents from Chinese natural medicines. XXII. Absolute structures of new megastigmane glycosides, sedumosides E1, E2, E3, F1, F2, and G, from Sedum sarmentosum (Crassulaceae).

PubMed

Morikawa, Toshio; Zhang, Yi; Nakamura, Seikou; Matsuda, Hisashi; Muraoka, Osamu; Yoshikawa, Masayuki

2007-03-01

Six new megastigmane glycosides, sedumosides E1, E2, E3, F1, F2, and G, were isolated from the whole plant of Sedum sarmentosum (Crassulaceae). The structures of new constituents including the absolute configuration were elucidated on the basis of chemical and physicochemical evidence.

Rotenone Induction of Hydrogen Peroxide Inhibits mTOR-mediated S6K1 and 4E-BP1/eIF4E Pathways, Leading to Neuronal Apoptosis

PubMed Central

Zhou, Qian; Liu, Chunxiao; Liu, Wen; Zhang, Hai; Zhang, Ruijie; Liu, Jia; Zhang, Jinfei; Xu, Chong; Liu, Lei; Huang, Shile; Chen, Long

2015-01-01

Rotenone, a common pesticide and inhibitor of mitochondrial complex I, induces loss of dopaminergic neurons and consequential aspects of Parkinson’s disease (PD). However, the exact mechanism of rotenone neurotoxicity is not fully elucidated. Here, we show that rotenone induced reactive oxygen species (ROS), leading to apoptotic cell death in PC12 cells and primary neurons. Pretreatment with catalase (CAT), a hydrogen peroxide-scavenging enzyme, attenuated rotenone-induced ROS and neuronal apoptosis, implying hydrogen peroxide (H2O2) involved, which was further verified by imaging intracellular H2O2 using a peroxide-selective probe H2DCFDA. Using thenoyltrifluoroacetone (TTFA), antimycin A, or Mito-TEMPO, we further demonstrated rotenone-induced mitochondrial H2O2-dependent neuronal apoptosis. Rotenone dramatically inhibited mTOR-mediated phosphorylation of S6K1 and 4E-BP1, which was also attenuated by CAT in the neuronal cells. Of interest, ectopic expression of wild-type mTOR or constitutively active S6K1, or downregulation of 4E-BP1 partially prevented rotenone-induced H2O2 and cell apoptosis. Furthermore, we noticed that rotenone-induced H2O2 was linked to the activation of caspase-3 pathway. This was evidenced by the finding that pretreatment with CAT partially blocked rotenone-induced cleavages of caspase-3 and poly (ADP-ribose) polymerase. Of note, zVAD-fmk, a pan caspase inhibitor, only partially prevented rotenone-induced apoptosis in PC12 cells and primary neurons. Expression of mTOR-wt, S6K1-ca, or silencing 4E-BP1 potentiated zVAD-fmk protection against rotenone-induced apoptosis in the cells. The results indicate that rotenone induction of H2O2 inhibits mTOR-mediated S6K1 and 4E-BP1/eIF4E pathways, resulting in caspase-dependent and -independent apoptosis in neuronal cells. Our findings suggest that rotenone-induced neuronal loss in PD may be prevented by activating mTOR signaling and/or administering antioxidants. PMID:25304210

4E-BP1 regulates the differentiation of white adipose tissue.

PubMed

Tsukiyama-Kohara, Kyoko; Katsume, Asao; Kimura, Kazuhiro; Saito, Masayuki; Kohara, Michinori

2013-07-01

4E Binding protein 1 (4E-BP1) suppresses translation initiation. The absence of 4E-BP1 drastically reduces the amount of adipose tissue in mice. To address the role of 4E-BP1 in adipocyte differentiation, we characterized 4E-BP1(-/-) mice in this study. The lack of 4E-BP1 decreased the amount of white adipose tissue and increased the amount of brown adipose tissue. In 4E-BP1(-/-) MEF cells, PPARγ coactivator 1 alpha (PGC-1α) expression increased and exogenous 4E-BP1 expression suppressed PGC-1α expression. The level of 4E-BP1 expression was higher in white adipocytes than in brown adipocytes and showed significantly greater up-regulation in white adipocytes than in brown adipocytes during preadipocyte differentiation into mature adipocytes. The amount of PGC-1α was consistently higher in HB cells (a brown preadipocyte cell line) than in HW cells (a white preadipocyte cell line) during differentiation. Moreover, the ectopic over-expression of 4E-BP1 suppressed PGC-1α expression in white adipocytes, but not in brown adipocytes. Thus, the results of our study indicate that 4E-BP1 may suppress brown adipocyte differentiation and PGC-1α expression in white adipose tissues. © 2013 The Authors Genes to Cells © 2013 by the Molecular Biology Society of Japan and Wiley Publishing Asia Pty Ltd.

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.

Proof without Words: (1 + 1/n)[superscript n] less than e less than (1 + 1/n)[superscript n+1

ERIC Educational Resources Information Center

Khattri, Sanjay Kumar

2008-01-01

We present a pictorial proof of the inequation (1 + 1/n)[superscript n] less than e less than (1 + 1/n)[superscript n+1]. The inequation is also confirmed through the Taylor expansion and alternating series theorem.

Helicopter Flying Qualities Characteristics-CH-46E. Volume 1

DTIC Science & Technology

1983-10-03

STABILITY 54 6.0 CONTROL SENSITIVITY 61 . ° i CONTENTS (continued) Page 7.0 TIME HISTORY DATA 65 7.1 GENERAL 65 7.2 DYNAMIC STABILITY 65 7.3 CONTROL...The CH-46E meets these requirements by comfortable margins at the conditions tested. 61 NADC-81118-60 Volume 1 CH-46E (FRB, CONTROL IESPON!SE CH-46E...0 4.- ip L% t ,. _ 5" * ,.4 ••t, EFIGURE 7-5 I 7 ----- 1 FIU- - IA2 NADC-81118-6 Volume I-- .4 . > b in Zh. JI - ag4A 19-- O e t’ c 4 a I,- t

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. © 2015 Wiley Periodicals, Inc.

Association of papillomavirus E6 proteins with either MAML1 or E6AP clusters E6 proteins by structure, function, and evolutionary relatedness

PubMed Central

Brimer, Nicole

2017-01-01

Papillomavirus E6 proteins bind to LXXLL peptide motifs displayed on targeted cellular proteins. Alpha genus HPV E6 proteins associate with the cellular ubiquitin ligase E6AP (UBE3A), by binding to an LXXLL peptide (ELTLQELLGEE) displayed by E6AP, thereby stimulating E6AP ubiquitin ligase activity. Beta, Gamma, and Delta genera E6 proteins bind a similar LXXLL peptide (WMSDLDDLLGS) on the cellular transcriptional co-activator MAML1 and thereby repress Notch signaling. We expressed 45 different animal and human E6 proteins from diverse papillomavirus genera to ascertain the overall preference of E6 proteins for E6AP or MAML1. E6 proteins from all HPV genera except Alpha preferentially interacted with MAML1 over E6AP. Among animal papillomaviruses, E6 proteins from certain ungulate (SsPV1 from pigs) and cetacean (porpoises and dolphins) hosts functionally resembled Alpha genus HPV by binding and targeting the degradation of E6AP. Beta genus HPV E6 proteins functionally clustered with Delta, Pi, Tau, Gamma, Chi, Mu, Lambda, Iota, Dyokappa, Rho, and Dyolambda E6 proteins to bind and repress MAML1. None of the tested E6 proteins physically and functionally interacted with both MAML1 and E6AP, indicating an evolutionary split. Further, interaction of an E6 protein was insufficient to activate degradation of E6AP, indicating that E6 proteins that target E6AP co-evolved to separately acquire both binding and triggering of ubiquitin ligase activation. E6 proteins with similar biological function clustered together in phylogenetic trees and shared structural features. This suggests that the divergence of E6 proteins from either MAML1 or E6AP binding preference is a major event in papillomavirus evolution. PMID:29281732

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

PubMed

Leung, Travis; Rajendran, Ramkumar; Singh, Subir; Garva, Richa; Krstic-Demonacos, Marija; Demonacos, Constantinos

2013-11-08

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. 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. 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. These results support the notion that CYP2E1 exerts an important role in mammary carcinogenesis, provide a potential link between ethanol metabolism and breast cancer and suggest that

Simultaneous Cocirculation of Both European Varicella-Zoster Virus Genotypes (E1 and E2) in Mexico City▿

PubMed Central

Rodríguez-Castillo, Araceli; Vaughan, Gilberto; Ramírez-González, José Ernesto; Escobar-Gutiérrez, Alejandro

2010-01-01

Full-length genome analysis of varicella-zoster virus (VZV) has shown that viral strains can be classified into seven different genotypes: European (E), Mosaic (M), and Japanese (J), and the E and M genotypes can be further subclassified into E1, E2, and M1 through 4, respectively. The distribution of the main VZV genotypes in Mexico was described earlier, demonstrating the predominance of E genotype, although other genotypes (M1 and M4) were also identified. However, no information regarding the circulation of either E genotype in the country is available. In the present study, we confirm the presence of both E1 and E2 genotypes in the country and explore the possibility of coinfection as the triggering factor for increased virulence among severe cases. A total of 61 different European VZV isolates collected in the Mexico City metropolitan area from 2005 to 2006 were typed by using a PCR method based on genotype-specific primer amplification. Fifty isolates belonged to the E1 genotype, and the eleven remaining samples were classified as E2 genotypes. No coinfection with both E genotypes was identified among these specimens. We provide here new information on the distribution of VZV genotypes circulating in Mexico City. PMID:20220168

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

Code of Federal Regulations, 2011 CFR

2011-04-01

... TAX (CONTINUED) INCOME TAXES (CONTINUED) Empowerment Zone Employment Credit § 1.1397E-1 Qualified zone... empowerment zone or enterprise community (as defined in section 1393), or there is a reasonable expectation... located in an empowerment zone or enterprise community for the entire term of the issue if the public...

Arginine methyltransferase inhibitor 1 inhibits gastric cancer by downregulating eIF4E and targeting PRMT5.

PubMed

Zhang, Baolai; Zhang, Su; Zhu, Lijuan; Chen, Xue; Zhao, Yunfeng; Chao, Li; Zhou, Juanping; Wang, Xing; Zhang, Xinyang; Ma, Nengqian

2017-12-01

Arginine methylation is carried out by protein arginine methyltransferase (PRMTs) family. Arginine methyltransferase inhibitor 1 (AMI-1) is mainly used to inhibit type I PRMT activity in vitro. However, the effects of AMI-1 on type II PRMT5 activity and gastric cancer (GC) remain unclear. In this study, we provided the first evidence that AMI-1 significantly inhibited GC cell proliferation and migration while induced GC cell apoptosis, and reduced the expression of PRMT5, eukaryotic translation initiation factor 4E (eIF4E), symmetric dimethylation of histone 3 (H3R8me2s) and histone 4 (H4R3me2s). In addition, AMI-1 inhibited tumor growth, downregulated eIF4E, H4R3me2s and H3R8me2s expression in mice xenografts model of GC. Collectively, our results suggest that AMI-1 inhibits GC by downregulating eIF4E and targeting type II PRMT5. Copyright © 2017 Elsevier Inc. All rights reserved.

Diabetes and exocrine pancreatic insufficiency in E2F1/E2F2 double-mutant mice.

PubMed

Iglesias, Ainhoa; Murga, Matilde; Laresgoiti, Usua; Skoudy, Anouchka; Bernales, Irantzu; Fullaondo, Asier; Moreno, Bernardino; Lloreta, José; Field, Seth J; Real, Francisco X; Zubiaga, Ana M

2004-05-01

E2F transcription factors are thought to be key regulators of cell growth control. Here we use mutant mouse strains to investigate the function of E2F1 and E2F2 in vivo. E2F1/E2F2 compound-mutant mice develop nonautoimmune insulin-deficient diabetes and exocrine pancreatic dysfunction characterized by endocrine and exocrine cell dysplasia, a reduction in the number and size of acini and islets, and their replacement by ductal structures and adipose tissue. Mutant pancreatic cells exhibit increased rates of DNA replication but also of apoptosis, resulting in severe pancreatic atrophy. The expression of genes involved in DNA replication and cell cycle control was upregulated in the E2F1/E2F2 compound-mutant pancreas, suggesting that their expression is repressed by E2F1/E2F2 activities and that the inappropriate cell cycle found in the mutant pancreas is likely the result of the deregulated expression of these genes. Interestingly, the expression of ductal cell and adipocyte differentiation marker genes was also upregulated, whereas expression of pancreatic cell marker genes were downregulated. These results suggest that E2F1/E2F2 activity negatively controls growth of mature pancreatic cells and is necessary for the maintenance of differentiated pancreatic phenotypes in the adult.

Adenovirus type 5 E1A and E6 proteins of low-risk cutaneous beta-human papillomaviruses suppress cell transformation through interaction with FOXK1/K2 transcription factors.

PubMed

Komorek, Jessica; Kuppuswamy, Mohan; Subramanian, T; Vijayalingam, S; Lomonosova, Elena; Zhao, Ling-Jun; Mymryk, Joe S; Schmitt, Kimberly; Chinnadurai, G

2010-03-01

The adenovirus (Adv) oncoprotein E1A stimulates cell proliferation and inhibits differentiation. These activities are primarily linked to the N-terminal region (exon 1) of E1A, which interacts with multiple cellular protein complexes. The C terminus (exon 2) of E1A antagonizes these processes, mediated in part through interaction with C-terminal binding proteins 1 and 2 (CtBP1/2). To identify additional cellular E1A targets that are involved in the modulation of E1A C-terminus-mediated activities, we undertook tandem affinity purification of E1A-associated proteins. Through mass spectrometric analysis, we identified several known E1A-interacting proteins as well as novel E1A targets, such as the forkhead transcription factors, FOXK1/K2. We identified a Ser/Thr-containing sequence motif in E1A that mediated interaction with FOXK1/K2. We demonstrated that the E6 proteins of two beta-human papillomaviruses (HPV14 and HPV21) associated with epidermodysplasia verruciformis also interacted with FOXK1/K2 through a motif similar to that of E1A. The E1A mutants deficient in interaction with FOXK1/K2 induced enhanced cell proliferation and oncogenic transformation. The hypertransforming activity of the mutant E1A was suppressed by HPV21 E6. An E1A-E6 chimeric protein containing the Ser/Thr domain of the E6 protein in E1A interacted efficiently with FOXK1/K2 and inhibited cell transformation. Our results suggest that targeting FOXK1/K2 may be a common mechanism for certain beta-HPVs and Adv5. E1A exon 2 mutants deficient in interaction with the dual-specificity kinases DYRK1A/1B and their cofactor HAN11 also induced increased cell proliferation and transformation. Our results suggest that the E1A C-terminal region may suppress cell proliferation and oncogenic transformation through interaction with three different cellular protein complexes: FOXK1/K2, DYRK(1A/1B)/HAN11, and CtBP1/2.

Adenovirus E1A and E1B-19K Proteins Protect Human Hepatoma Cells from Transforming Growth Factor β1-induced Apoptosis

PubMed Central

Tarakanova, Vera L.; Wold, William S. M.

2009-01-01

Primary and some transformed hepatocytes undergo apoptosis in response to transforming growth factor β1 (TGFβ). We report that infection with species C human adenovirus conferred resistance to TGFβ-induced apoptosis in human hepatocellular carcinoma cells (Huh-7). Protection against TGFβ-mediated cell death in adenovirus-infected cells correlated with the maintenance of normal nuclear morphology, lack of pro-caspases 8 and 3 processing, maintenance of the mitochondrial membrane potential, and lack of cellular DNA degradation. The TGFβ pro-apoptotic signaling pathway was blocked upstream of mitochondria in adenovirus-infected cells. Both the N-terminal sequences of the E1A proteins and the E1B-19K protein were necessary to protect infected cells against TGFβ-induced apoptosis. PMID:19854227

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. Section...

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. Section...

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. Section...

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. Section...

Melanocortin 1 Receptor Deficiency Promotes Atherosclerosis in Apolipoprotein E-/- Mice.

PubMed

Rinne, Petteri; Kadiri, James J; Velasco-Delgado, Mauricio; Nuutinen, Salla; Viitala, Miro; Hollmén, Maija; Rami, Martina; Savontaus, Eriika; Steffens, Sabine

2018-02-01

The MC1-R (melanocortin 1 receptor) is expressed by monocytes and macrophages where it mediates anti-inflammatory actions. MC1-R also protects against macrophage foam cell formation primarily by promoting cholesterol efflux through the ABCA1 (ATP-binding cassette transporter subfamily A member 1) and ABCG1 (ATP-binding cassette transporter subfamily G member 1). In this study, we aimed to investigate whether global deficiency in MC1-R signaling affects the development of atherosclerosis. Apoe -/- (apolipoprotein E deficient) mice were crossed with recessive yellow (Mc1r e/e ) mice carrying dysfunctional MC1-R and fed a high-fat diet to induce atherosclerosis. Apoe -/- Mc1r e/e mice developed significantly larger atherosclerotic lesions in the aortic sinus and in the whole aorta compared with Apoe -/- controls. In terms of plaque composition, MC1-R deficiency was associated with less collagen and smooth muscle cells and increased necrotic core, indicative of more vulnerable lesions. These changes were accompanied by reduced Abca1 and Abcg1 expression in the aorta. Furthermore, Apoe -/- Mc1r e/e mice showed a defect in bile acid metabolism that aggravated high-fat diet-induced hypercholesterolemia and hepatic lipid accumulation. Flow cytometric analysis of leukocyte profile revealed that dysfunctional MC1-R enhanced arterial accumulation of classical Ly6C high monocytes and macrophages, effects that were evident in mice fed a normal chow diet but not under high-fat diet conditions. In support of enhanced arterial recruitment of Ly6C high monocytes, these cells had increased expression of L-selectin and P-selectin glycoprotein ligand 1. The present study highlights the importance of MC1-R in the development of atherosclerosis. Deficiency in MC1-R signaling exacerbates atherosclerosis by disturbing cholesterol handling and by increasing arterial monocyte accumulation. © 2017 The Authors.

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

Occupational Survey Report AFSC 3E6X1; Operations Management

DTIC Science & Technology

2004-02-01

Lt Bryan Pickett Feb 04 Occupational Survey Report AFSC 3E6X1 Operations Management I n t e g r i t y - S e r v i c e - E x c e l l e n c e...Report AFSC 3E6X1 Operations Management 5a. CONTRACT NUMBER 5b. GRANT NUMBER 5c. PROGRAM ELEMENT NUMBER 6. AUTHOR(S) 5d. PROJECT NUMBER 5e. TASK...Nellis AFB NV (5) • Fairchild AFB WA (5) • Hurlburt Field FL (6) • Eglin AFB FL (4) • Ramstein AB (5) Operations Management 3E6X1 February 2004 (Approved

Swift XRT Observation of 1E 1841-045

NASA Astrophysics Data System (ADS)

Archibald, Robert; Scholz, Paul; Kaspi, Victoria

2013-09-01

We report on Swift XRT observations made following an X-ray/soft gamma-ray burst detected by Fermi-GBM on 13 September 2013 (GCNs 15245, 15228) from the direction of magnetar 1E 1841-045 in the supernova remnant Kes 73. As part of an ongoing monitoring campaign of 1E 1841-045, as well as several other magnetars with the Swift XRT, we observed the source on 16 September 2013 for 4.4 ks. We detect no significant change in the X-ray flux relative to pre-burst epochs: we measure an absorbed 2-10 keV flux of 2.48^(+0.07)_(-0.09) E-11 ergs/s/cm^2 for the 16 September 2013 observation, compared with an average of 2.484^(+0.006)_(-0.06) E-11 ergs/s/cm^2 for observations for the 2 years prior, or 2.39^(+0.10)_(-0.17) E-11 ergs/s/cm^2 for the prior observation on 24 August, 2013.

The BPS spectrum of the 4d {N}=2 SCFT's H 1, H 2, D 4, E 6, E 7, E 8

NASA Astrophysics Data System (ADS)

Cecotti, Sergio; Del Zotto, Michele

2013-06-01

Extending results of 1112.3984, we show that all rank 1 {N}=2 SCFT's in the sequence H 1, H 2, D 4 E 6, E 7, E 8 have canonical finite BPS chambers containing precisely 2 h(F) = 12(∆ - 1) hypermultiplets. The BPS spectrum of the canonical BPS chambers saturates the conformal central charge c, and satisfies some intriguing numerology.

RP-1 delivered to E-1 Test Stand

NASA Image and Video Library

2010-03-30

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.

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

PubMed

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

2014-02-01

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. 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. 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. 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. © 2013 The British Pharmacological Society.

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

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

The translation elongation factor eEF1A1 couples transcription to translation during heat shock response

PubMed Central

Vera, Maria; Pani, Bibhusita; Griffiths, Lowri A; Muchardt, Christian; Abbott, Catherine M; Singer, Robert H; Nudler, Evgeny

2014-01-01

Translation elongation factor eEF1A has a well-defined role in protein synthesis. In this study, we demonstrate a new role for eEF1A: it participates in the entire process of the heat shock response (HSR) in mammalian cells from transcription through translation. Upon stress, isoform 1 of eEF1A rapidly activates transcription of HSP70 by recruiting the master regulator HSF1 to its promoter. eEF1A1 then associates with elongating RNA polymerase II and the 3′UTR of HSP70 mRNA, stabilizing it and facilitating its transport from the nucleus to active ribosomes. eEF1A1-depleted cells exhibit severely impaired HSR and compromised thermotolerance. In contrast, tissue-specific isoform 2 of eEF1A does not support HSR. By adjusting transcriptional yield to translational needs, eEF1A1 renders HSR rapid, robust, and highly selective; thus, representing an attractive therapeutic target for numerous conditions associated with disrupted protein homeostasis, ranging from neurodegeneration to cancer. DOI: http://dx.doi.org/10.7554/eLife.03164.001 PMID:25233275

Therapeutic Strategies against Cyclin E1-Amplified Ovarian Cancers

DTIC Science & Technology

2016-10-01

AWARD NUMBER: W81XWH-15-1-0564 TITLE : Therapeutic Strategies against Cyclin E1-Amplified Ovarian Cancers PRINCIPAL INVESTIGATOR: Panagiotis A...4. TITLE AND SUBTITLE 5a. CONTRACT NUMBER Therapeutic Strategies against Cyclin E1-Amplified Ovarian Cancers 5b. GRANT NUMBER W81XWH-15-1-0564 5c... box protein M1, Retinoblastoma 16. SECURITY CLASSIFICATION OF: 17. LIMITATION OF ABSTRACT 18. NUMBER OF PAGES 19a. NAME OF RESPONSIBLE PERSON

X-1E on Display Stand at Dryden

NASA Technical Reports Server (NTRS)

1996-01-01

The Bell Aircraft Corporation X-1E is shown in this artistic night photo taken in February 1996. This aircraft is displayed on a pedestal in front of the main building (4800) at NASA Dryden Flight Research Center, Edwards, California. There were four versions of the Bell X-1 rocket-powered research aircraft that flew at the NACA High-Speed Flight Research Station, Edwards, California. The bullet-shaped X-1 aircraft were built by Bell Aircraft Corporation, Buffalo, N.Y. for the U.S. Army Air Forces (after 1947, U.S. Air Force) and the National Advisory Committee for Aeronautics (NACA). The X-1 Program was originally designated the XS-1 for EXperimental Supersonic. The X-1's mission was to investigate the transonic speed range (speeds from just below to just above the speed of sound) and, if possible, to break the 'sound barrier.' Three different X-1s were built and designated: X-1-1, X-1-2 (later modified to become the X-1E), and X-1-3. The basic X-1 aircraft were flown by a large number of different pilots from 1946 to 1951. The X-1 Program not only proved that humans could go beyond the speed of sound, it reinforced the understanding that technological barriers could be overcome. The X-1s pioneered many structural and aerodynamic advances including extremely thin, yet extremely strong wing sections; supersonic fuselage configurations; control system requirements; powerplant compatibility; and cockpit environments. The X-1 aircraft were the first transonic-capable aircraft to use an all-moving stabilizer. The flights of the X-1s opened up a new era in aviation. The first X-1 was air-launched unpowered from a Boeing B-29 Superfortress on January 25, 1946. Powered flights began in December 1946. On October 14, 1947, the X-1-1, piloted by Air Force Captain Charles 'Chuck' Yeager, became the first aircraft to exceed the speed of sound, reaching about 700 miles per hour (Mach 1.06) and an altitude of 43,000 feet. The number 2 X-1 was modified and redesignated the X-1E

E2f1–3 Are Critical for Myeloid Development*

PubMed Central

Trikha, Prashant; Sharma, Nidhi; Opavsky, Rene; Reyes, Andres; Pena, Clarissa; Ostrowski, Michael C.; Roussel, Martine F.; Leone, Gustavo

2011-01-01

Hematopoietic development involves the coordinated activity of differentiation and cell cycle regulators. In current models of mammalian cell cycle control, E2f activators (E2f1, E2f2, and E2f3) are portrayed as the ultimate transcriptional effectors that commit cells to enter and progress through S phase. Using conditional gene knock-out strategies, we show that E2f1–3 are not required for the proliferation of early myeloid progenitors. Rather, these E2fs are critical for cell survival and proliferation at two distinct steps of myeloid development. First, E2f1–3 are required as transcriptional repressors for the survival of CD11b+ myeloid progenitors, and then they are required as activators for the proliferation of CD11b+ macrophages. In bone marrow macrophages, we show that E2f1–3 respond to CSF1-Myc mitogenic signals and serve to activate E2f target genes and promote their proliferation. Together, these findings expose dual functions for E2f1–3 at distinct stages of myeloid development in vivo, first as repressors in cell survival and then as activators in cell proliferation. In summary, this work places E2f1–3 in a specific signaling cascade that is critical for myeloid development in vivo. PMID:21115501

Word Frequency Analysis. MOS: 62E. Skill Levels 1 & 2.

DTIC Science & Technology

1981-05-01

j., JtU I J ’I JT S EiM. ’J~VERSA. 5 *J (Jtl~iL I ’JSJP 1 J LUSE t J &USED 5 1 .3 u51’G 3 J .3 UTZLI ATEICN 2 J & VALVE 1 0 J3 EVISUAL I .3 fVj5JLY 2...ECOVERY I 7TI;1Irk 9 REVI1VE I RECTLY 2 PEO 4 i:’ 1 R.EC13CED ? Rr.~vl I IlFr~k 44 RcFFZ(E: ; I Ff ," TF1 ’. I 1 !E* ST EPS 4 1 RELL..SSD 7 PkLIF I RELI~E t

Physicochemical and biological characterization of 1E10 Anti-Idiotype vaccine

PubMed Central

2011-01-01

Background 1E10 monoclonal antibody is a murine anti-idiotypic antibody that mimics N-glycolyl-GM3 gangliosides. This antibody has been tested as an anti-idiotypic cancer vaccine, adjuvated in Al(OH)3, in several clinical trials for melanoma, breast, and lung cancer. During early clinical development this mAb was obtained in vivo from mice ascites fluid. Currently, the production process of 1E10 is being transferred from the in vivo to a bioreactor-based method. Results Here, we present a comprehensive molecular and immunological characterization of 1E10 produced by the two different production processes in order to determine the impact of the manufacturing process in vaccine performance. We observed differences in glycosylation pattern, charge heterogeneity and structural stability between in vivo-produced 1E10 and bioreactor-obtained 1E10. Interestingly, these modifications had no significant impact on the immune responses elicited in two different animal models. Conclusions Changes in 1E10 primary structure like glycosylation; asparagine deamidation and oxidation affected 1E10 structural stability but did not affect the immune response elicited in mice and chickens when compared to 1E10 produced in mice. PMID:22108317

Impotence evaluated by the use of prostaglandin E1

DOE Office of Scientific and Technical Information (OSTI.GOV)

Hwang, T.I.; Yang, C.R.; Wang, S.J.

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 lessmore » 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.« less

Dominant role of cytochrome P-450 2E1 in human hepatic microsomal oxidation of the CFC-substitute 1,1,1,2-tetrafluoroethane.

PubMed

Surbrook, S E; Olson, M J

1992-01-01

The chlorofluorocarbon substitute 1,1,1,2-tetrafluoroethane (HFC-134a) is subject to metabolism by cytochrome P-450 in hepatic microsomes from rat, rabbit, and human. In rat and rabbit, the P-450 form 2E1 is a predominant low-KM, high-rate catalyst of HFC-134a biotransformation and is prominently involved in the metabolism of other tetrahaloalkanes of greater toxicity than HFC-134a [e.g. 1,2-dichloro-1,1-difluoroethane (HCFC-132b)]. In this study, we determined that the human ortholog of P-450 2E1 plays a role of similar importance in the metabolism of HFC-134a. In human hepatic microsomes from 12 individuals, preparations from subjects with relatively high P-450 2E1 levels were shown to metabolize HFC-134a at rates 5- to 10-fold greater than microsomes of individuals with lower levels of this enzyme; the increased rate of metabolism of HFC-134a was specifically linked to increased expression of P-450 2E1. The primary evidence for this conclusion is drawn from studies using mechanism-based inactivation of P-450 2E1 by diethyldithiocarbamate, competitive inhibition of HFC-134a oxidation by p-nitrophenol (a high-affinity substrate for P-450 2E1), strong positive correlation of rates of HFC-134a defluorination with p-nitrophenol hydroxylation in the study population, and correlation of P-450 2E1 levels with rates of halocarbon oxidation. Thus, our findings support the conclusion that human metabolism of HFC-134a is qualitatively similar to that of the species (rat and rabbit) used for toxicological assessment of this halocarbon.(ABSTRACT TRUNCATED AT 250 WORDS)

Evidence supporting a role for TopBP1 and Brd4 in the initiation but not continuation of human papillomavirus 16 E1/E2-mediated DNA replication.

PubMed

Gauson, Elaine J; Donaldson, Mary M; Dornan, Edward S; Wang, Xu; Bristol, Molly; Bodily, Jason M; Morgan, Iain M

2015-05-01

To replicate the double-stranded human papillomavirus 16 (HPV16) DNA genome, viral proteins E1 and E2 associate with the viral origin of replication, and E2 can also regulate transcription from adjacent promoters. E2 interacts with host proteins in order to regulate both transcription and replication; TopBP1 and Brd4 are cellular proteins that interact with HPV16 E2. Previous work with E2 mutants demonstrated the Brd4 requirement for the transactivation properties of E2, while TopBP1 is required for DNA replication induced by E2 from the viral origin of replication in association with E1. More-recent studies have also implicated Brd4 in the regulation of DNA replication by E2 and E1. Here, we demonstrate that both TopBP1 and Brd4 are present at the viral origin of replication and that interaction with E2 is required for optimal initiation of DNA replication. Both cellular proteins are present in E1-E2-containing nuclear foci, and the viral origin of replication is required for the efficient formation of these foci. Short hairpin RNA (shRNA) against either TopBP1 or Brd4 destroys the E1-E2 nuclear bodies but has no effect on E1-E2-mediated levels of DNA replication. An E2 mutation in the context of the complete HPV16 genome that compromises Brd4 interaction fails to efficiently establish episomes in primary human keratinocytes. Overall, the results suggest that interactions between TopBP1 and E2 and between Brd4 and E2 are required to correctly initiate DNA replication but are not required for continuing DNA replication, which may be mediated by alternative processes such as rolling circle amplification and/or homologous recombination. Human papillomavirus 16 (HPV16) is causative in many human cancers, including cervical and head and neck cancers, and is responsible for the annual deaths of hundreds of thousands of people worldwide. The current vaccine will save lives in future generations, but antivirals targeting HPV16 are required for the alleviation of disease

Evidence Supporting a Role for TopBP1 and Brd4 in the Initiation but Not Continuation of Human Papillomavirus 16 E1/E2-Mediated DNA Replication

PubMed Central

Gauson, Elaine J.; Donaldson, Mary M.; Dornan, Edward S.; Wang, Xu; Bristol, Molly; Bodily, Jason M.

2015-01-01

ABSTRACT To replicate the double-stranded human papillomavirus 16 (HPV16) DNA genome, viral proteins E1 and E2 associate with the viral origin of replication, and E2 can also regulate transcription from adjacent promoters. E2 interacts with host proteins in order to regulate both transcription and replication; TopBP1 and Brd4 are cellular proteins that interact with HPV16 E2. Previous work with E2 mutants demonstrated the Brd4 requirement for the transactivation properties of E2, while TopBP1 is required for DNA replication induced by E2 from the viral origin of replication in association with E1. More-recent studies have also implicated Brd4 in the regulation of DNA replication by E2 and E1. Here, we demonstrate that both TopBP1 and Brd4 are present at the viral origin of replication and that interaction with E2 is required for optimal initiation of DNA replication. Both cellular proteins are present in E1-E2-containing nuclear foci, and the viral origin of replication is required for the efficient formation of these foci. Short hairpin RNA (shRNA) against either TopBP1 or Brd4 destroys the E1-E2 nuclear bodies but has no effect on E1-E2-mediated levels of DNA replication. An E2 mutation in the context of the complete HPV16 genome that compromises Brd4 interaction fails to efficiently establish episomes in primary human keratinocytes. Overall, the results suggest that interactions between TopBP1 and E2 and between Brd4 and E2 are required to correctly initiate DNA replication but are not required for continuing DNA replication, which may be mediated by alternative processes such as rolling circle amplification and/or homologous recombination. IMPORTANCE Human papillomavirus 16 (HPV16) is causative in many human cancers, including cervical and head and neck cancers, and is responsible for the annual deaths of hundreds of thousands of people worldwide. The current vaccine will save lives in future generations, but antivirals targeting HPV16 are required for the

Gender difference in NASH susceptibility: Roles of hepatocyte Ikkβ and Sult1e1

PubMed Central

Matsushita, Noriko; Hassanein, Mohamed T.; Martinez-Clemente, Marcos; Lazaro, Raul; French, Samuel W.; Xie, Wen; Lai, Keane; Karin, Michael; Tsukamoto, Hidekazu

2017-01-01

Myeloid cell and hepatocyte IKKβ may mediate the genesis of obesity and insulin resistance in mice fed high fat diet. However, their gender-specific roles in the pathogenesis of non-alcoholic steatohepatitis (NASH) are not known. Here we demonstrate myeloid IKKβ deficiency prevents Western diet-induced obesity and visceral adiposity in females but not in males, and attenuates hyperglycemia, global IR, and NASH in both genders. In contrast, all metabolic sequela including NASH are aggravated by hepatocyte IKKβ deficiency (IkbkbΔhep) in male but not female mice. Gene profiling identifies sulfotransferase family 1E (Sult1e1), which encodes a sulfotransferase E1 responsible for inactivation of estrogen, as a gene upregulated in NASH in both genders and most conspicuously in male IkbkbΔhep mice having worst NASH and lowest plasma estradiol levels. LXRα is enriched to LXRE on Sult1e1 promoter in male WT and IkbkbΔhep mice with NASH, and a Sult1e1 promoter activity is increased by LXRα and its ligand and augmented by expression of a S32A mutant of IκBα. These results demonstrate striking gender differences in regulation by IKKβ of high cholesterol saturated fat diet-induced metabolic changes including NASH and suggest hepatocyte IKKβ is protective in male due at least in part to its ability to repress LXR-induced Sult1e1. Our findings also raise a caution for systemic IKK inhibition for the treatment of NASH as it may exacerbate the disease in male patients. PMID:28797077

Ultrafast transient photocarrier dynamics of the bulk-insulating topological insulator B i1.5S b0.5T e1.7S e1.3

NASA Astrophysics Data System (ADS)

Choi, Young Gwan; Zhung, Chan June; Park, Sun-Hee; Park, Joonbum; Kim, Jun Sung; Kim, Seongheun; Park, Jaehun; Lee, J. S.

2018-02-01

Using optical-pump terahertz-probe spectroscopy, we investigated an ultrafast photocarrier relaxation behavior in a B i1.5S b0.5T e1.7S e1.3 (BSTS) single crystal, which is one of the most bulk-insulating topological insulators. Compared to n -type bulk-metallic B i2S e3 , we found that BSTS endows distinct behaviors in its photocarrier dynamics; the relaxation time turns out to be an order of magnitude longer, and the transient conductance spectrum exhibits a nonlinear increase as a function of the pumping power. Also, we observed an abrupt reduction of the photocarrier scattering rate in several picoseconds after the initial photoexcitation. We discuss these intriguing experimental observations based on a bulk-to-surface carrier injection assisted by the built-in electric field near the surface and electron-phonon scattering.

eIF4E/Fmr1 double mutant mice display cognitive impairment in addition to ASD-like behaviors.

PubMed

Huynh, Thu N; Shah, Manan; Koo, So Yeon; Faraud, Kirsten S; Santini, Emanuela; Klann, Eric

2015-11-01

Autism spectrum disorder (ASD) is a group of heritable disorders with complex and unclear etiology. Classic ASD symptoms include social interaction and communication deficits as well as restricted, repetitive behaviors. In addition, ASD is often comorbid with intellectual disability. Fragile X syndrome (FXS) is the leading genetic cause of ASD, and is the most commonly inherited form of intellectual disability. Several mouse models of ASD and FXS exist, however the intellectual disability observed in ASD patients is not well modeled in mice. Using the Fmr1 knockout mouse and the eIF4E transgenic mouse, two previously characterized mouse models of fragile X syndrome and ASD, respectively, we generated the eIF4E/Fmr1 double mutant mouse. Our study shows that the eIF4E/Fmr1 double mutant mice display classic ASD behaviors, as well as cognitive dysfunction. Importantly, the learning impairments displayed by the double mutant mice spanned multiple cognitive tasks. Moreover, the eIF4E/Fmr1 double mutant mice display increased levels of basal protein synthesis. The results of our study suggest that the eIF4E/Fmr1 double mutant mouse may be a reliable model to study cognitive dysfunction in the context of ASD. Copyright © 2015 Elsevier Inc. All rights reserved.

CYP2E1 hydroxylation of aniline involves negative cooperativity.

PubMed

Hartman, Jessica H; Knott, Katie; Miller, Grover P

2014-02-01

CYP2E1 plays a role in the metabolic activation and elimination of aniline, yet there are conflicting reports on its mechanism of action, and hence relevance, in aniline metabolism. Based on our work with similar compounds, we hypothesized that aniline binds two CYP2E1 sites during metabolism resulting in cooperative reaction kinetics and tested this hypothesis through rigorous in vitro studies. The kinetic profile for recombinant CYP2E1 demonstrated significant negative cooperativity based on a fit of data to the Hill equation (n=0.56). Mechanistically, the data were best explained through a two-binding site cooperative model in which aniline binds with high affinity (K(s)=30 μM) followed by a second weaker binding event (K(ss)=1100 uM) resulting in a threefold increase in the oxidation rate. Binding sites for aniline were confirmed by inhibition studies with 4-methylpyrazole. Inhibitor phenotyping experiments with human liver microsomes validated the central role for CYP2E1 in aniline hydroxylation and indicated minor roles for CYP2A6 and CYP2C9. Importantly, inhibition of minor metabolic pathways resulted in a kinetic profile for microsomal CYP2E1 that replicated the preferred mechanism and parameters observed with the recombinant enzyme. Scaled modeling of in vitro CYP2E1 metabolism of aniline to in vivo clearance, especially at low aniline levels, led to significant deviations from the traditional model based on non-cooperative, Michaelis-Menten kinetics. These findings provide a critical mechanistic perspective on the potential importance of CYP2E1 in the metabolic activation and elimination of aniline as well as the first experimental evidence of a negatively cooperative metabolic reaction catalyzed by CYP2E1. Copyright © 2013 Elsevier Inc. All rights reserved.

Therapeutic Strategies Against Cyclin E1 Amplified Ovarian Cancers

DTIC Science & Technology

2017-10-01

interaction and suppression of E2F- dependent oncogenic activity resulting in activity against CCNE1-amplified cells. In the third aim, we hypothesize...tumors which are dependent on hyperactive HR and are sensitive to suppression of BRCA1. 15. SUBJECT TERMS Ovarian Cancer, CCNE1 amplification...suppression of E2F- dependent oncogenic activity resulting in activity against CCNE1-amplified cells. In the third aim, we hypothesize that miR-1255b, miR

Na(+) transport, and the E(1)P-E(2)P conformational transition of the Na(+)/K(+)-ATPase.

PubMed Central

Babes, A; Fendler, K

2000-01-01

We have used admittance analysis together with the black lipid membrane technique to analyze electrogenic reactions within the Na(+) branch of the reaction cycle of the Na(+)/K(+)-ATPase. ATP release by flash photolysis of caged ATP induced changes in the admittance of the compound membrane system that are associated with partial reactions of the Na(+)/K(+)-ATPase. Frequency spectra and the Na(+) dependence of the capacitive signal are consistent with an electrogenic or electroneutral E(1)P <--> E(2)P conformational transition which is rate limiting for a faster electrogenic Na(+) dissociation reaction. We determine the relaxation rate of the rate-limiting reaction and the equilibrium constants for both reactions at pH 6.2-8.5. The relaxation rate has a maximum value at pH 7.4 (approximately 320 s(-1)), which drops to acidic (approximately 190 s(-1)) and basic (approximately 110 s(-1)) pH. The E(1)P <--> E(2)P equilibrium is approximately at a midpoint position at pH 6.2 (equilibrium constant approximately 0.8) but moves more to the E(1)P side at basic pH 8.5 (equilibrium constant approximately 0.4). The Na(+) affinity at the extracellular binding site decreases from approximately 900 mM at pH 6.2 to approximately 200 mM at pH 8.5. The results suggest that during Na(+) transport the free energy supplied by the hydrolysis of ATP is mainly used for the generation of a low-affinity extracellular Na(+) discharge site. Ionic strength and lyotropic anions both decrease the relaxation rate. However, while ionic strength does not change the position of the conformational equilibrium E(1)P <--> E(2)P, lyotropic anions shift it to E(1)P. PMID:11053130

E1a is an exogenous in vivo tumour suppressor.

PubMed

Cimas, Francisco J; Callejas-Valera, Juan L; García-Olmo, Dolores C; Hernández-Losa, Javier; Melgar-Rojas, Pedro; Ruiz-Hidalgo, María J; Pascual-Serra, Raquel; Ortega-Muelas, Marta; Roche, Olga; Marcos, Pilar; Garcia-Gil, Elena; Fernandez-Aroca, Diego M; Ramón Y Cajal, Santiago; Gutkind, J Silvio; Sanchez-Prieto, Ricardo

2017-07-28

The E1a gene from adenovirus has become a major tool in cancer research. Since the discovery of E1a, it has been proposed to be an oncogene, becoming a key element in the model of cooperation between oncogenes. However, E1a's in vivo behaviour is consistent with a tumour suppressor gene, due to the block/delay observed in different xenograft models. To clarify this interesting controversy, we have evaluated the effect of the E1a 13s isoform from adenovirus 5 in vivo. Initially, a conventional xenograft approach was performed using previously unreported HCT116 and B16-F10 cells, showing a clear anti-tumour effect regardless of the mouse's immunological background (immunosuppressed/immunocompetent). Next, we engineered a transgenic mouse model in which inducible E1a 13s expression was under the control of cytokeratin 5 to avoid side effects during embryonic development. Our results show that E1a is able to block chemical skin carcinogenesis, showing an anti-tumour effect. The present report demonstrates the in vivo anti-tumour effect of E1a, showing that the in vitro oncogenic role of E1a cannot be extrapolated in vivo, supporting its future use in gene therapy approaches. Copyright © 2017 Elsevier B.V. All rights reserved.

cea-kil operon of the ColE1 plasmid.

PubMed Central

Sabik, J F; Suit, J L; Luria, S E

1983-01-01

We isolated a series of Tn5 transposon insertion mutants and chemically induced mutants with mutations in the region of the ColE1 plasmid that includes the cea (colicin) and imm (immunity) genes. Bacterial cells harboring each of the mutant plasmids were tested for their response to the colicin-inducing agent mitomycin C. All insertion mutations within the cea gene failed to bring about cell killing after mitomycin C treatment. A cea- amber mutation exerted a polar effect on killing by mitomycin C. Two insertions beyond the cea gene but within or near the imm gene also prevented the lethal response to mitomycin C. These findings suggest the presence in the ColE1 plasmid of an operon containing the cea and kil genes whose product is needed for mitomycin C-induced lethality. Bacteria carrying ColE1 plasmids with Tn5 inserted within the cea gene produced serologically cross-reacting fragments of the colicin E1 molecule, the lengths of which were proportional to the distance between the insertion and the promoter end of the cea gene. Images PMID:6298187

Evaluation of anti-melanoma activities of (1S,2E,4R,6E,8R,11S,12R)-8,12-epoxy-2,6-cembradiene-4,11-diol, (1S,2E,4R,6E,8S,11R,12S)-8,11-epoxy-4,12-epoxy-2,6-cembradiene and (1S,4R,13S)-cembra-2E,7E,11E-trien-4,13-diol from the Red Sea soft coral Sarcophyton glaucum.

PubMed

Szymanski, Pawel T; Ahmed, Safwat A; Radwan, Mohamed M; Khalifa, Sherief I; Fahmy, Hesham

2014-08-01

Three natural cembranoids from the Red Sea soft coral Sarcophyton glaucum namely (1S,2E,4R,6E,8R,11S,12R)-8,12-epoxy-2,6-cembradiene-4,11-diol, (1S,2E,4R,6E,8S,11R,12S)-8,11-epoxy-4,12-epoxy-2,6-cembradiene and (1S,4R,13S)-cembra-2E,7E,11E-trien-4,13-diol were evaluated for their inhibitory effects on mouse melanoma B16F10 cell growth. Results show that all the cembranoids strongly inhibit viability of melanoma cells particularly during 48 -72 hrs treatment and also inhibit de novo DNA synthesis and PARP activity and stimulate fragmentation of DNA. (1S,2E,4R,6E,8R,11S,12R)-8,12-epoxy-2,6-cembradiene-4,11-diol was not cytotoxic to monkey kidney CV-1 cells at the concentration that produces the anti-melanoma effects which indicates that this compound may be a good candidate for further development. (1S,2E,4R,6E,8S,11R,12S)-8,11-epoxy-4,12-epoxy-2,6-cembradiene and (1S,4R,13S)-cembra-2E,7E,11E-trien-4,13-diol were found to be cytotoxic to healthy cells.

Core 1-derived O-glycans are essential E-selectin ligands on neutrophils.

PubMed

Yago, Tadayuki; Fu, Jianxin; McDaniel, J Michael; Miner, Jonathan J; McEver, Rodger P; Xia, Lijun

2010-05-18

Neutrophils roll on E-selectin in inflamed venules through interactions with cell-surface glycoconjugates. The identification of physiologic E-selectin ligands on neutrophils has been elusive. Current evidence suggests that P-selectin glycoprotein ligand-1 (PSGL-1), E-selectin ligand-1 (ESL-1), and CD44 encompass all glycoprotein ligands for E-selectin; that ESL-1 and CD44 use N-glycans to bind to E-selectin; and that neutrophils lacking core 2 O-glycans have partially defective interactions with E-selectin. These data imply that N-glycans on ESL-1 and CD44 and O-glycans on PSGL-1 constitute all E-selectin ligands, with neither glycan subset having a dominant role. The enzyme T-synthase transfers Gal to GalNAcalpha1-Ser/Thr to form the core 1 structure Galbeta1-3GalNAcalpha1-Ser/Thr, a precursor for core 2 and extended core 1 O-glycans that might serve as selectin ligands. Here, using mice lacking T-synthase in endothelial and hematopoietic cells, we found that E-selectin bound to CD44 and ESL-1 in lysates of T-synthase-deficient neutrophils. However, the cells exhibited markedly impaired rolling on E-selectin in vitro and in vivo, failed to activate beta2 integrins while rolling, and did not emigrate into inflamed tissues. These defects were more severe than those of neutrophils lacking PSGL-1, CD44, and the mucin CD43. Our results demonstrate that core 1-derived O-glycans are essential E-selectin ligands; that some of these O-glycans are on protein(s) other than PSGL-1, CD44, and CD43; and that PSGL-1, CD44, and ESL-1 do not constitute all glycoprotein ligands for E-selectin.

The E3Σ1+ (63S1) ← A3Π0+(53P1) transition in CdAr revisited: The spectrum and new analysis of the E3Σ1+ Rydberg state interatomic potential

NASA Astrophysics Data System (ADS)

Urbańczyk, T.; Krośnicki, M.; Kędziorski, A.; Koperski, J.

2018-05-01

Revisited study of the E3Σ1+ (63S1) ← A3Π0+(53P1) transition in CdAr using both theoretical and experimental approach is presented. Systematic detection of the E3Σ1+in,υ' ← A3Π0+,υ″ = 6 transition frequencies with higher accuracy and spectrally narrower laser extended and improved analysis and simulation of the LIF excitation spectrum. More consistent characterization of the E3Σ1+in-Rydberg state inner well using inversed perturbation approach methodology was achieved. Free ← bound transitions in the E3Σ1+in ← A3Π0+,υ″ = 6 excitation were taken into account in the analysis and simulation of the recorded spectrum. The updated spectroscopic characterization of the A3Π0+ state was also revisited.

49 CFR Figure 3 to Subpart E of... - Trailer Car End Structure Conceptual Implementation 1-to Subpart E

Code of Federal Regulations, 2010 CFR

2010-10-01

... 49 Transportation 4 2010-10-01 2010-10-01 false Trailer Car End Structure Conceptual Implementation 1-to Subpart E 3 Figure 3 to Subpart E of Part 238 Transportation Other Regulations Relating to... to Subpart E of Part 238—Trailer Car End Structure Conceptual Implementation 1—to Subpart E ER12MY99...

49 CFR Figure 3 to Subpart E of... - Trailer Car End Structure Conceptual Implementation 1-to Subpart E

Code of Federal Regulations, 2014 CFR

2014-10-01

... 49 Transportation 4 2014-10-01 2014-10-01 false Trailer Car End Structure Conceptual Implementation 1-to Subpart E 3 Figure 3 to Subpart E of Part 238 Transportation Other Regulations Relating to... to Subpart E of Part 238—Trailer Car End Structure Conceptual Implementation 1—to Subpart E ER12MY99...

Pressure-induced magnetic collapse and metallization of TlF e1.6S e2

NASA Astrophysics Data System (ADS)

Naumov, P. G.; Filsinger, K.; Shylin, S. I.; Barkalov, O. I.; Ksenofontov, V.; Qi, Y.; Palasyuk, T.; Schnelle, W.; Medvedev, S. A.; Greenblatt, M.; Felser, C.

2017-08-01

The crystal structure, magnetic ordering, and electrical resistivity of TlF e1.6S e2 were studied at high pressures. Below ˜7 GPa , TlF e1.6S e2 is an antiferromagnetically ordered semiconductor with a ThC r2S i2 -type structure. The insulator-to-metal transformation observed at a pressure of ˜7 GPa is accompanied by a loss of magnetic ordering and an isostructural phase transition. In the pressure range ˜7.5 -11 GPa a remarkable downturn in resistivity, which resembles a superconducting transition, is observed below 15 K. We discuss this feature as the possible onset of superconductivity originating from a phase separation in a small fraction of the sample in the vicinity of the magnetic transition.

40 CFR 721.4575 - L-aspartic acid, N,N′- [(1E) - 1,2 - ethenediylbis[(3-sulfo-4, 1-phenylene)imino [6-(phenylamino...

Code of Federal Regulations, 2010 CFR

2010-07-01

... 40 Protection of Environment 30 2010-07-01 2010-07-01 false L-aspartic acid, N,Nâ²- [(1E) - 1,2... Substances § 721.4575 L-aspartic acid, N,N′- [(1E) - 1,2 - ethenediylbis[(3-sulfo-4, 1-phenylene)imino [6... uses subject to reporting. (1) The chemical substance identified as l-aspartic acid, N,N′- [(1E) - 1,2...

Human papillomavirus type 16 E7 oncoprotein mediates CCNA1 promoter methylation.

PubMed

Chalertpet, Kanwalat; Pakdeechaidan, Watcharapong; Patel, Vyomesh; Mutirangura, Apiwat; Yanatatsaneejit, Pattamawadee

2015-10-01

Human papillomavirus (HPV) oncoproteins drive distinctive promoter methylation patterns in cancer. However, the underlying mechanism remains to be elucidated. Cyclin A1 (CCNA1) promoter methylation is strongly associated with HPV-associated cancer. CCNA1 methylation is found in HPV-associated cervical cancers, as well as in head and neck squamous cell cancer. Numerous pieces of evidence suggest that E7 may drive CCNA1 methylation. First, the CCNA1 promoter is methylated in HPV-positive epithelial lesions after transformation. Second, the CCNA1 promoter is methylated at a high level when HPV is integrated into the human genome. Finally, E7 has been shown to interact with DNA methyltransferase 1 (Dnmt1). Here, we sought to determine the mechanism by which E7 increases methylation in cervical cancer by using CCNA1 as a gene model. We investigated whether E7 induces CCNA1 promoter methylation, resulting in the loss of expression. Using both E7 knockdown and overexpression approaches in SiHa and C33a cells, our data showed that CCNA1 promoter methylation decreases with a corresponding increase in expression in E7 siRNA-transfected cells. By contrast, CCNA1 promoter methylation was augmented with a corresponding reduction in expression in E7-overexpressing cells. To confirm whether the binding of the E7-Dnmt1 complex to the CCNA1 promoter induced methylation and loss of expression, ChIP assays were carried out in E7-, del CR3-E7 and vector control-overexpressing C33a cells. The data showed that E7 induced CCNA1 methylation by forming a complex with Dnmt1 at the CCNA1 promoter, resulting in the subsequent reduction of expression in cancers. It is interesting to further explore the genome-wide mechanism of E7 oncoprotein-mediated DNA methylation. © 2015 The Authors. Cancer Science published by Wiley Publishing Asia Pty Ltd on behalf of Japanese Cancer Association.

B1 Cell IgE Impedes Mast Cell-Mediated Enhancement of Parasite Expulsion through B2 IgE Blockade.

PubMed

Martin, Rebecca K; Damle, Sheela R; Valentine, Yolander A; Zellner, Matthew P; James, Briana N; Lownik, Joseph C; Luker, Andrea J; Davis, Elijah H; DeMeules, Martha M; Khandjian, Laura M; Finkelman, Fred D; Urban, Joseph F; Conrad, Daniel H

2018-02-13

Helminth infection is known for generating large amounts of poly-specific IgE. Here we demonstrate that innate-like B1 cells are responsible for this IgE production during infection with the nematode parasites Nippostrongylus brasiliensis and Heligmosomoides polygyrus bakeri. In vitro analysis of B1 cell immunoglobulin class switch recombination to IgE demonstrated a requirement for anti-CD40 and IL-4 that was further enhanced when IL-5 was added or when the B1 source was helminth infected mice. An IL-25-induced upregulation of IgE in B1 cells was also demonstrated. In T cell-reconstituted RAG1 -/- mice, N. brasiliensis clearance was enhanced with the addition of B2 cells in an IgE-dependent manner. This enhanced clearance was impeded by reconstitution with IgE sufficient B1 cells. Mucosal mast cells mediated the B2 cell enhancement of clearance in the absence of B1 cells. The data support B1 cell IgE secretion as a regulatory response exploited by the helminth. Copyright © 2018 The Author(s). Published by Elsevier Inc. All rights reserved.

The E3Σ1+ (63S1)←A3Π0+(53P1) transition in CdAr revisited: The spectrum and new analysis of the E3Σ1+ Rydberg state interatomic potential.

PubMed

Urbańczyk, T; Krośnicki, M; Kędziorski, A; Koperski, J

2018-05-05

Revisited study of the E 3 Σ 1 + (6 3 S 1 )←A 3 Π 0+ (5 3 P 1 ) transition in CdAr using both theoretical and experimental approach is presented. Systematic detection of the E 3 Σ 1 + in ,υ'←A 3 Π 0+ ,υ″=6 transition frequencies with higher accuracy and spectrally narrower laser extended and improved analysis and simulation of the LIF excitation spectrum. More consistent characterization of the E 3 Σ 1 + in -Rydberg state inner well using inversed perturbation approach methodology was achieved. Free←bound transitions in the E 3 Σ 1 + in ←A 3 Π 0+ ,υ″=6 excitation were taken into account in the analysis and simulation of the recorded spectrum. The updated spectroscopic characterization of the A 3 Π 0+ state was also revisited. Copyright © 2018 Elsevier B.V. All rights reserved.

[Construction and eukaryotic expression of PVAX1-hPV58mE6E7fcGB composite gene vaccine].

PubMed

Wang, He; Yu, Jiyun; Li, Li

2013-10-01

To construct and express a composite gene vaccine for human papillomavirus 58(HPV58)-associated cervical cancer, we inserted HPV58mE6E7 fusion gene into pCI-Fc-GPI eukaryotic expression vector, constructing a recombinant plasmid named pCI-sig-HPV58mE6E7-Fc-GPI. Then we further inserted fragment of sig-HPV58mE6E7Fc-GPI into the novel vaccine vector PVAX1-IRES-GM/B7, constructing PVAX1-HPV58mE6E7FcGB composite gene vaccine. PVAX1-HPV58mE6E7FcGB vaccine was successfully constructed and identified by restriction endonuclease and sequencing analysis. Eukaryotic expression of fusion antigen sig-HPV58mE6E7-Fc-GPI and molecular ad-juvant GM-CSF and B7. 1 were proved to be realized at the same time by flow cytometry and immunofluorescence. So PVAX1-HPV58mE6E7FcGB can be taken as a candidate of therapeutic vaccine for HPV58-associated tumors and their precancerous transformations.

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

DOE Office of Scientific and Technical Information (OSTI.GOV)

Chuang, J.L.; Fisher, C.R.; Chuang, D.T.

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 insertionmore » 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.« less

E4orf1 Enhances Glucose Uptake Independent of Proximal Insulin Signaling

PubMed Central

Na, Ha-Na; Hegde, Vijay; Dubuisson, Olga; Dhurandhar, Nikhil V.

2016-01-01

Impaired proximal insulin signaling is often present in diabetes. Hence, approaches to enhance glucose disposal independent of proximal insulin signaling are desirable. Evidence indicates that Adenovirus-derived E4orf1 protein may offer such an approach. This study determined if E4orf1 improves insulin sensitivity and downregulates proximal insulin signaling in vivo and enhances cellular glucose uptake independent of proximal insulin signaling in vitro. High fat fed mice were injected with a retrovirus plasmid expressing E4orf1, or a null vector. E4orf1 significantly improved insulin sensitivity in response to a glucose load. Yet, their proximal insulin signaling in fat depots was impaired, as indicated by reduced tyrosine phosphorylation of insulin receptor (IR), and significantly increased abundance of ectonucleotide pyrophosphatase/phosphodiesterase-1 (ENPP1). In 3T3-L1 pre-adipocytes E4orf1 expression impaired proximal insulin signaling. Whereas, treatment with rosiglitazone reduced ENPP1 abundance. Unaffected by IR-KD (insulin receptor knockdown) with siRNA, E4orf1 significantly up-regulated distal insulin signaling pathway and enhanced cellular glucose uptake. In vivo, E4orf1 impairs proximal insulin signaling in fat depots yet improves glycemic control. This is probably explained by the ability of E4orf1 to promote cellular glucose uptake independent of proximal insulin signaling. E4orf1 may provide a therapeutic template to enhance glucose disposal in the presence of impaired proximal insulin signaling. PMID:27537838

E4orf1 Enhances Glucose Uptake Independent of Proximal Insulin Signaling.

PubMed

Na, Ha-Na; Hegde, Vijay; Dubuisson, Olga; Dhurandhar, Nikhil V

2016-01-01

Impaired proximal insulin signaling is often present in diabetes. Hence, approaches to enhance glucose disposal independent of proximal insulin signaling are desirable. Evidence indicates that Adenovirus-derived E4orf1 protein may offer such an approach. This study determined if E4orf1 improves insulin sensitivity and downregulates proximal insulin signaling in vivo and enhances cellular glucose uptake independent of proximal insulin signaling in vitro. High fat fed mice were injected with a retrovirus plasmid expressing E4orf1, or a null vector. E4orf1 significantly improved insulin sensitivity in response to a glucose load. Yet, their proximal insulin signaling in fat depots was impaired, as indicated by reduced tyrosine phosphorylation of insulin receptor (IR), and significantly increased abundance of ectonucleotide pyrophosphatase/phosphodiesterase-1 (ENPP1). In 3T3-L1 pre-adipocytes E4orf1 expression impaired proximal insulin signaling. Whereas, treatment with rosiglitazone reduced ENPP1 abundance. Unaffected by IR-KD (insulin receptor knockdown) with siRNA, E4orf1 significantly up-regulated distal insulin signaling pathway and enhanced cellular glucose uptake. In vivo, E4orf1 impairs proximal insulin signaling in fat depots yet improves glycemic control. This is probably explained by the ability of E4orf1 to promote cellular glucose uptake independent of proximal insulin signaling. E4orf1 may provide a therapeutic template to enhance glucose disposal in the presence of impaired proximal insulin signaling.

Neutronic analysis of the 1D and 1E banks reflux detection system

DOE Office of Scientific and Technical Information (OSTI.GOV)

Blanchard, A.

1999-12-21

Two H Canyon neutron monitoring systems for early detection of postulated abnormal reflux conditions in the Second Uranium Cycle 1E and 1D Mixer-Settle Banks have been designed and built. Monte Carlo neutron transport simulations using the general purpose, general geometry, n-particle MCNP code have been performed to model expected response of the monitoring systems to varying conditions.The confirmatory studies documented herein conclude that the 1E and 1D neutron monitoring systems are able to achieve adequate neutron count rates for various neutron source and detector configurations, thereby eliminating excessive integration count time. Neutron count rate sensitivity studies are also performed. Conversely,more » the transport studies concluded that the neutron count rates are statistically insensitive to nitric acid content in the aqueous region and to the transition region length. These studies conclude that the 1E and 1D neutron monitoring systems are able to predict the postulated reflux conditions for all examined perturbations in the neutron source and detector configurations. In the cases examined, the relative change in the neutron count rates due to postulated transitions from normal {sup 235}U concentration levels to reflux levels remain satisfactory detectable.« less

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

1,3-Butadiene-Induced Mitochondrial Dysfunction is Correlated with Mitochondrial CYP2E1 Activity in Collaborative Cross Mice

PubMed Central

Hartman, Jessica H.; Miller, Grover P.; Caro, Andres A.; Byrum, Stephanie D.; Orr, Lisa M.; Mackintosh, Samuel G.; Tackett, Alan J.; MacMillan-Crow, Lee Ann; Hallberg, Lance M.; Ameredes, Bill T.; Boysen, Gunnar

2017-01-01

Cytochrome P450 2E1 (CYP2E1) metabolizes low molecular weight hydrophobic compounds, including 1,3-butadiene, which is converted by CYP2E1 to electrophilic epoxide metabolites that covalently modify cellular proteins and DNA. Previous CYP2E1 studies have mainly focused on the enzyme localized in the endoplasmic reticulum (erCYP2E1); however, active CYP2E1 also localizes in mitochondria (mtCYP2E1) and the distribution of CYP2E1 between organelles can influence an individual's response to exposure. Relatively few studies have focused on the contribution of mtCYP2E1 to activation of chemical toxicants. We hypothesized that CYP2E1 bioactivation of butadiene within mitochondria adversely affects mitochondrial respiratory complexes I-IV. A population of Collaborative Cross mice were exposed to air (control) or 200 ppm butadiene. Subcellular fractions (mitochondria, DNA, and microsomes) were collected from frozen livers and CYP2E1 activity was measured in microsomes and mitochondria. Individual activities of mitochondrial respiratory complexes I-IV were measured using in vitro assays with purified mitochondrial fractions. In air- and butadiene-exposed mouse samples, mtDNA copy numbers were assessed by RT-PCR, and mtDNA integrity was assessed through a PCR-based assay. No significant change in mtDNA copy number or integrity were observed; however, there was a decrease in overall activity of mitochondrial respiratory complexes I, II, and IV after butadiene exposure. Additionally, higher mtCYP2E1 (but not erCYP2E1) activity was correlated with decreased mitochondrial respiratory complex activity (in complexes I-IV) in the butadiene-exposed (not control) animals. Together, these results represent the first in vivo link between mitochondrial CYP2E1 activity and mitochondrial toxicity. PMID:28082109

Aviation Structural Mechanic E 1 & C.

ERIC Educational Resources Information Center

Naval Education and Training Command, Pensacola, FL.

The rate training manual is one of a series of training handbooks prepared for enlisted personnel of the Navy and Naval Reserve who are studying for advancement in the Aviation Structural Mechanic E (AME) rating. The manual is based on the professional qualifications for the rates AME1 and AMEC. Chapters are organized according to specific job…

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.

Ad E1A 243R oncoprotein promotes association of proto-oncogene product MYC with the NuA4/Tip60 complex via the E1A N-terminal repression domain.

PubMed

Zhao, Ling-Jun; Loewenstein, Paul M; Green, Maurice

2016-12-01

The adenovirus E1A 243R oncoprotein targets TRRAP, a scaffold protein that assembles histone acetyltransferase (HAT) complexes, such as the NuA4/Tip60 complex which mediates transcriptional activity of the proto-oncogene MYC and helps determine the cancer cell phenotype. How E1A transforms cells through TRRAP remains obscure. We performed proteomic analysis with the N-terminal transcriptional repression domain of E1A 243R (E1A 1-80) and showed that E1A 1-80 interacts with TRRAP, p400, and three other members of the NuA4 complex - DMAP1, RUVBL1 and RUVBL2 - not previously shown to associate with E1A 243R. E1A 1-80 interacts with these NuA4 components and MYC through the E1A TRRAP-targeting domain. E1A 243R association with the NuA4 complex was demonstrated by co-immunoprecipitation and analysis with DMAP1, Tip60, and MYC. Significantly, E1A 243R promotes association of MYC/MAX with the NuA4/Tip60 complex, implicating the importance of the MYC/NuA4 pathway in cellular transformation by both MYC and E1A. Copyright © 2016 Elsevier Inc. All rights reserved.

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... religious orders, and Christian Science practitioners for self-employment coverage. (a) In general. Any... order) or (2) a Christian Science practitioner may elect to have the Federal old-age, survivors, and...

Structural and electrochemical studies of TiO2 complexes with (4,4'-((1E,1'E)-(2,5-bis(octyloxy)-1,4-phenylene)bis(ethene-2,1-diyl))bis-(E)-N-(2,5-bis(octyloxy)benzylidene)) imine derivative bases towards organic devices.

PubMed

Rozycka, Anna; Iwan, Agnieszka; Bogdanowicz, Krzysztof Artur; Filapek, Michal; Górska, Natalia; Hreniak, Agnieszka; Marzec, Monika

2018-06-12

Three (4,4'-((1E,1'E)-(2,5-bis(octyloxy)-1,4-phenylene)bis(ethene-2,1-diyl))bis-(E)-N-(2,5-bis(octyloxy)benzylidene)) imine derivatives were synthesized via a condensation reaction with p-toluenesulfonic acid as a catalyst. The effects of the end groups and vinylene (-HC[double bond, length as m-dash]CH-) moieties on the structural, thermal, optical, electrochemical and photovoltaic properties of imines were investigated to check the influence of TiO2 on the imine properties. The thermal behavior of imines and their complexes with TiO2 was widely investigated using FT-IR, XRD, DSC and POM methods in order to determine the order type in the imine structure. All imines present the highest occupied molecular orbital (HOMO) levels of about -5.39 eV (SAI1 and SAI2) and -5.27 eV (SAI3) and the lowest unoccupied molecular orbital (LUMO) levels at about -3.17 eV. The difference of the end groups in the imines in each case did not affect redox properties. Generally, both oxidation and reduction are easier after TiO2 addition and it also changes the HOMO-LUMO levels of imines. Moreover, changes in the characteristic bands for imines in the region 1500-1700 cm-1 observed as a drastic decrease of intensity or even disappearance of bands in the imine : TiO2 mixture suggest the formation of a complex (C[double bond, length as m-dash]N)-TiO2. Organic devices with the configuration of ITO/TiO2/SAIx (or SAIx : TiO2)/Au were fabricated and investigated in the presence and absence of visible light irradiation with an intensity of 93 mW cm-2. In all imines and complexes with TiO2, the generation of the photocurrent indicates their use as photodiodes and the best result was observed for SAI3 : TiO2 complexes.

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..., members of religious orders, and Christian Science practitioners for self-employment coverage. (a) In... member of such order) or (2) a Christian Science practitioner may elect to have the Federal old-age...

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..., members of religious orders, and Christian Science practitioners for self-employment coverage. (a) In... member of such order) or (2) a Christian Science practitioner may elect to have the Federal old-age...

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..., members of religious orders, and Christian Science practitioners for self-employment coverage. (a) In... member of such order) or (2) a Christian Science practitioner may elect to have the Federal old-age...

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..., members of religious orders, and Christian Science practitioners for self-employment coverage. (a) In... member of such order) or (2) a Christian Science practitioner may elect to have the Federal old-age...

Demonstration of PDC-E1 subunits as major antigens in the complement-fixing fraction M4 and re-evaluation of PDC-E1-specific antibodies in PBC patients.

PubMed

Berg, Christoph P; Stein, Gerburg M; Klein, Reinhild; Pascu, Maria; Berg, Thomas; Kammer, Winfried; Priemer, Martin; Nordheim, Alfred; Schulze-Osthoff, Klaus; Gregor, Michael; Wesselborg, Sebastian; Berg, Peter A

2006-09-01

Primary biliary cirrhosis (PBC) is characterized by the presence of antimitochondrial antibodies (AMA). Autoantibodies specific for the mitochondrial M4 antigen can be detected by a complement fixation test (CFT) but not by immunoblotting. The aim of this study was to elucidate the identity of the M4 antigen. M4 proteins were purified by affinity chromatography using IgG fractions of PBC marker sera being CFT positive (n=5) or negative (n=5) and identified by Western blotting, silver staining and sequence analysis. Further, a cohort of 57 PBC patients was tested for the reactivity to M4 and pyruvate dehydrogenase complex (PDC). Two AMA patterns of the marker sera were visualized: CFT-positive sera were defined as PDC-E2(+)/E1(+) and the CFT-negative sera as PDC-E2(+)/E1(-). The major proteins in the M4 fraction could be related to the PDC-E1 subunits. A clear-cut association between anti-M4 reactivity in the CFT and the reactivity to both PDC subunits could also be documented in the cohort of 57 PBC patients showing anti-PDC-E1alpha and E1beta antibodies at a frequency of 74% and 67%. CFT reactivity against M4 antigens could be preferentially identified as a reaction against PDC-E1. As PDC-E1 subunits as compared with PDC-E2 lack lipoyl-binding sites, they probably have to be considered as an independent and important target.

26 CFR 1.1402(e)(4)-1 - Treatment of certain remuneration paid in 1955 and 1956 as wages.

Code of Federal Regulations, 2010 CFR

2010-04-01

... and 1956 as wages. 1.1402(e)(4)-1 Section 1.1402(e)(4)-1 Internal Revenue INTERNAL REVENUE SERVICE, DEPARTMENT OF THE TREASURY (CONTINUED) INCOME TAX (CONTINUED) INCOME TAXES Tax on Self-Employment Income § 1.1402(e)(4)-1 Treatment of certain remuneration paid in 1955 and 1956 as wages. If in 1955 or 1956 an...

Search for high-mass e+e- resonances in pp collisions at sqrt[s]=1.96 TeV.

PubMed

Aaltonen, T; Adelman, J; Akimoto, T; Albrow, M G; Alvarez González, B; Amerio, S; Amidei, D; Anastassov, A; Annovi, A; Antos, J; Apollinari, G; Apresyan, A; Arisawa, T; Artikov, A; Ashmanskas, W; Attal, A; Aurisano, A; Azfar, F; Azzurri, P; Badgett, W; Barbaro-Galtieri, A; Barnes, V E; Barnett, B A; Bartsch, V; Bauer, G; Beauchemin, P-H; Bedeschi, F; Beecher, D; Behari, S; Bellettini, G; Bellinger, J; Benjamin, D; Beretvas, A; Beringer, J; Bhatti, A; Binkley, M; Bisello, D; Bizjak, I; Blair, R E; Blocker, C; Blumenfeld, B; Bocci, A; Bodek, A; Boisvert, V; Bolla, G; Bortoletto, D; Boudreau, J; Boveia, A; Brau, B; Bridgeman, A; Brigliadori, L; Bromberg, C; Brubaker, E; Budagov, J; Budd, H S; Budd, S; Burke, S; Burkett, K; Busetto, G; Bussey, P; Buzatu, A; Byrum, K L; Cabrera, S; Calancha, C; Campanelli, M; Campbell, M; Canelli, F; Canepa, A; Carls, B; Carlsmith, D; Carosi, R; Carrillo, S; Carron, S; Casal, B; Casarsa, M; Castro, A; Catastini, P; Cauz, D; Cavaliere, V; Cavalli-Sforza, M; Cerri, A; Cerrito, L; Chang, S H; Chen, Y C; Chertok, M; Chiarelli, G; Chlachidze, G; Chlebana, F; Cho, K; Chokheli, D; Chou, J P; Choudalakis, G; Chuang, S H; Chung, K; Chung, W H; Chung, Y S; Chwalek, T; Ciobanu, C I; Ciocci, M A; Clark, A; Clark, D; Compostella, G; Convery, M E; Conway, J; Cordelli, M; Cortiana, G; Cox, C A; Cox, D J; Crescioli, F; Cuenca Almenar, C; Cuevas, J; Culbertson, R; Cully, J C; Dagenhart, D; Datta, M; Davies, T; de Barbaro, P; De Cecco, S; Deisher, A; De Lorenzo, G; Dell'Orso, M; Deluca, C; Demortier, L; Deng, J; Deninno, M; Derwent, P F; di Giovanni, G P; Dionisi, C; Di Ruzza, B; Dittmann, J R; D'Onofrio, M; Donati, S; Dong, P; Donini, J; Dorigo, T; Dube, S; Efron, J; Elagin, A; Erbacher, R; Errede, D; Errede, S; Eusebi, R; Fang, H C; Farrington, S; Fedorko, W T; Feild, R G; Feindt, M; Fernandez, J P; Ferrazza, C; Field, R; Flanagan, G; Forrest, R; Frank, M J; Franklin, M; Freeman, J C; Furic, I; Gallinaro, M; Galyardt, J; Garberson, F; Garcia, J E; Garfinkel, A F; Genser, K; Gerberich, H; Gerdes, D; Gessler, A; Giagu, S; Giakoumopoulou, V; Giannetti, P; Gibson, K; Gimmell, J L; Ginsburg, C M; Giokaris, N; Giordani, M; Giromini, P; Giunta, M; Giurgiu, G; Glagolev, V; Glenzinski, D; Gold, M; Goldschmidt, N; Golossanov, A; Gomez, G; Gomez-Ceballos, G; Goncharov, M; González, O; Gorelov, I; Goshaw, A T; Goulianos, K; Gresele, A; Grinstein, S; Grosso-Pilcher, C; Grundler, U; Guimaraes da Costa, J; Gunay-Unalan, Z; Haber, C; Hahn, K; Hahn, S R; Halkiadakis, E; Han, B-Y; Han, J Y; Happacher, F; Hara, K; Hare, D; Hare, M; Harper, S; Harr, R F; Harris, R M; Hartz, M; Hatakeyama, K; Hays, C; Heck, M; Heijboer, A; Heinrich, J; Henderson, C; Herndon, M; Heuser, J; Hewamanage, S; Hidas, D; Hill, C S; Hirschbuehl, D; Hocker, A; Hou, S; Houlden, M; Hsu, S-C; Huffman, B T; Hughes, R E; Husemann, U; Huston, J; Incandela, J; Introzzi, G; Iori, M; Ivanov, A; James, E; Jayatilaka, B; Jeon, E J; Jha, M K; Jindariani, S; Johnson, W; Jones, M; Joo, K K; Jun, S Y; Jung, J E; Junk, T R; Kamon, T; Kar, D; Karchin, P E; Kato, Y; Kephart, R; Keung, J; Khotilovich, V; Kilminster, B; Kim, D H; Kim, H S; Kim, H W; Kim, J E; Kim, M J; Kim, S B; Kim, S H; Kim, Y K; Kimura, N; Kirsch, L; Klimenko, S; Knuteson, B; Ko, B R; Kondo, K; Kong, D J; Konigsberg, J; Korytov, A; Kotwal, A V; Kreps, M; Kroll, J; Krop, D; Krumnack, N; Kruse, M; Krutelyov, V; Kubo, T; Kuhr, T; Kulkarni, N P; Kurata, M; Kusakabe, Y; Kwang, S; Laasanen, A T; Lami, S; Lammel, S; Lancaster, M; Lander, R L; Lannon, K; Lath, A; Latino, G; Lazzizzera, I; LeCompte, T; Lee, E; Lee, H S; Lee, S W; Leone, S; Lewis, J D; Lin, C-S; Linacre, J; Lindgren, M; Lipeles, E; Lister, A; Litvintsev, D O; Liu, C; Liu, T; Lockyer, N S; Loginov, A; Loreti, M; Lovas, L; Lucchesi, D; Luci, C; Lueck, J; Lujan, P; Lukens, P; Lungu, G; Lyons, L; Lys, J; Lysak, R; Macqueen, D; Madrak, R; Maeshima, K; Makhoul, K; Maki, T; Maksimovic, P; Malde, S; Malik, S; Manca, G; Manousakis-Katsikakis, A; Margaroli, F; Marino, C; Marino, C P; Martin, A; Martin, V; Martínez, M; Martínez-Ballarín, R; Maruyama, T; Mastrandrea, P; Masubuchi, T; Mathis, M; Mattson, M E; Mazzanti, P; McFarland, K S; McIntyre, P; McNulty, R; Mehta, A; Mehtala, P; Menzione, A; Merkel, P; Mesropian, C; Miao, T; Miladinovic, N; Miller, R; Mills, C; Milnik, M; Mitra, A; Mitselmakher, G; Miyake, H; Moggi, N; Moon, C S; Moore, R; Morello, M J; Morlok, J; Movilla Fernandez, P; Mülmenstädt, J; Mukherjee, A; Muller, Th; Mumford, R; Murat, P; Mussini, M; Nachtman, J; Nagai, Y; Nagano, A; Naganoma, J; Nakamura, K; Nakano, I; Napier, A; Necula, V; Nett, J; Neu, C; Neubauer, M S; Neubauer, S; Nielsen, J; Nodulman, L; Norman, M; Norniella, O; Nurse, E; Oakes, L; Oh, S H; Oh, Y D; Oksuzian, I; Okusawa, T; Orava, R; Griso, S Pagan; Palencia, E; Papadimitriou, V; Papaikonomou, A; Paramonov, A A; Parks, B; Pashapour, S; Patrick, J; Pauletta, G; Paulini, M; Paus, C; Peiffer, T; Pellett, D E; Penzo, A; Phillips, T J; Piacentino, G; Pianori, E; Pinera, L; Pitts, K; Plager, C; Pondrom, L; Poukhov, O; Pounder, N; Prakoshyn, F; Pronko, A; Proudfoot, J; Ptohos, F; Pueschel, E; Punzi, G; Pursley, J; Rademacker, J; Rahaman, A; Ramakrishnan, V; Ranjan, N; Redondo, I; Rekovic, V; Renton, P; Renz, M; Rescigno, M; Richter, S; Rimondi, F; Ristori, L; Robson, A; Rodrigo, T; Rodriguez, T; Rogers, E; Rolli, S; Roser, R; Rossi, M; Rossin, R; Roy, P; Ruiz, A; Russ, J; Rusu, V; Safonov, A; Sakumoto, W K; Saltó, O; Santi, L; Sarkar, S; Sartori, L; Sato, K; Savoy-Navarro, A; Schlabach, P; Schmidt, A; Schmidt, E E; Schmidt, M A; Schmidt, M P; Schmitt, M; Schwarz, T; Scodellaro, L; Scribano, A; Scuri, F; Sedov, A; Seidel, S; Seiya, Y; Semenov, A; Sexton-Kennedy, L; Sforza, F; Sfyrla, A; Shalhout, S Z; Shears, T; Shepard, P F; Shimojima, M; Shiraishi, S; Shochet, M; Shon, Y; Shreyber, I; Sidoti, A; Sinervo, P; Sisakyan, A; Slaughter, A J; Slaunwhite, J; Sliwa, K; Smith, J R; Snider, F D; Snihur, R; Soha, A; Somalwar, S; Sorin, V; Spalding, J; Spreitzer, T; Squillacioti, P; Stanitzki, M; St Denis, R; Stelzer, B; Stelzer-Chilton, O; Stentz, D; Strologas, J; Strycker, G L; Stuart, D; Suh, J S; Sukhanov, A; Suslov, I; Suzuki, T; Taffard, A; Takashima, R; Takeuchi, Y; Tanaka, R; Tecchio, M; Teng, P K; Terashi, K; Thom, J; Thompson, A S; Thompson, G A; Thomson, E; Tipton, P; Ttito-Guzmán, P; Tkaczyk, S; Toback, D; Tokar, S; Tollefson, K; Tomura, T; Tonelli, D; Torre, S; Torretta, D; Totaro, P; Tourneur, S; Trovato, M; Tsai, S-Y; Tu, Y; Turini, N; Ukegawa, F; Vallecorsa, S; van Remortel, N; Varganov, A; Vataga, E; Vázquez, F; Velev, G; Vellidis, C; Veszpremi, V; Vidal, M; Vidal, R; Vila, I; Vilar, R; Vine, T; Vogel, M; Volobouev, I; Volpi, G; Wagner, P; Wagner, R G; Wagner, R L; Wagner, W; Wagner-Kuhr, J; Wakisaka, T; Wallny, R; Wang, C; Wang, S M; Warburton, A; Waters, D; Weinberger, M; Weinelt, J; Wester, W C; Whitehouse, B; Whiteson, D; Wicklund, A B; Wicklund, E; Wilbur, S; Williams, G; Williams, H H; Wilson, P; Winer, B L; Wittich, P; Wolbers, S; Wolfe, C; Wright, T; Wu, X; Würthwein, F; Wynne, S M; Xie, S; Yagil, A; Yamamoto, K; Yamaoka, J; Yang, U K; Yang, Y C; Yao, W M; Yeh, G P; Yoh, J; Yorita, K; Yoshida, T; Yu, G B; Yu, I; Yu, S S; Yun, J C; Zanello, L; Zanetti, A; Zhang, X; Zheng, Y; Zucchelli, S

2009-01-23

A search for high-mass resonances in the e+e- final state is presented based on 2.5 fb(-1) of sqrt[s]=1.96 TeV pp collision data from the CDF II detector at the Fermilab Tevatron. The largest excess over the standard model prediction is at an e+e- invariant mass of 240 GeV/c2. The probability of observing such an excess arising from fluctuations in the standard model anywhere in the mass range of 150-1000 GeV/c2 is 0.6% (equivalent to 2.5sigma). We exclude the standard model coupling Z' and the Randall-Sundrum graviton for k/MPl=0.1 with masses below 963 and 848 GeV/c2 at the 95% credibility level, respectively.

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. Section...

Word Frequency Analysis MOS: 15E. Skill Levels 1 and 2.

DTIC Science & Technology

1982-05-01

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11 CFR 101.1 - Candidate designations (2 U.S.C. 432(e)(1)).

Code of Federal Regulations, 2010 CFR

2010-01-01

... 11 Federal Elections 1 2010-01-01 2010-01-01 false Candidate designations (2 U.S.C. 432(e)(1)). 101.1 Section 101.1 Federal Elections FEDERAL ELECTION COMMISSION GENERAL CANDIDATE STATUS AND... and address, party affiliation, and office sought, the District and State in which Federal office is...

A series of substituted (2E)-3-(2-fluoro-4-phenoxyphenyl)-1-phenylprop-2-en-1-ones.

PubMed

Chopra, Deepak; Mohan, T P; Vishalakshi, B; Row, T N Guru

2007-12-01

In the molecular structures of a series of substituted chalcones, namely (2E)-3-(2-fluoro-4-phenoxyphenyl)-1-phenylprop-2-en-1-one, C21H15FO2, (I), (2E)-3-(2-fluoro-4-phenoxyphenyl)-1-(4-fluorophenyl)prop-2-en-1-one, C21H14F2O2, (II), (2E)-1-(4-chlorophenyl)-3-(2-fluoro-4-phenoxyphenyl)prop-2-en-1-one, C21H14ClFO2, (III), (2E)-3-(2-fluoro-4-phenoxyphenyl)-1-(4-methylphenyl)prop-2-en-1-one, C22H17FO2, (IV), and (2E)-3-(2-fluoro-4-phenoxyphenyl)-1-(4-methoxyphenyl)prop-2-en-1-one, C22H17FO3, (V), the configuration of the keto group with respect to the olefinic double bond is s-cis. The molecules pack utilizing weak C-H...O and C-H...pi intermolecular contacts. Identical packing motifs involving C-H...O interactions, forming both chains and dimers, along with C-H...pi dimers and pi-pi aromatic interactions are observed in the fluoro, chloro and methyl derivatives.

E2F1 and E2F2 prevent replicative stress and subsequent p53-dependent organ involution.

PubMed

Iglesias-Ara, A; Zenarruzabeitia, O; Buelta, L; Merino, J; Zubiaga, A M

2015-10-01

Tissue homeostasis requires tight regulation of cellular proliferation, differentiation and apoptosis. E2F1 and E2F2 transcription factors share a critical role in tissue homeostasis, since their combined inactivation results in overall organ involution, specially affecting the pancreatic gland, which subsequently triggers diabetes. We have examined the mechanism by which these E2Fs regulate tissue homeostasis. We show that pancreas atrophy in E2F1/E2F2 double-knockout (DKO) mice is associated with mitochondrial apoptosis and activation of the p53 pathway in young animals, before the development of diabetes. A deregulated expression of E2F target genes was detected in pancreatic cells of young DKO animals, along with unscheduled DNA replication and activation of a DNA damage response. Importantly, suppression of DNA replication in vivo with aphidicolin led to a significant inhibition of the p53 pathway in DKO pancreas, implying a causal link between DNA replication stress and p53 activation in this model. We further show that activation of the p53 pathway has a key role in the aberrant phenotype of DKO mice, since targeted inactivation of p53 gene abrogated cellular apoptosis and prevented organ involution and insulin-dependent diabetes in mice lacking E2F1/E2F2. Unexpectedly, p53 inactivation unmasked oncogenic features of E2F1/E2F2-depleted cells, as evidenced by an accelerated tumor development in triple-knockout mice compared with p53(-/-) mice. Collectively, our data reveal a role for E2F1 and E2F2 as suppressors of replicative stress in differentiating cells, and uncover the existence of a robust E2F-p53 regulatory axis to enable tissue homeostasis and prevent tumorigenesis. These findings have implications in the design of approaches targeting E2F for cancer therapy.

Observation of e + e → Φ χ c 1 and Φ χ c 2 at s = 4.600 GeV

DOE Office of Scientific and Technical Information (OSTI.GOV)

Ablikim, M.; Achasov, M. N.; Ahmed, S.

Using a data sample collected with the BESIII detector operating at the BEPCII storage ring at a centerof- mass energy of √s = 4.600 GeV, we search for the production of e +e - → φχ c0,1,2. A search is also performed for the charmonium-like state X(4140) in the radiative transition e +e - → γX(4140) with X(4140) subsequently decaying into φJ/ψ. The processes e +e - → φχ c1 and φχ c2 are observed for the first time, each with a statistical significance ofmore than 10σ, and the Born cross sections are measuredmore » to be (4.2+1.7 -1.0±0.3) pb and (6.7+3.4 -1.7 ± 0.5) pb, respectively, where the first uncertainties are statistical and the second systematic. No significant signals are observed for e +e - → φχ c0 and e +e - → γX(4140) and upper limits on the Born cross sections at 90% confidence level are provided at √s = 4.600 GeV.« less

Observation of e + e → Φ χ c 1 and Φ χ c 2 at s = 4.600 GeV

DOE PAGES

Ablikim, M.; Achasov, M. N.; Ahmed, S.; ...

2018-02-12

Using a data sample collected with the BESIII detector operating at the BEPCII storage ring at a centerof- mass energy of √s = 4.600 GeV, we search for the production of e +e - → φχ c0,1,2. A search is also performed for the charmonium-like state X(4140) in the radiative transition e +e - → γX(4140) with X(4140) subsequently decaying into φJ/ψ. The processes e +e - → φχ c1 and φχ c2 are observed for the first time, each with a statistical significance ofmore than 10σ, and the Born cross sections are measuredmore » to be (4.2+1.7 -1.0±0.3) pb and (6.7+3.4 -1.7 ± 0.5) pb, respectively, where the first uncertainties are statistical and the second systematic. No significant signals are observed for e +e - → φχ c0 and e +e - → γX(4140) and upper limits on the Born cross sections at 90% confidence level are provided at √s = 4.600 GeV.« less

Elimination of both E1 and E2 from adenovirus vectors further improves prospects for in vivo human gene therapy.

PubMed Central

Gorziglia, M I; Kadan, M J; Yei, S; Lim, J; Lee, G M; Luthra, R; Trapnell, B C

1996-01-01

A novel recombinant adenovirus vector, Av3nBg, was constructed with deletions in adenovirus E1, E2a, and E3 regions and expressing a beta-galactosidase reporter gene. Av3nBg can be propagated at a high titer in a corresponding A549-derived cell line, AE1-2a, which contains the adenovirus E1 and E2a region genes inducibly expressed from separate glucocorticoid-responsive promoters. Av3nBg demonstrated gene transfer and expression comparable to that of Av1nBg, a first-generation adenovirus vector with deletions in E1 and E3. Several lines of evidence suggest that this vector is significantly more attenuated than E1 and E3 deletion vectors. Metabolic DNA labeling studies showed no detectable de novo vector DNA synthesis or accumulation, and metabolic protein labeling demonstrated no detectable de novo hexon protein synthesis for Av3nBg in naive A549 cells even at a multiplicity of infection of up to 3,000 PFU per cell. Additionally, naive A549 cells infected by Av3nBg did not accumulate infectious virions. In contrast, both Av1nBg and Av2Lu vectors showed DNA replication and hexon protein synthesis at multiplicities of infection of 500 PFU per cell. Av2Lu has a deletion in E1 and also carries a temperature-sensitive mutation in E2a. Thus, molecular characterization has demonstrated that the Av3nBg vector is improved with respect to the potential for vector DNA replication and hexon protein expression compared with both first-generation (Av1nBg) and second-generation (Av2Lu) adenoviral vectors. These observations may have important implications for potential use of adenovirus vectors in human gene therapy. PMID:8648763

E2F1 and E2F2 induction in response to DNA damage preserves genomic stability in neuronal cells.

PubMed

Castillo, Daniela S; Campalans, Anna; Belluscio, Laura M; Carcagno, Abel L; Radicella, J Pablo; Cánepa, Eduardo T; Pregi, Nicolás

2015-01-01

E2F transcription factors regulate a wide range of biological processes, including the cellular response to DNA damage. In the present study, we examined whether E2F family members are transcriptionally induced following treatment with several genotoxic agents, and have a role on the cell DNA damage response. We show a novel mechanism, conserved among diverse species, in which E2F1 and E2F2, the latter specifically in neuronal cells, are transcriptionally induced after DNA damage. This upregulation leads to increased E2F1 and E2F2 protein levels as a consequence of de novo protein synthesis. Ectopic expression of these E2Fs in neuronal cells reduces the level of DNA damage following genotoxic treatment, while ablation of E2F1 and E2F2 leads to the accumulation of DNA lesions and increased apoptotic response. Cell viability and DNA repair capability in response to DNA damage induction are also reduced by the E2F1 and E2F2 deficiencies. Finally, E2F1 and E2F2 accumulate at sites of oxidative and UV-induced DNA damage, and interact with γH2AX DNA repair factor. As previously reported for E2F1, E2F2 promotes Rad51 foci formation, interacts with GCN5 acetyltransferase and induces histone acetylation following genotoxic insult. The results presented here unveil a new mechanism involving E2F1 and E2F2 in the maintenance of genomic stability in response to DNA damage in neuronal cells.

E2F1 and E2F2 induction in response to DNA damage preserves genomic stability in neuronal cells

PubMed Central

Castillo, Daniela S; Campalans, Anna; Belluscio, Laura M; Carcagno, Abel L; Radicella, J Pablo; Cánepa, Eduardo T; Pregi, Nicolás

2015-01-01

E2F transcription factors regulate a wide range of biological processes, including the cellular response to DNA damage. In the present study, we examined whether E2F family members are transcriptionally induced following treatment with several genotoxic agents, and have a role on the cell DNA damage response. We show a novel mechanism, conserved among diverse species, in which E2F1 and E2F2, the latter specifically in neuronal cells, are transcriptionally induced after DNA damage. This upregulation leads to increased E2F1 and E2F2 protein levels as a consequence of de novo protein synthesis. Ectopic expression of these E2Fs in neuronal cells reduces the level of DNA damage following genotoxic treatment, while ablation of E2F1 and E2F2 leads to the accumulation of DNA lesions and increased apoptotic response. Cell viability and DNA repair capability in response to DNA damage induction are also reduced by the E2F1 and E2F2 deficiencies. Finally, E2F1 and E2F2 accumulate at sites of oxidative and UV-induced DNA damage, and interact with γH2AX DNA repair factor. As previously reported for E2F1, E2F2 promotes Rad51 foci formation, interacts with GCN5 acetyltransferase and induces histone acetylation following genotoxic insult. The results presented here unveil a new mechanism involving E2F1 and E2F2 in the maintenance of genomic stability in response to DNA damage in neuronal cells. PMID:25892555

Structure, Dynamics, and Interaction of Mycobacterium tuberculosis (Mtb) DprE1 and DprE2 Examined by Molecular Modeling, Simulation, and Electrostatic Studies

PubMed Central

Bhutani, Isha; Loharch, Saurabh; Gupta, Pawan; Madathil, Rethi; Parkesh, Raman

2015-01-01

The enzymes decaprenylphosphoryl-β-D-ribose oxidase (DprE1) and decaprenylphosphoryl-β-D-ribose-2-epimerase (DprE2) catalyze epimerization of decaprenylphosporyl ribose (DPR) todecaprenylphosporyl arabinose (DPA) and are critical for the survival of Mtb. Crystal structures of DprE1 so far reported display significant disordered regions and no structural information is known for DprE2. We used homology modeling, protein threading, molecular docking and dynamics studies to investigate the structural and dynamic features of Mtb DprE1 and DprE2 and DprE1-DprE2 complex. A three-dimensional model for DprE2 was generated using the threading approach coupled with ab initio modeling. A 50 ns simulation of DprE1 and DprE2 revealed the overall stability of the structures. Principal Component Analysis (PCA) demonstrated the convergence of sampling in both DprE1 and DprE2. In DprE1, residues in the 269–330 area showed considerable fluctuation in agreement with the regions of disorder observed in the reported crystal structures. In DprE2, large fluctuations were detected in residues 95–113, 146–157, and 197–226. The study combined docking and MD simulation studies to map and characterize the key residues involved in DprE1-DprE2 interaction. A 60 ns MD simulation for DprE1-DprE2 complex was also performed. Analysis of data revealed that the docked complex is stabilized by H-bonding, hydrophobic and ionic interactions. The key residues of DprE1 involved in DprE1-DprE2 interactions belong to the disordered region. We also examined the docked complex of DprE1-BTZ043 to investigate the binding pocket of DprE1 and its interactions with the inhibitor BTZ043. In summary, we hypothesize that DprE1-DprE2 interaction is crucial for the synthesis of DPA and DprE1-DprE2 complex may be a new therapeutic target amenable to pharmacological validation. The findings have important implications in tuberculosis (TB) drug discovery and will facilitate drug development efforts against TB

Structure, dynamics, and interaction of Mycobacterium tuberculosis (Mtb) DprE1 and DprE2 examined by molecular modeling, simulation, and electrostatic studies.

PubMed

Bhutani, Isha; Loharch, Saurabh; Gupta, Pawan; Madathil, Rethi; Parkesh, Raman

2015-01-01

The enzymes decaprenylphosphoryl-β-D-ribose oxidase (DprE1) and decaprenylphosphoryl-β-D-ribose-2-epimerase (DprE2) catalyze epimerization of decaprenylphosporyl ribose (DPR) todecaprenylphosporyl arabinose (DPA) and are critical for the survival of Mtb. Crystal structures of DprE1 so far reported display significant disordered regions and no structural information is known for DprE2. We used homology modeling, protein threading, molecular docking and dynamics studies to investigate the structural and dynamic features of Mtb DprE1 and DprE2 and DprE1-DprE2 complex. A three-dimensional model for DprE2 was generated using the threading approach coupled with ab initio modeling. A 50 ns simulation of DprE1 and DprE2 revealed the overall stability of the structures. Principal Component Analysis (PCA) demonstrated the convergence of sampling in both DprE1 and DprE2. In DprE1, residues in the 269-330 area showed considerable fluctuation in agreement with the regions of disorder observed in the reported crystal structures. In DprE2, large fluctuations were detected in residues 95-113, 146-157, and 197-226. The study combined docking and MD simulation studies to map and characterize the key residues involved in DprE1-DprE2 interaction. A 60 ns MD simulation for DprE1-DprE2 complex was also performed. Analysis of data revealed that the docked complex is stabilized by H-bonding, hydrophobic and ionic interactions. The key residues of DprE1 involved in DprE1-DprE2 interactions belong to the disordered region. We also examined the docked complex of DprE1-BTZ043 to investigate the binding pocket of DprE1 and its interactions with the inhibitor BTZ043. In summary, we hypothesize that DprE1-DprE2 interaction is crucial for the synthesis of DPA and DprE1-DprE2 complex may be a new therapeutic target amenable to pharmacological validation. The findings have important implications in tuberculosis (TB) drug discovery and will facilitate drug development efforts against TB.

Vitamin E supplementation ameliorates aflatoxin B1-induced nephrotoxicity in rats.

PubMed

Abdel-Hamid, Ahmed A M; Firgany, Alaa El-Din L

2015-10-01

Fungal toxins in nutrition can cause organ dysfunction or even failure. Aflatoxin B1 (AFB1)-induced renal impairment is not sufficiently studied regarding its extent and prevention. The aim of this experiment was to study the effect of AFB1 on renal cortical tissue and whether its possible harmful effect could be prevented by the conventional economical antioxidant, vitamin E. Forty rats were divided into four groups; I-IV. Group I represented the control while the others received vitamin E (Vit E), AFB1 and AFB1+Vit E, respectively. Renal cortex specimens were taken from each group after 25 days. Then, specimens were prepared for histological study by hematoxlyin and eosin (H&E), Masson's trichrome, caspase-3 as well as for ultrastructural examination and oxidative stress parameters evaluation. Data were morphometrically and statistically analyzed. In AFB1-treated group, focal tubulo-interstitial affection in the form of tubular cytoplasmic vacuolation, mitochondrial disruption, numerous lysosomes, marked increase in collagen deposition and in caspase-3 expression were observed. Glomerular impairment in the form of fusion of podocytes enlarged foot processes and thickening of the glomerular basement membrane (GBM) with loss of its trilaminar appearance were detected. In the group treated by AFB1+Vit E, there were minimal affection of the histological structure of the renal cortex as well as significant increase in the anti-oxidative parameters which were significantly decreased in the AFB1-treated group. Therefore, Vit E could be considered in wide experimental studies to be a first choice antioxidant of high cost-effectiveness in prevention of fungal toxins pro-oxidant-induced renal impairment. Copyright © 2015 Elsevier GmbH. All rights reserved.

A non-redundant function of cyclin E1 in hematopoietic stem cells.

PubMed

Campaner, Stefano; Viale, Andrea; De Fazio, Serena; Doni, Mirko; De Franco, Francesca; D'Artista, Luana; Sardella, Domenico; Pelicci, Pier Giuseppe; Amati, Bruno

2013-12-01

A precise balance between quiescence and proliferation is crucial for the lifelong function of hematopoietic stem cells (HSCs). Cyclins E1 and E2 regulate exit from quiescence in fibroblasts, but their role in HSCs remains unknown. Here, we report a non-redundant role for cyclin E1 in mouse HSCs. A long-term culture-initiating cell (LTC-IC) assay indicated that the loss of cyclin E1, but not E2, compromised the colony-forming activity of primitive hematopoietic progenitors. Ccne1(-/-) mice showed normal hematopoiesis in vivo under homeostatic conditions but a severe impairment following myeloablative stress induced by 5-fluorouracil (5-FU). Under these conditions, Ccne1(-/-) HSCs were less efficient in entering the cell cycle, resulting in decreased hematopoiesis and reduced survival of mutant mice upon weekly 5-FU treatment. The role of cyclin E1 in homeostatic conditions became apparent in aged mice, where HSC quiescence was increased in Ccne1(-/-) animals. On the other hand, loss of cyclin E1 provided HSCs with a competitive advantage in bone marrow serial transplantation assays, suggesting that a partial impairment of cell cycle entry may exert a protective role by preventing premature depletion of the HSC compartment. Our data support a role for cyclin E1 in controlling the exit from quiescence in HSCs. This activity, depending on the physiological context, can either jeopardize or protect the maintenance of hematopoiesis.

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

2015-12-02

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. Copyright © 2015 Elsevier Ireland Ltd

High-efficiency inverted metamorphic 1.7/1.1 eV GaInAsP/GaInAs dual-junction solar cells

NASA Astrophysics Data System (ADS)

Jain, Nikhil; Schulte, Kevin L.; Geisz, John F.; Friedman, Daniel J.; France, Ryan M.; Perl, Emmett E.; Norman, Andrew G.; Guthrey, Harvey L.; Steiner, Myles A.

2018-01-01

Photovoltaic conversion efficiencies of 32.6 ± 1.4% under the AM1.5 G173 global spectrum, and 35.5% ± 1.2% at 38-suns concentration under the direct spectrum, are demonstrated for a monolithic, dual-junction 1.7/1.1 eV solar cell. The tandem cell consists of a 1.7 eV GaInAsP top-junction grown lattice-matched to a GaAs substrate, followed by a metamorphic 1.1 eV GaInAs junction grown on a transparent, compositionally graded metamorphic AlGaInAs buffer. This bandgap combination is much closer to the dual-junction optimum and offers headroom for absolute 3% improvement in efficiency, in comparison to the incumbent lattice-matched GaInP/GaAs (˜1.86/1.41 eV) solar cells. The challenge of growing a high-quality 1.7 eV GaInAsP solar cell is the propensity for phase separation in the GaInAsP alloy. The challenge of lattice-mismatched GaInAs solar cell growth is that it requires minimizing the residual dislocation density during the growth of a transparent compositionally graded buffer to enable efficient metamorphic tandem cell integration. Transmission electron microscopy reveals relatively weak composition fluctuation present in the 1.7 eV GaInAsP alloy, attained through growth control. The threading dislocation density of the GaInAs junction is ˜1 × 106 cm-2, as determined from cathodoluminescence measurements, highlighting the quality of the graded buffer. These material advances have enabled the performance of both junctions to reach over 80% of their Shockley-Queisser limiting efficiencies, with both the subcells demonstrating a bandgap-voltage offset, WOC (=Eg/q-VOC), of ˜0.39 V.

High-efficiency inverted metamorphic 1.7/1.1 eV GaInAsP/GaInAs dual-junction solar cells

DOE PAGES

Jain, Nikhil; Schulte, Kevin L.; Geisz, John F.; ...

2018-01-29

Photovoltaic conversion efficiencies of 32.6 +/- 1.4% under the AM1.5 G173 global spectrum, and 35.5 +/- 1.2% at 38-suns concentration under the direct spectrum, are demonstrated for a monolithic, dual-junction 1.7/1.1 eV solar cell. The tandem cell consists of a 1.7 eV GaInAsP top-junction grown lattice-matched to a GaAs substrate, followed by a metamorphic 1.1 eV GaInAs junction grown on a transparent, compositionally graded metamorphic AlGaInAs buffer. This bandgap combination is much closer to the dual-junction optimum and offers headroom for absolute 3% improvement in efficiency, in comparison to the incumbent lattice-matched GaInP/GaAs (~1.86/1.41 eV) solar cells. The challenge ofmore » growing a high-quality 1.7 eV GaInAsP solar cell is the propensity for phase separation in the GaInAsP alloy. The challenge of lattice-mismatched GaInAs solar cell growth is that it requires minimizing the residual dislocation density during the growth of a transparent compositionally graded buffer to enable efficient metamorphic tandem cell integration. Transmission electron microscopy reveals relatively weak composition fluctuation present in the 1.7 eV GaInAsP alloy, attained through growth control. The threading dislocation density of the GaInAs junction is ~1 x 10^6 cm-2, as determined from cathodoluminescence measurements, highlighting the quality of the graded buffer. These material advances have enabled the performance of both junctions to reach over 80% of their Shockley-Queisser limiting efficiencies, with both the subcells demonstrating a bandgap-voltage offset, WOC (=Eg/q-VOC), of ~0.39 V.« less

High-efficiency inverted metamorphic 1.7/1.1 eV GaInAsP/GaInAs dual-junction solar cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Jain, Nikhil; Schulte, Kevin L.; Geisz, John F.

Photovoltaic conversion efficiencies of 32.6 +/- 1.4% under the AM1.5 G173 global spectrum, and 35.5 +/- 1.2% at 38-suns concentration under the direct spectrum, are demonstrated for a monolithic, dual-junction 1.7/1.1 eV solar cell. The tandem cell consists of a 1.7 eV GaInAsP top-junction grown lattice-matched to a GaAs substrate, followed by a metamorphic 1.1 eV GaInAs junction grown on a transparent, compositionally graded metamorphic AlGaInAs buffer. This bandgap combination is much closer to the dual-junction optimum and offers headroom for absolute 3% improvement in efficiency, in comparison to the incumbent lattice-matched GaInP/GaAs (~1.86/1.41 eV) solar cells. The challenge ofmore » growing a high-quality 1.7 eV GaInAsP solar cell is the propensity for phase separation in the GaInAsP alloy. The challenge of lattice-mismatched GaInAs solar cell growth is that it requires minimizing the residual dislocation density during the growth of a transparent compositionally graded buffer to enable efficient metamorphic tandem cell integration. Transmission electron microscopy reveals relatively weak composition fluctuation present in the 1.7 eV GaInAsP alloy, attained through growth control. The threading dislocation density of the GaInAs junction is ~1 x 10^6 cm-2, as determined from cathodoluminescence measurements, highlighting the quality of the graded buffer. These material advances have enabled the performance of both junctions to reach over 80% of their Shockley-Queisser limiting efficiencies, with both the subcells demonstrating a bandgap-voltage offset, WOC (=Eg/q-VOC), of ~0.39 V.« less

The zebrafish orphan nuclear receptor genes nr2e1 and nr2e3 are expressed in developing eye and forebrain.

PubMed

Kitambi, Satish Srinivas; Hauptmann, Giselbert

2007-02-01

Mammalian Nr2e1 (Tailless, Mtll or Tlx) and Nr2e3 (photoreceptor-specific nuclear receptor, Pnr) are highly related orphan nuclear receptors, that are expressed in eye and forebrain-derived structures. In this study, we analyzed the developmental expression patterns of zebrafish nr2e1 and nr2e3. RT-PCR analysis showed that nr2e1 and nr2e3 are both expressed during embryonic and post-embryonic development. To examine the spatial distribution of nr2e1 and nr2e3 during development whole-mount in situ hybridization was performed. At tailbud stage, initial nr2e1 expression was localized to the rostral brain rudiment anterior to pax2.1 and eng2 expression at the prospective midbrain-hindbrain boundary. During subsequent stages, nr2e1 became widely expressed in fore- and midbrain primordia, eye and olfactory placodes. At 24hpf, strong nr2e1 expression was detected in telencephalon, hypothalamus, dorsal thalamus, pretectum, midbrain tectum, and retina. At 2dpf, the initially widespread nr2e1 expression became more restricted to distinct regions within the fore- and midbrain and to the retinal ciliary margin, the germinal zone which gives rise to retina and presumptive iris. Expression of nr2e3 was exclusively found in the developing retina and epiphysis. In both structures, nr2e3 expression was found in photoreceptor cells. The developmental expression profile of zebrafish nr2e1 and nr2e3 is consistent with evolutionary conserved functions in eye and rostral brain structures.

Assignment of the gene encoding the 5-HT{sub 1E} serotonin receptor (S31) (locus HTR1E) to human chromosome 6q14-q15

DOE Office of Scientific and Technical Information (OSTI.GOV)

Levy, F.O.; Tasken, K.; Solberg, R.

1994-08-01

The human gene for the 5-HT{sub 1E} serotonin receptor was recently cloned, but no chromosomal assignment has yet been given to this gene (locus HTR1E). In this work, we demonstrate by two independent polymerase chain reactions on a panel of human-hamster somatic cell hybrid genomic DNA that the 5-HT{sub 1E} serotonin receptor gene is localized on human chromosome 6. Furthermore, by means of in situ hybridization to human metaphase chromosomes, using the cloned 5-HT{sub 1E} receptor gene (phage clone {lambda}-S31) as a probe, we demonstrate that this gene is localized to the q14-q15 region on chromosome 6. Screening of genomicmore » DNA from 15 unrelated Caucasian individuals, using as a probe the open reading frame of the cloned 5-HT{sub 1E} receptor gene, did not reveal any restriction fragment length polymorphisms with the enzymes BamHI, BanII, BglII, EcoRI, HincII, HindIII, HinfI, MspI, PstI, and PvuII. Since the 5-HT{sub 1E} receptor is found mainly in the cerebral cortex and abnormal function of the serotonergic system has been implicated in a variety of neurologic and psychiatric diseases, the precise chromosomal assignment of the 5-HT{sub 1E} receptor gene is the crucial first step toward the evaluation of this locus as a candidate for mutations in such syndromes. 28 refs., 2 figs., 2 tabs.« less

Deciphering the molecular mechanisms underlying the binding of the TWIST1/E12 complex to regulatory E-box sequences

PubMed Central

Bouard, Charlotte; Terreux, Raphael; Honorat, Mylène; Manship, Brigitte; Ansieau, Stéphane; Vigneron, Arnaud M.; Puisieux, Alain; Payen, Léa

2016-01-01

Abstract The TWIST1 bHLH transcription factor controls embryonic development and cancer processes. Although molecular and genetic analyses have provided a wealth of data on the role of bHLH transcription factors, very little is known on the molecular mechanisms underlying their binding affinity to the E-box sequence of the promoter. Here, we used an in silico model of the TWIST1/E12 (TE) heterocomplex and performed molecular dynamics (MD) simulations of its binding to specific (TE-box) and modified E-box sequences. We focused on (i) active E-box and inactive E-box sequences, on (ii) modified active E-box sequences, as well as on (iii) two box sequences with modified adjacent bases the AT- and TA-boxes. Our in silico models were supported by functional in vitro binding assays. This exploration highlighted the predominant role of protein side-chain residues, close to the heart of the complex, at anchoring the dimer to DNA sequences, and unveiled a shift towards adjacent ((-1) and (-1*)) bases and conserved bases of modified E-box sequences. In conclusion, our study provides proof of the predictive value of these MD simulations, which may contribute to the characterization of specific inhibitors by docking approaches, and their use in pharmacological therapies by blocking the tumoral TWIST1/E12 function in cancers. PMID:27151200

Prevalence of genes encoding virulence factors among Escherichia coli with K1 antigen and non-K1 E. coli strains.

PubMed

Kaczmarek, Agnieszka; Budzynska, Anna; Gospodarek, Eugenia

2012-10-01

Multiplex PCR was used to detect genes encoding selected virulence determinants associated with strains of Escherichia coli with K1 antigen (K1(+)) and non-K1 E. coli (K1(-)). The prevalence of the fimA, fimH, sfa/foc, ibeA, iutA and hlyF genes was studied for 134 (67 K1(+) and 67 K1(-)) E. coli strains isolated from pregnant women and neonates. The fimA gene was present in 83.6 % of E. coli K1(+) and in 86.6 % of E. coli K1(-) strains. The fimH gene was present in all tested E. coli K1(+) strains and in 97.0 % of non-K1 strains. E. coli K1(+) strains were significantly more likely to possess the following genes than E. coli K1(-) strains: sfa/foc (37.3 vs 16.4 %, P = 0.006), ibeA (35.8 vs 4.5 %, P<0.001), iutA (82.1 vs 35.8 %, P<0.001) and hlyF (28.4 vs 6.0 %, P<0.001). In conclusion, E. coli K1(+) seems to be more virulent than E. coli K1(-) strains in developing severe infections, thereby increasing possible sepsis or neonatal bacterial meningitis.

E2F1 and E2F2 prevent replicative stress and subsequent p53-dependent organ involution

PubMed Central

Iglesias-Ara, A; Zenarruzabeitia, O; Buelta, L; Merino, J; Zubiaga, A M

2015-01-01

Tissue homeostasis requires tight regulation of cellular proliferation, differentiation and apoptosis. E2F1 and E2F2 transcription factors share a critical role in tissue homeostasis, since their combined inactivation results in overall organ involution, specially affecting the pancreatic gland, which subsequently triggers diabetes. We have examined the mechanism by which these E2Fs regulate tissue homeostasis. We show that pancreas atrophy in E2F1/E2F2 double-knockout (DKO) mice is associated with mitochondrial apoptosis and activation of the p53 pathway in young animals, before the development of diabetes. A deregulated expression of E2F target genes was detected in pancreatic cells of young DKO animals, along with unscheduled DNA replication and activation of a DNA damage response. Importantly, suppression of DNA replication in vivo with aphidicolin led to a significant inhibition of the p53 pathway in DKO pancreas, implying a causal link between DNA replication stress and p53 activation in this model. We further show that activation of the p53 pathway has a key role in the aberrant phenotype of DKO mice, since targeted inactivation of p53 gene abrogated cellular apoptosis and prevented organ involution and insulin-dependent diabetes in mice lacking E2F1/E2F2. Unexpectedly, p53 inactivation unmasked oncogenic features of E2F1/E2F2-depleted cells, as evidenced by an accelerated tumor development in triple-knockout mice compared with p53−/− mice. Collectively, our data reveal a role for E2F1 and E2F2 as suppressors of replicative stress in differentiating cells, and uncover the existence of a robust E2F-p53 regulatory axis to enable tissue homeostasis and prevent tumorigenesis. These findings have implications in the design of approaches targeting E2F for cancer therapy. PMID:25656653

Role of SKP1-CUL1-F-Box-Protein (SCF) E3 Ubiquitin Ligases in Skin Cancer

PubMed Central

Xie, Chuan-Ming; Wei, Wenyi; Sun, Yi

2013-01-01

Many biological processes such as cell proliferation, differentiation, and cell death depend precisely on the timely synthesis and degradation of key regulatory proteins. While protein synthesis can be regulated at multiple levels, protein degradation is mainly controlled by the ubiquitin—proteasome system (UPS), which consists of two distinct steps: (1) ubiquitylation of targeted protein by E1 ubiquitin-activating enzyme, E2 ubiquitin-conjugating enzyme and E3 ubiquitin ligase, and (2) subsequent degradation by the 26S proteasome. Among all E3 ubiquitin ligases, the SCF (SKP1-CUL1-F-box protein) E3 ligases are the largest family and are responsible for the turnover of many key regulatory proteins. Aberrant regulation of SCF E3 ligases is associated with various human diseases, such as cancers, including skin cancer. In this review, we provide a comprehensive overview of all currently published data to define a promoting role of SCF E3 ligases in the development of skin cancer. The future directions in this area of research are also discussed with an ultimate goal to develop small molecule inhibitors of SCF E3 ligases as a novel approach for the treatment of human skin cancer. Furthermore, altered components or substrates of SCF E3 ligases may also be developed as the biomarkers for early diagnosis or predicting prognosis. PMID:23522382

E2/M1 and C2/M1 at Elsa

NASA Astrophysics Data System (ADS)

Gothe, R. W.

2003-07-01

At the ELectron Stretcher Accelerator ELSA the four momentum transfer dependence of the N to Δ transition has been investigated by measuring the \\vartheta {π }{*} and \\varphi {π }{*} angular distribution of the double differential pion production cross sections in a series of electron scattering coincidence experiments on hydrogen in the -K2-range from 0.04 GeV2 to 0.8 GeV2. The azimuthal angular dependence of the hadronic cross section is based on the polarization of the virtual photon and separates the response functions RT + ∈LRL, RLT and RTT. Whereas the individual \\vartheta {π }{*} -dependences of these response functions allow to form specific ratios, which are sensitive either to the electric transverse (E2 or E1+) or the Coulomb (C2 or S1+) quadrupole transition amplitudes. Preliminary results on the extraction of such multipole ratios have been previously reported in the NSTAR 2000 proceedings1. First results for a -K2 of 0.638 GeV2 obtained after radiative and resolution dependent corrections are described here and compared to other most recent experimental results.

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

Code of Federal Regulations, 2011 CFR

2011-04-01

....1402(e)-4A relate to section 1402(e) as amended by section 115(b)(2) of the Social Security Amendments... changes made by section 1704(a) of the Tax Reform Act of 1986 (100 Stat. 2085, 2779) and applies to... 1402(e) (as in effect prior to amendment by the Social Security Amendments of 1967) applicable to...

SIRT1 reduces endothelial activation without affecting vascular function in ApoE-/- mice

PubMed Central

Stein, Sokrates; Schäfer, Nicola; Breitenstein, Alexander; Besler, Christian; Winnik, Stephan; Lohmann, Christine; Heinrich, Kathrin; Brokopp, Chad E.; Handschin, Christoph; Landmesser, Ulf; Tanner, Felix C.; Lüscher, Thomas F.; Matter, Christian M.

2010-01-01

Excessive production of reactive oxygen species (ROS) contributes to progression of atherosclerosis, at least in part by causing endothelial dysfunction and inflammatory activation. The class III histone deacetylase SIRT1 has been implicated in extension of lifespan. In the vasculature,SIRT1 gain-of-function using SIRT1 overexpression or activation has been shown to improve endothelial function in mice and rats via stimulation of endothelial nitric oxide (NO) synthase (eNOS). However, the effects of SIRT1 loss-of-function on the endothelium in atherosclerosis remain to be characterized. Thus, we have investigated the endothelial effects of decreased endogenous SIRT1 in hypercholesterolemic ApoE-/- mice. We observed no difference in endothelial relaxation and eNOS (Ser1177) phosphorylation between 20-week old male atherosclerotic ApoE-/- SIRT1+/- and ApoE-/- SIRT1+/+ mice. However, SIRT1 prevented endothelial superoxide production, inhibited NF-κB signaling, and diminished expression of adhesion molecules. Treatment of young hypercholesterolemic ApoE-/- SIRT1+/- mice with lipopolysaccharide to boost NF-κB signaling led to a more pronounced endothelial expression of ICAM-1 and VCAM-1 as compared to ApoE-/- SIRT1+/+ mice. In conclusion, endogenous SIRT1 diminishes endothelial activation in ApoE-/- mice, but does not affect endothelium-dependent vasodilatation. PMID:20606253

26 CFR 31.3231(e)-1 - Compensation.

Code of Federal Regulations, 2010 CFR

2010-04-01

... Internal Revenue INTERNAL REVENUE SERVICE, DEPARTMENT OF THE TREASURY (CONTINUED) EMPLOYMENT TAXES AND COLLECTION OF INCOME TAX AT SOURCE EMPLOYMENT TAXES AND COLLECTION OF INCOME TAX AT SOURCE Railroad Retirement Tax Act (Chapter 22, Internal Revenue Code of 1954) General Provisions § 31.3231(e)-1 Compensation...

26 CFR 31.3402(e)-1 - Included and excluded wages.

Code of Federal Regulations, 2010 CFR

2010-04-01

... 26 Internal Revenue 15 2010-04-01 2010-04-01 false Included and excluded wages. 31.3402(e)-1... SOURCE Collection of Income Tax at Source § 31.3402(e)-1 Included and excluded wages. (a) If a portion of... not more than 31 consecutive days constitutes wages, and the remainder does not constitute wages, all...

PED/PEA-15 inhibits hydrogen peroxide-induced apoptosis in Ins-1E pancreatic beta-cells via PLD-1.

PubMed

Fiory, Francesca; Parrillo, Luca; Raciti, Gregory Alexander; Zatterale, Federica; Nigro, Cecilia; Mirra, Paola; Falco, Roberta; Ulianich, Luca; Di Jeso, Bruno; Formisano, Pietro; Miele, Claudia; Beguinot, Francesco

2014-01-01

The small scaffold protein PED/PEA-15 is involved in several different physiologic and pathologic processes, such as cell proliferation and survival, diabetes and cancer. PED/PEA-15 exerts an anti-apoptotic function due to its ability to interfere with both extrinsic and intrinsic apoptotic pathways in different cell types. Recent evidence shows that mice overexpressing PED/PEA-15 present larger pancreatic islets and increased beta-cells mass. In the present work we investigated PED/PEA-15 role in hydrogen peroxide-induced apoptosis in Ins-1E beta-cells. In pancreatic islets isolated from Tg(PED/PEA-15) mice hydrogen peroxide-induced DNA fragmentation was lower compared to WT islets. TUNEL analysis showed that PED/PEA-15 overexpression increases the viability of Ins-1E beta-cells and enhances their resistance to apoptosis induced by hydrogen peroxide exposure. The activity of caspase-3 and the cleavage of PARP-1 were markedly reduced in Ins-1E cells overexpressing PED/PEA-15 (Ins-1E(PED/PEA-15)). In parallel, we observed a decrease of the mRNA levels of pro-apoptotic genes Bcl-xS and Bad. In contrast, the expression of the anti-apoptotic gene Bcl-xL was enhanced. Accordingly, DNA fragmentation was higher in control cells compared to Ins-1E(PED/PEA-15) cells. Interestingly, the preincubation with propranolol, an inhibitor of the pathway of PLD-1, a known interactor of PED/PEA-15, responsible for its deleterious effects on glucose tolerance, abolishes the antiapoptotic effects of PED/PEA-15 overexpression in Ins-1E beta-cells. The same results have been obtained by inhibiting PED/PEA-15 interaction with PLD-1 in Ins-1E(PED/PEA-15). These results show that PED/PEA-15 overexpression is sufficient to block hydrogen peroxide-induced apoptosis in Ins-1E cells through a PLD-1 mediated mechanism.

The pig CYP2E1 promoter is activated by COUP-TF1 and HNF-1 and is inhibited by androstenone.

PubMed

Tambyrajah, Winston S; Doran, Elena; Wood, Jeffrey D; McGivan, John D

2004-11-15

Functional analysis of the pig cytochrome P4502E1 (CYP2E1) promoter identified two major activating elements. One corresponded to the hepatic nuclear factor 1 (HNF-1) consensus binding sequence at nucleotides -128/-98 and the other was located in the region -292/-266. The binding of proteins in pig liver nuclear extracts to a synthetic double-stranded oligonucleotide corresponding to this more distal activating sequence was studied by electrophoretic mobility shift assay. The minimum protein binding sequence was identified as TGTTCTGACCTCTGGG. Gel super-shift assays identified the protein binding to this site as chick ovalbumin upstream promoter transcription factor 1 (COUP-TF1). Androstenone inhibited promoter activity in transfection experiments only with constructs which included the COUP-TF1 binding site. Androstenone inhibited COUP-TF1 binding to synthetic oligonucleotides but did not affect HNF-1 binding. The results offer an explanation for the inhibition of CYP2E1 protein expression by androstenone in isolated pig hepatocytes and may be relevant to the low expression of hepatic CYP2E1 in those pigs which accumulate high levels of androstenone in vivo.

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.

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

Code of Federal Regulations, 2010 CFR

2010-04-01

... 26 Internal Revenue 2 2010-04-01 2010-04-01 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 in...

Electrostatic interactions of colicin E1 with the surface of Escherichia coli total lipid.

PubMed

Tian, Chunhong; Tétreault, Elaine; Huang, Christopher K; Dahms, Tanya E S

2006-06-01

The surface properties of colicin E1, a 522-amino acid protein, and its interaction with monolayers of Escherichia coli (E. coli) total lipid and 1,2-Dimyristoyl-sn-Glycero-3-Phosphocholine (DOPC) were studied using the Langmuir-Blodgett (LB) technique. Colicin E1 is amphiphilic, forming a protein monolayer at the air/buffer interface. The protein is thought to interact with the E. coli total lipid head groups through electrostatic interactions, followed by its insertion into the lipid monolayers. Supported lipid bilayers (SLBs) of E. coli total lipid and DOPC, deposited onto mica at the cell membrane equivalence pressure for E. coli and incubated with colicin E1, were imaged by contact mode atomic force microscopy (CM-AFM). Colicin E1 formed protein aggregates on DOPC SLBs, while E. coli total lipid SLB was deformed following its incubation with colicin E1. Corresponding lateral force images, along with electrostatic surface potentials for colicin E1 P190, imply a direct interaction of colicin E1 with lipid head groups facilitating their charge neutralization.

Unphosphorylated HSP27 (HSPB1) regulates the translation initiation process via a direct association with eIF4E in osteoblasts.

PubMed

Kuroyanagi, Gen; Tokuda, Haruhiko; Yamamoto, Naohiro; Matsushima-Nishiwaki, Rie; Kozawa, Osamu; Otsuka, Takanobu

2015-09-01

Heat-shock protein 27 (HSP27/HSPB1) and its phosphorylation are implicated in multiple physiological and pathophysiological cell functions. Our previous study reported that unphosphorylated HSP27 has an inhibitory role in triiodothyronine (T(3))‑induced osteocalcin (OC) synthesis in osteoblasts. However, the mechanisms behind the HSP27‑mediated effects on osteoblasts remain to be clarified. In the present study, to investigate the exact mechanism of HSP27 and its phosphorylation in osteoblasts, the molecular targets of HSP27 were explored using osteoblast‑like MC3T3‑E1 cells. The levels of OC mRNA induced by T(3) in the HSP27‑overexpressing cells did not show any significant differences compared with those in the control empty vector‑transfected cells. Therefore, the interactions between HSP27 and translational molecules were focused on, including eukaryotic translation initiation factor 4E (eIF4E), eIF4G and 4E‑binding protein 1 (4E‑BP1). The HSP27 protein in the unstimulated cells co‑immunoprecipitated with eIF4E, but not eIF4G or 4E‑BP1. In addition, the association of eIF4E with 4E‑BP1 was observed in the HSP27‑overexpressing cells, as well as in the control cells. Under T(3) stimulation, the binding of eIF4E to eIF4G was markedly attenuated in the HSP27‑overexpressing cells compared with the control cells. In addition, the binding of HSP27 to eIF4E in the unstimulated cells was diminished by the phosphorylation of HSP27. In response to T(3) stimulation, the association of eIF4E with eIF4G in the unphosphorylatable HSP27‑overexpressing cells was markedly reduced compared with the phospho‑mimic HSP27‑overexpressing cells. Taken together, these findings strongly suggest that unphosphorylated HSP27 associates with eIF4E in osteoblasts and suppresses the translation initiation process.

Structure of the eukaryotic translation initiation factor eIF4E in complex with 4EGI-1 reveals an allosteric mechanism for dissociating eIF4G.

PubMed

Papadopoulos, Evangelos; Jenni, Simon; Kabha, Eihab; Takrouri, Khuloud J; Yi, Tingfang; Salvi, Nicola; Luna, Rafael E; Gavathiotis, Evripidis; Mahalingam, Poornachandran; Arthanari, Haribabu; Rodriguez-Mias, Ricard; Yefidoff-Freedman, Revital; Aktas, Bertal H; Chorev, Michael; Halperin, Jose A; Wagner, Gerhard

2014-08-05

The interaction of the eukaryotic translation initiation factor eIF4E with the initiation factor eIF4G recruits the 40S ribosomal particle to the 5' end of mRNAs, facilitates scanning to the AUG start codon, and is crucial for eukaryotic translation of nearly all genes. Efficient recruitment of the 40S particle is particularly important for translation of mRNAs encoding oncoproteins and growth-promoting factors, which often harbor complex 5' UTRs and require efficient initiation. Thus, inhibiting the eIF4E/eIF4G interaction has emerged as a previously unpursued route for developing anticancer agents. Indeed, we discovered small-molecule inhibitors of this eIF4E/eIF4G interaction (4EGIs) that inhibit translation initiation both in vitro and in vivo and were used successfully in numerous cancer-biology and neurobiology studies. However, their detailed molecular mechanism of action has remained elusive. Here, we show that the eIF4E/eIF4G inhibitor 4EGI-1 acts allosterically by binding to a site on eIF4E distant from the eIF4G binding epitope. Data from NMR mapping and high-resolution crystal structures are congruent with this mechanism, where 4EGI-1 attaches to a hydrophobic pocket of eIF4E between β-sheet2 (L60-T68) and α-helix1 (E69-N77), causing localized conformational changes mainly in the H78-L85 region. It acts by unfolding a short 310-helix (S82-L85) while extending α-helix1 by one turn (H78-S82). This unusual helix rearrangement has not been seen in any previous eIF4E structure and reveals elements of an allosteric inhibition mechanism leading to the dislocation of eIF4G from eIF4E.

Observation of e+e-→ϕ χc 1 and ϕ χc 2 at √{s }=4.600 GeV

NASA Astrophysics Data System (ADS)

Ablikim, M.; Achasov, M. N.; Ahmed, S.; Albrecht, M.; Amoroso, A.; An, F. F.; An, Q.; Bai, J. Z.; Bai, Y.; Bakina, O.; Baldini Ferroli, R.; Ban, Y.; Bennett, D. W.; Bennett, J. V.; Berger, N.; Bertani, M.; Bettoni, D.; Bian, J. M.; Bianchi, F.; Boger, E.; Boyko, I.; Briere, R. A.; Cai, H.; Cai, X.; Cakir, O.; Calcaterra, A.; Cao, G. F.; Cetin, S. A.; Chai, J.; Chang, J. F.; Chelkov, G.; Chen, G.; Chen, H. S.; Chen, J. C.; Chen, M. L.; Chen, P. L.; Chen, S. J.; Chen, X. R.; Chen, Y. B.; Chu, X. K.; Cibinetto, G.; Dai, H. L.; Dai, J. P.; Dbeyssi, A.; Dedovich, D.; Deng, Z. Y.; Denig, A.; Denysenko, I.; Destefanis, M.; de Mori, F.; Ding, Y.; Dong, C.; Dong, J.; Dong, L. Y.; Dong, M. Y.; Dou, Z. L.; Du, S. X.; Duan, P. F.; Fang, J.; Fang, S. S.; Fang, Y.; Farinelli, R.; Fava, L.; Fegan, S.; Feldbauer, F.; Felici, G.; Feng, C. Q.; Fioravanti, E.; Fritsch, M.; Fu, C. D.; Gao, Q.; Gao, X. L.; Gao, Y.; Gao, Y. G.; Gao, Z.; Garzia, I.; Goetzen, K.; Gong, L.; Gong, W. X.; Gradl, W.; Greco, M.; Gu, M. H.; Gu, Y. T.; Guo, A. Q.; Guo, R. P.; Guo, Y. P.; Haddadi, Z.; Han, S.; Hao, X. Q.; Harris, F. A.; He, K. L.; He, X. Q.; Heinsius, F. H.; Held, T.; Heng, Y. K.; Holtmann, T.; Hou, Z. L.; Hu, H. M.; Hu, T.; Hu, Y.; Huang, G. S.; Huang, J. S.; Huang, X. T.; Huang, X. Z.; Huang, Z. L.; Hussain, T.; Ikegami Andersson, W.; Ji, Q.; Ji, Q. P.; Ji, X. B.; Ji, X. L.; Jiang, X. S.; Jiang, X. Y.; Jiao, J. B.; Jiao, Z.; Jin, D. P.; Jin, S.; Jin, Y.; Johansson, T.; Julin, A.; Kalantar-Nayestanaki, N.; Kang, X. L.; Kang, X. S.; Kavatsyuk, M.; Ke, B. C.; Khan, T.; Khoukaz, A.; Kiese, P.; Kliemt, R.; Koch, L.; Kolcu, O. B.; Kopf, B.; Kornicer, M.; Kuemmel, M.; Kuessner, M.; Kuhlmann, M.; Kupsc, A.; Kühn, W.; Lange, J. S.; Lara, M.; Larin, P.; Lavezzi, L.; Leithoff, H.; Leng, C.; Li, C.; Li, Cheng; Li, D. M.; Li, F.; Li, F. Y.; Li, G.; Li, H. B.; Li, H. J.; Li, J. C.; Li, Jin; Li, K. J.; Li, Kang; Li, Ke; Li, Lei; Li, P. L.; Li, P. R.; Li, Q. Y.; Li, W. D.; Li, W. G.; Li, X. L.; Li, X. N.; Li, X. Q.; Li, Z. B.; Liang, H.; Liang, Y. F.; Liang, Y. T.; Liao, G. R.; Lin, D. X.; Liu, B.; Liu, B. J.; Liu, C. X.; Liu, D.; Liu, F. H.; Liu, Fang; Liu, Feng; Liu, H. B.; Liu, H. M.; Liu, Huanhuan; Liu, Huihui; Liu, J. B.; Liu, J. P.; Liu, J. Y.; Liu, K.; Liu, K. Y.; Liu, Ke; Liu, L. D.; Liu, P. L.; Liu, Q.; Liu, S. B.; Liu, X.; Liu, Y. B.; Liu, Z. A.; Liu, Zhiqing; Long, Y. F.; Lou, X. C.; Lu, H. J.; Lu, J. G.; Lu, Y.; Lu, Y. P.; Luo, C. L.; Luo, M. X.; Luo, X. L.; Lyu, X. R.; Ma, F. C.; Ma, H. L.; Ma, L. L.; Ma, M. M.; Ma, Q. M.; Ma, T.; Ma, X. N.; Ma, X. Y.; Ma, Y. M.; Maas, F. E.; Maggiora, M.; Malik, Q. A.; Mao, Y. J.; Mao, Z. P.; Marcello, S.; Meng, Z. X.; Messchendorp, J. G.; Mezzadri, G.; Min, J.; Min, T. J.; Mitchell, R. E.; Mo, X. H.; Mo, Y. J.; Morales Morales, C.; Muchnoi, N. Yu.; Muramatsu, H.; Musiol, P.; Mustafa, A.; Nefedov, Y.; Nerling, F.; Nikolaev, I. B.; Ning, Z.; Nisar, S.; Niu, S. L.; Niu, X. Y.; Olsen, S. L.; Ouyang, Q.; Pacetti, S.; Pan, Y.; Papenbrock, M.; Patteri, P.; Pelizaeus, M.; Pellegrino, J.; Peng, H. P.; Peters, K.; Pettersson, J.; Ping, J. L.; Ping, R. G.; Pitka, A.; Poling, R.; Prasad, V.; Qi, H. R.; Qi, M.; Qian, S.; Qiao, C. F.; Qin, N.; Qin, X. S.; Qin, Z. H.; Qiu, J. F.; Rashid, K. H.; Redmer, C. F.; Richter, M.; Ripka, M.; Rolo, M.; Rong, G.; Rosner, Ch.; Sarantsev, A.; Savrié, M.; Schnier, C.; Schoenning, K.; Shan, W.; Shao, M.; Shen, C. P.; Shen, P. X.; Shen, X. Y.; Sheng, H. Y.; Song, J. J.; Song, W. M.; Song, X. Y.; Sosio, S.; Sowa, C.; Spataro, S.; Sun, G. X.; Sun, J. F.; Sun, L.; Sun, S. S.; Sun, X. H.; Sun, Y. J.; Sun, Y. K.; Sun, Y. Z.; Sun, Z. J.; Sun, Z. T.; Tang, C. J.; Tang, G. Y.; Tang, X.; Tapan, I.; Tiemens, M.; Tsednee, B.; Uman, I.; Varner, G. S.; Wang, B.; Wang, B. L.; Wang, D.; Wang, D. Y.; Wang, Dan; Wang, K.; Wang, L. L.; Wang, L. S.; Wang, M.; Wang, Meng; Wang, P.; Wang, P. L.; Wang, W. P.; Wang, X. F.; Wang, Y.; Wang, Y. D.; Wang, Y. F.; Wang, Y. Q.; Wang, Z.; Wang, Z. G.; Wang, Z. Y.; Wang, Zongyuan; Weber, T.; Wei, D. H.; Weidenkaff, P.; Wen, S. P.; Wiedner, U.; Wolke, M.; Wu, L. H.; Wu, L. J.; Wu, Z.; Xia, L.; Xia, Y.; Xiao, D.; Xiao, H.; Xiao, Y. J.; Xiao, Z. J.; Xie, Y. G.; Xie, Y. H.; Xiong, X. A.; Xiu, Q. L.; Xu, G. F.; Xu, J. J.; Xu, L.; Xu, Q. J.; Xu, Q. N.; Xu, X. P.; Yan, L.; Yan, W. B.; Yan, W. C.; Yan, Y. H.; Yang, H. J.; Yang, H. X.; Yang, L.; Yang, Y. H.; Yang, Y. X.; Ye, M.; Ye, M. H.; Yin, J. H.; You, Z. Y.; Yu, B. X.; Yu, C. X.; Yu, J. S.; Yuan, C. Z.; Yuan, Y.; Yuncu, A.; Zafar, A. A.; Zeng, Y.; Zeng, Z.; Zhang, B. X.; Zhang, B. Y.; Zhang, C. C.; Zhang, D. H.; Zhang, H. H.; Zhang, H. Y.; Zhang, J.; Zhang, J. L.; Zhang, J. Q.; Zhang, J. W.; Zhang, J. Y.; Zhang, J. Z.; Zhang, K.; Zhang, L.; Zhang, S. Q.; Zhang, X. Y.; Zhang, Y. H.; Zhang, Y. T.; Zhang, Yang; Zhang, Yao; Zhang, Yu; Zhang, Z. H.; Zhang, Z. P.; Zhang, Z. Y.; Zhao, G.; Zhao, J. W.; Zhao, J. Y.; Zhao, J. Z.; Zhao, Lei; Zhao, Ling; Zhao, M. G.; Zhao, Q.; Zhao, S. J.; Zhao, T. C.; Zhao, Y. B.; Zhao, Z. G.; Zhemchugov, A.; Zheng, B.; Zheng, J. P.; Zheng, W. J.; Zheng, Y. H.; Zhong, B.; Zhou, L.; Zhou, X.; Zhou, X. K.; Zhou, X. R.; Zhou, X. Y.; Zhou, Y. X.; Zhu, J.; Zhu, J.; Zhu, K.; Zhu, K. J.; Zhu, S.; Zhu, S. H.; Zhu, X. L.; Zhu, Y. C.; Zhu, Y. S.; Zhu, Z. A.; Zhuang, J.; Zou, B. S.; Zou, J. H.; Besiii Collaboration

2018-02-01

Using a data sample collected with the BESIII detector operating at the BEPCII storage ring at a center-of-mass energy of √{s }=4.600 GeV , we search for the production of e+e-→ϕ χc 0 ,1 ,2 . A search is also performed for the charmonium-like state X (4140 ) in the radiative transition e+e-→γ X (4140 ) with X (4140 ) subsequently decaying into ϕ J /ψ . The processes e+e-→ϕ χc 1 and ϕ χc 2 are observed for the first time, each with a statistical significance of more than 10 σ , and the Born cross sections are measured to be (4. 2-1.0+1.7±0.3 ) and (6. 7-1.7+3.4±0.5 ) pb , respectively, where the first uncertainties are statistical and the second systematic. No significant signals are observed for e+e-→ϕ χc 0 and e+e-→γ X (4140 ) and upper limits on the Born cross sections at 90% C.L. are provided at √{s }=4.600 GeV .

Angular distributions in the reactions pp-->χ1,2-->γψ-->γe+e-

NASA Astrophysics Data System (ADS)

Baglin, C.; Baird, S.; Bassompierre, G.; Borreani, G.; Brient, J.-C.; Broll, C.; Brom, J.-M.; Bugge, L.; Buran, T.; Burq, J.-P.; Bussière, A.; Buzzo, A.; Cester, R.; Chemarin, M.; Chevallier, M.; Escoubes, B.; Fay, J.; Ferroni, S.; Gracco, V.; Guillad, J.-P.; Khan-Aronsen, E.; Kirsebom, K.; Kylling, A.; Ille, B.; Lambert, M.; Leistam, L.; Lundby, A.; Macri, M.; Marchetto, F.; Menichetti, E.; Morch, C.; Mouëllic, B.; Olsen, D.; Pastrone, N.; Petrillo, L.; Pia, M. G.; Poole, J.; Poulet, M.; Rinaudo, G.; Santroni, A.; Severi, M.; Skjevling, G.; Stugu, B.

1987-08-01

In the experiment R704 at the CERN Intersecting Storage Rings, the two p-wave charmonium states χ1 and χ2 were formed directly in proton-antiproton annihilation, and detected through the decay chain χj-->γ+J/ψ-->ψ+e+e-. The angular d istributions of the events found are studied here. A maximum likehood analysis shows that χ1 radiative transition to the J/ψ is compatible with a pure dipole. Indications of a nonzero, positive quadropole contribution to the χ2 radiative transition are found. Finally, it is found that the χ2 data are consistent with the conventional assumption that a single quark radiates the photon in the transition from the χ2 to the J/ψ.

Arginine methylation-dependent reader-writer interplay governs growth control by E2F-1

PubMed Central

Zheng, Shunsheng; Moehlenbrink, Jutta; Lu, Yi-Chien; Zalmas, Lykourgos-Panagiotis; Sagum, Cari A.; Carr, Simon; McGouran, Joanna F.; Alexander, Leila; Fedorov, Oleg; Munro, Shonagh; Kessler, Benedikt; Bedford, Mark T.; Yu, Qiang; La Thangue, Nicholas B.

2014-01-01

Summary The mechanisms that underlie and dictate the different biological outcomes of E2F-1 activity have yet to be elucidated. We describe the residue-specific methylation of E2F-1 by the asymmetric dimethylating protein arginine methyltransferase (PRMT) 1 and symmetric dimethylating PRMT5, and relate the marks to different functional consequences of E2F-1 activity. Methylation by PRMT1 hinders methylation by PRMT5, which augments E2F-1-dependent apoptosis, whereas PRMT5-dependent methylation favours proliferation by antagonising methylation by PRMT1. The ability of E2F-1 to prompt apoptosis in DNA damaged cells coincides with enhanced PRMT1 methylation. In contrast, cyclin A binding to E2F-1 impedes PRMT1 methylation and augments PRMT5 methylation, thus ensuring that E2F-1 is locked into its cell cycle progression mode. The Tudor domain protein p100-TSN reads the symmetric methylation mark, and binding of p100-TSN down-regulates E2F-1 apoptotic activity. Our results define an exquisite level of precision in the reader-writer interplay that governs the biological outcome of E2F-1 activity. PMID:24076217

Crystal structure of the Agrobacterium virulence complex VirE1-VirE2 reveals a flexible protein that can accommodate different partners.

PubMed

Dym, Orly; Albeck, Shira; Unger, Tamar; Jacobovitch, Jossef; Branzburg, Anna; Michael, Yigal; Frenkiel-Krispin, Daphna; Wolf, Sharon Grayer; Elbaum, Michael

2008-08-12

Agrobacterium tumefaciens infects its plant hosts by a mechanism of horizontal gene transfer. This capability has led to its widespread use in artificial genetic transformation. In addition to DNA, the bacterium delivers an abundant ssDNA binding protein, VirE2, whose roles in the host include protection from cytoplasmic nucleases and adaptation for nuclear import. In Agrobacterium, VirE2 is bound to its acidic chaperone VirE1. When expressed in vitro in the absence of VirE1, VirE2 is prone to oligomerization and forms disordered filamentous aggregates. These filaments adopt an ordered solenoidal form in the presence of ssDNA, which was characterized previously by electron microscopy and three-dimensional image processing. VirE2 coexpressed in vitro with VirE1 forms a soluble heterodimer. VirE1 thus prevents VirE2 oligomerization and competes with its binding to ssDNA. We present here a crystal structure of VirE2 in complex with VirE1, showing that VirE2 is composed of two independent domains presenting a novel fold, joined by a flexible linker. Electrostatic interactions with VirE1 cement the two domains of VirE2 into a locked form. Comparison with the electron microscopy structure indicates that the VirE2 domains adopt different relative orientations. We suggest that the flexible linker between the domains enables VirE2 to accommodate its different binding partners.

A single mouse monoclonal antibody, E58 modulates multiple IgE epitopes on group 1 cedar pollen allergens

PubMed Central

Goldblum, Randall M.; Ning, Bo; Judy, Barbara. M.; Holthauzen, Luis Marcelo F.; van Bavel, Julius; Kamijo, Atsushi; Midoro-Horiuti, Terumi

2016-01-01

We recently described a dominant role for conformational epitopes on the group 1 allergen of the mountain cedar (Juniperus ashei, Cupressaceae), Jun a 1, in pollen hypersensitivity in South Central U.S.A. Since these epitopes are surface exposed and are likely to be flexible, they may be susceptible to molecular or physical perturbations. This may make Jun a 1 a potential target for new forms of therapy for cedar pollinosis. Here, we describe a mouse monoclonal antibody, termed E58, which binds to the group 1 allergens of cedar pollens from three highly populated regions of the world (central U.S.A., France and Japan). Upon binding to these allergens, E58 strongly reduces the binding of patient’s IgE antibodies to these dominant allergens. This characteristic of E58, and potentially other similar antibodies, suggests an opportunity to identify preventative or therapeutic agents that may inhibit cedar pollen sensitization or prevent the allergic reactions. PMID:27174188

Measurement of the methyl cyanide E/A ratio in TMC-1

NASA Technical Reports Server (NTRS)

Minh, Y. C.; Irvine, W. M.; Ohishi, M.; Ishikawa, S.; Saito, S.; Kaifu, N.

1993-01-01

We have observed the methyl cyanide (CH3CN) J = 2-1 K = 0 and 1 transitions toward the cyanopolyyne peak of TMC-1 and have derived an E/A (ortho/para)abundance ratio N(E)/N(A) = 0.75 +/- 0.10. The total methyl cyanide column density is N(total) = 5 x 10 exp 12/sq cm toward TMC-1, in agreement with earlier results from the J = 1-0 lines.

PGC-1α dictates endothelial function through regulation of eNOS expression

PubMed Central

Craige, Siobhan M.; Kröller-Schön, Swenja; Li, Chunying; Kant, Shashi; Cai, Shenghe; Chen, Kai; Contractor, Mayur M.; Pei, Yongmei; Schulz, Eberhard; Keaney, John F.

2016-01-01

Endothelial dysfunction is a characteristic of many vascular related diseases such as hypertension. Peroxisome proliferator activated receptor gamma, coactivator 1α (PGC-1α) is a unique stress sensor that largely acts to promote adaptive responses. Therefore, we sought to define the role of endothelial PGC-1α in vascular function using mice with endothelial specific loss of function (PGC-1α EC KO) and endothelial specific gain of function (PGC-1α EC TG). Here we report that endothelial PGC-1α is suppressed in angiotensin-II (ATII)-induced hypertension. Deletion of endothelial PGC-1α sensitized mice to endothelial dysfunction and hypertension in response to ATII, whereas PGC-1α EC TG mice were protected. Mechanistically, PGC-1α promotes eNOS expression and activity, which is necessary for protection from ATII-induced dysfunction as mice either treated with an eNOS inhibitor (LNAME) or lacking eNOS were no longer responsive to transgenic endothelial PGC-1α expression. Finally, we determined that the orphan nuclear receptor, estrogen related receptor α (ERRα) is required to coordinate the PGC-1α -induced eNOS expression. In conclusion, endothelial PGC-1α expression protects from vascular dysfunction by promoting NO• bioactivity through ERRα induced expression of eNOS. PMID:27910955

Protein tyrosine phosphatase 1B (PTP1B) is dispensable for IgE-mediated cutaneous reaction in vivo.

PubMed

Yang, Ting; Xie, Zhongping; Li, Hua; Yue, Lei; Pang, Zheng; MacNeil, Adam J; Tremblay, Michel L; Tang, Jin-Tian; Lin, Tong-Jun

2016-01-01

Mast cells play a critical role in allergic reactions. The cross-linking of FcεRI-bound IgE with multivalent antigen initiates a cascade of signaling events leading to mast cell activation. It has been well-recognized that cross linking of FcεRI mediates tyrosine phosphorylation. However, the mechanism involved in tyrosine dephosphorylation in mast cells is less clear. Here we demonstrated that protein tyrosine phosphatase 1B (PTP1B)-deficient mast cells showed increased IgE-mediated phosphorylation of the signal transducer and activator of transcription 5 (STAT5) and enhanced production of CCL9 (MIP-1γ) and IL-6 in IgE-mediated mast cells activation in vitro. However, IgE-mediated calcium mobilization, β-hexaosaminidase release (degranulation), and phosphorylation of IκB and MAP kinases were not affected by PTP1B deficiency. Furthermore, PTP1B deficient mice showed normal IgE-dependent passive cutaneous anaphylaxis and late phase cutaneous reactions in vivo. Thus, PTP1B specifically regulates IgE-mediated STAT5 pathway, but is redundant in influencing mast cell function in vivo. Copyright © 2016 Elsevier Inc. All rights reserved.

Aberrant signature methylome by DNMT1 hot spot mutation in hereditary sensory and autonomic neuropathy 1E.

PubMed

Sun, Zhifu; Wu, Yanhong; Ordog, Tamas; Baheti, Saurabh; Nie, Jinfu; Duan, Xiaohui; Hojo, Kaori; Kocher, Jean-Pierre; Dyck, Peter J; Klein, Christopher J

2014-08-01

DNA methyltransferase 1 (DNMT1) is essential for DNA methylation, gene regulation and chromatin stability. We previously discovered DNMT1 mutations cause hereditary sensory and autonomic neuropathy type 1 with dementia and hearing loss (HSAN1E; OMIM 614116). HSAN1E is the first adult-onset neurodegenerative disorder caused by a defect in a methyltransferase gene. HSAN1E patients appear clinically normal until young adulthood, then begin developing the characteristic symptoms involving central and peripheral nervous systems. Some HSAN1E patients also develop narcolepsy and it has recently been suggested that HSAN1E is allelic to autosomal dominant cerebellar ataxia, deafness, with narcolepsy (ADCA-DN; OMIM 604121), which is also caused by mutations in DNMT1. A hotspot mutation Y495C within the targeting sequence domain of DNMT1 has been identified among HSAN1E patients. The mutant DNMT1 protein shows premature degradation and reduced DNA methyltransferase activity. Herein, we investigate genome-wide DNA methylation at single-base resolution through whole-genome bisulfite sequencing of germline DNA in 3 pairs of HSAN1E patients and their gender- and age-matched siblings. Over 1 billion 75-bp single-end reads were generated for each sample. In the 3 affected siblings, overall methylation loss was consistently found in all chromosomes with X and 18 being most affected. Paired sample analysis identified 564,218 differentially methylated CpG sites (DMCs; P<0.05), of which 300 134 were intergenic and 264 084 genic CpGs. Hypomethylation was predominant in both genic and intergenic regions, including promoters, exons, most CpG islands, L1, L2, Alu, and satellite repeats and simple repeat sequences. In some CpG islands, hypermethylated CpGs outnumbered hypomethylated CpGs. In 201 imprinted genes, there were more DMCs than in non-imprinted genes and most were hypomethylated. Differentially methylated region (DMR) analysis identified 5649 hypomethylated and 1872

Aberrant signature methylome by DNMT1 hot spot mutation in hereditary sensory and autonomic neuropathy 1E

PubMed Central

Sun, Zhifu; Wu, Yanhong; Ordog, Tamas; Baheti, Saurabh; Nie, Jinfu; Duan, Xiaohui; Hojo, Kaori; Kocher, Jean-Pierre; Dyck, Peter J; Klein, Christopher J

2014-01-01

DNA methyltransferase 1 (DNMT1) is essential for DNA methylation, gene regulation and chromatin stability. We previously discovered DNMT1 mutations cause hereditary sensory and autonomic neuropathy type 1 with dementia and hearing loss (HSAN1E; OMIM 614116). HSAN1E is the first adult-onset neurodegenerative disorder caused by a defect in a methyltransferase gene. HSAN1E patients appear clinically normal until young adulthood, then begin developing the characteristic symptoms involving central and peripheral nervous systems. Some HSAN1E patients also develop narcolepsy and it has recently been suggested that HSAN1E is allelic to autosomal dominant cerebellar ataxia, deafness, with narcolepsy (ADCA-DN; OMIM 604121), which is also caused by mutations in DNMT1. A hotspot mutation Y495C within the targeting sequence domain of DNMT1 has been identified among HSAN1E patients. The mutant DNMT1 protein shows premature degradation and reduced DNA methyltransferase activity. Herein, we investigate genome-wide DNA methylation at single-base resolution through whole-genome bisulfite sequencing of germline DNA in 3 pairs of HSAN1E patients and their gender- and age-matched siblings. Over 1 billion 75-bp single-end reads were generated for each sample. In the 3 affected siblings, overall methylation loss was consistently found in all chromosomes with X and 18 being most affected. Paired sample analysis identified 564,218 differentially methylated CpG sites (DMCs; P < 0.05), of which 300 134 were intergenic and 264 084 genic CpGs. Hypomethylation was predominant in both genic and intergenic regions, including promoters, exons, most CpG islands, L1, L2, Alu, and satellite repeats and simple repeat sequences. In some CpG islands, hypermethylated CpGs outnumbered hypomethylated CpGs. In 201 imprinted genes, there were more DMCs than in non-imprinted genes and most were hypomethylated. Differentially methylated region (DMR) analysis identified 5649 hypomethylated and 1872

Proteomic analysis of trichloroethylene-induced alterations in expression, distribution, and interactions of SET/TAF-Iα and two SET/TAF-Iα-binding proteins, eEF1A1 and eEF1A2, in hepatic L-02 cells.

PubMed

Hong, Wen-Xu; Yang, Liang; Chen, Moutong; Yang, Xifei; Ren, Xiaohu; Fang, Shisong; Ye, Jinbo; Huang, Haiyan; Peng, Chaoqiong; Zhou, Li; Huang, Xinfeng; Yang, Fan; Wu, Desheng; Zhuang, Zhixiong; Liu, Jianjun

2012-09-01

Emerging evidence indicates that trichloroethylene (TCE) exposure causes severe hepatotoxicity. However, the mechanisms of TCE hepatotoxicity remain unclear. Recently, we reported that TCE exposure up-regulated the expression of the oncoprotein SET/TAF-Iα and SET knockdown attenuated TCE-induced cytotoxicity in hepatic L-02 cells. To decipher the function of SET/TAF-Iα and its contributions to TCE-induced hepatotoxicity, we employed a proteomic analysis of SET/TAF-Iα with tandem affinity purification to identify SET/TAF-Iα-binding proteins. We identified 42 novel Gene Ontology co-annotated SET/TAF-Iα-binding proteins. The identifications of two of these proteins (eEF1A1, elongation factor 1-alpha 1; eEF1A2, elongation factor 1-alpha 2) were confirmed by Western blot analysis and co-immunoprecipitation (Co-IP). Furthermore, we analyzed the effects of TCE on the expression, distribution and interactions of eEF1A1, eEF1A2 and SET in L-02 cells. Western blot analysis reveals a significant up-regulation of eEF1A1, eEF1A2 and two isoforms of SET, and immunocytochemical analysis reveals that eEF1A1 and SET is redistributed by TCE. SET is redistributed from the nucleus to the cytoplasm, while eFE1A1 is translocated from the cytoplasm to the nucleus. Moreover, we find by Co-IP that TCE exposure significantly increases the interaction of SET with eEF1A2. Our data not only provide insights into the physiological functions of SET/TAF-Iα and complement the SET interaction networks, but also demonstrate that TCE exposure induces alterations in the expression, distribution and interactions of SET and its binding partners. These alterations may constitute the mechanisms of TCE cytotoxicity. Copyright © 2012 Elsevier Inc. All rights reserved.

Vaccination with Eimeria tenella elongation factor-1α recombinant protein induces protective immunity against E. tenella and E. maxima infections.

PubMed

Lin, Rui-Qing; Lillehoj, Hyun S; Lee, Seung Kyoo; Oh, Sungtaek; Panebra, Alfredo; Lillehoj, Erik P

2017-08-30

Avian coccidiosis is caused by multiple species of the apicomplexan protozoan, Eimeria, and is one of the most economically devastating enteric diseases for the poultry industry worldwide. Host immunity to Eimeria infection, however, is relatively species-specific. The ability to immunize chickens against different species of Eimeria using a single vaccine will have a major beneficial impact on commercial poultry production. In this paper, we describe the molecular cloning, purification, and vaccination efficacy of a novel Eimeria vaccine candidate, elongation factor-1α (EF-1α). One day-old broiler chickens were given two subcutaneous immunizations one week apart with E. coli-expressed E. tenella recombinant (r)EF-1α protein and evaluated for protection against challenge infection with E. tenella or E. maxima. rEF-1α-vaccinated chickens exhibited increased body weight gains, decreased fecal oocyst output, and greater serum anti-EF-1α antibody levels following challenge infection with either E. tenella or E. maxima compared with unimmunized controls. Vaccination with EF-1α may represent a new approach to inducing cross-protective immunity against avian coccidiosis in the field. Published by Elsevier B.V.

E2F1 transcription is induced by genotoxic stress through ATM/ATR activation.

PubMed

Carcagno, Abel L; Ogara, María F; Sonzogni, Silvina V; Marazita, Mariela C; Sirkin, Pablo F; Ceruti, Julieta M; Cánepa, Eduardo T

2009-05-01

E2F1, a member of the E2F family of transcription factors, plays a critical role in controlling both cell cycle progression and apoptotic cell death in response to DNA damage and oncogene activation. Following genotoxic stresses, E2F1 protein is stabilized by phosphorylation and acetylation driven to its accumulation. The aim of the present work was to examine whether the increase in E2F1 protein levels observed after DNA damage is only a reflection of an increase in E2F1 protein stability or is also the consequence of enhanced transcription of the E2F1 gene. The data presented here demonstrates that UV light and other genotoxics induce the transcription of E2F1 gene in an ATM/ATR dependent manner, which results in increasing E2F1 mRNA and protein levels. After genotoxic stress, transcription of cyclin E, an E2F1 target gene, was significantly induced. This induction was the result of two well-differentiated effects, one of them dependent on de novo protein synthesis and the other on the protein stabilization. Our results strongly support a transcriptional effect of DNA damaging agents on E2F1 expression. The results presented herein uncover a new mechanism involving E2F1 in response to genotoxic stress.

Chimeras of Bet v 1 and Api g 1 reveal heterogeneous IgE responses in patients with birch pollen allergy

PubMed Central

Gepp, Barbara; Lengger, Nina; Bublin, Merima; Hemmer, Wolfgang; Breiteneder, Heimo; Radauer, Christian

2014-01-01

Background Characterization of IgE-binding epitopes of allergens and determination of their patient-specific relevance is crucial for the diagnosis and treatment of allergy. Objective We sought to assess the contribution of specific surface areas of the major birch pollen allergen Bet v 1.0101 to binding IgE of individual patients. Methods Four distinct areas of Bet v 1 representing in total 81% of its surface were grafted onto the scaffold of its homolog, Api g 1.0101, to yield the chimeras Api-Bet-1 to Api-Bet-4. The chimeras were expressed in Escherichia coli and purified. IgE binding of 64 sera from Bet v 1–sensitized subjects with birch pollen allergy was determined by using direct ELISA. Specificity was assessed by means of inhibition ELISA. Results rApi g 1.0101, Api-Bet-1, Api-Bet-2, Api-Bet-3, and Api-Bet-4 bound IgE from 44%, 89%, 80%, 78%, and 48% of the patients, respectively. By comparing the amount of IgE binding to the chimeras and to rApi g 1.0101, 81%, 70%, 75%, and 45% of the patients showed significantly enhanced IgE binding to Api-Bet-1, Api-Bet-2, Api-Bet-3, and Api-Bet-4, respectively. The minority (8%) of the sera revealed enhanced IgE binding exclusively to a single chimera, whereas 31% showed increased IgE binding to all 4 chimeras compared with rApi g 1.0101. The chimeras inhibited up to 70% of IgE binding to rBet v 1.0101, confirming the specific IgE recognition of the grafted regions. Conclusion The Bet v 1–specific IgE response is polyclonal, and epitopes are spread across the entire Bet v 1 surface. Furthermore, the IgE recognition profile of Bet v 1 is highly patient specific. PMID:24529686

Chimeras of Bet v 1 and Api g 1 reveal heterogeneous IgE responses in patients with birch pollen allergy.

PubMed

Gepp, Barbara; Lengger, Nina; Bublin, Merima; Hemmer, Wolfgang; Breiteneder, Heimo; Radauer, Christian

2014-07-01

Characterization of IgE-binding epitopes of allergens and determination of their patient-specific relevance is crucial for the diagnosis and treatment of allergy. We sought to assess the contribution of specific surface areas of the major birch pollen allergen Bet v 1.0101 to binding IgE of individual patients. Four distinct areas of Bet v 1 representing in total 81% of its surface were grafted onto the scaffold of its homolog, Api g 1.0101, to yield the chimeras Api-Bet-1 to Api-Bet-4. The chimeras were expressed in Escherichia coli and purified. IgE binding of 64 sera from Bet v 1-sensitized subjects with birch pollen allergy was determined by using direct ELISA. Specificity was assessed by means of inhibition ELISA. rApi g 1.0101, Api-Bet-1, Api-Bet-2, Api-Bet-3, and Api-Bet-4 bound IgE from 44%, 89%, 80%, 78%, and 48% of the patients, respectively. By comparing the amount of IgE binding to the chimeras and to rApi g 1.0101, 81%, 70%, 75%, and 45% of the patients showed significantly enhanced IgE binding to Api-Bet-1, Api-Bet-2, Api-Bet-3, and Api-Bet-4, respectively. The minority (8%) of the sera revealed enhanced IgE binding exclusively to a single chimera, whereas 31% showed increased IgE binding to all 4 chimeras compared with rApi g 1.0101. The chimeras inhibited up to 70% of IgE binding to rBet v 1.0101, confirming the specific IgE recognition of the grafted regions. The Bet v 1-specific IgE response is polyclonal, and epitopes are spread across the entire Bet v 1 surface. Furthermore, the IgE recognition profile of Bet v 1 is highly patient specific. Copyright © 2014 The Authors. Published by Mosby, Inc. All rights reserved.

IgG1 memory B cells keep the memory of IgE responses.

PubMed

He, Jin-Shu; Subramaniam, Sharrada; Narang, Vipin; Srinivasan, Kandhadayar; Saunders, Sean P; Carbajo, Daniel; Wen-Shan, Tsao; Hidayah Hamadee, Nur; Lum, Josephine; Lee, Andrea; Chen, Jinmiao; Poidinger, Michael; Zolezzi, Francesca; Lafaille, Juan J; Curotto de Lafaille, Maria A

2017-09-21

The unique differentiation of IgE cells suggests unconventional mechanisms of IgE memory. IgE germinal centre cells are transient, most IgE cells are plasma cells, and high affinity IgE is produced by the switching of IgG1 cells to IgE. Here we investigate the function of subsets of IgG1 memory B cells in IgE production and find that two subsets of IgG1 memory B cells, CD80 + CD73 + and CD80 - CD73 - , contribute distinctively to the repertoires of high affinity pathogenic IgE and low affinity non-pathogenic IgE. Furthermore, repertoire analysis indicates that high affinity IgE and IgG1 plasma cells differentiate from rare CD80 + CD73 + high affinity memory clones without undergoing further mutagenesis. By identifying the cellular origin of high affinity IgE and the clonal selection of high affinity memory B cells into the plasma cell fate, our findings provide fundamental insights into the pathogenesis of allergies, and on the mechanisms of antibody production in memory B cell responses.IgE is an important mediator of protective immunity as well as allergic reaction, but how high affinity IgE antibodies are produced in memory responses is not clear. Here the authors show that IgE can be generated via class-switch recombination in IgG1 memory B cells without additional somatic hypermutation.

MKS1 regulates ciliary INPP5E levels in Joubert syndrome

PubMed Central

Slaats, Gisela G.; Isabella, Christine R.; Kroes, Hester Y.; Dempsey, Jennifer C.; Gremmels, Hendrik; Monroe, Glen R.; Phelps, Ian G.; Duran, Karen J.; Adkins, Jonathan; Kumar, Sairam A.; Knutzen, Dana M.; Knoers, Nine V.; Mendelsohn, Nancy J.; Neubauer, David; Mastroyianni, Sotiria D.; Vogt, Julie; Worgan, Lisa; Karp, Natalya; Bowdin, Sarah; Glass, Ian A.; Parisi, Melissa A.; Otto, Edgar A.; Johnson, Colin A.; Hildebrandt, Friedhelm; van Haaften, Gijs; Giles, Rachel H.; Doherty, Dan

2016-01-01

Background Joubert syndrome (JS) is a recessive ciliopathy characterized by a distinctive brain malformation “the molar tooth sign”. Mutations in >27 genes cause JS, and mutations in 12 of these genes also cause Meckel syndrome (MKS). The goals of this work are to describe the clinical features of MKS1-related JS and determine whether disease causing MKS1 mutations affect cellular phenotypes such as cilium number, length and protein content as potential mechanisms underlying JS. Methods We measured cilium number, length and protein content (ARL13B and INPP5E) by immunofluorescence in fibroblasts from individuals with MKS1-related JS and in a 3D spheroid rescue assay to test the effects of disease-related MKS1 mutations. Results We report MKS1 mutations (eight of them previously unreported) in nine individuals with JS. A minority of the individuals with MKS1-related JS have MKS features. In contrast to the truncating mutations associated with MKS, all of the individuals with MKS1-related JS carry ≥1 non-truncating mutation. Fibroblasts from individuals with MKS1-related JS make normal or fewer cilia than control fibroblasts, their cilia are more variable in length than controls, and show decreased ciliary ARL13B and INPP5E. Additionally, MKS1 mutant alleles have similar effects in 3D spheroids. Conclusions MKS1 functions in the transition zone at the base of the cilium to regulate ciliary INPP5E content, through an ARL13B-dependent mechanism. Mutations in INPP5E also cause JS, so our findings in patient fibroblasts support the notion that loss of INPP5E function, due to either mutation or mislocalization, is a key mechanism underlying JS, downstream of MKS1 and ARL13B. PMID:26490104

Anti-E1E2 antibodies do predict response to triple therapy in treatment-experienced Hepatitis C Virus-cirrhosis cases.

PubMed

Petit, Marie-Anne; Berthillon, Pascale; Pradat, Pierre; Arnaud, Clémence; Bordes, Isabelle; Virlogeux, Victor; Maynard, Marianne; Bailly, François; Zoulim, Fabien; Chemin, Isabelle; Trépo, Christian

2015-12-01

We previously showed that pre-treatment serum anti-E1E2 predicted hepatitis C virus (HCV) RNA viral kinetics (VKs) and treatment outcome in patients with chronic hepatitis C receiving pegylated interferon/ribavirin (Peg-IFN/RBV) double therapy. Here, we determined whether baseline anti-E1E2 was correlated with the on-treatment VK and could predict virological outcome in treatment-experienced HCV-infected cirrhotic patients receiving protease inhibitor-based triple therapy. Sera from 19 patients with HCV genotype 1 infection and compensated cirrhosis who failed to respond to a prior course of Peg-IFN/RBV were selected at time 0 before starting triple therapy with boceprevir or telaprevir. We assessed patients with sustained viral response 12 weeks after the end of triple therapy (SVR12) by analyzing VKs at weeks 4, 12, 24, 36, 48 (end of treatment) and 60. Patients baseline characteristics were similar to the well-defined CUPIC cohort (age, HCV subtype, baseline viremia, and treatment history). Among the 19 patients, 11 achieved an SVR12. Fifteen patients were positive for pre-treatment anti-E1E2 and all of them achieved SVR12. Moreover, anti-E1E2 and SVR12 correlated with prior response to IFN/RBV therapy (relapse, partial or null response). Baseline anti-E1E2 could be considered as a new biomarker to predict SVR12 after triple therapy in this most difficult-to-treat population. These results warrant further validation on larger cohorts including patients receiving highly effective direct-acting antivirals to explore whether this test could help in better defining treatment duration for these very costly molecules. Copyright © 2015 Elsevier Masson SAS. All rights reserved.

Highly efficient focus formation by Rous sarcoma virus on adenovirus type 12 E1A-transformed rat 3Y1 cells.

PubMed Central

Shiroki, K; Hamaguchi, M; Kawai, S

1992-01-01

When rat 3Y1 cells were infected with Rous sarcoma virus (RSV) variant SR-RSV-D(H), many 3Y1 cells acquired a stable provirus but only few of them formed transformed foci. In contrast, 12E1AY cells (3Y1 cells expressing the adenovirus type 12 [Ad12] E1A protein) formed transformed foci upon RSV infection with the same high frequency as did chicken embryo fibroblast cells. This enhancement of focus-forming efficiency was specifically observed in 3Y1 cells expressing Ad12 E1A protein but was not observed in 3Y1 cells expressing simian virus 40 T, c-myc, p53, c-fos, or v-fos protein. This enhancement was not evident in 5E1AY cells (3Y1 cells expressing the Ad5 E1A protein). Judging from the experiment using Ad12-Ad5 hybird E1A DNAs, the N-terminal half of the Ad12 E1A protein was responsible for this enhancement. The promoter activity of the RSV long terminal repeat measured by pLTR-CAT did not correlate to the efficiency of focus formation by RSV in these 3Y1 cells. Moreover, RSV containing the neo gene instead of the src gene produced G418-resistant cells equally efficiently among 3Y1, E1AY, and chicken embryo fibroblast cells. These results suggest that the enhancement of focus formation by RSV is not due to the increased expression of the src gene by the E1A protein. src mRNA and src protein were lower in RSV-transformed E1AY (RSVE1AY) cells than in RSV-transformed 3Y1 (RSV3Y1) cells. The phosphotyrosine-containing proteins were also less abundant in RSVE1AY cells than in RSV3Y1 cells, suggesting that E1AY cells require a lower threshold dose of p60v-src for transformation than do 3Y1 cells. E1AY cells were found to be more sensitive to lysis by detergents. The results suggest that the enhancement is due to changes in membrane structures in E1AY cells. Images PMID:1310757

Structural model of the hUbA1-UbcH10 quaternary complex: in silico and experimental analysis of the protein-protein interactions between E1, E2 and ubiquitin.

PubMed

Correale, Stefania; de Paola, Ivan; Morgillo, Carmine Marco; Federico, Antonella; Zaccaro, Laura; Pallante, Pierlorenzo; Galeone, Aldo; Fusco, Alfredo; Pedone, Emilia; Luque, F Javier; Catalanotti, Bruno

2014-01-01

UbcH10 is a component of the Ubiquitin Conjugation Enzymes (Ubc; E2) involved in the ubiquitination cascade controlling the cell cycle progression, whereby ubiquitin, activated by E1, is transferred through E2 to the target protein with the involvement of E3 enzymes. In this work we propose the first three dimensional model of the tetrameric complex formed by the human UbA1 (E1), two ubiquitin molecules and UbcH10 (E2), leading to the transthiolation reaction. The 3D model was built up by using an experimentally guided incremental docking strategy that combined homology modeling, protein-protein docking and refinement by means of molecular dynamics simulations. The structural features of the in silico model allowed us to identify the regions that mediate the recognition between the interacting proteins, revealing the active role of the ubiquitin crosslinked to E1 in the complex formation. Finally, the role of these regions involved in the E1-E2 binding was validated by designing short peptides that specifically interfere with the binding of UbcH10, thus supporting the reliability of the proposed model and representing valuable scaffolds for the design of peptidomimetic compounds that can bind selectively to Ubcs and inhibit the ubiquitylation process in pathological disorders.

20 CFR 655.700 - What statutory provisions govern the employment of H-1B, H-1B1, and E-3 nonimmigrants and how do...

Code of Federal Regulations, 2012 CFR

2012-04-01

... employment of H-1B, H-1B1, and E-3 nonimmigrants and how do employers apply for H-1B, H-1B1, and E-3 visas... Requirements for Employers Seeking To Employ Nonimmigrants on H-1b Visas in Specialty Occupations and as Fashion Models, and Requirements for Employers Seeking To Employ Nonimmigrants on H-1b1 and E-3 Visas in...

20 CFR 655.700 - What statutory provisions govern the employment of H-1B, H-1B1, and E-3 nonimmigrants and how do...

Code of Federal Regulations, 2013 CFR

2013-04-01

... employment of H-1B, H-1B1, and E-3 nonimmigrants and how do employers apply for H-1B, H-1B1, and E-3 visas... Requirements for Employers Seeking To Employ Nonimmigrants on H-1b Visas in Specialty Occupations and as Fashion Models, and Requirements for Employers Seeking To Employ Nonimmigrants on H-1b1 and E-3 Visas in...

20 CFR 655.700 - What statutory provisions govern the employment of H-1B, H-1B1, and E-3 nonimmigrants and how do...

Code of Federal Regulations, 2014 CFR

2014-04-01

... employment of H-1B, H-1B1, and E-3 nonimmigrants and how do employers apply for H-1B, H-1B1, and E-3 visas... Requirements for Employers Seeking To Employ Nonimmigrants on H-1b Visas in Specialty Occupations and as Fashion Models, and Requirements for Employers Seeking To Employ Nonimmigrants on H-1b1 and E-3 Visas in...

20 CFR 655.700 - What statutory provisions govern the employment of H-1B, H-1B1, and E-3 nonimmigrants and how do...

Code of Federal Regulations, 2011 CFR

2011-04-01

... employment of H-1B, H-1B1, and E-3 nonimmigrants and how do employers apply for H-1B, H-1B1, and E-3 visas... Requirements for Employers Seeking To Employ Nonimmigrants on H-1b Visas in Specialty Occupations and as Fashion Models, and Requirements for Employers Seeking To Employ Nonimmigrants on H-1b1 and E-3 Visas in...

20 CFR 655.700 - What statutory provisions govern the employment of H-1B, H-1B1, and E-3 nonimmigrants and how do...

Code of Federal Regulations, 2010 CFR

2010-04-01

... employment of H-1B, H-1B1, and E-3 nonimmigrants and how do employers apply for H-1B, H-1B1, and E-3 visas... Requirements for Employers Seeking To Employ Nonimmigrants on H-1b Visas in Specialty Occupations and as Fashion Models, and Requirements for Employers Seeking To Employ Nonimmigrants on H-1b1 and E-3 Visas in...

ITOS D AND E system design report, volume 1

NASA Technical Reports Server (NTRS)

1970-01-01

The configuration and functions of the ITOS D and E system are described. The system will expand the operational capability of the basic TIROS M/ITOS system. The ITOS D and E mission will utilize the capabilities of the two-stage DSV 3N-6 Delta launch vehicle to place the ITOS D and E spacecraft into a circular, near-polar, sun synchronous orbit at 790 nautical miles altitude. The ITOS D and E will provide the following primary data: (1) visible daytime observations of cloud cover, (2) daytime and nighttime observations of cloud cover as detected from radiance in infrared spectrum, and (3) vertical temperature profile of the atmosphere on a global basis for data processing. In addition, the ITOS D and E system will provide secondary data comprising solar proton density measurements obtained throughout the orbit.

Resolvin E1-induced intestinal alkaline phosphatase promotes resolution of inflammation through LPS detoxification.

PubMed

Campbell, Eric L; MacManus, Christopher F; Kominsky, Douglas J; Keely, Simon; Glover, Louise E; Bowers, Brittelle E; Scully, Melanie; Bruyninckx, Walter J; Colgan, Sean P

2010-08-10

Resolvin-E1 (RvE1) has been demonstrated to promote inflammatory resolution in numerous disease models. Given the importance of epithelial cells to coordination of mucosal inflammation, we hypothesized that RvE1 elicits an epithelial resolution signature. Initial studies revealed that the RvE1-receptor (ChemR23) is expressed on intestinal epithelial cells (IECs) and that microarray profiling of cells exposed to RvE1 revealed regulation of inflammatory response gene expression. Notably, RvE1 induced intestinal alkaline phosphatase (ALPI) expression and significantly enhanced epithelial ALPI enzyme activity. One role recently attributed to ALPI is the detoxification of bacterial LPS. In our studies, RvE1-exposed epithelia detoxified LPS (assessed by attenuation of NF-kappaB signaling). Furthermore, in epithelial-bacterial interaction assays, we determined that ALPI retarded the growth of Escherichia coli. To define these features in vivo, we used a murine dextran sulfate sodium (DSS) model of colitis. Compared with vehicle controls, administration of RvE1 resulted in significant improvement of disease activity indices (e.g., body weight, colon length) concomitant with increased ALPI expression in the intestinal epithelium. Moreover, inhibition of ALPI activity resulted in increased severity of colitis in DSS-treated animals and partially abrogated the protective influence of RvE1. Together, these data implicate a previously unappreciated role for ALPI in RvE1-mediated inflammatory resolution.

The anti-tumor activity of E1A and its implications in cancer therapy.

PubMed

Chang, Yi-Wen; Hung, Mien-Chie; Su, Jen-Liang

2014-06-01

The adenovirus type 5 E1A protein (E1A) plays a critical role in anti-cancer gene therapy and has been tested in clinical trials. The expression of E1A significantly reduces tumorigenesis, promotes cell death, and inhibits cancer cell mobility. Chemosensitization is one of the anti-tumor effects of E1A, increasing in vitro and in vivo sensitization of anti-cancer drugs, including cisplatin, gemcitabine, etoposide, doxorubicin, paclitaxel, and tumor necrosis factor-related apoptosis-inducing ligand and histone deacetylase inhibitors in different types of cancer cells. E1A also demonstrates anti-metastasis activity through various molecular mechanisms such as the repression of protease expression, suppression of HER2/neu and downregulation of microRNA (miR-520h). Moreover, E1A has been reported to reprogram transcription in tumor cells and stabilize tumor suppressors such as PP2A/C, p21 and p53. Because E1A plays a potentially significant role in anti-tumor therapy, there exists an urgent need to study the anti-cancer activities of E1A. This paper presents a review of our current understanding of the tumor-suppressive functions and molecular regulation of E1A, as well as the potential clinical applications of E1A.

Interaction of Adenovirus E1A with the HHV8 Promoter of Latent Genes: E1A Proteins are Able to Activate the HHV-8 LANAp in MV3 Reporter Cells

PubMed Central

Koehler-Hansner, Karin; Flore, Ornella; Opalka, Bertram; Hengge, Ulrich R

2008-01-01

Human herpesvirus 8 (HHV-8) is associated with Kaposi’s sarcoma, body cavity-based lymphoma, and Castleman’s disease. Adenoviral (Ad) E1A proteins regulate the activity of cellular and viral promoters/enhancers and transcription factors and can suppress tumorigenicity of human cancers. As (i) HHV-8 and Ad may co-exist in immunocompromised patients and (ii) E1A might be considered as therapeutic transgene for HHV-8-associated neoplasms we investigated whether the promoter of the latency-associated nuclear antigen (LANAp) controlling expression of vCyclin, vFLIP, and LANA proteins required for latent type infection is regulated by E1A. Transfection experiments in MV3 melanoma cells revealed activation of the LANAp by Ad5 E1A constructs containing an intact N terminus (aa 1-119). In particular, an Ad12 E1A mutant, Spm2, lacking six consecutive alanine residues in the “spacer” region activated the HHV-8 promoter about 15-fold compared to vector controls. In summary, we report the activation of the LANAp by E1A as a novel interaction of E1A with a viral promoter. These data may have relevance for the management of viral infections in immunocompromised patients. A role for E1A as a therapeutic in this context remains to be defined. PMID:19440465

Evaluation and Validation (E&V) Team Public Report. Volume 1.

DTIC Science & Technology

1984-11-30

Components. .......... K-109 *Table K-6. Possible Approaches to Integrating Methodology Components Across Life Cycle Phases. .........K-109 SECTION I...provide a detailed and organized approach to the development of technology which will be used as a basis for the E&V of APSEs. The E&V Plan which is...A-4 1.2 Background ....... .................. A-6 2. SCOPE ...... ....................... A-8 3. E&V TECHNICAL APPROACH

C 1 s ionization in C sub 2 H sub 2 studied by asymmetric ( e ,2 e ) experiments

DOE Office of Scientific and Technical Information (OSTI.GOV)

Avaldi, L.; Camilloni, R.; Stefani, G.

1990-01-01

The dynamics of core ionization by electron impact is investigated through the measurement of the triply differential cross section of the C {sigma}1{ital s} orbital in the molecule C{sub 2}H{sub 2}. The ({ital e},2{ital e}) experiments have been performed under asymmetric conditions and at small scattering angles, with a scattered electron energy of 1500 eV and low energies of the ejected electrons (9.6 and 41.0 eV). The measured angular distributions are characterized by large-size recoil lobes, breaking of the symmetry around the momentum-transfer direction, and unusual deviations of the maxima of the recoil peaks towards smaller deflection angles. In themore » ({ital e},2{ital e}) energy spectrum a shift is observed in the position of the C {sigma}1{ital s} peak with respect to the expected value as measured by x-ray photoelectron spectroscopy. The amplitude of the shift amounts to 0.46{plus minus}0.23 eV at 9.6 eV excess energy, and it is too large to be explained only in terms of postcollision interactions.« less

Virasoro constraints for D 2n + 1 -, E 6 -, E 7 -, E 8 -type minimal models coupled to 2D gravity

NASA Astrophysics Data System (ADS)

Yen, Tim

1990-12-01

We find Virasoro constraints for D 2 n + 1 -, E 6 -, E 7 -, E 8 -type models analogous to the recently discovered Virasoro constraints for A n-type models by Fukuma et al., and Dijkgraaf et al. We verify that the proposed Virasoro constraints give operator scaling dimensions identical to those found by Kostov. We check that these Virasoro constraints and, more generally, W-algebra constraints can be used to express correlation functions with non-primary operator in terms of correlation functions of primary operators only.

Decoy Wnt receptor (sLRP6E1E2)-expressing adenovirus induces anti-fibrotic effect via inhibition of Wnt and TGF-β signaling.

PubMed

Lee, Won Jai; Lee, Jung-Sun; Ahn, Hyo Min; Na, Youjin; Yang, Chae Eun; Lee, Ju Hee; Hong, JinWoo; Yun, Chae-Ok

2017-11-08

Aberrant activation of the canonical Wingless type (Wnt) signaling pathway plays a key role in the development of hypertrophic scars and keloids, and this aberrant activation of Wnt pathway can be a potential target for the development of novel anti-fibrotic agents. In this study, we evaluated the anti-fibrotic potential of a soluble Wnt decoy receptor (sLRP6E1E2)-expressing non-replicating adenovirus (Ad; dE1-k35/sLRP6E1E2) on human dermal fibroblasts (HDFs), keloid fibroblasts (KFs), and keloid tissue explants. Higher Wnt3a and β-catenin expression was observed in the keloid region compared to the adjacent normal tissues. The activity of β-catenin and mRNA expression of type-I and -III collagen were significantly decreased following treatment with dE1-k35/sLRP6E1E2 in HDFs and KFs. The expression of LRP6, β-catenin, phosphorylated glycogen synthase kinase 3 beta, Smad 2/3 complex, and TGF-β1 were decreased in Wnt3a- or TGF-β1-activated HDFs, following administration of dE1-k35/sLRP6E1E2. Moreover, dE1-k35/sLRP6E1E2 markedly inhibited nuclear translocation of both β-catenin and Smad 2/3 complex. The expression levels of type-I and -III collagen, fibronectin, and elastin were also significantly reduced in keloid tissue explants after treatment with dE1-k35/sLRP6E1E2. These results indicate that Wnt decoy receptor-expressing Ad can degrade extracellular matrix in HDFs, KFs, and primary keloid tissue explants, and thus it may be beneficial for treatment of keloids.

Lettuce and rhizosphere microbiome responses to growth promoting Pseudomonas species under field conditions.

PubMed

Cipriano, Matheus A P; Lupatini, Manoeli; Lopes-Santos, Lucilene; da Silva, Márcio J; Roesch, Luiz F W; Destéfano, Suzete A L; Freitas, Sueli S; Kuramae, Eiko E

2016-12-01

Plant growth promoting rhizobacteria are well described and recommended for several crops worldwide. However, one of the most common problems in research into them is the difficulty in obtaining reproducible results. Furthermore, few studies have evaluated plant growth promotion and soil microbial community composition resulting from bacterial inoculation under field conditions. Here we evaluated the effect of 54 Pseudomonas strains on lettuce (Lactuca sativa) growth. The 12 most promising strains were phylogenetically and physiologically characterized for plant growth-promoting traits, including phosphate solubilization, hormone production and antagonism to pathogen compounds, and their effect on plant growth under farm field conditions. Additionally, the impact of beneficial strains on the rhizospheric bacterial community was evaluated for inoculated plants. The strains IAC-RBcr4 and IAC-RBru1, with different plant growth promoting traits, improved lettuce plant biomass yields up to 30%. These two strains also impacted rhizosphere bacterial groups including Isosphaera and Pirellula (phylum Planctomycetes) and Acidothermus, Pseudolabrys and Singusphaera (phylum Actinobacteria). This is the first study to demonstrate consistent results for the effects of Pseudomonas strains on lettuce growth promotion for seedlings and plants grown under tropical field conditions. © FEMS 2016. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

Molecular Architecture of the 40S⋅eIF1⋅eIF3 Translation Initiation Complex

PubMed Central

Erzberger, Jan P.; Stengel, Florian; Pellarin, Riccardo; Zhang, Suyang; Schaefer, Tanja; Aylett, Christopher H.S.; Cimermančič, Peter; Boehringer, Daniel; Sali, Andrej; Aebersold, Ruedi; Ban, Nenad

2014-01-01

Summary Eukaryotic translation initiation requires the recruitment of the large, multiprotein eIF3 complex to the 40S ribosomal subunit. We present X-ray structures of all major components of the minimal, six-subunit Saccharomyces cerevisiae eIF3 core. These structures, together with electron microscopy reconstructions, cross-linking coupled to mass spectrometry, and integrative structure modeling, allowed us to position and orient all eIF3 components on the 40S⋅eIF1 complex, revealing an extended, modular arrangement of eIF3 subunits. Yeast eIF3 engages 40S in a clamp-like manner, fully encircling 40S to position key initiation factors on opposite ends of the mRNA channel, providing a platform for the recruitment, assembly, and regulation of the translation initiation machinery. The structures of eIF3 components reported here also have implications for understanding the architecture of the mammalian 43S preinitiation complex and the complex of eIF3, 40S, and the hepatitis C internal ribosomal entry site RNA. PMID:25171412

E2F1-mediated human POMC expression in ectopic Cushing's syndrome.

PubMed

Araki, Takako; Liu, Ning-Ai; Tone, Yukiko; Cuevas-Ramos, Daniel; Heltsley, Roy; Tone, Masahide; Melmed, Shlomo

2016-11-01

Cushing's syndrome is caused by excessive adrenocorticotropic hormone (ACTH) secretion derived from pituitary corticotroph tumors (Cushing disease) or from non-pituitary tumors (ectopic Cushing's syndrome). Hypercortisolemic features of ectopic Cushing's syndrome are severe, and no definitive treatment for paraneoplastic ACTH excess is available. We aimed to identify subcellular therapeutic targets by elucidating transcriptional regulation of the human ACTH precursor POMC (proopiomelanocortin) and ACTH production in non-pituitary tumor cells and in cell lines derived from patients with ectopic Cushing's syndrome. We show that ectopic hPOMC transcription proceeds independently of pituitary-specific Tpit/Pitx1 and demonstrate a novel E2F1-mediated transcriptional mechanism regulating hPOMC We identify an E2F1 cluster binding to the proximal hPOMC promoter region (-42 to +68), with DNA-binding activity determined by the phosphorylation at Ser-337. hPOMC mRNA expression in cancer cells was upregulated (up to 40-fold) by the co-expression of E2F1 and its heterodimer partner DP1. Direct and indirect inhibitors of E2F1 activity suppressed hPOMC gene expression and ACTH by modifying E2F1 DNA-binding activity in ectopic Cushing's cell lines and primary tumor cells, and also suppressed paraneoplastic ACTH and cortisol levels in xenografted mice. E2F1-mediated hPOMC transcription is a potential target for suppressing ACTH production in ectopic Cushing's syndrome. © 2016 Society for Endocrinology.

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

2016-06-22

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.