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Sample records for programming enzyme specificity

  1. Motif-directed redesign of enzyme specificity.

    PubMed

    Borgo, Benjamin; Havranek, James J

    2014-03-01

    Computational protein design relies on several approximations, including the use of fixed backbones and rotamers, to reduce protein design to a computationally tractable problem. However, allowing backbone and off-rotamer flexibility leads to more accurate designs and greater conformational diversity. Exhaustive sampling of this additional conformational space is challenging, and often impossible. Here, we report a computational method that utilizes a preselected library of native interactions to direct backbone flexibility to accommodate placement of these functional contacts. Using these native interaction modules, termed motifs, improves the likelihood that the interaction can be realized, provided that suitable backbone perturbations can be identified. Furthermore, it allows a directed search of the conformational space, reducing the sampling needed to find low energy conformations. We implemented the motif-based design algorithm in Rosetta, and tested the efficacy of this method by redesigning the substrate specificity of methionine aminopeptidase. In summary, native enzymes have evolved to catalyze a wide range of chemical reactions with extraordinary specificity. Computational enzyme design seeks to generate novel chemical activities by altering the target substrates of these existing enzymes. We have implemented a novel approach to redesign the specificity of an enzyme and demonstrated its effectiveness on a model system.

  2. Functional Representation of Enzymes by Specific Peptides

    PubMed Central

    Kunik, Vered; Meroz, Yasmine; Solan, Zach; Sandbank, Ben; Weingart, Uri; Ruppin, Eytan; Horn, David

    2007-01-01

    Predicting the function of a protein from its sequence is a long-standing goal of bioinformatic research. While sequence similarity is the most popular tool used for this purpose, sequence motifs may also subserve this goal. Here we develop a motif-based method consisting of applying an unsupervised motif extraction algorithm (MEX) to all enzyme sequences, and filtering the results by the four-level classification hierarchy of the Enzyme Commission (EC). The resulting motifs serve as specific peptides (SPs), appearing on single branches of the EC. In contrast to previous motif-based methods, the new method does not require any preprocessing by multiple sequence alignment, nor does it rely on over-representation of motifs within EC branches. The SPs obtained comprise on average 8.4 ± 4.5 amino acids, and specify the functions of 93% of all enzymes, which is much higher than the coverage of 63% provided by ProSite motifs. The SP classification thus compares favorably with previous function annotation methods and successfully demonstrates an added value in extreme cases where sequence similarity fails. Interestingly, SPs cover most of the annotated active and binding site amino acids, and occur in active-site neighboring 3-D pockets in a highly statistically significant manner. The latter are assumed to have strong biological relevance to the activity of the enzyme. Further filtering of SPs by biological functional annotations results in reduced small subsets of SPs that possess very large enzyme coverage. Overall, SPs both form a very useful tool for enzyme functional classification and bear responsibility for the catalytic biological function carried out by enzymes. PMID:17722976

  3. Double clicking for site-specific coupling of multiple enzymes.

    PubMed

    Lim, Sung In; Cho, Jinhwan; Kwon, Inchan

    2015-09-14

    A method to site-specifically couple multiple enzymes is reported. The approach is based on the site-specific incorporation of a clickable non-natural amino acid into enzymes and two compatible click reactions. The multi-enzyme reaction system exhibited enhanced catalytic efficiency over the respective free enzymes.

  4. Monobody-mediated alteration of enzyme specificity.

    PubMed

    Tanaka, Shun-Ichi; Takahashi, Tetsuya; Koide, Akiko; Ishihara, Satoru; Koikeda, Satoshi; Koide, Shohei

    2015-10-01

    Current methods for engineering enzymes modify enzymes themselves and require a detailed mechanistic understanding or a high-throughput assay. Here, we describe a new approach where catalytic properties are modulated with synthetic binding proteins, termed monobodies, directed to an unmodified enzyme. Using the example of a β-galactosidase from Bacillus circulans, we efficiently identified monobodies that restricted its substrates for its transgalactosylation reaction and selectively enhanced the production of small oligosaccharide prebiotics.

  5. Enzyme

    MedlinePlus

    Enzymes are complex proteins that cause a specific chemical change in all parts of the body. For ... use them. Blood clotting is another example of enzymes at work. Enzymes are needed for all body ...

  6. Performance Specifications for Occupational Programs.

    ERIC Educational Resources Information Center

    Maryland State Dept. of Education, Baltimore. Div. of Career Technology and Adult Learning.

    This document lists and discusses the development of Maryland's performance specifications for occupational programs. The introduction explains the process used to develop performance standards and specifications for 10 career cluster majors that were identified by a task force of educators and employers as high-demand occupational areas in…

  7. Logic programming and metadata specifications

    NASA Technical Reports Server (NTRS)

    Lopez, Antonio M., Jr.; Saacks, Marguerite E.

    1992-01-01

    Artificial intelligence (AI) ideas and techniques are critical to the development of intelligent information systems that will be used to collect, manipulate, and retrieve the vast amounts of space data produced by 'Missions to Planet Earth.' Natural language processing, inference, and expert systems are at the core of this space application of AI. This paper presents logic programming as an AI tool that can support inference (the ability to draw conclusions from a set of complicated and interrelated facts). It reports on the use of logic programming in the study of metadata specifications for a small problem domain of airborne sensors, and the dataset characteristics and pointers that are needed for data access.

  8. O-GlcNAc processing enzymes: catalytic mechanisms, substrate specificity, and enzyme regulation.

    PubMed

    Vocadlo, David J

    2012-12-01

    The addition of N-acetylglucosamine (GlcNAc) O-linked to serine and threonine residues of proteins is known as O-GlcNAc. This post-translational modification is found within multicellular eukaryotes on hundreds of nuclear and cytoplasmic proteins. O-GlcNAc transferase (OGT) installs O-GlcNAc onto target proteins and O-GlcNAcase (OGA) removes O-GlcNAc. Their combined action makes O-GlcNAc reversible and serves to regulate cellular O-GlcNAc levels. Here I review select recent literature on the catalytic mechanism of these enzymes and studies on the molecular basis by which these enzymes identify and process their substrates. Molecular level understanding of how these enzymes work, and the basis for their specificity, should aid understanding how O-GlcNAc contributes to diverse cellular processes ranging from cellular signaling through to transcriptional regulation.

  9. Substrate specificity and enzyme recycling using chitosan immobilized laccase.

    PubMed

    Skoronski, Everton; Fernandes, Mylena; Magalhães, Maria de Lourdes Borba; da Silva, Gustavo Felippe; João, Jair Juarez; Soares, Carlos Henrique Lemos; Júnior, Agenor Fúrigo

    2014-10-17

    The immobilization of laccase (Aspergillus sp.) on chitosan by cross-linking and its application in bioconversion of phenolic compounds in batch reactors were studied. Investigation was performed using laccase immobilized via chemical cross-linking due to the higher enzymatic operational stability of this method as compared to immobilization via physical adsorption. To assess the influence of different substrate functional groups on the enzyme's catalytic efficiency, substrate specificity was investigated using chitosan-immobilized laccase and eighteen different phenol derivatives. It was observed that 4-nitrophenol was not oxidized, while 2,5-xylenol, 2,6-xylenol, 2,3,5-trimethylphenol, syringaldazine, 2,6-dimetoxyphenol and ethylphenol showed reaction yields up 90% at 40 °C. The kinetic of process, enzyme recyclability and operational stability were studied. In batch reactors, it was not possible to reuse the enzyme when it was applied to syringaldazne bioconversion. However, when the enzyme was applied to bioconversion of 2,6-DMP, the activity was stable for eight reaction batches.

  10. Enzyme Substrate Specificity Conferred by Distinct Conformational Pathways.

    PubMed

    Rago, Florencia; Saltzberg, Daniel; Allen, Karen N; Tolan, Dean R

    2015-11-04

    Substrate recognition is one of the hallmarks of enzyme catalysis. Enzyme conformational changes have been linked to selectivity between substrates with little direct evidence. Aldolase, a glycolytic enzyme, must distinguish between two physiologically important substrates, fructose 1-phosphate and fructose 1,6-bisphosphate, and provides an excellent model system for the study of this question. Previous work has shown that isozyme specific residues (ISRs) distant from the active site are responsible for kinetic distinction between these substrates. Notably, most of the ISRs reside in a cluster of five surface α-helices, and the carboxyl-terminal region (CTR), and cooperative interactions among these helices have been demonstrated. To test the hypothesis that conformational changes are at the root of these changes, single surface-cysteine variants were created with the cysteine located on helices of the cluster and CTR. This allowed for site-specific labeling with an environmentally sensitive fluorophore, and subsequent monitoring of conformational changes by fluorescence emission spectrophotometry. These labeled variants revealed different spectra in the presence of saturating amounts of each substrate, which suggested the occurrence of different conformations. Emission spectra collected at various substrate concentrations showed a concentration dependence of the fluorescence spectra, consistent with binding events. Lastly, stopped-flow fluorescence spectrophotometry showed that the rate of these fluorescence changes was on the same time-scale as catalysis, thus suggesting a link between the different fluorescence changes and events during catalysis. On the basis of these results, we propose that different conformational changes may be a common mechanism for dictating substrate specificity in other enzymes with multiple substrates.

  11. Site-specific DNA transesterification catalyzed by a restriction enzyme

    PubMed Central

    Sasnauskas, Giedrius; Connolly, Bernard A.; Halford, Stephen E.; Siksnys, Virginijus

    2007-01-01

    Most restriction endonucleases use Mg2+ to hydrolyze phosphodiester bonds at specific DNA sites. We show here that BfiI, a metal-independent restriction enzyme from the phospholipase D superfamily, catalyzes both DNA hydrolysis and transesterification reactions at its recognition site. In the presence of alcohols such as ethanol or glycerol, it attaches the alcohol covalently to the 5′ terminus of the cleaved DNA. Under certain conditions, the terminal 3′-OH of one DNA strand can attack the target phosphodiester bond in the other strand to create a DNA hairpin. Transesterification reactions on DNA with phosphorothioate linkages at the target bond proceed with retention of stereoconfiguration at the phosphorus, indicating, uniquely for a restriction enzyme, a two-step mechanism. We propose that BfiI first makes a covalent enzyme–DNA intermediate, and then it resolves it by a nucleophilic attack of water or an alcohol, to yield hydrolysis or transesterification products, respectively. PMID:17267608

  12. Structural studies of the Trypanosoma cruzi Old Yellow Enzyme: insights into enzyme dynamics and specificity.

    PubMed

    Murakami, Mário T; Rodrigues, Nathalia C; Gava, Lisandra M; Honorato, Rodrigo V; Canduri, Fernanda; Barbosa, Leandro R S; Oliva, Glaucius; Borges, Júlio C

    2013-12-31

    The flavoprotein old yellow enzyme of Trypanosoma cruzi (TcOYE) is an oxidoreductase that uses NAD(P)H as cofactor. This enzyme is clinically relevant due to its role in the action mechanism of some trypanocidal drugs used in the treatment of Chagas' disease by producing reactive oxygen species. In this work, the recombinant enzyme TcOYE was produced and collectively, X-ray crystallography, small angle X-ray scattering, analytical ultracentrifugation and molecular dynamics provided a detailed description of its structure, specificity and hydrodynamic behavior. The crystallographic structure at 1.27Å showed a classical (α/β)8 fold with the FMN prosthetic group buried at the positively-charged active-site cleft. In solution, TcOYE behaved as a globular monomer, but it exhibited a molecular envelope larger than that observed in the crystal structure, suggesting intrinsic protein flexibility. Moreover, the binding mode of β-lapachone, a trypanocidal agent, and other naphthoquinones was investigated by molecular docking and dynamics suggesting that their binding to TcOYE are stabilized mainly by interactions with the isoalloxazine ring from FMN and residues from the active-site pocket.

  13. Sensitivity and Specificity of Histoplasma Antigen Detection by Enzyme Immunoassay.

    PubMed

    Cunningham, Lauren; Cook, Audrey; Hanzlicek, Andrew; Harkin, Kenneth; Wheat, Joseph; Goad, Carla; Kirsch, Emily

    2015-01-01

    The objective of this study was to evaluate the sensitivity and specificity of an antigen enzyme immunoassay (EIA) on urine samples for the diagnosis of histoplasmosis in dogs. This retrospective medical records review included canine cases with urine samples submitted for Histoplasma EIA antigen assay between 2007 and 2011 from three veterinary institutions. Cases for which urine samples were submitted for Histoplasma antigen testing were reviewed and compared to the gold standard of finding Histoplasma organisms or an alternative diagnosis on cytology or histopathology. Sensitivity, specificity, negative predictive value, positive predictive value, and the kappa coefficient and associated confidence interval were calculated for the EIA-based Histoplasma antigen assay. Sixty cases met the inclusion criteria. Seventeen cases were considered true positives based on identification of the organism, and 41 cases were considered true negatives with an alternative definitive diagnosis. Two cases were considered false negatives, and there were no false positives. Sensitivity was 89.47% and the negative predictive value was 95.35%. Specificity and the positive predictive value were both 100%. The kappa coefficient was 0.9207 (95% confidence interval, 0.8131-1). The Histoplasma antigen EIA test demonstrated high specificity and sensitivity for the diagnosis of histoplasmosis in dogs.

  14. Understanding the Specificity and Random Collision of Enzyme-Substrate Interaction

    ERIC Educational Resources Information Center

    Kin, Ng Hong; Ling, Tan Aik

    2016-01-01

    The concept of specificity of enzyme action can potentially be abstract for some students as they fail to appreciate how the three-dimensional configuration of enzymes and the active sites confer perfect fit for specific substrates. In science text books, the specificity of enzyme-substrate binding is typically likened to the action of a lock and…

  15. Energy Landscape Topography Reveals the Underlying Link Between Binding Specificity and Activity of Enzymes.

    PubMed

    Chu, Wen-Ting; Wang, Jin

    2016-06-14

    Enzyme activity (often quantified by kcat/Km) is the main function of enzyme when it is active against the specific substrate. Higher or lower activities are highly desired for the design of novel enzyme and drug resistance. However, it is difficult to measure the activities of all possible variants and find the "hot-spot" within the limit of experimental time. In this study, we explore the underlying energy landscape of enzyme-substrate interactions and introduce the intrinsic specificity ratio (ISR), which reflects the landscape topography. By studying two concrete systems, we uncover the statistical correlation between the intrinsic specificity and the enzyme activity kcat/Km. This physics-based concept and method show that the energy landscape topography is valuable for understanding the relationship between enzyme specificity and activity. In addition, it can reveal the underlying mechanism of enzyme-substrate actions and has potential applications on enzyme design.

  16. Energy Landscape Topography Reveals the Underlying Link Between Binding Specificity and Activity of Enzymes

    NASA Astrophysics Data System (ADS)

    Chu, Wen-Ting; Wang, Jin

    2016-06-01

    Enzyme activity (often quantified by kcat/Km) is the main function of enzyme when it is active against the specific substrate. Higher or lower activities are highly desired for the design of novel enzyme and drug resistance. However, it is difficult to measure the activities of all possible variants and find the “hot-spot” within the limit of experimental time. In this study, we explore the underlying energy landscape of enzyme-substrate interactions and introduce the intrinsic specificity ratio (ISR), which reflects the landscape topography. By studying two concrete systems, we uncover the statistical correlation between the intrinsic specificity and the enzyme activity kcat/Km. This physics-based concept and method show that the energy landscape topography is valuable for understanding the relationship between enzyme specificity and activity. In addition, it can reveal the underlying mechanism of enzyme-substrate actions and has potential applications on enzyme design.

  17. Energy Landscape Topography Reveals the Underlying Link Between Binding Specificity and Activity of Enzymes

    PubMed Central

    Chu, Wen-Ting; Wang, Jin

    2016-01-01

    Enzyme activity (often quantified by kcat/Km) is the main function of enzyme when it is active against the specific substrate. Higher or lower activities are highly desired for the design of novel enzyme and drug resistance. However, it is difficult to measure the activities of all possible variants and find the “hot-spot” within the limit of experimental time. In this study, we explore the underlying energy landscape of enzyme-substrate interactions and introduce the intrinsic specificity ratio (ISR), which reflects the landscape topography. By studying two concrete systems, we uncover the statistical correlation between the intrinsic specificity and the enzyme activity kcat/Km. This physics-based concept and method show that the energy landscape topography is valuable for understanding the relationship between enzyme specificity and activity. In addition, it can reveal the underlying mechanism of enzyme-substrate actions and has potential applications on enzyme design. PMID:27298067

  18. Automatic program generation from specifications using Prolog

    NASA Technical Reports Server (NTRS)

    Pelin, Alex; Morrow, Paul

    1987-01-01

    An automatic program generator which creates Prolog programs from input/output specifications is presented. The generator takes as input descriptions of the input and output data types, a set of tests, a set of transformations and the input/out relation. Abstract data types are used as models. The tests, the transformations and the input/out relation are also specified by equations. The heuristics used by the automatic propram generator in building Prolog programs are discussed.

  19. Aptamer capturing of enzymes on magnetic beads to enhance assay specificity and sensitivity.

    PubMed

    Zhao, Qiang; Li, Xing-Fang; Le, X Chris

    2011-12-15

    Activity and specificity of enzyme molecules are important to enzymatic reactions and enzyme assays. We describe an aptamer capturing approach that improves the specificity and the sensitivity of enzyme detection. An aptamer recognizing the target enzyme molecule is conjugated on a magnetic bead, increasing the local concentration, and serves as an affinity probe to capture and separate minute amounts of the enzyme. The captured enzymes catalyze the subsequent conversion of fluorogenic substrate to fluorescent products, enabling a sensitive measure of the active enzyme. The feasibility of this technique is demonstrated through assays for human alpha thrombin and human neutrophil elastase (HNE), two important enzymes. Thrombin (2 fM) and 100 fM HNE can be detected. The incorporation of two binding events, substrate recognition and aptamer binding, greatly improves assay specificity. With its simplicity, this approach is applicable to biosensing and detection of disease biomarkers.

  20. Site-specific immobilization of enzymes on magnetic nanoparticles and their use in organic synthesis.

    PubMed

    Yu, Ching-Ching; Kuo, Yu-Ying; Liang, Chien-Fu; Chien, Wei-Ting; Wu, Huan-Ting; Chang, Tsung-Che; Jan, Fan-Dan; Lin, Chun-Cheng

    2012-04-18

    Magnetic nanoparticles (MNPs) are attractive materials that serve as a support for enzyme immobilization and facilitate separations by applying an external magnetic field; this could facilitate the recycling of enzymes and broaden their applications in organic synthesis. Herein, we report the methods for the immobilization of water-soluble and membrane-bound enzymes, and the activity difference between free and immobilized enzymes is discussed. Sialyltransferase (PmST1, from Pasteurella multocida ) and cytidine monophosphate (CMP)-sialic acid synthetase (CSS, from Neisseria meningitides ) were chosen as water-soluble enzymes and expressed using an intein expression system. The enzymes were site-specifically and covalently immobilized on PEGylated-N-terminal cysteine MNPs through native chemical ligation (NCL). Increasing the length of the PEG linker between the enzyme and the MNP surface increased the activity of the immobilized enzymes relative to the free parent enzymes. In addition, the use of a fluorescent acceptor tag for PmST1 affected enzyme kinetics. In contrast, sialyltransferase from Neisseria gonorrheae (NgST, a membrane-bound enzyme) was modified with a biotin-labeled cysteine at the C-terminus using NCL, and the enzyme was then assembled on streptavidin-functionalized MNPs. Using a streptavidin-biotin interaction, it was possible to immobilize NgST on a solid support under mild ligation conditions, which prevented the enzyme from high-temperature decomposition and provided an approximately 2-fold increase in activity compared to other immobilization methods on MNPs. Finally, the ganglioside GM3-derivative (sialyl-lactose derivative) was synthesized in a one-pot system by combining the use of immobilized PmST1 and CSS. The enzymes retained 50% activity after being reused ten times. Furthermore, the results obtained using the one-pot two-immobilized-enzyme system demonstrated that it can be applied to large-scale reactions with acceptable yields and

  1. Formal Specification and Verification of Concurrent Programs

    DTIC Science & Technology

    1993-02-01

    specification and verification en- stand the effect of his or her own program. It is even vironments is included. Theoretical work is de- more difficult for...others, such as the client and the scribed to the extent that it provides the logical basis maintainer, to understand this effect . for practical work...objects properties are those that can be expressed thusly * ( communications channels included) the’ no process * P is true in all snapshots, i.e., Vs in C(P

  2. Vacuolar processing enzyme in plant programmed cell death

    PubMed Central

    Hatsugai, Noriyuki; Yamada, Kenji; Goto-Yamada, Shino; Hara-Nishimura, Ikuko

    2015-01-01

    Vacuolar processing enzyme (VPE) is a cysteine proteinase originally identified as the proteinase responsible for the maturation and activation of vacuolar proteins in plants, and it is known to be an ortholog of animal asparaginyl endopeptidase (AEP/VPE/legumain). VPE has been shown to exhibit enzymatic properties similar to that of caspase 1, which is a cysteine protease that mediates the programmed cell death (PCD) pathway in animals. Although there is limited sequence identity between VPE and caspase 1, their predicted three-dimensional structures revealed that the essential amino-acid residues for these enzymes form similar pockets for the substrate peptide YVAD. In contrast to the cytosolic localization of caspases, VPE is localized in vacuoles. VPE provokes vacuolar rupture, initiating the proteolytic cascade leading to PCD in the plant immune response. It has become apparent that the VPE-dependent PCD pathway is involved not only in the immune response, but also in the responses to a variety of stress inducers and in the development of various tissues. This review summarizes the current knowledge on the contribution of VPE to plant PCD and its role in vacuole-mediated cell death, and it also compares VPE with the animal cell death executor caspase 1. PMID:25914711

  3. Homology models guide discovery of diverse enzyme specificities among dipeptide epimerases in the enolase superfamily

    PubMed Central

    Lukk, Tiit; Sakai, Ayano; Kalyanaraman, Chakrapani; Brown, Shoshana D.; Imker, Heidi J.; Song, Ling; Fedorov, Alexander A.; Fedorov, Elena V.; Toro, Rafael; Hillerich, Brandan; Seidel, Ronald; Patskovsky, Yury; Vetting, Matthew W.; Nair, Satish K.; Babbitt, Patricia C.; Almo, Steven C.; Gerlt, John A.; Jacobson, Matthew P.

    2012-01-01

    The rapid advance in genome sequencing presents substantial challenges for protein functional assignment, with half or more of new protein sequences inferred from these genomes having uncertain assignments. The assignment of enzyme function in functionally diverse superfamilies represents a particular challenge, which we address through a combination of computational predictions, enzymology, and structural biology. Here we describe the results of a focused investigation of a group of enzymes in the enolase superfamily that are involved in epimerizing dipeptides. The first members of this group to be functionally characterized were Ala-Glu epimerases in Eschericiha coli and Bacillus subtilis, based on the operon context and enzymological studies; these enzymes are presumed to be involved in peptidoglycan recycling. We have subsequently studied more than 65 related enzymes by computational methods, including homology modeling and metabolite docking, which suggested that many would have divergent specificities;, i.e., they are likely to have different (unknown) biological roles. In addition to the Ala-Phe epimerase specificity reported previously, we describe the prediction and experimental verification of: (i) a new group of presumed Ala-Glu epimerases; (ii) several enzymes with specificity for hydrophobic dipeptides, including one from Cytophaga hutchinsonii that epimerizes D-Ala-D-Ala; and (iii) a small group of enzymes that epimerize cationic dipeptides. Crystal structures for certain of these enzymes further elucidate the structural basis of the specificities. The results highlight the potential of computational methods to guide experimental characterization of enzymes in an automated, large-scale fashion. PMID:22392983

  4. Specificity of Cryptococcus neoformans factor sera determined by enzyme-linked immunosorbent assay and dot enzyme assay.

    PubMed Central

    Belay, T; Cherniak, R; Shinoda, T

    1993-01-01

    An indirect enzyme-linked immunosorbent assay (ELISA) and a dot enzyme assay (DEA) were used to determine the specificities of Cryptococcus neoformans factor sera to serotype type-specific capsular polysaccharides, glucuronoxylomannans (GXMs). Pure and chemically characterized GXMs were obtained from representative isolates of C. neoformans serotypes A, B, C, and D. Distinctive specificity patterns and quantitative differences were observed for each factor serum when the selected GXMs were studied by ELISA. The specificity patterns for each factor serum determined by DEA almost completely paralleled the ELISA results. The serotype specificities demonstrated by ELISA and DEA were similar to previously reported results that were obtained by slide agglutination studies of whole cells. On the basis of the ELISA and DEA activity patterns, factor sera 5, 6, and 8 were specific for serotypes B, C, and D, respectively; factor serum 1 was strongly reactive to all serotypes; factor serum 2 was specific for serotypes A, B, and D; factor serum 3 was specific for serotypes A and D; and factor serum 4 was specific for serotypes B and C. The specificity of factor serum 7 for serotype A was demonstrated by DEA only. Structural variation was indicated among the serotype C isolates studied because a unique activity pattern versus factor serum 6 was observed for each isolate. The quantitative differences in the activity of the GXMs from five serotype C isolates suggest that mannopyranoside residues substituted O-2 and O-4 with xylose are essential elements of the determinant responsible for the observed activity of factor 6. No significant differences in activity patterns and specificities of factor serum 6 were observed when O-deacetylated GXMs were substituted for the native GXMs. Our results show that ELISA and DEA are valuable techniques for the serological analysis of cryptococcal factor sera and GXMs. Images PMID:7685739

  5. Testing of concurrent programs and partial specifications

    SciTech Connect

    Hamlet, D.

    1982-12-01

    The testing problems of concurrent systems include those of sequential programs, but there are two additional difficulties: the scheduling of tasks may alter the behavior, making tests misleading; testing may be conducted at an early stage of development, by users who are not software experts. Concurrent process systems can be modeled by a collection of finite-state transducers, in a way that displays their unique problems. The specification languages PAISLey and Gist approach the definition of concurrent systems differently, but both permit users to execute partially defined systems. The declarative language PROLOG, although not explicitly designed for concurrent programming, exhibits similar characteristics. Prototype execution has some unexpected implications for testing, and for final implementation.

  6. The programming language HAL: A specification

    NASA Technical Reports Server (NTRS)

    1971-01-01

    HAL accomplishes three significant objectives: (1) increased readability, through the use of a natural two-dimensional mathematical format; (2) increased reliability, by providing for selective recognition of common data and subroutines, and by incorporating specific data-protect features; (3) real-time control facility, by including a comprehensive set of real-time control commands and signal conditions. Although HAL is designed primarily for programming on-board computers, it is general enough to meet nearly all the needs in the production, verification and support of aerospace, and other real-time applications.

  7. MAIL LOG, program summary and specifications

    NASA Technical Reports Server (NTRS)

    Harris, D. K.

    1979-01-01

    The summary and specifications to obtain the software package, MAIL LOG, developed for the Scout Project Automatic Data System, SPADS are provided. The MAIL LOG program has four modes of operation: (1) input - putting new records into the data base; (2) revise - changing or modifying existing records in the data base; (3) search - finding special records existing in the data base; and (4) archive - store or put away existing records in the data base. The output includes special printouts of records in the data base and results from the input and search modes.

  8. Relationship of sequence and structure to specificity in the alpha-amylase family of enzymes.

    PubMed

    MacGregor, E A; Janecek, S; Svensson, B

    2001-03-09

    The hydrolases and transferases that constitute the alpha-amylase family are multidomain proteins, but each has a catalytic domain in the form of a (beta/alpha)(8)-barrel, with the active site being at the C-terminal end of the barrel beta-strands. Although the enzymes are believed to share the same catalytic acids and a common mechanism of action, they have been assigned to three separate families - 13, 70 and 77 - in the classification scheme for glycoside hydrolases and transferases that is based on amino acid sequence similarities. Each enzyme has one glutamic acid and two aspartic acid residues necessary for activity, while most enzymes of the family also contain two histidine residues critical for transition state stabilisation. These five residues occur in four short sequences conserved throughout the family, and within such sequences some key amino acid residues are related to enzyme specificity. A table is given showing motifs distinctive for each specificity as extracted from 316 sequences, which should aid in identifying the enzyme from primary structure information. Where appropriate, existing problems with identification of some enzymes of the family are pointed out. For enzymes of known three-dimensional structure, action is discussed in terms of molecular architecture. The sequence-specificity and structure-specificity relationships described may provide useful pointers for rational protein engineering.

  9. Integrating Proteomics and Enzyme Kinetics Reveals Tissue-Specific Types of the Glycolytic and Gluconeogenic Pathways.

    PubMed

    Wiśniewski, Jacek R; Gizak, Agnieszka; Rakus, Dariusz

    2015-08-07

    Glycolysis is the core metabolic pathway supplying energy to cells. Whereas the vast majority of studies focus on specific aspects of the process, global analyses characterizing simultaneously all enzymes involved in the process are scarce. Here, we demonstrate that quantitative label- and standard-free proteomics allows accurate determination of titers of metabolic enzymes and enables simultaneous measurements of titers and maximal enzymatic activities (Amax) of all glycolytic enzymes and the gluconeogenic fructose 1,6-bisphosphatase in mouse brain, liver and muscle. Despite occurrence of tissue-specific isoenzymes bearing different kinetic properties, the enzyme titers often correlated well with the Amax values. To provide a more general picture of energy metabolism, we analyzed titers of the enzymes in additional 7 mouse organs and in human cells. Across the analyzed samples, we identified two basic profiles: a "fast glucose uptake" one in brain and heart, and a "gluconeogenic rich" one occurring in liver. In skeletal muscles and other organs, we found intermediate profiles. Obtained data highlighted the glucose-flux-limiting role of hexokinase which activity was always 10- to 100-fold lower than the average activity of all other glycolytic enzymes. A parallel determination of enzyme titers and maximal enzymatic activities allowed determination of kcat values without enzyme purification. Results of our in-depth proteomic analysis of the mouse organs did not support the concepts of regulation of glycolysis by lysine acetylation.

  10. Interconversion of the Specificities of Human Lysosomal Enzymes Associated with Fabry and Schindler Diseases

    SciTech Connect

    Tomasic, Ivan B.; Metcalf, Matthew C.; Guce, Abigail I.; Clark, Nathaniel E.; Garman, Scott C.

    2010-09-03

    The human lysosomal enzymes {alpha}-galactosidase ({alpha}-GAL, EC 3.2.1.22) and {alpha}-N-acetylgalactosaminidase ({alpha}-NAGAL, EC 3.2.1.49) share 46% amino acid sequence identity and have similar folds. The active sites of the two enzymes share 11 of 13 amino acids, differing only where they interact with the 2-position of the substrates. Using a rational protein engineering approach, we interconverted the enzymatic specificity of {alpha}-GAL and {alpha}-NAGAL. The engineered {alpha}-GAL (which we call {alpha}-GALSA) retains the antigenicity of {alpha}-GAL but has acquired the enzymatic specificity of {alpha}-NAGAL. Conversely, the engineered {alpha}-NAGAL (which we call {alpha}-NAGAL{sup EL}) retains the antigenicity of {alpha}-NAGAL but has acquired the enzymatic specificity of the {alpha}-GAL enzyme. Comparison of the crystal structures of the designed enzyme {alpha}-GAL{sup SA} to the wild-type enzymes shows that active sites of {alpha}-GAL{sup SA} and {alpha}-NAGAL superimpose well, indicating success of the rational design. The designed enzymes might be useful as non-immunogenic alternatives in enzyme replacement therapy for treatment of lysosomal storage disorders such as Fabry disease.

  11. Design of Peptide Substrate for Sensitively and Specifically Detecting Two Aβ-Degrading Enzymes: Neprilysin and Angiotensin-Converting Enzyme.

    PubMed

    Chen, Po-Ting; Chen, Chao-Long; Lin, Lilian Tsai-Wei; Lo, Chun-Hsien; Hu, Chaur-Jong; Chen, Rita P-Y; Wang, Steven S-S

    2016-01-01

    Upregulation of neprilysin (NEP) to reduce Aβ accumulation in the brain is a promising strategy for the prevention of Alzheimer's disease (AD). This report describes the design and synthesis of a quenched fluorogenic peptide substrate qf-Aβ(12-16)AAC (with the sequence VHHQKAAC), which has a fluorophore, Alexa-350, linked to the side-chain of its C-terminal cysteine and a quencher, Dabcyl, linked to its N-terminus. This peptide emitted strong fluorescence upon cleavage. Our results showed that qf-Aβ(12-16)AAC is more sensitive to NEP than the previously reported peptide substrates, so that concentrations of NEP as low as 0.03 nM could be detected at peptide concentration of 2 μM. Moreover, qf-Aβ(12-16)AAC had superior enzymatic specificity for both NEP and angiotensin-converting enzyme (ACE), but was inert with other Aβ-degrading enzymes. This peptide, used in conjunction with a previously reported peptide substrate qf-Aβ(1-7)C [which is sensitive to NEP and insulin-degrading enzyme (IDE)], could be used for high-throughput screening of compounds that only upregulate NEP. The experimental results of cell-based activity assays using both qf-Aβ(1-7)C and qf-Aβ(12-16)AAC as the substrates confirm that somatostatin treatment most likely upregulates IDE, but not NEP, in neuroblastoma cells.

  12. Design of Peptide Substrate for Sensitively and Specifically Detecting Two Aβ-Degrading Enzymes: Neprilysin and Angiotensin-Converting Enzyme

    PubMed Central

    Chen, Po-Ting; Chen, Chao-Long; Lin, Lilian Tsai-Wei; Lo, Chun-Hsien; Hu, Chaur-Jong; Chen, Rita P.-Y.; Wang, Steven S.-S.

    2016-01-01

    Upregulation of neprilysin (NEP) to reduce Aβ accumulation in the brain is a promising strategy for the prevention of Alzheimer’s disease (AD). This report describes the design and synthesis of a quenched fluorogenic peptide substrate qf-Aβ(12–16)AAC (with the sequence VHHQKAAC), which has a fluorophore, Alexa-350, linked to the side-chain of its C-terminal cysteine and a quencher, Dabcyl, linked to its N-terminus. This peptide emitted strong fluorescence upon cleavage. Our results showed that qf-Aβ(12–16)AAC is more sensitive to NEP than the previously reported peptide substrates, so that concentrations of NEP as low as 0.03 nM could be detected at peptide concentration of 2 μM. Moreover, qf-Aβ(12–16)AAC had superior enzymatic specificity for both NEP and angiotensin-converting enzyme (ACE), but was inert with other Aβ-degrading enzymes. This peptide, used in conjunction with a previously reported peptide substrate qf-Aβ(1–7)C [which is sensitive to NEP and insulin-degrading enzyme (IDE)], could be used for high-throughput screening of compounds that only upregulate NEP. The experimental results of cell-based activity assays using both qf-Aβ(1–7)C and qf-Aβ(12–16)AAC as the substrates confirm that somatostatin treatment most likely upregulates IDE, but not NEP, in neuroblastoma cells. PMID:27096746

  13. Simplified assays of lipolysis enzymes for drug discovery and specificity assessment of known inhibitors.

    PubMed

    Iglesias, Jose; Lamontagne, Julien; Erb, Heidi; Gezzar, Sari; Zhao, Shangang; Joly, Erik; Truong, Vouy Linh; Skorey, Kathryn; Crane, Sheldon; Madiraju, S R Murthy; Prentki, Marc

    2016-01-01

    Lipids are used as cellular building blocks and condensed energy stores and also act as signaling molecules. The glycerolipid/ fatty acid cycle, encompassing lipolysis and lipogenesis, generates many lipid signals. Reliable procedures are not available for measuring activities of several lipolytic enzymes for the purposes of drug screening, and this resulted in questionable selectivity of various known lipase inhibitors. We now describe simple assays for lipolytic enzymes, including adipose triglyceride lipase (ATGL), hormone sensitive lipase (HSL), sn-1-diacylglycerol lipase (DAGL), monoacylglycerol lipase, α/β-hydrolase domain 6, and carboxylesterase 1 (CES1) using recombinant human and mouse enzymes either in cell extracts or using purified enzymes. We observed that many of the reported inhibitors lack specificity. Thus, Cay10499 (HSL inhibitor) and RHC20867 (DAGL inhibitor) also inhibit other lipases. Marked differences in the inhibitor sensitivities of human ATGL and HSL compared with the corresponding mouse enzymes was noticed. Thus, ATGListatin inhibited mouse ATGL but not human ATGL, and the HSL inhibitors WWL11 and Compound 13f were effective against mouse enzyme but much less potent against human enzyme. Many of these lipase inhibitors also inhibited human CES1. Results describe reliable assays for measuring lipase activities that are amenable for drug screening and also caution about the specificity of the many earlier described lipase inhibitors.

  14. Substrate Specificity and Diastereoselectivity of Strictosidine Glucosidase, a Key Enzyme in Monoterpene Indole Alkaloid Biosynthesis

    PubMed Central

    Yerkes, Nancy; Wu, Jia; McCoy, Elizabeth; Galan, M. Carmen; Chen, Shi; O’Connor, Sarah E.

    2008-01-01

    Strictosidine glucosidase (SGD) from Catharanthus roseus catalyzes the deglycosylation of strictosidine, an intermediate from which thousands of monoterpene indole alkaloids are derived. The steady state kinetics of SGD with a variety of strictosidine analogs revealed the substrate preferences of this enzyme at two key positions of the strictosidine substrate. Additionally, SGD from C. roseus turns over both strictosidine and its stereoisomer vincoside, indicating that although this enzyme prefers the naturally occurring diastereomer, the enzyme is not completely diastereoselective. The implications of the substrate specificity of SGD in metabolic engineering efforts of C. roseus are highlighted. PMID:18061449

  15. Substrate specificity and diastereoselectivity of strictosidine glucosidase, a key enzyme in monoterpene indole alkaloid biosynthesis.

    PubMed

    Yerkes, Nancy; Wu, Jia Xin; McCoy, Elizabeth; Galan, M Carmen; Chen, Shi; O'Connor, Sarah E

    2008-05-15

    Strictosidine glucosidase (SGD) from Catharanthus roseus catalyzes the deglycosylation of strictosidine, an intermediate from which thousands of monoterpene indole alkaloids are derived. The steady-state kinetics of SGD with a variety of strictosidine analogs revealed the substrate preferences of this enzyme at two key positions of the strictosidine substrate. Additionally, SGD from C. roseus turns over both strictosidine and its stereoisomer vincoside, indicating that although this enzyme prefers the naturally occurring diastereomer, the enzyme is not completely diastereoselective. The implications of the substrate specificity of SGD in metabolic engineering efforts of C. roseus are highlighted.

  16. Prediction of detailed enzyme functions and identification of specificity determining residues by random forests.

    PubMed

    Nagao, Chioko; Nagano, Nozomi; Mizuguchi, Kenji

    2014-01-01

    Determining enzyme functions is essential for a thorough understanding of cellular processes. Although many prediction methods have been developed, it remains a significant challenge to predict enzyme functions at the fourth-digit level of the Enzyme Commission numbers. Functional specificity of enzymes often changes drastically by mutations of a small number of residues and therefore, information about these critical residues can potentially help discriminate detailed functions. However, because these residues must be identified by mutagenesis experiments, the available information is limited, and the lack of experimentally verified specificity determining residues (SDRs) has hindered the development of detailed function prediction methods and computational identification of SDRs. Here we present a novel method for predicting enzyme functions by random forests, EFPrf, along with a set of putative SDRs, the random forests derived SDRs (rf-SDRs). EFPrf consists of a set of binary predictors for enzymes in each CATH superfamily and the rf-SDRs are the residue positions corresponding to the most highly contributing attributes obtained from each predictor. EFPrf showed a precision of 0.98 and a recall of 0.89 in a cross-validated benchmark assessment. The rf-SDRs included many residues, whose importance for specificity had been validated experimentally. The analysis of the rf-SDRs revealed both a general tendency that functionally diverged superfamilies tend to include more active site residues in their rf-SDRs than in less diverged superfamilies, and superfamily-specific conservation patterns of each functional residue. EFPrf and the rf-SDRs will be an effective tool for annotating enzyme functions and for understanding how enzyme functions have diverged within each superfamily.

  17. Cryoenzymology in mixed solvents without cosolvent effects on enzyme specific activity.

    PubMed Central

    Douzou, P; Balny, C

    1977-01-01

    Water-soluble polyelectrolytes in interaction with proteins are described. These polyelectrolytes make it possible to investigate enzyme-catalyzed reactions in cooled mixed solvents without the usual effects of their organic solvent component on enzyme specific activity. The applicability of techniques developed is illustrated by results obtained on several systems. The possibility of an electrostatic "sorting out" of solvents and its potentialities in cryoenzymology are discussed. PMID:18734

  18. Directed evolution of cytochrome P450 enzymes for biocatalysis: exploiting the catalytic versatility of enzymes with relaxed substrate specificity.

    PubMed

    Behrendorff, James B Y H; Huang, Weiliang; Gillam, Elizabeth M J

    2015-04-01

    Cytochrome P450 enzymes are renowned for their ability to insert oxygen into an enormous variety of compounds with a high degree of chemo- and regio-selectivity under mild conditions. This property has been exploited in Nature for an enormous variety of physiological functions, and representatives of this ancient enzyme family have been identified in all kingdoms of life. The catalytic versatility of P450s makes them well suited for repurposing for the synthesis of fine chemicals such as drugs. Although these enzymes have not evolved in Nature to perform the reactions required for modern chemical industries, many P450s show relaxed substrate specificity and exhibit some degree of activity towards non-natural substrates of relevance to applications such as drug development. Directed evolution and other protein engineering methods can be used to improve upon this low level of activity and convert these promiscuous generalist enzymes into specialists capable of mediating reactions of interest with exquisite regio- and stereo-selectivity. Although there are some notable successes in exploiting P450s from natural sources in metabolic engineering, and P450s have been proven repeatedly to be excellent material for engineering, there are few examples to date of practical application of engineered P450s. The purpose of the present review is to illustrate the progress that has been made in altering properties of P450s such as substrate range, cofactor preference and stability, and outline some of the remaining challenges that must be overcome for industrial application of these powerful biocatalysts.

  19. Combining structure and sequence information allows automated prediction of substrate specificities within enzyme families.

    PubMed

    Röttig, Marc; Rausch, Christian; Kohlbacher, Oliver

    2010-01-08

    An important aspect of the functional annotation of enzymes is not only the type of reaction catalysed by an enzyme, but also the substrate specificity, which can vary widely within the same family. In many cases, prediction of family membership and even substrate specificity is possible from enzyme sequence alone, using a nearest neighbour classification rule. However, the combination of structural information and sequence information can improve the interpretability and accuracy of predictive models. The method presented here, Active Site Classification (ASC), automatically extracts the residues lining the active site from one representative three-dimensional structure and the corresponding residues from sequences of other members of the family. From a set of representatives with known substrate specificity, a Support Vector Machine (SVM) can then learn a model of substrate specificity. Applied to a sequence of unknown specificity, the SVM can then predict the most likely substrate. The models can also be analysed to reveal the underlying structural reasons determining substrate specificities and thus yield valuable insights into mechanisms of enzyme specificity. We illustrate the high prediction accuracy achieved on two benchmark data sets and the structural insights gained from ASC by a detailed analysis of the family of decarboxylating dehydrogenases. The ASC web service is available at http://asc.informatik.uni-tuebingen.de/.

  20. Specific point mutations in key redox enzymes are associated with chemoresistance in epithelial ovarian cancer.

    PubMed

    Fletcher, Nicole M; Belotte, Jimmy; Saed, Mohammed G; Memaj, Ira; Diamond, Michael P; Morris, Robert T; Saed, Ghassan M

    2017-01-01

    Oxidative stress plays an important role in the pathophysiology of ovarian cancer. Resistance to chemotherapy presents a significant challenge for ovarian cancer treatment. Specific single nucleotide polymorphisms (SNPs) in key redox enzymes have been associated with ovarian cancer survival and progression. The objective of this study was to determine whether chemotherapy induces point mutations in key redox enzymes that lead to the acquisition of chemoresistance in epithelial ovarian cancer (EOC). Human EOC cell lines and their chemoresistant counterpart were utilized for this study. Specific SNPs in key redox enzymes were analyzed by TaqMan SNP Genotyping. Activities and levels of key redox enzymes were determined by real-time RT-PCR, ELISA and a greiss assay. Point mutations in key redox enzymes were introduced into sensitive EOC cells via the CRISPR/Cas9 system. Cell viability and IC50 for cisplatin were determined by the MTT Cell Proliferation Assay. Data was analyzed with SPSS using Student's two-tailed t-tests and One-way ANOVA followed by Dunnett's or Tukey's post hoc tests, p<0.05. Here, we demonstrate that chemoresistant EOC cells are characterized by a further enhancement in oxidative stress as compared to sensitive counterparts. Additionally, chemoresistant EOC cells manifested specific point mutations, which are associated with altered enzymatic activity, in key redox enzymes that are not detected in sensitive counterparts. Supplementation of an antioxidant was able to successfully sensitize EOC cells to chemotherapeutics. Causality was established by the induction of these point mutations in sensitive EOC cells, which resulted in a significant increase in the level of chemoresistance. These findings indicate that chemotherapy induces specific point mutations in key redox enzymes that contribute to the acquisition of chemoresistance in EOC cells, highlighting a potential novel mechanism. Identification of targets for chemoresistance with either

  1. Mechanism of sirtuin inhibition by nicotinamide: altering the NAD(+) cosubstrate specificity of a Sir2 enzyme.

    PubMed

    Avalos, José L; Bever, Katherine M; Wolberger, Cynthia

    2005-03-18

    Sir2 enzymes form a unique class of NAD(+)-dependent deacetylases required for diverse biological processes, including transcriptional silencing, regulation of apoptosis, fat mobilization, and lifespan regulation. Sir2 activity is regulated by nicotinamide, a noncompetitive inhibitor that promotes a base-exchange reaction at the expense of deacetylation. To elucidate the mechanism of nicotinamide inhibition, we determined ternary complex structures of Sir2 enzymes containing nicotinamide. The structures show that free nicotinamide binds in a conserved pocket that participates in NAD(+) binding and catalysis. Based on our structures, we engineered a mutant that deacetylates peptides by using nicotinic acid adenine dinucleotide (NAAD) as a cosubstrate and is inhibited by nicotinic acid. The characteristics of the altered specificity enzyme establish that Sir2 enzymes contain a single site that participates in catalysis and nicotinamide regulation and provides additional insights into the Sir2 catalytic mechanism.

  2. Broad specification fuels combustion technology program

    NASA Technical Reports Server (NTRS)

    Dodds, W. J.; Ekstedt, E. E.

    1984-01-01

    Design and development efforts to evolve promising aircraft gas turbine combustor configurations for burning broadened-properties fuels were discussed. Design and experimental evaluations of three different combustor concepts in sector combustor rig tests was conducted. The combustor concepts were a state of the art single-annular combustor, a staged double-annular combustor, and a short single-annular combustor with variable geometry to control primary zone stoichiometry. A total of 25 different configurations of the three combustor concepts were evaluated. Testing was conducted over the full range of CF6-80A engine combustor inlet conditions, using four fuels containing between 12% and 14% hydrogen by weight. Good progress was made toward meeting specific program emissions and performance goals with each of the three combustor concepts. The effects of reduced fuel hydrogen content, including increased flame radiation, liner metal temperature, smoke, and NOx emissions were documented. The most significant effect on the baseline combustor was a projected 33% life reduction, for a reduction from 14% to 13% fuel hydrogen content, due to increased liner temperatures.

  3. Substrate Specificity Profiling of Histone-Modifying Enzymes by Peptide Microarray.

    PubMed

    Cornett, E M; Dickson, B M; Vaughan, R M; Krishnan, S; Trievel, R C; Strahl, B D; Rothbart, S B

    2016-01-01

    The dynamic addition and removal of covalent posttranslational modifications (PTMs) on histone proteins serves as a major mechanism regulating chromatin-templated biological processes in eukaryotic genomes. Histone PTMs and their combinations function by directly altering the physical structure of chromatin and as rheostats for effector protein interactions. In this chapter, we detail microarray-based methods for analyzing the substrate specificity of lysine methyltransferase and demethylase enzymes on immobilized synthetic histone peptides. Consistent with the "histone code" hypothesis, we reveal a strong influence of adjacent and, surprisingly, distant histone PTMs on the ability of histone-modifying enzymes to methylate or demethylate their substrates. This platform will greatly facilitate future investigations into histone substrate specificity and mechanisms of PTM signaling that regulate the catalytic properties of histone-modifying enzymes.

  4. Substrate Specificity Profiling of Histone-Modifying Enzymes by Peptide Microarray

    PubMed Central

    Cornett, E.M.; Dickson, B.M.; Vaughan, R.M.; Krishnan, S.; Trievel, R.C.; Strahl, B.D.; Rothbart, S.B.

    2017-01-01

    The dynamic addition and removal of covalent posttranslational modifications (PTMs) on histone proteins serves as a major mechanism regulating chromatin-templated biological processes in eukaryotic genomes. Histone PTMs and their combinations function by directly altering the physical structure of chromatin and as rheostats for effector protein interactions. In this chapter, we detail microarray-based methods for analyzing the substrate specificity of lysine methyltransferase and demethylase enzymes on immobilized synthetic histone peptides. Consistent with the “histone code” hypothesis, we reveal a strong influenceof adjacent and,surprisingly,distant histonePTMs onthe ability of histone-modifying enzymes to methylate or demethylate their substrates. This platform will greatly facilitate future investigations into histone substrate specificity and mechanisms of PTM signaling that regulate the catalytic properties of histone-modifying enzymes. PMID:27423856

  5. Substrate specificity determinants of the methanogen homoaconitase enzyme: structure and function of the small subunit.

    PubMed

    Jeyakanthan, Jeyaraman; Drevland, Randy M; Gayathri, Dasara Raju; Velmurugan, Devadasan; Shinkai, Akeo; Kuramitsu, Seiki; Yokoyama, Shigeyuki; Graham, David E

    2010-03-30

    The aconitase family of hydro-lyase enzymes includes three classes of proteins that catalyze the isomerization of alpha-hydroxy acids to beta-hydroxy acids. Besides aconitase, isopropylmalate isomerase (IPMI) proteins specifically catalyze the isomerization of alpha,beta-dicarboxylates with hydrophobic gamma-chain groups, and homoaconitase (HACN) proteins catalyze the isomerization of tricarboxylates with variable chain length gamma-carboxylate groups. These enzymes' stereospecific hydro-lyase activities make them attractive catalysts to produce diastereomers from unsaturated precursors. However, sequence similarity and convergent evolution among these proteins lead to widespread misannotation and uncertainty about gene function. To find the substrate specificity determinants of homologous IPMI and HACN proteins from Methanocaldococcus jannaschii, the small-subunit HACN protein (MJ1271) was crystallized for X-ray diffraction. The structural model showed characteristic residues in a flexible loop region between alpha2 and alpha3 that distinguish HACN from IPMI and aconitase proteins. Site-directed mutagenesis of MJ1271 produced loop-region variant proteins that were reconstituted with wild-type MJ1003 large-subunit protein. The heteromers formed promiscuous hydro-lyases with reduced activity but broader substrate specificity. Both R26K and R26V variants formed relatively efficient IPMI enzymes, while the T27A variant had uniformly lower specificity constants for both IPMI and HACN substrates. The R26V T27Y variant resembles the MJ1277 IPMI small subunit in its flexible loop sequence but demonstrated the broad substrate specificity of the R26V variant. These mutations may reverse the evolution of HACN activity from an ancestral IPMI gene, demonstrating the evolutionary potential for promiscuity in hydro-lyase enzymes. Understanding these specificity determinants enables the functional reannotation of paralogous HACN and IPMI genes in numerous genome sequences. These

  6. Development of an Enzyme Linked Immunosorbent Assay to Detect Chicken Parvovirus Specific Antibodies

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Here we report the development and application of an enzyme linked immunosorbent assay to detect parvovirus-specific antibodies in chicken sera. We used an approach previously described for other parvoviruses to clone and express viral structural proteins in insect cells from recombinant baculovirus...

  7. Structure, specificity and function of cyclomaltodextrinase, a multispecific enzyme of the alpha-amylase family.

    PubMed

    Park, K H; Kim, T J; Cheong, T K; Kim, J W; Oh, B H; Svensson, B

    2000-05-23

    Cyclomaltodextrinase (CDase, EC 3.2.1.54), maltogenic amylase (EC 3. 2.1.133), and neopullulanase (EC 3.2.1.135) are reported to be capable of hydrolyzing all or two of the following three types of substrates: cyclomaltodextrins (CDs); pullulan; and starch. These enzymes hydrolyze CDs and starch to maltose and pullulan to panose by cleavage of alpha-1,4 glycosidic bonds whereas alpha-amylases essentially lack activity on CDs and pullulan. They also catalyze transglycosylation of oligosaccharides to the C3-, C4- or C6-hydroxyl groups of various acceptor sugar molecules. The present review surveys the biochemical, enzymatic, and structural properties of three types of such enzymes as defined based on the substrate specificity toward the CDs: type I, cyclomaltodextrinase and maltogenic amylase that hydrolyze CDs much faster than pullulan and starch; type II, Thermoactinomyces vulgaris amylase II (TVA II) that hydrolyzes CDs much less efficiently than pullulan; and type III, neopullulanase that hydrolyzes pullulan efficiently, but remains to be reported to hydrolyze CDs. These three types of enzymes exhibit 40-60% amino acid sequence identity. They occur in the cytoplasm of bacteria and have molecular masses from 62 to 90 kDa which are slightly larger than those of most alpha-amylases. Multiple amino acid sequence alignment and crystal structures of maltogenic amylase and TVA II reveal the presence of an N-terminal extension of approximately 130 residues not found in alpha-amylases. This unique N-terminal domain as seen in the crystal structures apparently contributes to the active site structure leading to the distinct substrate specificity through a dimer formation. In aqueous solution, most of these enzymes show a monomer-dimer equilibrium. The present review discusses the multiple specificity in the light of the oligomerization and the molecular structures arriving at a clarified enzyme classification. Finally, a physiological role of the enzymes is proposed.

  8. A computer program for enzyme kinetics that combines model discrimination, parameter refinement and sequential experimental design.

    PubMed Central

    Franco, R; Gavaldà, M T; Canela, E I

    1986-01-01

    A method of model discrimination and parameter estimation in enzyme kinetics is proposed. The experimental design and analysis of the model are carried out simultaneously and the stopping rule for experimentation is deduced by the experimenter when the probabilities a posteriori indicate that one model is clearly superior to the rest. A FORTRAN77 program specifically developed for joint designs is given. The method is very powerful, as indicated by its usefulness in the discrimination between models. For example, it has been successfully applied to three cases of enzyme kinetics (a single-substrate Michaelian reaction with product inhibition, a single-substrate complex reaction and a two-substrate reaction). By using this method the most probable model and the estimates of the parameters can be obtained in one experimental session. The FORTRAN77 program is deposited as Supplementary Publication SUP 50134 (19 pages) at the British Library (Lending Division), Boston Spa, Wetherby, West Yorkshire LS23 7BQ, U.K., from whom copies can be obtained on the terms indicated in Biochem. J. (1986) 233, 5. PMID:3800965

  9. Specification and Error Pattern Based Program Monitoring

    NASA Technical Reports Server (NTRS)

    Havelund, Klaus; Johnson, Scott; Rosu, Grigore; Clancy, Daniel (Technical Monitor)

    2001-01-01

    We briefly present Java PathExplorer (JPAX), a tool developed at NASA Ames for monitoring the execution of Java programs. JPAX can be used not only during program testing to reveal subtle errors, but also can be applied during operation to survey safety critical systems. The tool facilitates automated instrumentation of a program in order to properly observe its execution. The instrumentation can be either at the bytecode level or at the source level when the source code is available. JPaX is an instance of a more general project, called PathExplorer (PAX), which is a basis for experiments rather than a fixed system, capable of monitoring various programming languages and experimenting with other logics and analysis techniques

  10. Biovar-specific epitopes of the urease enzyme of Ureaplasma urealyticum.

    PubMed

    MacKenzie, C R; Henrich, B; Hadding, U

    1996-11-01

    The importance of Ureaplasma urealyticum as a pathogen in premature neonates and patients with a profound defect in humoral immunity has, over the last few years, become well recognised. U. urealyticum is unique amongst the Mycoplasmataceae for its use of urea metabolism as an essential source of energy. The urease enzyme responsible for this is, therefore, of prime importance and any variability in expression of this enzyme may play a role in virulence of the organism. U. urealyticum is divided into 14 serovars comprising two biovars -- the parvo-biovar and T960-biovar. In this study monoclonal antibodies (MAbs) were produced against the urease enzyme. Two distinct epitopes of the 72-kDa alpha-subunit were recognised by three different MAbs. Under denaturing conditions both epitopes were shown to be specific for the parvo-biovar.

  11. SigrafW: An Easy-to-Use Program for Fitting Enzyme Kinetic Data

    ERIC Educational Resources Information Center

    Leone, Francisco Assis; Baranauskas, Jose Augusto; Furriel, Rosa Prazeres Melo; Borin, Ivana Aparecida

    2005-01-01

    SigrafW is Windows-compatible software developed using the Microsoft[R] Visual Basic Studio program that uses the simplified Hill equation for fitting kinetic data from allosteric and Michaelian enzymes. SigrafW uses a modified Fibonacci search to calculate maximal velocity (V), the Hill coefficient (n), and the enzyme-substrate apparent…

  12. Positive dermal hypersensitivity and specific antibodies in workers exposed to bio-engineered enzymes

    SciTech Connect

    Biagini, R.E.; Henningsen, G.M.; Driscoll, R.; MacKenzie, B.A.; Wilcox, T.; Scinto, J.D.; Bernstein, D.M.; Swanson, M. Mayo Clinic, Rochester, MN )

    1991-03-15

    Thirty-six employees who produced industrial enzymes from bio-engineered strains of bacteria and fungi were evaluated by skin prick testing and enzyme linked immunosorbent assays for specific IgE and IgG antibodies. The workers complained of asthma- and flu-like' symptoms which generally lessened away from work. The enzymes evaluated were {alpha}-amylase from A. niger (ind-AAN), B. licheniformis (ind-AAL) and B. subtilis (ind-AAS); purified {alpha}-amylase from B. subtilis (AAS) and A. niger (AAN); alkaline protease from B. licheniformis (ind-APL) and purified alkaline protease (APL); amylase glucosidase from A. niger (ind-AGN) and purified amylase glucosidase (AGN). Significantly positive skin tests were found for APL, AGN and ind-AAN. Significantly elevated specific IgE results were observed for AAN, AGN, and ind-AAN; elevated specific IgGs were observed for AAN, ind-AAN, ind-AAS, ind-AAL and ind-AGN. Radioimmunoassays of air filter samples (using sera with high Ab titers) for 4 of the ind-enzymes showed only ind-AAN at extremely high environmental levels. These results indicate that occupational exposure to some ind-enzymes causes immediate onset dermal hypersensitivity reactions. The results are equivocal as to whether these reactions are IgE mediated, as IgE titers were low. Contrary to this, IgG titers were extremely high and suggest that these biomarkers can be used as indicators of both individual exposure and environmental analyses.

  13. Substrate specificities of hybrid naphthalene and 2,4-dinitrotoluene dioxygenase enzyme systems.

    PubMed

    Parales, R E; Emig, M D; Lynch, N A; Gibson, D T

    1998-05-01

    Bacterial three-component dioxygenase systems consist of reductase and ferredoxin components which transfer electrons from NAD(P)H to a terminal oxygenase. In most cases, the oxygenase consists of two different subunits (alpha and beta). To assess the contributions of the alpha and beta subunits of the oxygenase to substrate specificity, hybrid dioxygenase enzymes were formed by coexpressing genes from two compatible plasmids in Escherichia coli. The activities of hybrid naphthalene and 2,4-dinitrotoluene dioxygenases containing four different beta subunits were tested with four substrates (indole, naphthalene, 2,4-dinitrotoluene, and 2-nitrotoluene). In the active hybrids, replacement of small subunits affected the rate of product formation but had no effect on the substrate range, regiospecificity, or enantiomeric purity of oxidation products with the substrates tested. These studies indicate that the small subunit of the oxygenase is essential for activity but does not play a major role in determining the specificity of these enzymes.

  14. Specifications and programs for computer software validation

    NASA Technical Reports Server (NTRS)

    Browne, J. C.; Kleir, R.; Davis, T.; Henneman, M.; Haller, A.; Lasseter, G. L.

    1973-01-01

    Three software products developed during the study are reported and include: (1) FORTRAN Automatic Code Evaluation System, (2) the Specification Language System, and (3) the Array Index Validation System.

  15. Specific detection of the cleavage activity of mycobacterial enzymes using a quantum dot based DNA nanosensor

    NASA Astrophysics Data System (ADS)

    Jepsen, Morten Leth; Harmsen, Charlotte; Godbole, Adwait Anand; Nagaraja, Valakunja; Knudsen, Birgitta R.; Ho, Yi-Ping

    2015-12-01

    We present a quantum dot based DNA nanosensor specifically targeting the cleavage step in the reaction cycle of the essential DNA-modifying enzyme, mycobacterial topoisomerase I. The design takes advantages of the unique photophysical properties of quantum dots to generate visible fluorescence recovery upon specific cleavage by mycobacterial topoisomerase I. This report, for the first time, demonstrates the possibility to quantify the cleavage activity of the mycobacterial enzyme without the pre-processing sample purification or post-processing signal amplification. The cleavage induced signal response has also proven reliable in biological matrices, such as whole cell extracts prepared from Escherichia coli and human Caco-2 cells. It is expected that the assay may contribute to the clinical diagnostics of bacterial diseases, as well as the evaluation of treatment outcomes.We present a quantum dot based DNA nanosensor specifically targeting the cleavage step in the reaction cycle of the essential DNA-modifying enzyme, mycobacterial topoisomerase I. The design takes advantages of the unique photophysical properties of quantum dots to generate visible fluorescence recovery upon specific cleavage by mycobacterial topoisomerase I. This report, for the first time, demonstrates the possibility to quantify the cleavage activity of the mycobacterial enzyme without the pre-processing sample purification or post-processing signal amplification. The cleavage induced signal response has also proven reliable in biological matrices, such as whole cell extracts prepared from Escherichia coli and human Caco-2 cells. It is expected that the assay may contribute to the clinical diagnostics of bacterial diseases, as well as the evaluation of treatment outcomes. Electronic supplementary information (ESI) available: Characterization of the QD-based DNA Nanosensor. See DOI: 10.1039/c5nr06326d

  16. Evolution of substrate specificity in a recipient's enzyme following horizontal gene transfer.

    PubMed

    Noda-García, Lianet; Camacho-Zarco, Aldo R; Medina-Ruíz, Sofía; Gaytán, Paul; Carrillo-Tripp, Mauricio; Fülöp, Vilmos; Barona-Gómez, Francisco

    2013-09-01

    Despite the prominent role of horizontal gene transfer (HGT) in shaping bacterial metabolism, little is known about the impact of HGT on the evolution of enzyme function. Specifically, what is the influence of a recently acquired gene on the function of an existing gene? For example, certain members of the genus Corynebacterium have horizontally acquired a whole l-tryptophan biosynthetic operon, whereas in certain closely related actinobacteria, for example, Mycobacterium, the trpF gene is missing. In Mycobacterium, the function of the trpF gene is performed by a dual-substrate (βα)8 phosphoribosyl isomerase (priA gene) also involved in l-histidine (hisA gene) biosynthesis. We investigated the effect of a HGT-acquired TrpF enzyme upon PriA's substrate specificity in Corynebacterium through comparative genomics and phylogenetic reconstructions. After comprehensive in vivo and enzyme kinetic analyses of selected PriA homologs, a novel (βα)8 isomerase subfamily with a specialized function in l-histidine biosynthesis, termed subHisA, was confirmed. X-ray crystallography was used to reveal active-site mutations in subHisA important for narrowing of substrate specificity, which when mutated to the naturally occurring amino acid in PriA led to gain of function. Moreover, in silico molecular dynamic analyses demonstrated that the narrowing of substrate specificity of subHisA is concomitant with loss of ancestral protein conformational states. Our results show the importance of HGT in shaping enzyme evolution and metabolism.

  17. Specific xyloglucanases as a new class of polysaccharide-degrading enzymes.

    PubMed

    Grishutin, Sergey G; Gusakov, Alexander V; Markov, Alexander V; Ustinov, Boris B; Semenova, Margarita V; Sinitsyn, Arkady P

    2004-11-01

    Three specific xyloglucanases (XGs) were isolated from Aspergillus japonicus (32 kDa, pI 2.8), Chrysosporium lucknowense (78 kDa, pI 3.8) and Trichoderma reesei (75-105 kDa, pI 4.1-4.3). The characteristic feature of these enzymes was their high specific activity toward tamarind xyloglucan, whereas the activity against carboxymethylcellulose (CMC) and barley beta-glucan was absent or very low. Peptide mass fingerprinting using MALDI-TOF mass spectrometry showed that the T. reesei XG represents Cel74A, whose gene has been discovered recently (GenBank accession no. AY281371 ), but the enzyme has not been characterized and described elsewhere. Tryptic peptides from A. japonicus and C. lucknowense xyloglucanases did not show any identity to those from known glycoside hydrolases. All enzymes produced XXXG, XXLG/XLXG and XLLG oligosaccharides as the end products of xyloglucan hydrolysis. A. japonicus XG displayed an endo-type of attack on the polymeric substrate, while the mode of action of two other xyloglucanases was similar to the exo-type, when oligosaccharides containing four glucose residues in the main chain were split off the ends of xyloglucan molecules. These results together with growing literature data allow concluding that specific xyloglucanases may represent a new class of glycoside hydrolases, which are different from regular endo-1,4-beta-glucanases.

  18. Demonstration of specific neuronal cell groups in rat brain by beta-galactosidase enzyme histochemistry.

    PubMed

    Hatton, J D; Lin, L

    1992-12-01

    beta-Galactosidase activity as illuminated by the indigogenic X-gal staining method has been used to demonstrate the presence of genetically modified cells carrying the reporter gene lacZ, coding for the E. coli enzyme. Endogenous activity has been assumed to be minimal since the pH optimum for the mammalian enzyme is 3.5-5.5, while the pH optimum for the E. coli enzyme (and thus of the staining procedure usually employed) is 7.3. Background staining has been reported to be limited to pericytes and a few specific neuronal cell groups. In contrast, our investigations of normal rat brain anatomy demonstrate that many specific neuronal cell groups possess endogenous beta-galactosidase activity when staining is performed at physiological pH. This suggests that background staining of endogenous beta-galactosidase activity in the rat brain has been underestimated. In addition, such specific activity would afford an additional means of identification and illustration of these cells.

  19. Prediction and experimental validation of enzyme substrate specificity in protein structures.

    PubMed

    Amin, Shivas R; Erdin, Serkan; Ward, R Matthew; Lua, Rhonald C; Lichtarge, Olivier

    2013-11-05

    Structural Genomics aims to elucidate protein structures to identify their functions. Unfortunately, the variation of just a few residues can be enough to alter activity or binding specificity and limit the functional resolution of annotations based on sequence and structure; in enzymes, substrates are especially difficult to predict. Here, large-scale controls and direct experiments show that the local similarity of five or six residues selected because they are evolutionarily important and on the protein surface can suffice to identify an enzyme activity and substrate. A motif of five residues predicted that a previously uncharacterized Silicibacter sp. protein was a carboxylesterase for short fatty acyl chains, similar to hormone-sensitive-lipase-like proteins that share less than 20% sequence identity. Assays and directed mutations confirmed this activity and showed that the motif was essential for catalysis and substrate specificity. We conclude that evolutionary and structural information may be combined on a Structural Genomics scale to create motifs of mixed catalytic and noncatalytic residues that identify enzyme activity and substrate specificity.

  20. Prediction and experimental validation of enzyme substrate specificity in protein structures

    PubMed Central

    Amin, Shivas R.; Erdin, Serkan; Ward, R. Matthew; Lua, Rhonald C.; Lichtarge, Olivier

    2013-01-01

    Structural Genomics aims to elucidate protein structures to identify their functions. Unfortunately, the variation of just a few residues can be enough to alter activity or binding specificity and limit the functional resolution of annotations based on sequence and structure; in enzymes, substrates are especially difficult to predict. Here, large-scale controls and direct experiments show that the local similarity of five or six residues selected because they are evolutionarily important and on the protein surface can suffice to identify an enzyme activity and substrate. A motif of five residues predicted that a previously uncharacterized Silicibacter sp. protein was a carboxylesterase for short fatty acyl chains, similar to hormone-sensitive-lipase–like proteins that share less than 20% sequence identity. Assays and directed mutations confirmed this activity and showed that the motif was essential for catalysis and substrate specificity. We conclude that evolutionary and structural information may be combined on a Structural Genomics scale to create motifs of mixed catalytic and noncatalytic residues that identify enzyme activity and substrate specificity. PMID:24145433

  1. Sex-specificity in transgenerational epigenetic programming.

    PubMed

    Dunn, Gregory A; Morgan, Christopher P; Bale, Tracy L

    2011-03-01

    Prenatal programming of the epigenome is a critical determinant in offspring outcome and stands at the interface between environment and genetics. Maternal experiences such as stress and obesity are associated with a host of neurodevelopmental and metabolic diseases, some of which have been characterized into the second and third generations. The mechanism through which determinants such as maternal diet or stress contribute to disease development likely involves a complex interaction between the maternal environment, placental changes, and epigenetic programming of the embryo. While we have begun to more fully appreciate and explore the epigenome in determination of disease risk, we know little as to the contribution embryo sex makes in epigenetic regulation. This review discusses the importance of sex differences in the transmission and inheritance of traits that are generated in the prenatal environment using models of maternal stress and diet.

  2. Structure of a Berberine Bridge Enzyme-Like Enzyme with an Active Site Specific to the Plant Family Brassicaceae

    PubMed Central

    Daniel, Bastian; Wallner, Silvia; Steiner, Barbara; Oberdorfer, Gustav; Kumar, Prashant; van der Graaff, Eric; Roitsch, Thomas; Sensen, Christoph W.; Gruber, Karl; Macheroux, Peter

    2016-01-01

    Berberine bridge enzyme-like (BBE-like) proteins form a multigene family (pfam 08031), which is present in plants, fungi and bacteria. They adopt the vanillyl alcohol-oxidase fold and predominantly show bi-covalent tethering of the FAD cofactor to a cysteine and histidine residue, respectively. The Arabidopsis thaliana genome was recently shown to contain genes coding for 28 BBE-like proteins, while featuring four distinct active site compositions. We determined the structure of a member of the AtBBE-like protein family (termed AtBBE-like 28), which has an active site composition that has not been structurally and biochemically characterized thus far. The most salient and distinguishing features of the active site found in AtBBE-like 28 are a mono-covalent linkage of a histidine to the 8α-position of the flavin-isoalloxazine ring and the lack of a second covalent linkage to the 6-position, owing to the replacement of a cysteine with a histidine. In addition, the structure reveals the interaction of a glutamic acid (Glu426) with an aspartic acid (Asp369) at the active site, which appear to share a proton. This arrangement leads to the delocalization of a negative charge at the active site that may be exploited for catalysis. The structure also indicates a shift of the position of the isoalloxazine ring in comparison to other members of the BBE-like family. The dioxygen surrogate chloride was found near the C(4a) position of the isoalloxazine ring in the oxygen pocket, pointing to a rapid reoxidation of reduced enzyme by dioxygen. A T-DNA insertional mutant line for AtBBE-like 28 results in a phenotype, that is characterized by reduced biomass and lower salt stress tolerance. Multiple sequence analysis showed that the active site composition found in AtBBE-like 28 is only present in the Brassicaceae, suggesting that it plays a specific role in the metabolism of this plant family. PMID:27276217

  3. Gamma-Glutamyl Compounds: Substrate Specificity of Gamma-Glutamyl Transpeptidase Enzymes

    PubMed Central

    Wickham, Stephanie; West, Matthew B.; Cook, Paul F.; Hanigan, Marie H.

    2011-01-01

    Gamma-glutamyl compounds include antioxidants, inflammatory molecules, drug metabolites and neuroactive compounds. Two cell surface enzymes have been identified that metabolize gamma-glutamyl compounds, gamma-glutamyl transpeptidase (GGT1) and gamma-glutamyl leukotrienase (GGT5). There is controversy in the literature regarding the substrate specificity of these enzymes. To address this issue, we have developed a method for comprehensive kinetics analysis of compounds as substrates for GGT enzymes. Our assay is sensitive, quantitative and is conducted at physiologic pH. We evaluated a series of gamma-glutamyl compounds as substrates for human GGT1 and human GGT5. The Kms for reduced glutathione were 11μM for both GGT1 and GGT5. However, the Km for oxidized glutathione was 9μM for GGT1 and 43μM for GGT5. Our data show that the Kms for leukotriene C4 are equivalent for GGT1 and GGT5 at 10.8μM and 10.2μM, respectively. This assay was also used to evaluate serine-borate, a well-known inhibitor of GGT1, which was 8-fold more potent in inhibiting GGT1 than inhibiting GGT5. These data provide essential information regarding the target enzymes for developing treatments for inflammatory diseases such as asthma and cardiovascular disease in humans. This assay is invaluable for studies of oxidative stress, drug metabolism and other pathways that involve gamma-glutamyl compounds. PMID:21447318

  4. Quantitative Site-Specific Phosphoproteomics of Trichoderma reesei Signaling Pathways upon Induction of Hydrolytic Enzyme Production.

    PubMed

    Nguyen, Elizabeth V; Imanishi, Susumu Y; Haapaniemi, Pekka; Yadav, Avinash; Saloheimo, Markku; Corthals, Garry L; Pakula, Tiina M

    2016-02-05

    The filamentous fungus Trichoderma reesei is used for industrial production of secreted enzymes including carbohydrate active enzymes, such as cellulases and hemicellulases. The production of many of these enzymes by T. reesei is influenced by the carbon source it grows on, where the regulation system controlling hydrolase genes involves various signaling pathways. T. reesei was cultivated in the presence of sorbitol, a carbon source that does not induce the production of cellulases and hemicellulases, and then exposed to either sophorose or spent-grain extract, which are efficient inducers of the enzyme production. Specific changes at phosphorylation sites were investigated in relation to the production of cellulases and hemicellulases using an MS-based framework. Proteome-wide phosphorylation following carbon source exchange was investigated in the early stages of induction: 0, 2, 5, and 10 min. The workflow involved sequential trypsin digestion, TiO2 enrichment, and MS analysis using a Q Exactive mass spectrometer. We report on the identification and quantitation of 1721 phosphorylation sites. Investigation of the data revealed a complex signaling network activated upon induction involving components related to light-mediated cellulase induction, osmoregulation, and carbon sensing. Changes in protein phosphorylation were detected in the glycolytic pathway, suggesting an inhibition of glucose catabolism at 10 min after the addition of sophorose and as early as 2 min after the addition of spent-grain extract. Differential phosphorylation of factors related to carbon storage, intracellular trafficking, cytoskeleton, and cellulase gene regulation were also observed.

  5. Induction Specificity and Catabolite Repression of the Early Enzymes in Camphor Degradation by Pseudomonas putida

    PubMed Central

    Hartline, Richard A.; Gunsalus, I. C.

    1971-01-01

    The ability of bornane and substituted bornanes to induce the early enzymes for d(+)-camphor degradation and control of these enzymes by catabolite repression were studied in a strain of a Pseudomonas putida. Bornane and 20 substituted bornane compounds showed induction. Of these 21 compounds, bornane and 8 of the substituted bornanes provided induction without supporting growth. Oxygen, but not nitrogen, enhanced the inductive potency of the unsubstituted bornane ring. All bornanedione isomers caused induction, and those with substituents on each of the three consecutive carbon atoms, including the methyl group at the bridgehead carbon, showed induction without supporting growth. Although it was not possible to obtain experimental data for a case of absolute gratuitous induction by compounds not supporting growth, indirect evidence in support of gratuitous induction is presented. It is proposed that the ability of P. putida to tolerate the unusually high degree of possible gratuitous induction observed for camphor catabolism may be related to the infrequent occurrence of bicyclic ring structures in nature. Survival of an organism with a broad specificity for gratuitous induction is discussed. Glucose and succinate, but not glutamate, produced catabolite repression of the early camphor-degrading enzymes. Pathway enzymes differ in their degree of sensitivity to succinate-provoked catabolite repression. The ability of a compound to produce catabolite repression is not, however, directly related to the duration of the lag period (diauxic lag) between growth on camphor and growth on the repressing compound. PMID:5573731

  6. Generating Data Flow Programs from Nonprocedural Specifications.

    DTIC Science & Technology

    1983-03-01

    Program Office of Naval Research Under Contract N00014-76-C-0416 DTIC Moore School Report ^ LECTE - S SEP 191983J D Aprow foIu~ 00k Dfat ..bu.. U...single proqram counter. Possible parallelism may be inferred to a limited extent from evaluation of the algorithm. However since wamory cells can hold...addresses . Inherent in the conventional model is the idea of an address [Arvi7S]. The address of a memory cell is invariant, while the value stored in the

  7. Broad specification fuels technology program, phase 1

    NASA Technical Reports Server (NTRS)

    Lohmann, R. P.; Jeroszko, R. A.

    1982-01-01

    An experimental evaluation was conducted to assess the impact of the use of broadened properties fuels on combustor design concepts. Emphasis was placed on establishing the viability of design modifications to current combustor concepts and the use of advanced technology concepts to facilitate operation on Experimental Referee Broad Specification (ERBS) fuel while meeting exhaust emissions and performance specifications and maintaining acceptable durability. Three different combustor concepts, representative of progressively more aggressive technology levels, were evaluated. When operated on ERBS rather than Jet A fuel, a single stage combustor typical of that in the most recent versions of the JT9D-7 engine was found to produce excess carbon monoxide emissions at idle and elevated liner temperatures at high power levels that were projected to reduced liner life by 13 percent. The introduction of improved component technology, such as refined fuel injectors and advanced liner cooling concepts were shown to have the potential of enhancing the fuel flexibility of the single stage combustor.

  8. Development of a Program Specific Locator Test. Final Report.

    ERIC Educational Resources Information Center

    Benn, Robert J.

    A project was undertaken to develop a series of program-specific vocational locator tests (PSVLTs) that would consist of subject-specific questions in three academic disciplines--writing, reading, and mathematics--for use in predicting vocational students' success in their vocational programs. As a prelude to constructing the tests, project staff…

  9. 42 CFR 457.1180 - Program specific review process: Notice.

    Code of Federal Regulations, 2010 CFR

    2010-10-01

    ... 42 Public Health 4 2010-10-01 2010-10-01 false Program specific review process: Notice. 457.1180... State Plan Requirements: Applicant and Enrollee Protections § 457.1180 Program specific review process: Notice. A State must provide enrollees and applicants timely written notice of any...

  10. Compartment-specific Control of Reactive Oxygen Species Scavenging by Antioxidant Pathway Enzymes.

    PubMed

    Dey, Swati; Sidor, Agnieszka; O'Rourke, Brian

    2016-05-20

    Oxidative stress arises from an imbalance in the production and scavenging rates of reactive oxygen species (ROS) and is a key factor in the pathophysiology of cardiovascular disease and aging. The presence of parallel pathways and multiple intracellular compartments, each having its own ROS sources and antioxidant enzymes, complicates the determination of the most important regulatory nodes of the redox network. Here we quantified ROS dynamics within specific intracellular compartments in the cytosol and mitochondria and determined which scavenging enzymes exert the most control over antioxidant fluxes in H9c2 cardiac myoblasts. We used novel targeted viral gene transfer vectors expressing redox-sensitive GFP fused to sensor domains to measure H2O2 or oxidized glutathione. Using genetic manipulation in heart-derived H9c2 cells, we explored the contribution of specific antioxidant enzymes to ROS scavenging and glutathione redox potential within each intracellular compartment. Our findings reveal that antioxidant flux is strongly dependent on mitochondrial substrate catabolism, with availability of NADPH as a major rate-controlling step. Moreover, ROS scavenging by mitochondria significantly contributes to cytoplasmic ROS handling. The findings provide fundamental information about the control of ROS scavenging by the redox network and suggest novel interventions for circumventing oxidative stress in cardiac cells.

  11. Carotenoids in Rhodoplanes species: variation of compositions and substrate specificity of predicted carotenogenesis enzymes.

    PubMed

    Takaichi, Shinichi; Sasikala, Ch; Ramana, Ch V; Okamura, Keiko; Hiraishi, Akira

    2012-08-01

    Phototrophic bacteria necessarily contain carotenoids for photosynthesis, and accumulate unusual carotenoids in some cases. The carotenoids in all established species of Rhodoplanes (Rpl.), a representative of phototrophic genera, were identified using spectroscopic methods. The major carotenoid was spirilloxanthin in Rpl. roseus and Rpl. serenus, and rhodopin in "Rpl. cryptolactis". Rpl. elegans contained rhodopin, anhydrorhodovibrin, and spirilloxanthin. Rpl. pokkaliisoli contained not only rhodopin but also 1,1'-dihydroxylycopene and 3,4,3',4'-tetrahydrospirilloxanthin. These variations in carotenoid composition suggested that Rpl. roseus and Rpl. serenus had normal substrate specificity of the carotenogenesis enzymes of CrtC (acyclic carotene 1,2-hydratase), CrtD (acyclic carotenoid 3,4-desaturase), and CrtF (acyclic 1-hydroxycarotenoid methyltransferase). On the other hand, CrtC of Rpl. elegans, CrtD of "Rpl. cryptolactis", and CrtC, CrtD, and CrtF of Rpl. pokkaliisoli might have different characteristics from the usual activity of these normal enzymes in the normal spirilloxanthin pathway. These results suggest that the variation of carotenoids among the species of Rhodoplanes results from modified substrate specificity of the carotenogenesis enzymes involved.

  12. Switching of self-assembly in a peptide nanostructure with a specific enzyme

    SciTech Connect

    Webber, Matthew J.; Newcomb, Christina J.; Bitton, Ronit; Stupp, Samuel I.

    2012-03-14

    Peptide self-assembly has been shown to be a useful tool for the preparation of bioactive nanostructures, and recent work has demonstrated their potential as therapies for regenerative medicine. In principle, one route to make these nanostructures more biomimetic would be to incorporate in their molecular design the capacity for biological sensing. We report here on the use of a reversible enzymatic trigger to control the assembly and disassembly of peptide amphiphile (PA) nanostructures. The PA used in these studies contained a consensus substrate sequence specific to protein kinase A (PKA), a biological enzyme important for intracellular signaling that has also been shown to be an extracellular cancer biomarker. Upon treatment with PKA, this PA molecule becomes phosphorylated causing the high aspect-ratio filamentous PA nanostructures to disassemble. Treatment with an enzyme to cleave the phosphate group results in reformation of the filamentous nanostructures. We also show that disassembly in the presence of PKA allows the enzyme-triggered release of an encapsulated cancer drug. In addition, these drug-loaded nanostructures were found to induce preferential cytotoxicity in a cancer cell line that is known to secrete high levels of PKA. This ability to control nanostructure through an enzymatic switch could allow for the preparation of highly sophisticated and biomimetic materials that incorporate a biological sensing capability to enable therapeutic specificity.

  13. The four aldehyde oxidases of Drosophila melanogaster have different gene expression patterns and enzyme substrate specificities.

    PubMed

    Marelja, Zvonimir; Dambowsky, Miriam; Bolis, Marco; Georgiou, Marina L; Garattini, Enrico; Missirlis, Fanis; Leimkühler, Silke

    2014-06-15

    In the genome of Drosophila melanogaster, four genes coding for aldehyde oxidases (AOX1-4) were identified on chromosome 3. Phylogenetic analysis showed that the AOX gene cluster evolved via independent duplication events in the vertebrate and invertebrate lineages. The functional role and the substrate specificity of the distinct Drosophila AOX enzymes is unknown. Two loss-of-function mutant alleles in this gene region, low pyridoxal oxidase (Po(lpo)) and aldehyde oxidase-1 (Aldox-1(n1)) are associated with a phenotype characterized by undetectable AOX enzymatic activity. However, the genes involved and the corresponding mutations have not yet been identified. In this study we characterized the activities, substrate specificities and expression profiles of the four AOX enzymes in D. melanogaster. We show that the Po(lpo)-associated phenotype is the consequence of a structural alteration of the AOX1 gene. We identified an 11-bp deletion in the Po(lpo) allele, resulting in a frame-shift event, which removes the molybdenum cofactor domain of the encoded enzyme. Furthermore, we show that AOX2 activity is detectable only during metamorphosis and characterize a Minos-AOX2 insertion in this developmental gene that disrupts its activity. We demonstrate that the Aldox-1(n1) phenotype maps to the AOX3 gene and AOX4 activity is not detectable in our assays.

  14. Genome-Scale Analysis of Cell-Specific Regulatory Codes Using Nuclear Enzymes.

    PubMed

    Baek, Songjoon; Sung, Myong-Hee

    2016-01-01

    High-throughput sequencing technologies have made it possible for biologists to generate genome-wide profiles of chromatin features at the nucleotide resolution. Enzymes such as nucleases or transposes have been instrumental as a chromatin-probing agent due to their ability to target accessible chromatin for cleavage or insertion. On the scale of a few hundred base pairs, preferential action of the nuclear enzymes on accessible chromatin allows mapping of cell state-specific accessibility in vivo. Such accessible regions contain functionally important regulatory sites, including promoters and enhancers, which undergo active remodeling for cells adapting in a dynamic environment. DNase-seq and the more recent ATAC-seq are two assays that are gaining popularity. Deep sequencing of DNA libraries from these assays, termed genomic footprinting, has been proposed to enable the comprehensive construction of protein occupancy profiles over the genome at the nucleotide level. Recent studies have discovered limitations of genomic footprinting which reduce the scope of detectable proteins. In addition, the identification of putative factors that bind to the observed footprints remains challenging. Despite these caveats, the methodology still presents significant advantages over alternative techniques such as ChIP-seq or FAIRE-seq. Here we describe computational approaches and tools for analysis of chromatin accessibility and genomic footprinting. Proper experimental design and assay-specific data analysis ensure the detection sensitivity and maximize retrievable information. The enzyme-based chromatin profiling approaches represent a powerful and evolving methodology which facilitates our understanding of how the genome is regulated.

  15. The four aldehyde oxidases of Drosophila melanogaster have different gene expression patterns and enzyme substrate specificities

    PubMed Central

    Marelja, Zvonimir; Dambowsky, Miriam; Bolis, Marco; Georgiou, Marina L.; Garattini, Enrico; Missirlis, Fanis; Leimkühler, Silke

    2014-01-01

    In the genome of Drosophila melanogaster, four genes coding for aldehyde oxidases (AOX1–4) were identified on chromosome 3. Phylogenetic analysis showed that the AOX gene cluster evolved via independent duplication events in the vertebrate and invertebrate lineages. The functional role and the substrate specificity of the distinct Drosophila AOX enzymes is unknown. Two loss-of-function mutant alleles in this gene region, low pyridoxal oxidase (Polpo) and aldehyde oxidase-1 (Aldox-1n1) are associated with a phenotype characterized by undetectable AOX enzymatic activity. However, the genes involved and the corresponding mutations have not yet been identified. In this study we characterized the activities, substrate specificities and expression profiles of the four AOX enzymes in D. melanogaster. We show that the Polpo-associated phenotype is the consequence of a structural alteration of the AOX1 gene. We identified an 11-bp deletion in the Polpo allele, resulting in a frame-shift event, which removes the molybdenum cofactor domain of the encoded enzyme. Furthermore, we show that AOX2 activity is detectable only during metamorphosis and characterize a Minos-AOX2 insertion in this developmental gene that disrupts its activity. We demonstrate that the Aldox-1n1 phenotype maps to the AOX3 gene and AOX4 activity is not detectable in our assays. PMID:24737760

  16. Evolution of substrate specificity in a retained enzyme driven by gene loss.

    PubMed

    Juárez-Vázquez, Ana Lilia; Edirisinghe, Janaka E; Verduzco-Castro, Ernesto A; Michalska, Karolina; Wu, Chenggang; Noda-García, Lianet; Babnigg, Gyorgy; Endres, Michael; Medina-Ruíz, Sofía; Santoyo-Flores, Julián; Carrillo-Tripp, Mauricio; Ton-That, Hung; Joachimiak, Andrzej; Henry, Christopher S; Barona-Gómez, Francisco

    2017-03-31

    The connection between gene loss and the functional adaptation of retained proteins is still poorly understood. We apply phylogenomics and metabolic modeling to detect bacterial species that are evolving by gene loss, with the finding that Actinomycetaceae genomes from human cavities are undergoing sizable reductions, including loss of L-histidine and L-tryptophan biosynthesis. We observe that the dual-substrate phosphoribosyl isomerase A or priA gene, at which these pathways converge, appears to coevolve with the occurrence of trp and his genes. Characterization of a dozen PriA homologs shows that these enzymes adapt from bifunctionality in the largest genomes, to a monofunctional, yet not necessarily specialized, inefficient form in genomes undergoing reduction. These functional changes are accomplished via mutations, which result from relaxation of purifying selection, in residues structurally mapped after sequence and X-ray structural analyses. Our results show how gene loss can drive the evolution of substrate specificity from retained enzymes.

  17. Nanobiosensors exploiting specific interactions between an enzyme and herbicides in atomic force spectroscopy.

    PubMed

    da Silva, Aline C N; Deda, Daiana K; Bueno, Carolina C; Moraes, Ariana S; Da Roz, Alessandra L; Yamaji, Fabio M; Prado, Rogilene A; Viviani, Vadim; Oliveira, Osvaldo N; Leite, Fábio L

    2014-09-01

    The development of sensitive methodologies for detecting agrochemicals has become important in recent years due to the increasingly indiscriminate use of these substances. In this context, nanosensors based on atomic force microscopy (AFM) tips are useful because they provide higher sensitivity with operation at the nanometer scale. In this paper we exploit specific interactions between AFM tips functionalized with the enzyme acetolactate synthase (ALS) to detect the ALS-inhibitor herbicides metsulfuron-methyl and imazaquin. Using atomic force spectroscopy (AFS) we could measure the adhesion force between tip and substrate, which was considerably higher when the ALS-functionalized tip (nanobiosensor) was employed. The increase was approximately 250% and 160% for metsulfuron-methyl and imazaquin, respectively, in comparison to unfunctionalized probes. We estimated the specific enzyme-herbicide force by assuming that the measured force comprises an adhesion force according to the Johnson-Kendall-Roberts (JKR) model, the capillary force and the specific force. We show that the specific, biorecognition force plays a crucial role in the higher sensitivity of the nanobiosensor, thus opening the way for the design of similarly engineered tips for detecting herbicides and other analytes.

  18. Formation of target-specific binding sites in enzymes: solid-phase molecular imprinting of HRP

    NASA Astrophysics Data System (ADS)

    Czulak, J.; Guerreiro, A.; Metran, K.; Canfarotta, F.; Goddard, A.; Cowan, R. H.; Trochimczuk, A. W.; Piletsky, S.

    2016-05-01

    Here we introduce a new concept for synthesising molecularly imprinted nanoparticles by using proteins as macro-functional monomers. For a proof-of-concept, a model enzyme (HRP) was cross-linked using glutaraldehyde in the presence of glass beads (solid-phase) bearing immobilized templates such as vancomycin and ampicillin. The cross-linking process links together proteins and protein chains, which in the presence of templates leads to the formation of permanent target-specific recognition sites without adverse effects on the enzymatic activity. Unlike complex protein engineering approaches commonly employed to generate affinity proteins, the method proposed can be used to produce protein-based ligands in a short time period using native protein molecules. These affinity materials are potentially useful tools especially for assays since they combine the catalytic properties of enzymes (for signaling) and molecular recognition properties of antibodies. We demonstrate this concept in an ELISA-format assay where HRP imprinted with vancomycin and ampicillin replaced traditional enzyme-antibody conjugates for selective detection of templates at micromolar concentrations. This approach can potentially provide a fast alternative to raising antibodies for targets that do not require high assay sensitivities; it can also find uses as a biochemical research tool, as a possible replacement for immunoperoxidase-conjugates.Here we introduce a new concept for synthesising molecularly imprinted nanoparticles by using proteins as macro-functional monomers. For a proof-of-concept, a model enzyme (HRP) was cross-linked using glutaraldehyde in the presence of glass beads (solid-phase) bearing immobilized templates such as vancomycin and ampicillin. The cross-linking process links together proteins and protein chains, which in the presence of templates leads to the formation of permanent target-specific recognition sites without adverse effects on the enzymatic activity. Unlike

  19. Development of a highly specific enzyme immunoassay for oxytocin and its use in plasma samples.

    PubMed

    Haraya, Shiomi; Karasawa, Koji; Sano, Yoshihiro; Ozawa, Kimiko; Kato, Nobumasa; Arakawa, Hidetoshi

    2017-01-01

    Background The peptide hormone oxytocin acts in the central nervous system and plays an important role in various complex social behaviours. We report the production of a high affinity and specificity antibody for oxytocin and its use in a highly sensitive enzyme immunoassay. Biotin that was chemically bound to oxytocin derivative containing zero to six lysines as bridge was the labelled antigen. Seven labelled antigens were used to develop a highly sensitive enzyme immunoassay. Methods Antioxytocin antiserum was obtained by immunization of oxytocin-bovine thyrogloblin conjugate to rabbit. Oxytocin sample was added to the second antibody-coated microtitre plate and allowed to react overnight at 4℃, then biotinylated oxytocin was added 1 h at 4℃, and horseradish peroxidase-labelled avidin was added and incubated for 1 h at room temperature. The plate was then washed. Horseradish peroxidase activity was measured by a colorimetric method using o-phenylenediamine (490 nm). Results The sensitivity of the enzyme immunoassay improved as the number of lysine residues increased; consequently, biotinylated oxytocin bridged with five lysines was used. A standard curve for oxytocin ranged from 1.0 to 1000 pg/assay. The detection limit of the assay was 2.36 pg, and the reproducibility was 3.6% as CV% ( n = 6). Cross-reactivity with vasopressin and vasotocin was less than 0.01%. Conclusion The sensitivity of the enzyme immunoassay could be improved by increasing the number of lysine residues on the biotin-labelled antigen. The proposed method is sensitive and more specific than conventional immunoassays for oxytocin and can be used to determine plasma oxytocin concentrations.

  20. Development of Diubiquitin‐Based FRET Probes To Quantify Ubiquitin Linkage Specificity of Deubiquitinating Enzymes

    PubMed Central

    van Tol, Bianca D. M.; van Dalen, Duco; Brundel, Paul J. G.; Mevissen, Tycho E. T.; Pruneda, Jonathan N.; Elliott, Paul R.; van Tilburg, Gabriëlle B. A.; Komander, David

    2016-01-01

    Abstract Deubiquitinating enzymes (DUBs) are proteases that fulfill crucial roles in the ubiquitin (Ub) system, by deconjugation of Ub from its targets and disassembly of polyUb chains. The specificity of a DUB towards one of the polyUb chain linkages largely determines the ultimate signaling function. We present a novel set of diubiquitin FRET probes, comprising all seven isopeptide linkages, for the absolute quantification of chain cleavage specificity of DUBs by means of Michaelis–Menten kinetics. Each probe is equipped with a FRET pair consisting of Rhodamine110 and tetramethylrhodamine to allow the fully synthetic preparation of the probes by SPPS and NCL. Our synthetic strategy includes the introduction of N,N′‐Boc‐protected 5‐carboxyrhodamine as a convenient building block in peptide chemistry. We demonstrate the value of our probes by quantifying the linkage specificities of a panel of nine DUBs in a high‐throughput manner. PMID:26996281

  1. Structural Basis of the Substrate Specificity and Enzyme Catalysis of a Papaver somniferum Tyrosine Decarboxylase.

    PubMed

    Guan, Huai; Song, Shuaibao; Robinson, Howard; Liang, Jing; Ding, Haizhen; Li, Jianyong; Han, Qian

    2017-01-01

    Tyrosine decarboxylase (TyDC), a type II pyridoxal 5'-phosphate decarboxylase, catalyzes the decarboxylation of tyrosine. Due to a generally high sequence identity to other aromatic amino acid decarboxylases (AAADs), primary sequence information is not enough to understand substrate specificities with structural information. In this study, we selected a typical TyDC from Papaver somniferum as a model to study the structural basis of AAAD substrate specificities. Analysis of the native P. somniferum TyDC crystal structure and subsequent molecular docking and dynamics simulation provide some structural bases that explain substrate specificity for tyrosine. The result confirmed the previous proposed mechanism for the enzyme selectivity of indolic and phenolic substrates. Additionally, this study yields the first crystal structure for a plant type II pyridoxal-5'-phosphate decarboxylase.

  2. Structural Basis of the Substrate Specificity and Enzyme Catalysis of a Papaver somniferum Tyrosine Decarboxylase

    PubMed Central

    Guan, Huai; Song, Shuaibao; Robinson, Howard; Liang, Jing; Ding, Haizhen; Li, Jianyong; Han, Qian

    2017-01-01

    Tyrosine decarboxylase (TyDC), a type II pyridoxal 5′-phosphate decarboxylase, catalyzes the decarboxylation of tyrosine. Due to a generally high sequence identity to other aromatic amino acid decarboxylases (AAADs), primary sequence information is not enough to understand substrate specificities with structural information. In this study, we selected a typical TyDC from Papaver somniferum as a model to study the structural basis of AAAD substrate specificities. Analysis of the native P. somniferum TyDC crystal structure and subsequent molecular docking and dynamics simulation provide some structural bases that explain substrate specificity for tyrosine. The result confirmed the previous proposed mechanism for the enzyme selectivity of indolic and phenolic substrates. Additionally, this study yields the first crystal structure for a plant type II pyridoxal-5'-phosphate decarboxylase. PMID:28232911

  3. A measure of the broad substrate specificity of enzymes based on 'duplicate' catalytic residues.

    PubMed

    Chakraborty, Sandeep; Ásgeirsson, Bjarni; Rao, Basuthkar J

    2012-01-01

    The ability of an enzyme to select and act upon a specific class of compounds with unerring precision and efficiency is an essential feature of life. Simultaneously, these enzymes often catalyze the reaction of a range of similar substrates of the same class, and also have promiscuous activities on unrelated substrates. Previously, we have established a methodology to quantify promiscuous activities in a wide range of proteins. In the current work, we quantitatively characterize the active site for the ability to catalyze distinct, yet related, substrates (BRASS). A protein with known structure and active site residues provides the framework for computing 'duplicate' residues, each of which results in slightly modified replicas of the active site scaffold. Such spatial congruence is supplemented by Finite difference Poisson Boltzmann analysis which filters out electrostatically unfavorable configurations. The congruent configurations are used to compute an index (BrassIndex), which reflects the broad substrate profile of the active site. We identify an acetylhydrolase and a methyltransferase as having the lowest and highest BrassIndex, respectively, from a set of non-homologous proteins extracted from the Catalytic Site Atlas. The acetylhydrolase, a regulatory enzyme, is known to be highly specific for platelet-activating factor. In the methyltransferase (PDB: 1QAM), various combinations of glycine (Gly38/40/42), asparagine (Asn101/11) and glutamic acid (Glu59/36) residues having similar spatial and electrostatic profiles with the specified scaffold (Gly38, Asn101 and Glu59) exemplifies the broad substrate profile such an active site may provide. 'Duplicate' residues identified by relaxing the spatial and/or electrostatic constraints can be the target of directed evolution methodologies, like saturation mutagenesis, for modulating the substrate specificity of proteins.

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

    PubMed

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

    2014-04-01

    α-Amylase (EC 3.2.1.1) represents the best known amylolytic enzyme. It catalyzes the hydrolysis of α-1,4-glucosidic bonds in starch and related α-glucans. In general, the α-amylase is an enzyme with a broad substrate preference and product specificity. In the sequence-based classification system of all carbohydrate-active enzymes, it is one of the most frequently occurring glycoside hydrolases (GH). α-Amylase is the main representative of family GH13, but it is probably also present in the families GH57 and GH119, and possibly even in GH126. Family GH13, known generally as the main α-amylase family, forms clan GH-H together with families GH70 and GH77 that, however, contain no α-amylase. Within the family GH13, the α-amylase specificity is currently present in several subfamilies, such as GH13_1, 5, 6, 7, 15, 24, 27, 28, 36, 37, and, possibly in a few more that are not yet defined. The α-amylases classified in family GH13 employ a reaction mechanism giving retention of configuration, share 4-7 conserved sequence regions (CSRs) and catalytic machinery, and adopt the (β/α)8-barrel catalytic domain. Although the family GH57 α-amylases also employ the retaining reaction mechanism, they possess their own five CSRs and catalytic machinery, and adopt a (β/α)7-barrel fold. These family GH57 attributes are likely to be characteristic of α-amylases from the family GH119, too. With regard to family GH126, confirmation of the unambiguous presence of the α-amylase specificity may need more biochemical investigation because of an obvious, but unexpected, homology with inverting β-glucan-active hydrolases.

  5. Structure of Human Dual Specificity Protein Phosphatase 23, VHZ, Enzyme-Substrate/Product Complex

    SciTech Connect

    Agarwal,R.; Burley, S.; Swaminathan, S.

    2008-01-01

    Protein phosphorylation plays a crucial role in mitogenic signal transduction and regulation of cell growth and differentiation. Dual specificity protein phosphatase 23 (DUSP23) or VHZ mediates dephosphorylation of phospho-tyrosyl (pTyr) and phospho-seryl/threonyl (pSer/pThr) residues in specific proteins. In vitro, it can dephosphorylate p44ERK1 but not p54SAPK-{beta} and enhance activation of c-Jun N-terminal kinase (JNK) and p38. Human VHZ, the smallest of the catalytically active protein-tyrosine phosphatases (PTP) reported to date (150 residues), is a class I Cys-based PTP and bears the distinctive active site signature motif HCXXGXXRS(T). We present the crystal structure of VHZ determined at 1.93 angstrom resolution. The polypeptide chain adopts the typical a{beta}a PTP fold, giving rise to a shallow active site cleft that supports dual phosphorylated substrate specificity. Within our crystals, the Thr-135-Tyr-136 from a symmetry-related molecule bind in the active site with a malate ion, where they mimic the phosphorylated TY motif of the MAPK activation loop in an enzyme-substrate/product complex. Analyses of intermolecular interactions between the enzyme and this pseudo substrate/product along with functional analysis of Phe-66, Leu-97, and Phe-99 residues provide insights into the mechanism of substrate binding and catalysis in VHZ.

  6. Formation of target-specific binding sites in enzymes: solid-phase molecular imprinting of HRP.

    PubMed

    Czulak, J; Guerreiro, A; Metran, K; Canfarotta, F; Goddard, A; Cowan, R H; Trochimczuk, A W; Piletsky, S

    2016-06-07

    Here we introduce a new concept for synthesising molecularly imprinted nanoparticles by using proteins as macro-functional monomers. For a proof-of-concept, a model enzyme (HRP) was cross-linked using glutaraldehyde in the presence of glass beads (solid-phase) bearing immobilized templates such as vancomycin and ampicillin. The cross-linking process links together proteins and protein chains, which in the presence of templates leads to the formation of permanent target-specific recognition sites without adverse effects on the enzymatic activity. Unlike complex protein engineering approaches commonly employed to generate affinity proteins, the method proposed can be used to produce protein-based ligands in a short time period using native protein molecules. These affinity materials are potentially useful tools especially for assays since they combine the catalytic properties of enzymes (for signaling) and molecular recognition properties of antibodies. We demonstrate this concept in an ELISA-format assay where HRP imprinted with vancomycin and ampicillin replaced traditional enzyme-antibody conjugates for selective detection of templates at micromolar concentrations. This approach can potentially provide a fast alternative to raising antibodies for targets that do not require high assay sensitivities; it can also find uses as a biochemical research tool, as a possible replacement for immunoperoxidase-conjugates.

  7. Reverse enzyme immunoassay for detection of specific anti-Toxoplasma immunoglobulin M antibodies.

    PubMed Central

    Franco, E L; Walls, K W; Sulzer, A J

    1981-01-01

    A reverse enzyme immunoassay (R-EIA) is described, in which polystyrene muplates are sensitized with anti-immunoglobulin M (IgM) (mu chain) antibodies and then sequentially allowed to react with patient's serum, peroxidase-labeled Toxoplasma gondii soluble antigen, and substrate. Measurement of activity of the solid-phase bound enzyme conjugate was done by colorimetric reading of the final developed color and kinetically by the initial rate of color development. This R-EIA allowed full resolution between absorbance values of a group of 36 sera which presented positive results in the Toxoplasma IgM immunofluorescence test and the remaining groups, which consisted of 39 normal individuals, 22 rheumatoid factor-positive sera, 8 Waldenstrom's macroglobulinemic sera, 3 infectious mononucleosis samples, and 6 high-titered IgG anti-T. gondii sera. No interference of rheumatoid factor IgM or inhibition by high-titered specific IgG was detected, even in the false IgM immunofluorescence-positive rheumatoid factor samples. Likewise, false-negative IgM immunofluorescence samples gave positive R-EIA even without adsorption with Staphylococcus aureus protein A. The possibility of direct tagging of the antigen with the enzyme eliminates the need for using antigen and anti-antigen conjugates as separate layers, therefore eliminating one step in the assay. PMID:7016911

  8. Effect of hydrogen peroxide on antioxidant enzyme activities in Saccharomyces cerevisiae is strain-specific.

    PubMed

    Bayliak, M; Semchyshyn, H; Lushchak, V

    2006-09-01

    The effect of hydrogen peroxide on the survival and activity of antioxidant and associated enzymes in Saccharomyces cerevisiae has been studied. A difference found in the response of wild-type yeast strains treated with hydrogen peroxide was probably related to the different protective effects of antioxidant enzymes in these strains. Exposure of wild-type YPH250 cells to 0.25 mM H(2)O(2) for 30 min increased activities of catalase and superoxide dismutase (SOD) by 3.4- and 2-fold, respectively. However, no activation of catalase in the EG103 strain, as well as of SOD in the YPH98 and EG103 wild strains was detected, which was in parallel to lower survival of these strains under oxidative stress. There is a strong positive correlation (R(2) = 0.95) between activities of catalase and SOD in YPH250 cells treated with different concentrations of hydrogen peroxide. It is conceivable that catalase would protect SOD against inactivation caused by oxidative stress and vice versa. Finally, yeast cell treatment with hydrogen peroxide can lead to either a H(2)O(2)-induced increase in activities of antioxidant and associated enzymes or their decrease depending on the H(2)O(20 concentration used or the yeast strain specificity.

  9. Closing the Gap Between Specification and Programming: VDM++ and SCALA

    NASA Technical Reports Server (NTRS)

    Havelund, Klaus

    2011-01-01

    We argue that a modern programming language such as Scala offers a level of succinctness, which makes it suitable for program and systems specification as well as for high-level programming. We illustrate this by comparing the language with the Vdm++ specification language. The comparison also identifies areas where Scala perhaps could be improved, inspired by Vdm++. We furthermore illustrate Scala's potential as a specification language by augmenting it with a combination of parameterized state machines and temporal logic, defined as a library, thereby forming an expressive but simple runtime verification framework.

  10. Protein Stabilization and Enzyme Activation in Ionic Liquids: Specific Ion Effects

    PubMed Central

    Zhao, Hua

    2015-01-01

    There are still debates on whether the hydration of ions perturbs the water structure, and what is the degree of such disturbance; therefore, the origin of Hofmeister effect on protein stabilization continues being questioned. For this reason, it is suggested to use the ‘specific ion effect’ instead of other misleading terms such as Hofmeister effect, Hofmeister series, lyotropic effect, and lyotropic series. In this review, we firstly discuss the controversial aspect of inorganic ion effects on water structures, and several possible contributors to the specific ion effect of protein stability. Due to recent overwhelming attraction of ionic liquids (ILs) as benign solvents in many enzymatic reactions, we further evaluate the structural properties and molecular-level interactions in neat ILs and their aqueous solutions. Next, we systematically compare the specific ion effects of ILs on enzyme stability and activity, and conclude that (a) the specificity of many enzymatic systems in diluted aqueous IL solutions is roughly in line with the traditional Hofmeister series albeit some exceptions; (b) however, the specificity follows a different track in concentrated or neat ILs because other factors (such as hydrogen-bond basicity, nucelophilicity, and hydrophobicity, etc) are playing leading roles. In addition, we demonstrate some examples of biocatalytic reactions in IL systems that are guided by the empirical specificity rule. PMID:26949281

  11. Optimization of a human papillomavirus-specific enzyme-linked immunosorbent assay.

    PubMed

    Karem, Kevin L; Poon, Alysia C; Bierl, Cynthia; Nisenbaum, Rosane; Unger, Elizabeth

    2002-05-01

    A strategy was developed for the control, standardization, and critical evaluation of an enzyme-linked immunosorbent assay (ELISA) for the detection of human papillomavirus-specific immunoglobulin G in human sera. Control human sera, polyclonal animal sera, and monoclonal antibodies were used to establish optimal assay parameters, including antigen coating, serum dilutions, and criteria for daily reproducibility, monitoring, and rejection of assays. Three evaluation techniques were used in parallel to define an optimal cutoff absorbance value that yields greater than 93% sensitivity and 98.5% specificity in the assay's ability to discriminate positive and negative control sera. This strategy provides an optimal method by which to determine cutoff absorbance values for ELISA.

  12. Optimization of a Human Papillomavirus-Specific Enzyme-Linked Immunosorbent Assay

    PubMed Central

    Karem, Kevin L.; Poon, Alysia C.; Bierl, Cynthia; Nisenbaum, Rosane; Unger, Elizabeth

    2002-01-01

    A strategy was developed for the control, standardization, and critical evaluation of an enzyme-linked immunosorbent assay (ELISA) for the detection of human papillomavirus-specific immunoglobulin G in human sera. Control human sera, polyclonal animal sera, and monoclonal antibodies were used to establish optimal assay parameters, including antigen coating, serum dilutions, and criteria for daily reproducibility, monitoring, and rejection of assays. Three evaluation techniques were used in parallel to define an optimal cutoff absorbance value that yields greater than 93% sensitivity and 98.5% specificity in the assay's ability to discriminate positive and negative control sera. This strategy provides an optimal method by which to determine cutoff absorbance values for ELISA. PMID:11986263

  13. Papain-catalyzed peptide bond formation: enzyme-specific activation with guanidinophenyl esters.

    PubMed

    de Beer, Roseri J A C; Zarzycka, Barbara; Amatdjais-Groenen, Helene I V; Jans, Sander C B; Nuijens, Timo; Quaedflieg, Peter J L M; van Delft, Floris L; Nabuurs, Sander B; Rutjes, Floris P J T

    2011-09-19

    The substrate mimetics approach is a versatile method for small-scale enzymatic peptide-bond synthesis in aqueous systems. The protease-recognized amino acid side chain is incorporated in an ester leaving group, the substrate mimetic. This shift of the specific moiety enables the acceptance of amino acids and peptide sequences that are normally not recognized by the enzyme. The guanidinophenyl group (OGp), a known substrate mimetic for the serine proteases trypsin and chymotrypsin, has now been applied for the first time in combination with papain, a cheap and commercially available cysteine protease. To provide insight in the binding mode of various Z-X(AA)-OGp esters, computational docking studies were performed. The results strongly point at enzyme-specific activation of the OGp esters in papain through a novel mode of action, rather than their functioning as mimetics. Furthermore, the scope of a model dipeptide synthesis was investigated with respect to both the amino acid donor and the nucleophile. Molecular dynamics simulations were carried out to prioritize 22 natural and unnatural amino acid donors for synthesis. Experimental results correlate well with the predicted ranking and show that nearly all amino acids are accepted by papain.

  14. Optimization of ultrasound-assisted extraction of pectinase enzyme from guava (Psidium guajava) peel: Enzyme recovery, specific activity, temperature, and storage stability.

    PubMed

    Amid, Mehrnoush; Murshid, Fara Syazana; Manap, Mohd Yazid; Islam Sarker, Zaidul

    2016-01-01

    This study aimed to investigate the effects of the ultrasound-assisted extraction conditions on the yield, specific activity, temperature, and storage stability of the pectinase enzyme from guava peel. The ultrasound variables studied were sonication time (10-30 min), ultrasound temperature (30-50 °C), pH (2.0-8.0), and solvent-to-sample ratio (2:1 mL/g to 6:1 mL/g). The main goal was to optimize the ultrasound-assisted extraction conditions to maximize the recovery of pectinase from guava peel with the most desirable enzyme-specific activity and stability. Under the optimum conditions, a high yield (96.2%), good specific activity (18.2 U/mg), temperature stability (88.3%), and storage stability (90.3%) of the extracted enzyme were achieved. The optimal conditions were 20 min sonication time, 40 °C temperature, at pH 5.0, using a 4:1 mL/g solvent-to-sample ratio. The study demonstrated that optimization of ultrasound-assisted process conditions for the enzyme extraction could improve the enzymatic characteristics and yield of the enzyme.

  15. Enzyme-linked immunosorbent assay for immunoglobulin M specific antibody for the diagnosis of melioidosis.

    PubMed Central

    Kunakorn, M; Boonma, P; Khupulsup, K; Petchclai, B

    1990-01-01

    Indirect hemagglutination (IHA) is commonly used for serodiagnosis of melioidosis. However, in endemic areas, high background titers in normal populations and occasional low titers in patients with septicemic melioidosis prompted a search for a more sensitive and more specific method of serodiagnosis. An indirect fluorescent-antibody test for immunoglobulin M (IgM) specific antibody to Pseudomonas pseudomallei was more sensitive and more specific, but fluorescence microscopes are rarely available in the endemic areas. An enzyme-linked immunosorbent assay (ELISA) for IgM antibody is an attractive alternative. An indirect ELISA for IgM antibody (IgM ELISA) and an IgM antibody capture ELISA for melioidosis were developed. Both tests, together with IHA, were evaluated for 153 serum specimens from blood donors and 16 serum specimens from 16 melioidosis patients. It was found that IHA, the IgM ELISA, and the IgM antibody capture ELISA had sensitivities of 88, 88, and 75%, respectively, with specificities of 97.4, 92.2, and 91.5%, respectively. When IHA was combined with IgM ELISA, a sensitivity of 100% and a specificity of 95.4% were obtained. The IgM ELISA and IHA should be used in combination for serodiagnosis of melioidosis. PMID:2199494

  16. Program Aids Specification Of Multiple-Block Grids

    NASA Technical Reports Server (NTRS)

    Sorenson, R. L.; Mccann, K. M.

    1993-01-01

    3DPREP computer program aids specification of multiple-block computational grids. Highly interactive graphical preprocessing program designed for use on powerful graphical scientific computer workstation. Divided into three main parts, each corresponding to principal graphical-and-alphanumerical display. Relieves user of some burden of collecting and formatting many data needed to specify blocks and grids, and prepares input data for NASA's 3DGRAPE grid-generating computer program.

  17. 13 CFR 130.350 - Specific program responsibilities.

    Code of Federal Regulations, 2010 CFR

    2010-01-01

    ... 13 Business Credit and Assistance 1 2010-01-01 2010-01-01 false Specific program responsibilities. 130.350 Section 130.350 Business Credit and Assistance SMALL BUSINESS ADMINISTRATION SMALL BUSINESS... Program policies and procedures to improve the delivery of services by SBDCs to the small...

  18. 14 CFR 91.1017 - Amending program manager's management specifications.

    Code of Federal Regulations, 2010 CFR

    2010-01-01

    ... 14 Aeronautics and Space 2 2010-01-01 2010-01-01 false Amending program manager's management... TRANSPORTATION (CONTINUED) AIR TRAFFIC AND GENERAL OPERATING RULES GENERAL OPERATING AND FLIGHT RULES Fractional... specifications will set a reasonable period (but not less than 7 days) within which the program manager...

  19. Approximated maximum adsorption of His-tagged enzyme/mutants on Ni2+-NTA for comparison of specific activities.

    PubMed

    Li, Yuanli; Long, Gaobo; Yang, Xiaolan; Hu, Xiaolei; Feng, Yiran; Tan, Deng; Xie, Yanling; Pu, Jun; Liao, Fei

    2015-03-01

    By approximating maximum activities of six-histidine (6His)-tagged enzyme/mutants adsorbed on Ni2+-NTA-magnetic-submicron-particle (Ni2+-NTA-MSP), a facile approach was tested for comparing enzyme specific activities in cell lysates. On a fixed quantity of Ni2+-NTA-MSP, the activity of an adsorbed 6His-tagged enzyme/mutant was measured via spectrophotometry; the activity after saturation adsorption (Vs) was predicted from response curve with quantities of total proteins from the same lysate as the predictor; Vs was equivalent of specific activity for comparison. This approach required abundance of a 6His-tagged enzyme/mutant over 3% among total proteins in lysate, an accurate series of quantities of total proteins from the same lysate, the largest activity generated by enzyme occupying over 85% binding sites on Ni2+-NTA-MSP and the minimum activity as absorbance change rates of 0.003 min(-1) for analysis. The prediction of Vs tolerated errors in concentrations of total proteins in lysates and was effective to 6His-tagged alkaline phosphatase and its 6His-tagged mutant in lysates. Notably, of those two 6His-tagged enzymes, Vs was effectively approximated with just one optimized quantity of lysates. Hence, this approach with Ni2+-NTA-MSP worked for comparison of specific activities of 6His-tagged enzyme/mutants in lysates when they had sufficient abundance among proteins and activities of adsorbed enzymes were measurable.

  20. Purification and characterization of cellulase from North Pacific krill (Euphausia pacifica). Analysis of cleavage specificity of the enzyme.

    PubMed

    Tsuji, Akihiko; Sato, Shiori; Kondo, Ayumi; Tominaga, Keiko; Yuasa, Keizo

    2012-01-01

    Krill are filter feeders that consume algae, plankton and detritus, indicating that krill possess an adequate cellulose digesting system. However, less is known about the enzymatic properties of crustacean cellulases compared to termite cellulases. In the present study, 48 kDa-cellulase was highly purified from krill (Euphausia pacifica) in an effort to determine the cleavage specificity of the enzyme. The most notable characteristic of the enzyme was its high activity against both lichenan and carboxymethyl cellulose. The enzyme hydrolyzed internal β-1,4 glycosidic bonds within lichenan as well as carboxymethyl cellulose to release oligosaccharides and glucose. The effects of pH and temperature on the activity and stability of both enzyme activities were almost identical. Cello-oligosaccharides with a degree of polymerization of 4-6 were hydrolyzed by the enzyme, and the same endo-products, cellotriose, cellobiose and glucose, were produced from these oligosaccharides. Neither cellotriose nor cellobiose was hydrolyzed by the enzyme. The enzyme digested filter paper and sea lettuce to produce cellobiose, cellotriose and glucose as major products. Although amino acid sequence homology of the enzyme with termite cellulases and the presence of oligosaccharides in the enzyme suggested that the enzyme is produced by krill itself, further analysis is necessary.

  1. Altered substrate specificity of the Pterygoplichthys sp. (Loricariidae) CYP1A enzyme.

    PubMed

    Parente, Thiago E M; Urban, Philippe; Pompon, Denis; Rebelo, Mauro F

    2014-09-01

    Ethoxyresorufin is a classical substrate for vertebrate CYP1A enzymes. In Pterygoplichthys sp. (Loricariidae) this enzyme possesses 48 amino acids substitutions compared to CYP1A sequences from other vertebrate species. These substitutions or a certain subset substitution are responsible for the non-detection of the EROD reaction in this species liver microsomes. In the present study, we investigated the catalytic activity of Pterygoplichthys sp. CYP1A toward 15 potential substrates in order to understand the substrate preferences of this modified CYP1A. The fish gene was expressed in yeast and the accumulation of the protein was confirmed by both the characteristic P450-CO absorbance spectra and by detection with monoclonal antibodies. Catalytic activities were assayed with yeast microsomes and four resorufin ethers, six coumarin derivates, three flavones, resveratrol and ethoxyfluoresceinethylester. Results demonstrated that the initial velocity pattern of this enzyme for the resorufin derivatives is different from the one described for most vertebrate CYP1As. The initial velocity for the activity with the coumarin derivatives is several orders of magnitude higher than with the resorufins, i.e. the turnover number (kcat) for ECOD is 400× higher than for EROD. Nonetheless, the specificity constant (kcat/km) for EROD is only slightly higher than for ECOD. EFEE is degraded at a rate comparable to the resorufins. Pterygoplichthys sp. CYP1A also degrades 7-methoxyflavone and β-naphthoflavone but not resveratrol and chrysin. These results indicate a divergent substrate preference for Pterygoplichthys sp. CYP1A, which may be involved in the adaptation of Loricariidae fish to their particular environment and feeding habits.

  2. Inhibition of phosphatidylinositol-specific phospholipase C: studies on synthetic substrates, inhibitors and a synthetic enzyme.

    PubMed

    Vizitiu, D; Kriste, A G; Campbell, A S; Thatcher, G R

    1996-01-01

    Enzyme inhibition studies on phosphatidylinositol-specific phospholipase C (PI-PLC) from B. Cereus were performed in order to gain an understanding of the mechanism of the PI-PLC family of enzymes and to aid inhibitor design. Inhibition studies on two synthetic cyclic phosphonate analogues (1,2) of inositol cyclic-1:2-monophosphate (cIP), glycerol-2-phosphate and vanadate were performed using natural phosphatidylinositol (PI) substrate in Triton X100 co-micelles and an NMR assay. Further inhibition studies on PI-PLC from B. Cereus were performed using a chromogenic, synthetic PI analogue (DPG-PI), an HPLC assay and Aerosol-OT (AOT), phytic acid and vanadate as inhibitors. For purposes of comparison, a model PI-PLC enzyme system was developed employing a synthetic Cu(II)-metallomicelle and a further synthetic PI analogue (IPP-PI). The studies employing natural PI substrate in Triton X100 co-micelles and synthetic DPG-PI in the absence of surfactant indicate three classes of PI-PLC inhibitors: (1) active-site directed inhibitors (e.g. 1,2); (2) water-soluble polyanions (e.g. tetravanadate, phytic acid); (3) surfactant anions (e.g. AOT). Three modes of molecular recognition are indicated to be important: (1) active site molecular recognition; (2) recognition at an anion-recognition site which may be the active site, and; (3) interfacial (or hydrophobic) recognition which may be exploited to increase affinity for the anion-recognition site in anionic surfactants such as AOT. The most potent inhibition of PI-PLC was observed by tetravanadate and AOT. The metallomicelle model system was observed to mimic PI-PLC in reproducing transesterification of the PI analogue substrate to yield cIP as product and in showing inhibition by phytic acid and AOT.

  3. Controlling substrate specificity and product regio- and stereo-selectivities of P450 enzymes without mutagenesis.

    PubMed

    Polic, Vanja; Auclair, Karine

    2014-10-15

    P450 enzymes (P450s) are well known for their ability to oxidize unactivated CH bonds with high regio- and stereoselectivity. Hence, there is emerging interest in exploiting P450s as potential biocatalysts. Although bacterial P450s typically show higher activity than their mammalian counterparts, they tend to be more substrate selective. Most drug-metabolizing P450s on the other hand, display remarkable substrate promiscuity, yet product prediction remains challenging. Protein engineering is one established strategy to overcome these issues. A less explored, yet promising alternative involves substrate engineering. This review discusses the use of small molecules for controlling the substrate specificity and product selectivity of P450s. The focus is on two approaches, one taking advantage of non-covalent decoy molecules, and the other involving covalent substrate modifications.

  4. Influence of fermentation conditions on specific activity of the enzymes alcohol and aldehyde dehydrogenase from yeasts.

    PubMed

    Mauricio, J C; Ortega, J M

    1993-01-01

    The effects of anaerobic, semi-aerobic and short aeration fermentation conditions and the addition of ergosterol and oleic acid to musts on the specific activity of alcohol and aldehyde dehydrogenase (ADH and ALDH) from two yeast species, Saccharomyces cerevisiae and Torulaspora delbrueckii, were studied. ADH I biosynthesis only occurred during the first few hours of fermentation. ADH II from S. cerevisiae and ALDH-NADP+ from the two yeast species behaved as constitutive enzymes under all fermentation conditions. ADH II from T. delbrueckii was only synthesized in small amounts, and its activity was always lower than in S. cerevisiae, where it was responsible for the termination of alcoholic fermentation during the steady growth phase.

  5. Aspects of Protein Chemistry. Part I: Some Recent Insights Into Enzyme Specificity

    ERIC Educational Resources Information Center

    Nixon, J. E.

    1976-01-01

    Describes some recent advances in enzyme structure and action, including a description of enzyme-substrate interaction. Discusses the methods for determination of amino acid sequences in proteins; the actions of chymotrypsin, trypsin, and elastase; and details of the enzyme-substrate complex derived from kinetic studies and x-ray diffraction…

  6. Development of species-specific enzyme-linked immunosorbent assay for diagnosis of Johne's disease in cattle.

    PubMed Central

    Vannuffel, P; Gilot, P; Limbourg, B; Naerhuyzen, B; Dieterich, C; Coene, M; Machtelinckx, L; Cocito, C

    1994-01-01

    The previously described (M. De Kesel, P. Gilot, M.-C. Misonne, M. Coene, and C. Cocito, J. Clin. Microbiol., 31:947-954, 1993) a362 recombinant polypeptide of Mycobacterium paratuberculosis was used as reagent for an enzyme-linked immunosorbent assay (ELISA). This ELISA, which is endowed with species specificity with respect to the other mycobacteria, was applied to the analysis of bovine paratuberculosis (Johne's disease), an endemic mycobacteriosis of cattle caused by M. paratuberculosis. The distribution of anti-a362 antibodies in the cattle population was analyzed by a computer program (mixture population model) to determine a cutoff value for the test. The prevalence of a362 seropositivity in the Belgian bovine population was estimated to be 12%. The sensitivity of the a362 assay was 70%, as determined with reference sera from the U.S. National Repository of Paratuberculosis Specimens. Some 40% of the animals in the herds with paratuberculosis analyzed were found to be positive by the a362 assay. The latter proved to be 95% specific with respect to both healthy and tuberculous cattle. PMID:8051246

  7. Enzyme-linked immunosorbent assays for detection of equine antibodies specific to Sarcocystis neurona surface antigens.

    PubMed

    Hoane, Jessica S; Morrow, Jennifer K; Saville, William J; Dubey, J P; Granstrom, David E; Howe, Daniel K

    2005-09-01

    Sarcocystis neurona is the primary causative agent of equine protozoal myeloencephalitis (EPM), a common neurologic disease of horses in the Americas. We have developed a set of enzyme-linked immunosorbent assays (ELISAs) based on the four major surface antigens of S. neurona (SnSAGs) to analyze the equine antibody response to S. neurona. The SnSAG ELISAs were optimized and standardized with a sample set of 36 equine sera that had been characterized by Western blotting against total S. neurona parasite antigen, the current gold standard for S. neurona serology. The recombinant SnSAG2 (rSnSAG2) ELISA showed the highest sensitivity and specificity at 95.5% and 92.9%, respectively. In contrast, only 68.2% sensitivity and 71.4% specificity were achieved with the rSnSAG1 ELISA, indicating that this antigen may not be a reliable serological marker for analyzing antibodies against S. neurona in horses. Importantly, the ELISA antigens did not show cross-reactivity with antisera to Sarcocystis fayeri or Neospora hughesi, two other equine parasites. The accuracy and reliability exhibited by the SnSAG ELISAs suggest that these assays will be valuable tools for examining the equine immune response against S. neurona infection, which may help in understanding the pathobiology of this accidental parasite-host interaction. Moreover, with modification and further investigation, the SnSAG ELISAs have potential for use as immunodiagnostic tests to aid in the identification of horses affected by EPM.

  8. NKT sublineage specification and survival requires the ubiquitin-modifying enzyme TNFAIP3/A20

    PubMed Central

    Verheugen, Eveline; Taghon, Tom; Lambrecht, Bart N.

    2016-01-01

    Natural killer T (NKT) cells are innate lymphocytes that differentiate into NKT1, NKT2, and NKT17 sublineages during development. However, the signaling events that control NKT sublineage specification and differentiation remain poorly understood. Here, we demonstrate that the ubiquitin-modifying enzyme TNFAIP3/A20, an upstream regulator of T cell receptor (TCR) signaling in T cells, is an essential cell-intrinsic regulator of NKT differentiation. A20 is differentially expressed during NKT cell development, regulates NKT cell maturation, and specifically controls the differentiation and survival of NKT1 and NKT2, but not NKT17, sublineages. Remaining A20-deficient NKT1 and NKT2 thymocytes are hyperactivated in vivo and secrete elevated levels of Th1 and Th2 cytokines after TCR ligation in vitro. Defective NKT development was restored by compound deficiency of MALT1, a key downstream component of TCR signaling in T cells. These findings therefore show that negative regulation of TCR signaling during NKT development controls the differentiation and survival of NKT1 and NKT2 cells. PMID:27551157

  9. Endotoxin reduces specific pulmonary uptake of radiolabeled monoclonal antibody to angiotensin-converting enzyme

    SciTech Connect

    Muzykantov, V.R.; Puchnina, E.A.; Atochina, E.N.; Hiemish, H.; Slinkin, M.A.; Meertsuk, F.E.; Danilov, S.M. )

    1991-03-01

    The biodistribution of radiolabeled monoclonal antibody (Mab) to angiotensin-converting enzyme (ACE) was examined in normal and endotoxin-treated rats. Endotoxin administration at a dose of 4 mg/kg induced mild or middle pulmonary edema. The ACE activity in lung homogenate remained virtually unchanged, while the activity of serum ACE increased 15 hr after endotoxin infusion. In normal rats, anti-ACE Mab accumulates specifically in the lung after i.v. injection. Endotoxin injection induces reduction of specific pulmonary uptake of this antibody. Even in non-edematous endotoxemia, the accumulation of anti-ACE Mab antibody (Mab 9B9) decreased from 19.02 to 11.91% of ID/g of tissue without any change in accumulation of control nonspecific IgG. The antibody distribution in other organs and its blood level were almost the same as in the control. In a case of endotoxemia accompanied by increased microvascular permeability, the lung accumulation of Mab 9B9 was reduced to 9.17% of ID/g of tissue, while the accumulation of nonspecific IgG increased to 1.44% versus 0.89% in the control.

  10. Lid domain plasticity and lipid flexibility modulate enzyme specificity in human monoacylglycerol lipase.

    PubMed

    Riccardi, Laura; Arencibia, Jose M; Bono, Luca; Armirotti, Andrea; Girotto, Stefania; De Vivo, Marco

    2017-01-12

    Human monoacylglycerol lipase (MAGL) is a membrane-interacting enzyme that generates pro-inflammatory signaling molecules. For this reason, MAGL inhibition is a promising strategy to treat pain, cancer, and neuroinflammatory diseases. MAGL can hydrolyze monoacylglycerols bearing an acyl chain of different lengths and degrees of unsaturation, cleaving primarily the endocannabinoid 2-arachidonoylglycerol. Importantly, the enzymatic binding site of MAGL is confined by a 75-amino-acid-long, flexible cap domain, named 'lid domain', which is structurally similar to that found in several other lipases. However, it is unclear how lid domain plasticity affects catalysis in MAGL. By integrating extensive molecular dynamics simulations and free-energy calculations with mutagenesis and kinetic experiments, we here define a lid-domain-mediated mechanism for substrate selection and binding in MAGL catalysis. In particular, we clarify the key role of Phe159 and Ile179, two conserved residues within the lid domain, in regulating substrate specificity in MAGL. We conclude by proposing that other structurally related lipases may share this lid-domain-mediated mechanism for substrate specificity.

  11. Northwest Energy Efficient Manufactured Housing Program Specification Development

    SciTech Connect

    Hewes, Tom; Peeks, Brady

    2013-02-01

    The DOE research team Building America Partnership for Improved Residential Construction (BA-PIRC), Bonneville Power Administration (BPA), and Northwest Energy Works (NEW), the current Northwest Energy Efficient Manufactured Home Program (NEEM) program administrator, collaborated to research a new specification that would reduce the energy requirements of a NEEM home.This research identified and developed combinations of cost-effective high performance building assemblies and mechanical systems that can readily can be deployed in the manufacturing setting that reduce energy used for space conditioning, water heating and lighting by 50% over the present NEEM specifications.

  12. Alterations in Circulatory and Renal Angiotensin-Converting Enzyme and Angiotensin-Converting Enzyme 2 in Fetal Programmed Hypertension

    PubMed Central

    Shaltout, Hossam A.; Figueroa, Jorge P.; Rose, James C.; Diz, Debra I.; Chappell, Mark C.

    2009-01-01

    Antenatal betamethasone treatment is a widely accepted therapy to accelerate lung development and improve survival in preterm infants. However, there are reports that infants who receive antenatal glucocorticoids exhibit higher systolic blood pressure in their early adolescent years. We have developed an experimental model of programming whereby the offspring of pregnant sheep administered clinically relevant doses of betamethasone exhibit elevated blood pressure. We tested the hypothesis as to whether alterations in angiotensin-converting enzyme (ACE), ACE2, and neprilysin in serum, urine, and proximal tubules are associated with this increase in mean arterial pressure. Male sheep were administered betamethasone (2 doses of 0.17 mg/kg, 24 hours apart) or vehicle at the 80th day of gestation and delivered at term. Sheep were instrumented at adulthood (1.8 years) for direct conscious recording of mean arterial pressure. Serum and urine were collected and proximal tubules isolated from the renal cortex. Betamethasone-treated animals had elevated mean arterial pressure (97±3 versus 83±2 mm Hg; P<0.05) and a 25% increase in serum ACE activity (48.4±7.0 versus 36.0±2.7 fmol/mL per minute) but a 40% reduction in serum ACE2 activity (18.8±1.2 versus 31.4±4.4 fmol/mL per minute). In isolated proximal tubules, ACE2 activity and expression were 50% lower in the treated sheep with no significant change in ACE or neprilysin activities. We conclude that antenatal steroid treatment results in the chronic alteration of ACE and ACE2 in the circulatory and tubular compartments, which may contribute to the higher blood pressure in this model of fetal programming-induced hypertension. PMID:19047579

  13. 38 CFR 41.235 - Program-specific audits.

    Code of Federal Regulations, 2010 CFR

    2010-07-01

    ... fiscal years beginning on or before June 30, 1998, the audit shall be completed and the required... 38 Pensions, Bonuses, and Veterans' Relief 2 2010-07-01 2010-07-01 false Program-specific audits... (CONTINUED) AUDITS OF STATES, LOCAL GOVERNMENTS, AND NON-PROFIT ORGANIZATIONS Audits § 41.235...

  14. AMPHION: Specification-based programming for scientific subroutine libraries

    NASA Technical Reports Server (NTRS)

    Lowry, Michael; Philpot, Andrew; Pressburger, Thomas; Underwood, Ian; Waldinger, Richard; Stickel, Mark

    1994-01-01

    AMPHION is a knowledge-based software engineering (KBSE) system that guides a user in developing a diagram representing a formal problem specification. It then automatically implements a solution to this specification as a program consisting of calls to subroutines from a library. The diagram provides an intuitive domain oriented notation for creating a specification that also facilitates reuse and modification. AMPHION'S architecture is domain independent. AMPHION is specialized to an application domain by developing a declarative domain theory. Creating a domain theory is an iterative process that currently requires the joint expertise of domain experts and experts in automated formal methods for software development.

  15. Podocyte-specific overexpression of human angiotensin-converting enzyme 2 attenuates diabetic nephropathy in mice.

    PubMed

    Nadarajah, Renisha; Milagres, Rosangela; Dilauro, Marc; Gutsol, Alex; Xiao, Fengxia; Zimpelmann, Joseph; Kennedy, Chris; Wysocki, Jan; Batlle, Daniel; Burns, Kevin D

    2012-08-01

    Angiotensin-converting enzyme 2 (ACE2) degrades angiotensin II to angiotensin-(1-7) and is expressed in podocytes. Here we overexpressed ACE2 in podocytes in experimental diabetic nephropathy using transgenic methods where a nephrin promoter drove the expression of human ACE2. Glomeruli from these mice had significantly increased mRNA, protein, and activity of ACE2 compared to wild-type mice. Male mice were treated with streptozotocin to induce diabetes. After 16 weeks, there was no significant difference in plasma glucose levels between wild-type and transgenic diabetic mice. Urinary albumin was significantly increased in wild-type diabetic mice at 4 weeks, whereas albuminuria in transgenic diabetic mice did not differ from wild-type nondiabetic mice. However, this effect was transient and by 16 weeks both transgenic and nontransgenic diabetic mice had similar rates of proteinuria. Compared to wild-type diabetic mice, transgenic diabetic mice had an attenuated increase in mesangial area, decreased glomerular area, and a blunted decrease in nephrin expression. Podocyte numbers decreased in wild-type diabetic mice at 16 weeks, but were unaffected in transgenic diabetic mice. At 8 weeks, kidney cortical expression of transforming growth factor-β1 was significantly inhibited in transgenic diabetic mice as compared to wild-type diabetic mice. Thus, the podocyte-specific overexpression of human ACE2 transiently attenuates the development of diabetic nephropathy.

  16. Single-dilution enzyme-linked immunosorbent assay for quantification of antigen-specific salmonid antibody

    USGS Publications Warehouse

    Alcorn, S.W.; Pascho, R.J.

    2000-01-01

    An enzyme-linked immunosorbent assay (ELISA) was developed on the basis of testing a single dilution of serum to quantify the level of antibody to the p57 protein of Renibaclerium salmoninarum in sockeye salmon (Oncorhynchus nerka). The levels of antibody were interpolated from a standard curve constructed by relating the optical densities (OD) produced by several dilutions of a high-titer rainbow trout (O. mykiss) antiserum to the p57 protein. The ELISA OD values produced by as many as 36 test sera on each microplate were compared with the standard curve to calculate the antigen-specific antibody activity. Repeated measurements of 36 samples on 3 microplates on each of 6 assay dates indicated that the mean intraassay coefficient of variation (CV) was 6.68% (range, 0-23%) and the mean interassay CV was 8.29% (range, 4-16%). The antibody levels determined for the serum sample from 24 sockeye salmon vaccinated with a recombinant p57 protein generally were correlated with the levels determined by endpoint titration (r2 = 0.936) and with results from another ELISA that was based on extrapolation of antibody levels from a standard curve (r2 = 0.956). The single-dilution antibody ELISA described here increases the number of samples that can be tested on each microplate compared with immunoassays based on analysis of several dilutions of each test serum. It includes controls for interassay standardization and can be used to test fish weighing <3 g.

  17. Inactivation of the ubiquitin-specific protease 19 deubiquitinating enzyme protects against muscle wasting.

    PubMed

    Bédard, Nathalie; Jammoul, Samer; Moore, Tamara; Wykes, Linda; Hallauer, Patricia L; Hastings, Kenneth E M; Stretch, Cynthia; Baracos, Vickie; Chevalier, Stéphanie; Plourde, Marie; Coyne, Erin; Wing, Simon S

    2015-09-01

    The ubiquitin system plays a critical role in muscle wasting. Previous work has focused on the roles of ubiquitination. However, a role for deubiquitination in this process has not been established. Because ubiquitin-specific protease (USP)19 deubiquitinating enzyme is induced in skeletal muscle in many catabolic conditions, we generated USP19 knockout (KO) mice. These mice lost less muscle mass than wild-type (WT) animals in response to glucocorticoids, a common systemic cause of muscle atrophy as well as in response to denervation, a model of disuse atrophy. KO mice retained more strength and had less myofiber atrophy with both type I and type IIb fibers being protected. Rates of muscle protein synthesis were similar in WT and KO mice, suggesting that the sparing of atrophy was attributed to suppressed protein degradation. Consistent with this, expression of the ubiquitin ligases MuRF1 and MAFbx/atrogin-1 as well as several autophagy genes was decreased in the muscles of catabolic KO mice. Expression of USP19 correlates with that of MuRF1 and MAFbx/atrogin-1 in skeletal muscles from patients with lung cancer or gastrointestinal cancer, suggesting that USP19 is involved in human muscle wasting. Inhibition of USP19 may be a useful approach to the treatment of many muscle-wasting conditions.

  18. Structural basis for specificity and promiscuity in a carrier protein/enzyme system from the sulfur cycle

    PubMed Central

    Grabarczyk, Daniel B.; Chappell, Paul E.; Johnson, Steven; Stelzl, Lukas S.; Berks, Ben C.

    2015-01-01

    The bacterial Sox (sulfur oxidation) pathway is an important route for the oxidation of inorganic sulfur compounds. Intermediates in the Sox pathway are covalently attached to the heterodimeric carrier protein SoxYZ through conjugation to a cysteine on a protein swinging arm. We have investigated how the carrier protein shuttles intermediates between the enzymes of the Sox pathway using the interaction between SoxYZ and the enzyme SoxB as our model. The carrier protein and enzyme interact only weakly, but we have trapped their complex by using a “suicide enzyme” strategy in which an engineered cysteine in the SoxB active site forms a disulfide bond with the incoming carrier arm cysteine. The structure of this trapped complex, together with calorimetric data, identifies sites of protein–protein interaction both at the entrance to the enzyme active site tunnel and at a second, distal, site. We find that the enzyme distinguishes between the substrate and product forms of the carrier protein through differences in their interaction kinetics and deduce that this behavior arises from substrate-specific stabilization of a conformational change in the enzyme active site. Our analysis also suggests how the carrier arm-bound substrate group is able to outcompete the adjacent C-terminal carboxylate of the carrier arm for binding to the active site metal ions. We infer that similar principles underlie carrier protein interactions with other enzymes of the Sox pathway. PMID:26655737

  19. A specific affinity reagent to distinguish aldehyde dehydrogenases and oxidases. Enzymes catalyzing aldehyde oxidation in an adult moth

    SciTech Connect

    Tasayco, M.L.; Prestwich, G.D. )

    1990-02-25

    Aldehyde dehydrogenase (ALDH) and oxidase (AO) enzymes from the tissue extracts of male and female tobacco budworm moth (Heliothis virescens) were identified after electrophoretic protein separation. AO activity was visualized using formazan- or horseradish peroxidase-mediated staining coupled to the AO-catalyzed oxidation of benzaldehyde. A set of six soluble AO enzymes with isoelectric points from pI 4.6 to 5.3 were detected primarily in the antennal extracts. Partially purified antennal AO enzymes also oxidized both (Z)-9-tetradecenal and (Z)-11-hexadecenal, the two major pheromone components of this moth. ALDH activity was detected using a tritium-labeled affinity reagent based on a known irreversible inhibitor of this enzyme. This labeled vinyl ketone, (3H)(Z)-1,11-hexadecadien-3-one, was synthesized and used to covalently modify the soluble ALDH enzymes from tissue extracts. Molecular subunits of potential ALDH enzymes were visualized in the fluorescence autoradiograms of sodium dodecyl sulfate-polyacrylamide gel electrophoresis-separated proteins of the antenna, head, and leg tissues. Covalent modification of these protein subunits decreased specifically in the presence of excess pheromone aldehyde or benzaldehyde. Labeled vinyl ketones are thus novel tools for the identification of molecular subunits of ALDH enzymes.

  20. Specific detection and properties of enzyme hydrolyzing phosphonate ester in serum.

    PubMed

    Han, G Y; Fan, X H; Jin, X B; Wang, D P

    1992-03-01

    An enzyme capable of hydrolyzing 4-methylumbelliferyl phenylphosphonate to 4-methylumbelliferone and phenylphosphonic acid has been detected in human serum. It has a Km value of 1.72 x 10(-4) mol/L, has an optimum pH of 8.8-9.1 in Tris buffer, and shows maximum activity at 60 degrees C (30 min). The enzymic activity can be inhibited by Na3PO4, EDTA, and cysteine. We saw no effect of CuSO4, adenosine, thymidine, NaN3, diethyl p-nitrophenyl phosphate, p-chloromercuribenzoate, isopropyl fluorophosphate, or eserine on the enzymic activity. The enzyme cannot hydrolyze substrates of phosphodiesterase I or alkaline phosphatase. The enzyme is considered a phosphonate esterase.

  1. Moving college students to a better understanding of substrate specificity of enzymes through utilizing multimedia pre-training and an interactive enzyme model

    NASA Astrophysics Data System (ADS)

    Saleh, Mounir R.

    Scientists' progress in understanding enzyme specificity uncovered a complex natural phenomenon. However, not all of the currently available biology textbooks seem to be up to date on this progress. Students' understanding of how enzymes work is a core requirement in biochemistry and biology tertiary education. Nevertheless, current pre-college science education does not provide students with enough biochemical background to enable them to understand complex material such as this. To bridge this gap, a multimedia pre-training presentation was prepared to fuel the learner's prior knowledge with discrete facts necessary to understand the presented concept. This treatment is also known to manage intrinsic cognitive load during the learning process. An interactive instructional enzyme model was also built to motivate students to learn about substrate specificity of enzymes. Upon testing the effect of this combined treatment on 111 college students, desirable learning outcomes were found in terms of cognitive load, motivation, and achievement. The multimedia pre-training group reported significantly less intrinsic cognitive load, higher motivation, and demonstrated higher transfer performance than the control and post-training groups. In this study, a statistical mediation model is also proposed to explain how cognitive load and motivation work in concert to foster learning from multimedia pre-training. This type of research goes beyond simple forms of "what works" to a deeper understanding of "how it works", thus enabling informed decisions for multimedia instructional design. Multimedia learning plays multiple roles in science education. Therefore, science learners would be some of the first to benefit from improving multimedia instructional design. Accordingly, complex scientific phenomena can be introduced to college students in a motivating, informative, and cognitively efficient learning environment.

  2. STAGE 64: OUTPUT PROCESSOR PROGRAMMING SPECIFICATIONS MANUAL, VOLUME I. HISTORY PROGRAMS.

    DTIC Science & Technology

    This volume contains a detailed description of the history programs used in the STAGE 64 Output Processor Programming Specifications. The STAGE ...Output Processor sorts, compiles tallies, and presents the output of the STAGE model in printed form. The complete program presently consists of 38...processes for each side, but in view of the fact that the processes are run under an executive control , new tasks can be added to the model as they are

  3. SigrafW: An easy-to-use program for fitting enzyme kinetic data.

    PubMed

    Leone, Francisco Assis; Baranauskas, José Augusto; Furriel, Rosa Prazeres Melo; Borin, Ivana Aparecida

    2005-11-01

    SigrafW is Windows-compatible software developed using the Microsoft® Visual Basic Studio program that uses the simplified Hill equation for fitting kinetic data from allosteric and Michaelian enzymes. SigrafW uses a modified Fibonacci search to calculate maximal velocity (V), the Hill coefficient (n), and the enzyme-substrate apparent dissociation constant (K). The estimation of V, K, and the sum of the squares of residuals is performed using a Wilkinson nonlinear regression at any Hill coefficient (n). In contrast to many currently available kinetic analysis programs, SigrafW shows several advantages for the determination of kinetic parameters of both hyperbolic and nonhyperbolic saturation curves. No initial estimates of the kinetic parameters are required, a measure of the goodness-of-the-fit for each calculation performed is provided, the nonlinear regression used for calculations eliminates the statistical bias inherent in linear transformations, and the software can be used for enzyme kinetic simulations either for educational or research purposes. Persons interested in receiving a free copy of the software should contact Dr. F. A. Leone.

  4. Detection, quantification, and glycotyping of prion protein in specifically activated enzyme-linked immunosorbent assay plates.

    PubMed

    Triantaphyllidou, I E; Sklaviadis, T; Vynios, D H

    2006-12-15

    The conversion of a normal glycoprotein, prion protein (PrP(C)), to its abnormal protease-resistant isoform (PrP(Sc)) seems to be one of the main factors underlying the pathogenesis of spongiform encephalopathies. There are many studies indicating that PrP interacts with glycosaminoglycans, and we exploited this interaction to develop a sensitive solid phase assay for detection of both PrP forms. Glycosaminoglycans, such as chondroitin sulfate and heparin, were immobilized by their negative charge to enzyme-linked immunosorbent assay (ELISA) plate wells activated by glutaraldehyde and spermine. PrP in the samples examined (recombinant PrP or tissue homogenate) was allowed to interact with glycans. The interaction of recombinant PrP was more efficient against immobilized chondroitin sulfate of type A, and a linear correlation with concentration was demonstrated. From this curve, the concentration of each one of the PrP isoforms in biological samples can be determined. In addition, and taking into account that glycosylation of prion protein is species specific, we used similarly activated ELISA plate wells to determine different PrP glycoforms. A monoclonal antibody against PrP was immobilized, and PrP present in the samples (brain homogenates) was bound and visualized by various lectins. The most interesting outcome of the study is the differential binding of ricinus communis agglutinin I to the normal and scrapie brain homogenates. Dattura stramonium lectin and wheat germ agglutinin seem to bind almost equally to both samples, and all three have an increased sensitivity to PrP(Sc) after proteinase K digestion.

  5. Human c-fgr induces a monocyte-specific enzyme in NIH 3T3 cells

    SciTech Connect

    Inoue, Kazushi; Akiyama, Tetsu; Toyoshima, Kumao ); Wongsasant, Budsaba )

    1991-12-01

    The mutant c-fgr protein (p58{sup c-fgr/F523}) containing Phe-523 instead of Tyr-523 exhibited transforming activity in NIH 3T3 cells like other protein-tyrosine kinases of the src family, but normal p58{sup c-fgr} (p58{sup c-fgr/wt}) did not. The mutant protein showed tyrosine kinase activity threefold higher than that of the normal protein in vitro. Surprisingly, transfection of the normal c-fgr gene into NIH 3T3 cells resulted in induction of sodium fluoride (NaF)-sensitive {alpha}-naphthyl butyrate esterase ({alpha}-NBE), marker enzyme of cells of monocytic origin, which was not induced in v-src-, v-fgr-, or lyn-transfected NIH 3T3 cells. The NaF-sensitive {alpha}-NBE induced in c-fgr transfectants was shown by isoelectric focusing to have a pI of 5.2 to 5.4, a range which was the same as those for thioglycolate-induced murine peritoneal macrophages and 1{alpha}, 25-dihydroxyvitamin D{sub 3}-treated WEHI-3B cells. Immunoblotting studies with antophosphotyrosine antibodies revealed that 58-, 62-, 75-, 120-, 200-, and 230-kDa proteins were commonly phosphorylated at tyrosine residues in NIH 3T3 cells transfected with normal and mutated c-fgr, while 95-kDa protein was significantly phosphorylated at tyrosine residues in NIH 3T3 cells transfected with normal and mutated c-fgr, while 95-kDa protein was significantly phosphorylated at tyrosine residues in cells transfected with the mutated c-fgr. These findings suggest that tyrosine phosphorylation of specific cellular substrate proteins is important in induction of NaF-sensitive {alpha}-NBE and cell transformation by p58{sup c-fgr}.

  6. Power-free chip enzyme immunoassay for detection of prostate specific antigen (PSA) in serum.

    PubMed

    Adel Ahmed, Heba; Azzazy, Hassan M E

    2013-11-15

    A power-free, portable "Chip EIA" was designed to render the popular Enzyme Linked Immunosorbent Assay (ELISA) more suitable for point-of-care testing. A number of microfluidic platforms have enabled miniaturization of the conventional microtitre plate ELISA, however, they require external pumping systems, valves, and electric power supply. The Chip EIA platform has eliminated the need for pumps and valves through utilizing a simple permanent magnet and magnetic nanoparticles. The magnetic nanoparticles act as solid support to capture the target and are then moved through chambers harboring different reagents necessary to perform a sandwich ELISA. The use of magnetic nanoparticles increases the volume-to-surface ratio reducing the assay time to 30 min. Changing the color of horseradish peroxidase (HRP) substrate to green indicates a positive result. In addition, a quantitative read-out was obtained through the use of cellphone camera imaging and analyzing the images using Matlab®. Cell phones, including smart ones, are readily available almost everywhere. The Chip EIA device was used to assay total prostate specific antigen (tPSA) in 19 serum samples. The PSA Chip EIA was tested for accuracy, precision, repeatability, and the results were correlated to the commercial Beckman Colter, Hybritech immunoassay® for determination of tPSA in serum samples with a Pearson correlation coefficient (R(2)=0.96). The lower detection limit of the PSA Chip EIA was 3.2 ng/mL. The assay has 88.9% recovery and good reproducibility (% CV of 6.5). We conclude that the developed Chip EIA can be used for detection of protein biomarkers in biological specimens.

  7. Evaluation of specificity of indirect enzyme-linked immunosorbent assay for diagnosis of human Q fever.

    PubMed Central

    Uhaa, I J; Fishbein, D B; Olson, J G; Rives, C C; Waag, D M; Williams, J C

    1994-01-01

    Ninety-five acute- and convalescent-phase serum specimens from 48 patients suspected of having rickettsial or Legionella infections were assayed for antibodies to Coxiella burnetii, the causative agent of Q fever. To evaluate the specificity of the indirect enzyme-linked immunosorbent assay (ELISA) for human Q fever, we compared the ELISA results with those of the indirect immunofluorescence antibody (IFA) test. The ELISA data were analyzed by two different criteria for a positive test. The first criterion for positive results by ELISA was based upon diagnostic titers established in a study of 150 subjects who had no demonstrable cellular or humoral immune responses to C. burnetii phase I or phase II whole cells or phase I lipopolysaccharide. The second criterion was based upon diagnostic antibody titers in a study of 51 subjects who had been diagnosed as having clinical Q fever and had fourfold or greater rises in humoral immune responses to C. burnetii phase I and phase II whole-cell antigens. A comparison of the ELISA and IFA test results of the 95 serum specimens indicated excellent agreement between the tests (Kappa = 92.9%; P < 0.05). None of the 38 patients whose etiologies were confirmed serologically as Legionnaires' disease or rickettsial diseases other than Q fever were classified as positive for C. burnetii by the ELISA. Only one patient identified by the IFA test as having Q fever was not scored positive by the ELISA. These results suggest that the ELISA is useful for epidemiologic screening and as a diagnostic test for human Q fever. PMID:8077404

  8. Broad Specification Fuels Combustion Technology Program, Phase 2

    NASA Technical Reports Server (NTRS)

    Lohmann, R. P.; Jeroszko, R. A.; Kennedy, J. B.

    1990-01-01

    An experimental evaluation of two advanced technology combustor concepts was conducted to evolve and assess their capability for operation on broadened properties fuels. The concepts were based on the results of Phase 1 of the Broad Specification Fuel Combustor Technology Program which indicated that combustors with variable geometry or staged combustion zones had a flexibility of operation that could facilitate operation on these fuels. Emphasis in defining these concepts included the use of single pipe as opposed to duplex or staged fuels systems to avoid the risk of coking associated with the reduction in thermal stability expected in broadened properties fuels. The first concept was a variable geometry combustor in which the airflow into the primary zone could be altered through valves on the front while the second was an outgrowth of the staged Vorbix combustor, evolved under the NASA/P&W ECCP and EEE programs incorporating simplified fuel and air introduction. The results of the investigation, which involved the use of Experimental Referee Broad Specification (ERBS) fuel, indicated that in the form initially conceived, both of these combustor concepts were deficient in performance relative to many of the program goals for performance emissions. However, variations of both combustors were evaluated that incorporated features to simulate conceptual enhancement to demonstrate the long range potential of the combustor. In both cases, significant improvements relative to the program goals were observed.

  9. A Study in Enzyme Kinetics Using an Ion-Specific Electrode.

    ERIC Educational Resources Information Center

    Turchi, Sandra; And Others

    1989-01-01

    Describes an undergraduate biochemistry laboratory experiment on enzyme kinetics using the D-amino acid oxidase system and an ammonia electrode. Preparation of an ammonia standard curve, a sample preparation, and inhibition studies are discussed. (YP)

  10. Biochemical characterization of plasmepsin V from Plasmodium vivax Thailand isolates: Substrate specificity and enzyme inhibition.

    PubMed

    Sappakhaw, Khomkrit; Takasila, Ratchaneekorn; Sittikul, Pichamon; Wattana-Amorn, Pakorn; Assavalapsakul, Wanchai; Boonyalai, Nonlawat

    2015-12-01

    Plasmepsin V (PMV) is a Plasmodium aspartic protease responsible for the cleavage of the Plasmodium export element (PEXEL) motif, which is an essential step for export of PEXEL containing proteins and crucial for parasite viability. Here we describe the genetic polymorphism of Plasmodium vivax PMV (PvPMV) Thailand isolates, followed by cloning, expression, purification and characterization of PvPMV-Thai, presenting the pro- and mature-form of PvPMV-Thai. With our refolding and purification method, approximately 1mg of PvPMV-Thai was obtained from 1g of washed inclusion bodies. Unlike PvPMV-Ind and PvPMV-Sal-1, PvPMV-Thai contains a four-amino acid insertion (SVSE) at residues 246-249. We have confirmed that this insertion did not interfere with the catalytic activity as it is located in the long loop (R241-E272) pointing away from the substrate-binding pocket. PvPMV-Thai exhibited similar activity to PfPMV counterparts in which PfEMP2 could be hydrolyzed more efficiently than HRPII. Substrate specificity studies at P1' showed that replacing Ser by Val or Glu of the PfEMP2 peptide markedly reduced the enzyme activity of PvPMV similar to that of PfPMV whereas replacing His by Val or Ser of the HRPII peptide increased the cleavage activity. However, the substitution of amino acids at the P2 position with Glu dramatically reduced the cleavage efficiency by 80% in PvPMV in contrast to 30% in PfPMV, indicating subtle differences around the S2 binding pocket of both PfPMV and PvPMV. Four inhibitors were also evaluated for PvPMV-Thai activity including PMSF, pepstatin A, nelfinavir, and menisporopsin A-a macrocyclic polylactone. We are the first to show that menisporopsin A partially inhibits the PvPMV-Thai activity at high concentration. Taken together, these findings provide insights into recombinant production, substrate specificity and inhibition of PvPMV-Thai.

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

    PubMed

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

    2012-01-01

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

  12. Responses of absolute and specific soil enzyme activities to long term additions of organic and mineral fertilizer.

    PubMed

    Zhang, Xinyu; Dong, Wenyi; Dai, Xiaoqin; Schaeffer, Sean; Yang, Fengting; Radosevich, Mark; Xu, Lili; Liu, Xiyu; Sun, Xiaomin

    2015-12-01

    Long-term phosphorus (P) and nitrogen (N) applications may seriously affect soil microbial activity. A long-term field fertilizer application trial was established on reddish paddy soils in the subtropical region of southern China in 1998. We assessed the effects of swine manure and seven different rates or ratios of NPK fertilizer treatments on (1) the absolute and specific enzyme activities per unit of soil organic carbon (SOC) or microbial biomass carbon (MBC) involved in C, N, and P transformations and (2) their relationships with soil environmental factors and soil microbial community structures. The results showed that manure applications led to increases in the absolute and specific activities of soil β-1,4-glucosidase(βG), β-1,4-N-acetylglucosaminidase (NAG), and leucine aminopeptidase (LAP). The absolute and specific acid phosphatase (AP) activities decreased as mineral P fertilizer application rates and ratios increased. Redundancy analysis (RDA) showed that there were negative correlations between absolute and specific AP activities, pH, and total P contents, while there were positive correlations between soil absolute and specific βG, NAG, and LAP enzyme activities, and SOC and total N contents. RDA showed that the contents of actinomycete and Gram-positive bacterium PLFA biomarkers are more closely related to the absolute and specific enzyme activities than the other PLFA biomarkers (P<0.01). Our results suggest that both the absolute and specific enzyme activities could be used as sensitive soil quality indicators that provide useful linkages with the microbial community structures and environmental factors. To maintain microbial activity and to minimize environmental impacts, P should be applied as a combination of inorganic and organic forms, and total P fertilizer application rates to subtropical paddy soils should not exceed 44 kg P ha(-1) year(-1).

  13. An analytical method for determining relative specificities for sequential reactions catalyzed by the same enzyme: general formulation.

    PubMed

    Mitchell, David Alexander; Carrière, Frédéric; Krieger, Nadia

    2008-04-01

    We present a general formulation of a model that can be used to analyze reaction profiles in systems in which a single enzyme catalyzes several sequential reactions with the same molecular backbone. The analysis of these so-called "repeated-attack systems" allows estimation of the specificities that the enzyme has for the various intermediate substrates that appear in the reaction mixture, relative to the specificity that it has for the initial substrate. Our analytical method has the important advantage that it is not affected by competitive or uncompetitive inhibition, nor by denaturation of the enzyme during the reaction. We carry out case studies in three different systems, the lipase-catalyzed alcoholysis of triacylglycerols, the phytase-catalyzed removal of phosphate groups from phytic acid and the beta-amylase-catalyzed removal of maltose units from maltoheptaose. Our model fits well to all reaction profiles in which the phenomenon of processivity does not occur. It can therefore be used as a general tool for characterizing the relative specificities of "repeated-attack enzymes".

  14. Structure of 5-hydroxymethylcytosine-specific restriction enzyme, AbaSI, in complex with DNA

    SciTech Connect

    Horton, John R.; Borgaro, Janine G.; Griggs, Rose M.; Quimby, Aine; Guan, Shengxi; Zhang, Xing; Wilson, Geoffrey G.; Zheng, Yu; Zhu, Zhenyu; Cheng, Xiaodong

    2014-07-03

    AbaSI, a member of the PvuRts1I-family of modification-dependent restriction endonucleases, cleaves DNA containing 5-hydroxymethylctosine (5hmC) and glucosylated 5hmC (g5hmC), but not DNA containing unmodified cytosine. AbaSI has been used as a tool for mapping the genomic locations of 5hmC, an important epigenetic modification in the DNA of higher organisms. Here we report the crystal structures of AbaSI in the presence and absence of DNA. These structures provide considerable, although incomplete, insight into how this enzyme acts. AbaSI appears to be mainly a homodimer in solution, but interacts with DNA in our structures as a homotetramer. Each AbaSI subunit comprises an N-terminal, Vsr-like, cleavage domain containing a single catalytic site, and a C-terminal, SRA-like, 5hmC-binding domain. Two N-terminal helices mediate most of the homodimer interface. Dimerization brings together the two catalytic sites required for double-strand cleavage, and separates the 5hmC binding-domains by ~ 70 Å, consistent with the known activity of AbaSI which cleaves DNA optimally between symmetrically modified cytosines ~ 22 bp apart. The eukaryotic SET and RING-associated (SRA) domains bind to DNA containing 5-methylcytosine (5mC) in the hemi-methylated CpG sequence. They make contacts in both the major and minor DNA grooves, and flip the modified cytosine out of the helix into a conserved binding pocket. In contrast, the SRA-like domain of AbaSI, which has no sequence specificity, contacts only the minor DNA groove, and in our current structures the 5hmC remains intra-helical. A conserved, binding pocket is nevertheless present in this domain, suitable for accommodating 5hmC and g5hmC. We consider it likely, therefore, that base-flipping is part of the recognition and cleavage mechanism of AbaSI, but that our structures represent an earlier, pre-flipped stage, prior to actual recognition.

  15. Specification for a Program for an Interative Aeroelastic Solution (PIAS)

    NASA Technical Reports Server (NTRS)

    Manro, M. E.; Donahue, M. J.; Dreisbach, R. L.; Bussoletti, J. E.

    1983-01-01

    An engineering and software specification which was written for a computer program to calculate aeroelastic structural loads including the effects of nonlinear aerodynamics is presented. The procedure used in the program for an iterative aeroelastic solution (PIAS) is to alternately execute two computer codes: one to calculate aerodynamic loads for a specific wing shape, and another to calculate the deflected shape caused by this loading. A significant advantage to the design of PIAS is that the initial aerodynamic module can be replaced with others. The leading edge vortex (LEV) program is used as the aerodynamic module in PIAS. This provides the capability to calculate aeroelastic loads, including the effects of a separation induced leading edge vortex. The finite element method available in ATLAS Integrated structural analysis and design system is used to determine the deflected wing shape for the applied aerodynamics and inertia loads. The data management capabilities in ATLAS are used by the execution control monitor (ECM) of PIAS to control the solution process.

  16. Degradation kinetics of forchlorfenuron in typical grapevine soils of India and its influence on specific soil enzyme activities.

    PubMed

    Banerjee, Kaushik; Dasgupta, Soma; Oulkar, Dasharath P; Patil, Sangram H; Adsule, Pandurang G

    2008-05-01

    The rate of degradation of forchlorfenuron, a cytokinin-based plant growth regulator (PGR) was explored in typical grapevine soils of India with simultaneous evaluation of its effect on biochemical attributes of the test soils in terms of the activities of specific soil microbial enzymes. In all the test soils, namely clay, sandy-loam and silty-clay, the dissipation rate was faster at the beginning, which slowed down with time, indicating a non-linear pattern of degradation. Degradation in soils could best be explained by two-compartment 1st+1st order kinetics with half-life ranging between 4-10 days. The results suggest that organic matter might be playing a major role in influencing the rate of degradation of forchlorfenuron in soil. The rate of degradation in sandy-loam soil was fastest followed by clay and silty-clay soils, respectively. Comparison of the rate of degradation in natural against sterilized soils suggests that microbial degradation might be the major pathway of residue dissipation. Changes in soil enzyme activities as a consequence of forchlorfenuron treatment were studied for extra-cellular enzymes namely acid phosphatase, alkaline phosphatase and beta -glucosidase and intracellular enzyme-dehydrogenase. Although small changes in enzyme activities were observed, forchlorfenuron did not have any significant deleterious effect on the enzymatic activity of the test soils. Simple correlation studies between degradation percentage and individual enzyme activities did not establish any significant relationships. The pattern and change of enzyme activity was primarily the effect of the incubation period rather than the effect of forchlorfenuron itself.

  17. Voltage-programming-based capillary gel electrophoresis for the fast detection of angiotensin-converting enzyme insertion/deletion polymorphism with high sensitivity.

    PubMed

    Woo, Nain; Kim, Su-Kang; Kang, Seong Ho

    2016-08-01

    A voltage-programming-based capillary gel electrophoresis method with a laser-induced fluorescence detector was developed for the fast and highly sensitive detection of DNA molecules related to angiotensin-converting enzyme insertion/deletion polymorphism, which has been reported to influence predisposition to various diseases such as cardiovascular disease, high blood pressure, myocardial infarction, and Alzheimer's disease. Various voltage programs were investigated for fast detection of specific DNA molecules of angiotensin-converting enzyme insertion/deletion polymorphism as a function of migration time and separation efficiency to establish the effect of voltage strength to resolution. Finally, the amplified products of the angiotensin-converting enzyme insertion/deletion polymorphism (190 and 490 bp DNA) were analyzed in 3.2 min without losing resolution under optimum voltage programming conditions, which were at least 75 times faster than conventional slab gel electrophoresis. In addition, the capillary gel electrophoresis method also successfully applied to the analysis of real human blood samples, although no polymorphism genes were detected by slab gel electrophoresis. Consequently, the developed voltage-programming capillary gel electrophoresis method with laser-induced fluorescence detection is an effective, rapid analysis technique for highly sensitive detection of disease-related specific DNA molecules.

  18. Amazing pancreas: specific regulation of pancreatic secretion of individual digestive enzymes in rats.

    PubMed

    Maouyo, D; Morisset, J

    1995-02-01

    We investigated the effects of somatostatin (SMS)-201-995, atropine, and MK-329 on the role of cholinergic- and cholecystokinin-related systems and on the secretory relationship between five pancreatic digestive enzymes in rats. Animals kept in restraint cages and provided with pancreatic, biliary, duodenal, and jugular vein cannulas were treated as follows: 1) 0.25 micrograms.kg-1.h-1 caerulein alone, 2) both 0.25 micrograms.kg-1.h-1 caerulein and 100 micrograms.kg-1.h-1 atropine, 3) both caerulein and 5 micrograms.kg-1.h-1 SMS, 4) 91.3 micrograms.kg-1.h-1 carbachol alone, 5) both carbachol and 0.5 mg.kg-1.h-1 MK-329, and 6) both carbachol and 5 micrograms.kg-1.h-1 SMS, respectively. Food, but not water, was denied rats starting 10 h before the experiment and throughout the 6-h experimental period. The secretory patterns over the 6-h experimental period showed noticeably independent regulation of pancreatic secretion of individual digestive enzymes. The relationship between paired enzymes significantly varied according to the treatment. The correlation between chymotrypsinogen and the other enzymes was markedly modulated by MK-329. Our results suggest that SMS is a major "gate-keeper" in the regulation of exocrine pancreatic secretion and that the secretion of each digestive enzyme is individually regulated. Furthermore, they suggest that cholecystokinin and acetylcholine and their respective agonists are essentially initiators of secretory processes of the pancreas. Therefore, the paradigms of the regulation of pancreatic secretion heretofore accepted should be reexamined.

  19. Screening glycosynthase libraries with a fluoride chemosensor assay independently of enzyme specificity: identification of a transitional hydrolase to synthase mutant.

    PubMed

    Andrés, Eduardo; Aragunde, Hugo; Planas, Antoni

    2014-03-01

    Glycosynthases have become efficient tools for the enzymatic synthesis of oligosaccharides, glycoconjugates and polysaccharides. Enzyme-directed evolution approaches are applied to improve the performance of current glycosynthases and engineer specificity for non-natural substrates. However, simple and general screening methods are required since most of the reported assays are specific for each particular enzyme. In the present paper, we report a general screening assay that is independent of enzyme specificity, and implemented in an HTS (high-throughput screening) format for the screening of cell extracts in directed evolution experiments. Fluoride ion is a general by-product released in all glycosynthase reactions with glycosyl fluoride donors. The new assay is based on the use of a specific chemical sensor (a silyl ether of a fluorogenic methylumbelliferone) to transduce fluoride concentration into a fluorescence signal. As a proof-of-concept, it has been applied to a nucleophile saturation mutant library of Bacillus licheniformis 1,3-1,4-β-glucanase. Beyond the expected mutations at the glutamic acid (catalytic) nucleophile, other variants have been shown to acquire glycosynthase activity. Surprisingly, an aspartic acid for glutamic acid replacement renders a highly active glycosynthase, but still retains low hydrolase activity. It appears as an intermediate state between glycosyl hydrolase and glycosynthase.

  20. Nitric oxide inhibits specific enzymes in the Krebs cycle and the respiratory chain of rat hepatocyte mitochondria

    SciTech Connect

    Stadler, J.; Billiar, T.R.; Curran, R.D.; Kim, R.; Simmons, R.L. )

    1990-02-26

    Nitric oxide (NO) is a highly-reactive molecule produced from L-arginine as recently described. In macrophages and tumor cells, NO inhibits specific mitochondrial enzymes presumably by attacking their intrinsic 4Fe-4S centers. The susceptible enzymes include aconitase of the Krebs cycle and oxidoreductase (complex II) of the electron transport chain. The authors have recently demonstrated that hepatocytes (HC) produce NO in large amounts in response to endotoxin and inflammatory cytokines. To determine whether HC suffer a similar enzyme inhibition, the authors exposed rat HC to increasing concentrations of NO solutions for 5 minutes. The activity of aconitase, complex 1, complex 2, and complex 4 (cytochrome oxidase) was determined by measuring O{sub 2} consumption after addition of enzyme-specific substrates. An NO concentration-dependent inhibition of aconitase, complex 1, and complex 2 was measured. After exposure to 0.6 mM solution, the activity of aconitase was blocked to non-measurable values while complex 1 was reduced to 11 + 8%, and complex 2 to 36 + 2% of the activity of control HC. Complex 4 of the respiratory chain remained intact at 100 + 8%. These data indicate that HC, like other cell types, are susceptible to inhibition of important steps of energy production by NO. As NO is produced in response to septic stimuli, this mechanism may play a role in the metabolic dysfunction of HC in sepsis.

  1. Space Research and Technology Program: Program and specific objectives, document approval

    NASA Technical Reports Server (NTRS)

    1982-01-01

    A detailed view of the Space Research and Technology program work breakdown structure is provided down to the specific objective level. Goals or objectives at each of these levels are set forth. The specific objective narratives are structured into several parts. First, a short paragraph statement of the specific objective is given. This is followed by a list of subobjectives. A list of targets is then provided for those areas of the specific objective that are amenable to a quantitative description of technical accomplishment and schedule. Fluid and thermal physics, materials and structures, computer science and electronics, space energy conversion, multidisciplinary research, controls and human factors, chemical propulsion, spacecraft systems, transportation systems, platform systems, and spacecraft systems technology comprise the principal research programs.

  2. Development of a highly efficient oil degumming process using a novel phosphatidylinositol-specific phospholipase C enzyme.

    PubMed

    Cerminati, Sebastián; Eberhardt, Florencia; Elena, Claudia E; Peirú, Salvador; Castelli, María E; Menzella, Hugo G

    2017-02-25

    Enzymatic degumming using phospholipase C (PLC) enzymes may be used in environmentally friendly processes with improved oil recovery yields. In this work, phosphatidylinositol-specific phospholipase C (PIPLC) candidates obtained from an in silico analysis were evaluated for oil degumming. A PIPLC from Lysinibacillus sphaericus was shown to efficiently remove phosphatidylinositol from crude oil, and when combined with a second phosphatidylcholine and phosphatidylethanolamine-specific phospholipase C, the three major phospholipids were completely hydrolyzed, providing an extra yield of oil greater than 2.1%, compared to standard methods. A remarkably efficient fed-batch Escherichia coli fermentation process producing ∼14 g/L of the recombinant PIPLC enzyme was developed, which may facilitate the adoption of this cost-effective oil-refining process.

  3. Retrostatin, a new specific enzyme inhibitor against avian myeloblastosis virus reverse transcriptase.

    PubMed

    Nishio, M; Kuroda, A; Suzuki, M; Ishimaru, K; Nakamura, S; Nomi, R

    1983-07-01

    A novel enzyme inhibitor against RNA-directed DNA polymerase of avian myeloblastosis virus was produced by an isolate of a new streptomycete for which the name Streptomyces retrostaticus is proposed. This enzyme inhibitor, which was named retrostatin, did not inhibit DNA-directed DNA polymerase of Escherichia coli and DNA-directed RNA polymerase of Ehrlich ascites tumor cells. Retrostatin was produced by the microorganism together with streptonigrin. These two substances were extracted from the culture broth with ethyl acetate at acidic pH. Retrostatin is an acidic pH indicator and the free acid was recovered as a red powder. Retrostatin had weak antibiotic activities against Gram-positive bacteria and yeasts.

  4. Characterization of Yeast Aspartic Protease 3 (A novel basic-residue specific prohormone processing enzyme)

    DTIC Science & Technology

    1995-05-16

    proteinases with naturallyw occurring inhibitors from aclinomycetes and ascaris lumbricoides . 1. Enzyme Inhibition, 1985. 1 : p. 77-82. 11 8...evident surrounding the periphery of the yeast cell (Fig.8.A and C). This staining pattern, described as a doughnut shaped pattern is characteristic ...number of peptide hormone substrates. it was necessary to develop a purification procedure in order to be able to verify some of these characteristics

  5. Molecular Basis for Lysine Specificity in the Yeast Ubiquitin-Conjugating Enzyme Cdc34 ▿

    PubMed Central

    Sadowski, Martin; Suryadinata, Randy; Lai, Xianning; Heierhorst, Jörg; Sarcevic, Boris

    2010-01-01

    Ubiquitin (Ub)-conjugating enzymes (E2s) and ubiquitin ligases (E3s) catalyze the attachment of Ub to lysine residues in substrates and Ub during monoubiquitination and polyubiquitination. Lysine selection is important for the generation of diverse substrate-Ub structures, which provides versatility to this pathway in the targeting of proteins to different fates. The mechanisms of lysine selection remain poorly understood, with previous studies suggesting that the ubiquitination site(s) is selected by the E2/E3-mediated positioning of a lysine(s) toward the E2/E3 active site. By studying the polyubiquitination of Sic1 by the E2 protein Cdc34 and the RING E3 Skp1/Cul1/F-box (SCF) protein, we now demonstrate that in addition to E2/E3-mediated positioning, proximal amino acids surrounding the lysine residues in Sic1 and Ub are critical for ubiquitination. This mechanism is linked to key residues composing the catalytic core of Cdc34 and independent of SCF. Changes to these core residues altered the lysine preference of Cdc34 and specified whether this enzyme monoubiquitinated or polyubiquitinated Sic1. These new findings indicate that compatibility between amino acids surrounding acceptor lysine residues and key amino acids in the catalytic core of ubiquitin-conjugating enzymes is an important mechanism for lysine selection during ubiquitination. PMID:20194622

  6. Filament formation by metabolic enzymes is a specific adaptation to an advanced state of cellular starvation

    PubMed Central

    Petrovska, Ivana; Nüske, Elisabeth; Munder, Matthias C; Kulasegaran, Gayathrie; Malinovska, Liliana; Kroschwald, Sonja; Richter, Doris; Fahmy, Karim; Gibson, Kimberley; Verbavatz, Jean-Marc; Alberti, Simon

    2014-01-01

    One of the key questions in biology is how the metabolism of a cell responds to changes in the environment. In budding yeast, starvation causes a drop in intracellular pH, but the functional role of this pH change is not well understood. Here, we show that the enzyme glutamine synthetase (Gln1) forms filaments at low pH and that filament formation leads to enzymatic inactivation. Filament formation by Gln1 is a highly cooperative process, strongly dependent on macromolecular crowding, and involves back-to-back stacking of cylindrical homo-decamers into filaments that associate laterally to form higher order fibrils. Other metabolic enzymes also assemble into filaments at low pH. Hence, we propose that filament formation is a general mechanism to inactivate and store key metabolic enzymes during a state of advanced cellular starvation. These findings have broad implications for understanding the interplay between nutritional stress, the metabolism and the physical organization of a cell. DOI: http://dx.doi.org/10.7554/eLife.02409.001 PMID:24771766

  7. Lung function, atopy, specific hypersensitivity, and smoking of workers in the enzyme detergent industry over 11 years.

    PubMed Central

    Flood, D F; Blofeld, R E; Bruce, C F; Hewitt, J I; Juniper, C P; Roberts, D M

    1985-01-01

    A study of 2800 workers employed in three factories of the two major manufacturers of enzymatic products in the United Kingdom covering 11 years of operation from 1969 to 1980 showed that 2344 workers had sufficient lung function data to meet the operational criteria and these were analysed in three separate groups by factory locations. Spirometry and prick tests for specific skin reactions to standardised enzyme were performed at six monthly intervals for the first six years of the study and then annually. Factory enzyme dust and total dust measurements were made to determine the degree of dust exposure of the subjects. The lung function of the factory groups was analysed for the effects of working in the detergent industry, the degree of exposure to enzymes, skin prick test positivity to enzymes, atopicity, and smoking. The 4.5% of workers who had experienced respiratory effects from enzymes were analysed separately. Exposure to the enzyme allergen has had no significant long term effect on the lung function of the detergent workers. A higher proportion of atopics than non-atopics became skin test positive to the allergen and more smokers than non-smokers were sensitised. The overall lung function of detergent workers showed 39 ml/year loss in FEV1 on the 11 year longitudinal study and 51 ml/year loss on the lateral (cross sectional) analysis with better lung function in the south east than the north west of England. In the development of the methodology for the study several potential problems were discovered that could remain unrecognised in a cross sectional analysis performed in isolation. PMID:3871157

  8. Structural Basis for Specificity of Propeptide-Enzyme Interaction in Barley C1A Cysteine Peptidases

    PubMed Central

    Cambra, Inés; Hernández, David; Diaz, Isabel; Martinez, Manuel

    2012-01-01

    C1A cysteine peptidases are synthesized as inactive proenzymes. Activation takes place by proteolysis cleaving off the inhibitory propeptide. The inhibitory capacity of propeptides from barley cathepsin L and B-like peptidases towards commercial and barley cathepsins has been characterized. Differences in selectivity have been found for propeptides from L-cathepsins against their cognate and non cognate enzymes. Besides, the propeptide from barley cathepsin B was not able to inhibit bovine cathepsin B. Modelling of their three-dimensional structures suggests that most propeptide inhibitory properties can be explained from the interaction between the propeptide and the mature cathepsin structures. Their potential use as biotechnological tools is discussed. PMID:22615948

  9. Development of a highly sensitive and specific blastomycosis antibody enzyme immunoassay using Blastomyces dermatitidis surface protein BAD-1.

    PubMed

    Richer, Sarah M; Smedema, Melinda L; Durkin, Michelle M; Brandhorst, T Tristan; Hage, Chadi A; Connolly, Patricia A; Leland, Diane S; Davis, Thomas E; Klein, Bruce S; Wheat, L Joseph

    2014-02-01

    Serologic tests for antibodies to Blastomyces dermatitidis are not thought to be useful for the diagnosis of blastomycosis, in part due to the low sensitivity of immunodiffusion and complement fixation. Earlier studies have shown that the enzyme immunoassay improves the sensitivity of antibody detection for the diagnosis of blastomycosis. Microplates coated with the B. dermatitidis surface protein BAD-1 were used for testing sera from patients with proven blastomycosis or histoplasmosis and controls. Semiquantification was accomplished by using standards containing human anti-B. dermatitidis antibodies. The antibodies were detected in 87.8% of the patients with blastomycosis by the enzyme immunoassay compared to 15.0% by immunodiffusion. The specificities were 99.2% for patients with nonfungal infections and healthy subjects and 94.0% for patients with histoplasmosis. The results were highly reproducible on repeat testing. When combined with antigen testing, antibody testing improved the sensitivity from 87.8% to 97.6%. Enzyme immunoassay detection of antibodies against BAD-1 is highly specific, has greatly improved sensitivity over immunodiffusion, and may identify cases with negative results by antigen testing. This assay has the potential to aid in the diagnosis of blastomycosis.

  10. Using site-saturation mutagenesis to explore mechanism and substrate specificity in thiamin diphosphate-dependent enzymes.

    PubMed

    Andrews, Forest H; McLeish, Michael J

    2013-12-01

    For almost 20 years, site-saturation mutagenesis (SSM) has been used to evolve stereoselective enzymes as catalysts for synthetic organic chemistry. Much of this work has focused on enzymes such as lipases and esterases, although the range is rapidly expanding. By contrast, using SSM to study enzyme mechanisms is much less common. Instead, site-directed mutagenesis is more generally employed, with a particular emphasis on alanine variants. In the present review, we provide examples of the growing use of SSM to study not only substrate and reaction selectivity, but also the reaction mechanism of thiamin diphosphate (ThDP)-dependent enzymes. We report that the use of SSM to examine the roles of the catalytic residues of benzoylformate decarboxylase gave rise to results that were at odds with earlier kinetic and structural studies using alanine substitutions and also questioned their conclusions. SSM was also employed to examine the long held tenet that a bulky hydrophobic residue provides a fulcrum by which the V-conformation of the ThDP cofactor is maintained. X-ray structures showed that ThDP stayed in the V-conformation even when the replacement residues were charged or did not contact the cofactor. We also summarize the results obtained when SSM was used to evolve new substrate specificity and/or enantioselectivity in ThDP-dependent enzymes such as benzoylformate decarboxylase, transketolase, 2-succinyl-5-enolpyruvyl-6-hydroxy-3-cyclohexene-1-carboxylate synthase and the E1 component of the 2-oxoglutarate dehydrogenase complex.

  11. Substrate orientation and specificity in xanthine oxidase: crystal structures of the enzyme in complex with indole-3-acetaldehyde and guanine.

    PubMed

    Cao, Hongnan; Hall, James; Hille, Russ

    2014-01-28

    Xanthine oxidase is a molybdenum-containing hydroxylase that catalyzes the hydroxylation of sp(2)-hybridized carbon centers in a variety of aromatic heterocycles as well as aldehydes. Crystal structures of the oxidase form of the bovine enzyme in complex with a poor substrate indole-3-acetaldehyde and the nonsubstrate guanine have been determined, both at a resolution of 1.6 Å. In each structure, a specific and unambiguous orientation of the substrate in the active site is observed in which the hydroxylatable site is oriented away from the active site molybdenum center. The orientation seen with indole-3-acetaldehyde has the substrate positioned with the indole ring rather than the exocyclic aldehyde nearest the molybdenum center, indicating that the substrate must rotate some 30° in the enzyme active site to permit hydroxylation of the aldehyde group (as observed experimentally), accounting for the reduced reactivity of the enzyme toward this substrate. The principal product of hydroxylation of indole-3-acetaldehyde by the bovine enzyme is confirmed to be indole-3-carboxylic acid based on its characteristic UV-vis spectrum, and the kinetics of enzyme reduction are reported. With guanine, the dominant orientation seen crystallographically has the C-8 position that might be hydroxylated pointed away from the active site molybdenum center, in a configuration resembling that seen previously with hypoxanthine (a substrate that is effectively hydroxylated at position 2). The ∼180° reorientation required to permit reaction is sterically prohibited, indicating that substrate (mis)orientation in the active site is a major factor precluding formation of the highly mutagenic 8-hydroxyguanine.

  12. Goal specificity and learning with a hypermedia program.

    PubMed

    Vollmeyer, Regina; Burns, Bruce D

    2002-01-01

    Problem solving research has found that a nonspecific goal (NSG) leads to better learning than a specific goal (SG). This effect can be understood in terms of dual-space search theories of problem solving. To apply the theory, we studied goal specificity effects with a hypermedia program in which participants had to learn about the outbreak of World War 1, either with the goal to find twenty dates (i.e., SG) or with the goal to explain the reasons for the war (i.e., NSG). As expected, compared to the SG-group, the NSG-group correctly answered more factual questions about the text during the task, spent more time on average per page, and more often looked for extra information. In a final questionnaire with factual and inferential questions, the NSG-group still performed better than the SG-group. The NSG-group may also show better transfer of what they had learnt to a new situation.

  13. Ghrelin O-acyltransferase (GOAT), a specific enzyme that modifies ghrelin with a medium-chain fatty acid.

    PubMed

    Kojima, Masayasu; Hamamoto, Akie; Sato, Takahiro

    2016-10-01

    In the gastric peptide hormone ghrelin, serine 3 (threonine 3 in frogs) is modified, primarily by n-octanoic acid; this modification is essential for ghrelin's activity. The enzyme that transfers n-octanoic acid to Ser3 of ghrelin is ghrelin O-acyltransferase (GOAT). GOAT, the only enzyme known to catalyze acyl modification of ghrelin, specifically modifies serine (or threonine) at the third position and does not modify other serine residues in ghrelin peptides. GOAT prefers n-hexanoyl-CoA over n-octanoyl-CoA as the acyl donor, although in the stomach the n-octanoyl form is the predominant form of acyl-modified ghrelin. GOAT is a promising target for drug development to treat metabolic diseases and eating disorders.

  14. Development of a Plate-Based Screening Assay to Investigate the Substrate Specificity of the PRMT Family of Enzymes.

    PubMed

    Nguyen, Hao C; Wang, Min; Salsburg, Andrew; Knuckley, Bryan

    2015-09-14

    There are nine protein arginine methyltransferases (PRMTs 1-9) expressed in humans that vary in both subcellular localization and substrate specificity. The variation in substrate specificity between isozymes leads to competing effects that result in either activation or repression of tumor suppressor genes. Current methods used to study substrate specificity for these enzymes utilize radioisotopic labeling of substrates, mass spectrometry analysis of complex samples, or coupled assays that monitor cofactor degradation. Herein, we report the development of a rapid, nonradioactive, and sensitive method for screening multiple peptides in parallel to gain insight into the substrate specificity of PRMT enzymes. Our assay provides a major advantage over other high-throughput screening assays (e.g., ELISA, AlphaScreen chemiluminescence) by eliminating the need for purification of individual peptides and provides a timesaving, cost-effective alternative to the traditional PRMT assays. A one-bead one-compound (OBOC) peptide library was synthesized and subsequently screened against PRMT1 in a 96-well plate. This screen resulted in identification of a novel PRMT1 substrate with kinetic parameters similar to histone H4-21 (e.g., the best-known PRMT1 peptide substrate).

  15. Crystal structures of Physcomitrella patens AOC1 and AOC2: insights into the enzyme mechanism and differences in substrate specificity.

    PubMed

    Neumann, Piotr; Brodhun, Florian; Sauer, Kristin; Herrfurth, Cornelia; Hamberg, Mats; Brinkmann, Jens; Scholz, Julia; Dickmanns, Achim; Feussner, Ivo; Ficner, Ralf

    2012-11-01

    In plants, oxylipins regulate developmental processes and defense responses. The first specific step in the biosynthesis of the cyclopentanone class of oxylipins is catalyzed by allene oxide cyclase (AOC) that forms cis(+)-12-oxo-phytodienoic acid. The moss Physcomitrella patens has two AOCs (PpAOC1 and PpAOC2) with different substrate specificities for C₁₈- and C₂₀-derived substrates, respectively. To better understand AOC's catalytic mechanism and to elucidate the structural properties that explain the differences in substrate specificity, we solved and analyzed the crystal structures of 36 monomers of both apo and ligand complexes of PpAOC1 and PpAOC2. From these data, we propose the following intermediates in AOC catalysis: (1) a resting state of the apo enzyme with a closed conformation, (2) a first shallow binding mode, followed by (3) a tight binding of the substrate accompanied by conformational changes in the binding pocket, and (4) initiation of the catalytic cycle by opening of the epoxide ring. As expected, the substrate dihydro analog cis-12,13S-epoxy-9Z,15Z-octadecadienoic acid did not cyclize in the presence of PpAOC1; however, when bound to the enzyme, it underwent isomerization into the corresponding trans-epoxide. By comparing complex structures of the C₁₈ substrate analog with in silico modeling of the C₂₀ substrate analog bound to the enzyme allowed us to identify three major molecular determinants responsible for the different substrate specificities (i.e. larger active site diameter, an elongated cavity of PpAOC2, and two nonidentical residues at the entrance of the active site).

  16. Synthesis of a Comprehensive Polyprenol Library for Evaluation of Bacterial Enzyme Lipid Substrate Specificity.

    PubMed

    Wu, Baolin; Woodward, Robert; Wen, Liuqing; Wang, Xuan; Zhao, Guohui; Wang, Peng George

    2013-12-01

    Polyprenols, a type of universal glycan lipid carrier, play important roles for glycan bio-assembly in wide variety of living systems. Chemical synthesis of natural polyisoprenols such as undecaprenol and dolichols, but especially their homologs, could serves as a powerful molecular tool to dissect and define the functions of enzymes involved in glycan biosynthesis. In this paper, we report an efficient and reliable method to construct this type of hydrophoic molecule through a base-mediated iterative coupling approach using a key bifunctional (Z, Z)-diisoprenyl building block. The ligation with N-acetyl-D-glactosamine (GalNAc) with a set of the synthesized lipid analogs forming polyprenol pyrophosphate linked GalNAc (GalNAc-PP-lipid) conjugates is also demonstrated.

  17. Dihydroflavonol 4-reductase genes encode enzymes with contrasting substrate specificity and show divergent gene expression profiles in Fragaria species.

    PubMed

    Miosic, Silvija; Thill, Jana; Milosevic, Malvina; Gosch, Christian; Pober, Sabrina; Molitor, Christian; Ejaz, Shaghef; Rompel, Annette; Stich, Karl; Halbwirth, Heidi

    2014-01-01

    During fruit ripening, strawberries show distinct changes in the flavonoid classes that accumulate, switching from the formation of flavan 3-ols and flavonols in unripe fruits to the accumulation of anthocyanins in the ripe fruits. In the common garden strawberry (Fragaria×ananassa) this is accompanied by a distinct switch in the pattern of hydroxylation demonstrated by the almost exclusive accumulation of pelargonidin based pigments. In Fragaria vesca the proportion of anthocyanins showing one (pelargonidin) and two (cyanidin) hydroxyl groups within the B-ring is almost equal. We isolated two dihydroflavonol 4-reductase (DFR) cDNA clones from strawberry fruits, which show 82% sequence similarity. The encoded enzymes revealed a high variability in substrate specificity. One enzyme variant did not accept DHK (with one hydroxyl group present in the B-ring), whereas the other strongly preferred DHK as a substrate. This appears to be an uncharacterized DFR variant with novel substrate specificity. Both DFRs were expressed in the receptacle and the achenes of both Fragaria species and the DFR2 expression profile showed a pronounced dependence on fruit development, whereas DFR1 expression remained relatively stable. There were, however, significant differences in their relative rates of expression. The DFR1/DFR2 expression ratio was much higher in the Fragaria×ananassa and enzyme preparations from F.×ananassa receptacles showed higher capability to convert DHK than preparations from F. vesca. Anthocyanin concentrations in the F.×ananassa cultivar were more than twofold higher and the cyanidin:pelargonidin ratio was only 0.05 compared to 0.51 in the F. vesca cultivar. The differences in the fruit colour of the two Fragaria species can be explained by the higher expression of DFR1 in F.×ananassa as compared to F. vesca, a higher enzyme efficiency (Kcat/Km values) of DFR1 combined with the loss of F3'H activity late in fruit development of F.×ananassa.

  18. Human and rodent carboxylesterases: immunorelatedness, overlapping substrate specificity, differential sensitivity to serine enzyme inhibitors, and tumor-related expression.

    PubMed

    Xie, Mingxing; Yang, Dongfang; Liu, Lan; Xue, Bob; Yan, Bingfang

    2002-05-01

    Carboxylesterases hydrolyze numerous endogenous and foreign compounds with diverse structures. Humans and rodents express multiple forms of carboxylesterases, which share a high degree of sequence identity (approximately 70%). Alignment analyses locate in carboxylesterases several functional subsites such the catalytic triad as seen in acetylcholinesterase. The aim of this study was to determine among human and rodent carboxylesterases the immunorelatedness, overlapping substrate specificity, differential sensitivity to serine enzyme inhibitors, tissue distribution, and tumor-related expression. Six antibodies against whole carboxylesterases or synthetic peptides were tested for their reactivity toward 11 human or rodent recombinant carboxylesterases. The antibodies against whole proteins generally exhibited a broader cross-reactivity than the anti-peptide antibodies. All carboxylesterases hydrolyzed para-nitrophenylacetate and para-nitrophenylbutyrate. However, the relative activity varied markedly from enzyme to enzyme (>20-fold), and some carboxylesterases showed a clear substrate preference. Carboxylesterases with the same functional subsites had a similar profile on substrate specificity and sensitivity toward phenylmethylsulfonyl fluoride (PMSF) and paraoxon, suggesting that these subsites play determinant roles in the recognition of substrates and inhibitors. Among three human carboxylesterases, HCE-1 hydrolyzed both substrates to a similar extent, whereas HCE-2 and HCE-3 showed an opposite substrate preference. All three enzymes were inhibited by PMSF and paraoxon, but they showed a marked difference in relative sensitivities. Based on immunoblotting analyses, HCE-1 was present in all tissues examined, whereas HCE-2 and HCE-3 were expressed in a tissue-restricted pattern. Colon carcinomas expressed slightly higher levels of HCE-1 and HCE-2 than the adjacent normal tissues, whereas the opposite was true with HCE-3.

  19. A method for predicting individual residue contributions to enzyme specificity and binding-site energies, and its application to MTH1.

    PubMed

    Stewart, James J P

    2016-11-01

    A new method for predicting the energy contributions to substrate binding and to specificity has been developed. Conventional global optimization methods do not permit the subtle effects responsible for these properties to be modeled with sufficient precision to allow confidence to be placed in the results, but by making simple alterations to the model, the precisions of the various energies involved can be improved from about ±2 kcal mol(-1) to ±0.1 kcal mol(-1). This technique was applied to the oxidized nucleotide pyrophosphohydrolase enzyme MTH1. MTH1 is unusual in that the binding and reaction sites are well separated-an advantage from a computational chemistry perspective, as it allows the energetics involved in docking to be modeled without the need to consider any issues relating to reaction mechanisms. In this study, two types of energy terms were investigated: the noncovalent interactions between the binding site and the substrate, and those responsible for discriminating between the oxidized nucleotide 8-oxo-dGTP and the normal dGTP. Both of these were investigated using the semiempirical method PM7 in the program MOPAC. The contributions of the individual residues to both the binding energy and the specificity of MTH1 were calculated by simulating the effect of mutations. Where comparisons were possible, all calculated results were in agreement with experimental observations. This technique provides fresh insight into the binding mechanism that enzymes use for discriminating between possible substrates.

  20. Determining the specificities of TALENs, Cas9, and other genome editing enzymes

    PubMed Central

    Pattanayak, Vikram; Guilinger, John P.; Liu, David R.

    2015-01-01

    The rapid development of programmable site-specific endonucleases has led to a dramatic increase in genome engineering activities for research and therapeutic purposes. Specific loci of interest in the genomes of a wide range of organisms including mammals can now be modified using zinc-finger nucleases (ZFNs), transcription activator-like endonucleases (TALENs), and CRISPR-associated Cas9 endonucleases in a site-specific manner, in some cases requiring relatively modest effort for endonuclease design, construction and application. While these technologies have made genome engineering widely accessible, the ability of programmable nucleases to cleave off-target sequences can limit their applicability and raise concerns about therapeutic safety. In this article we review methods to evaluate and improve the DNA cleavage activity of programmable site-specific endonucleases and describe a procedure for a comprehensive off-target profiling method based on the in vitro selection of very large (~1012-membered) libraries of potential nuclease substrates. PMID:25398335

  1. 24 CFR 200.936 - Supplementary specific procedural requirements under HUD building products certification program...

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... requirements under HUD building products certification program for solid fuel type room heaters and fireplace... Supplementary specific procedural requirements under HUD building products certification program for solid fuel... fireplace stoves certified under the HUD Building Products Certification Program shall be...

  2. 24 CFR 200.936 - Supplementary specific procedural requirements under HUD building products certification program...

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... requirements under HUD building products certification program for solid fuel type room heaters and fireplace... Supplementary specific procedural requirements under HUD building products certification program for solid fuel... fireplace stoves certified under the HUD Building Products Certification Program shall be...

  3. A family of phase-variable restriction enzymes with differing specificities generated by high-frequency gene rearrangements.

    PubMed

    Dybvig, K; Sitaraman, R; French, C T

    1998-11-10

    The hsd genes of Mycoplasma pulmonis encode restriction and modification enzymes exhibiting a high degree of sequence similarity to the type I enzymes of enteric bacteria. The S subunits of type I systems dictate the DNA sequence specificity of the holoenzyme and are required for both the restriction and the modification reactions. The M. pulmonis chromosome has two hsd loci, both of which contain two hsdS genes each and are complex, site-specific DNA inversion systems. Embedded within the coding region of each hsdS gene are a minimum of three sites at which DNA inversions occur to generate extensive amino acid sequence variations in the predicted S subunits. We show that the polymorphic hsdS genes produced by gene rearrangement encode a family of functional S subunits with differing DNA sequence specificities. In addition to creating polymorphisms in hsdS sequences, DNA inversions regulate the phase-variable production of restriction activity because the other genes required for restriction activity (hsdR and hsdM) are expressed only from loci that are oriented appropriately in the chromosome relative to the hsd promoter. These data cast doubt on the prevailing paradigms that restriction systems are either selfish or function to confer protection from invasion by foreign DNA.

  4. Simple dual-spotting procedure enhances nLC-MALDI MS/MS analysis of digests with less specific enzymes.

    PubMed

    Baeumlisberger, Dominic; Rohmer, Marion; Arrey, Tabiwang N; Mueller, Benjamin F; Beckhaus, Tobias; Bahr, Ute; Barka, Guenes; Karas, Michael

    2011-06-03

    The beneficial effect of high mass accuracy in mass spectrometry is especially pronounced when using less specific enzymes as the number of theoretically possible peptides increases dramatically without any cleavage specificity defined. Together with a preceding chromatographic separation, high-resolution mass spectrometers such as the MALDI-LTQ-Orbitrap are therefore well suited for the analysis of protein digests with less specific enzymes. A combination with fast, automated, and informative MALDI-TOF/TOF analysis has already been shown to yield increased total peptide and protein identifications. Here, a simple method for nLC separation and subsequent alternating spotting on two targets for both a MALDI-LTQ-Orbitrap and a MALDI-TOF/TOF instrument is introduced. This allows for simultaneous measurements on both instruments and subsequent combination of both data sets by an in-house written software tool. The performance of this procedure was evaluated using a mixture of four standard proteins digested with elastase. Three replicate runs were examined concerning repeatability and the total information received from both instruments. A cytosolic extract of C. glutamicum was used to demonstrate the applicability to more complex samples. Database search results showed that an additional 32.3% of identified peptides were found using combined data sets in comparison to MALDI-TOF/TOF data sets.

  5. Specific capture of the hydrolysate on magnetic beads for sensitive detecting plant vacuolar processing enzyme activity.

    PubMed

    Zhou, Jun; Cheng, Meng; Zeng, Lizhang; Liu, Weipeng; Zhang, Tao; Xing, Da

    2016-05-15

    Conventional plant protease detection always suffers from high background interference caused by the complex coloring metabolites in plant cells. In this study, a bio-modified magnetic beads-based strategy was developed for sensitive and quantitative detection of plant vacuolar processing enzyme (VPE) activity. Cleavage of the peptide substrate (ESENCRK-FITC) after asparagine residue by VPE resulted in the 2-cyano-6-amino-benzothiazole (CABT)-functionalized magnetic beads capture of the severed substrate CRK-FITC via a condensation reaction between CABT and cysteine (Cys). The catalytic activity was subsequently obtained by the confocal microscopy imaging and flow cytometry quantitative analysis. The sensor system integrated advantages of (i) the high efficient enrichment and separation capabilities of magnetic beads and (ii) the catalyst-free properties of the CABT-Cys condensation reaction. It exhibited a linear relationship between the fluorescence signal and the concentration of severed substrate in the range of 10-600 pM. The practical results showed that, compared with normal growth conditions, VPE activity was increased by 2.7-fold (307.2 ± 25.3 μM min(-1)g(-1)) upon cadmium toxicity stress. This platform effectively overcame the coloring metabolites-caused background interference, showing fine applicability for the detection of VPE activity in real samples. The strategy offers great sensitivity and may be further extended to other protease activity detection.

  6. The fungal cultivar of leaf-cutter ants produces specific enzymes in response to different plant substrates

    SciTech Connect

    Khadempour, Lily; Burnum-Johnson, Kristin E.; Baker, Erin S.; Nicora, Carrie D.; Webb-Robertson, Bobbie-Jo M.; White, Richard A.; Monroe, Matthew E.; Huang, Eric L.; Smith, Richard D.; Currie, Cameron R.

    2016-10-26

    Herbivores use symbiotic microbes to help gain access to energy and nutrients from plant material. Leaf-cutter ants are a paradigmatic example, having tremendous impact on their ecosystems as dominant generalist herbivores through cultivation of a fungus, Leucoagaricus gongylophorous. Here we examine how this mutualism could facilitate the flexible substrate incorporation of the ants by providing leaf-cutter ant subcolonies four substrate types: leaves, flowers, oats, and a mixture of all three. Through metaproteomic analysis of the fungus gardens, we were able to identify and quantify 1766 different fungal proteins, including 161 biomass-degrading enzymes. This analysis revealed that fungal protein profiles were significantly different between subcolonies fed different substrates with the highest abundance of cellulolytic enzymes observed in the leaf and flower treatments. When the fungus garden is provided with leaves and flowers, which contain the majority of their energy in recalcitrant material, it increases its production of proteins that break down cellulose: endoglucanases, exoglucanase and β-glucosidase. Further, the complete metaproteomes for the leaves and flowers treatments were very similar, the mixed treatment closely resembled the treatment with oats alone. This suggests that when provided a mixture of substrates, the fungus garden preferentially produces enzymes necessary for breakdown of simpler, more digestible substrates. This flexible, substrate-specific response of the fungal cultivar allows the leaf-cutter ants to derive energy from a wide range of substrates, which may contribute to their ability to be dominant generalist herbivores.

  7. Identification of the Elusive Mammalian Enzyme Phospatidylcholine-Specific Phospholipase C

    DTIC Science & Technology

    2015-01-01

    mammalian protein, phosphatidycholine- specific phospholipase C (PC-PLC) in the inflammatory processes involved in progression of rheumatoid arthritis (RA...serum, rheumatoid arthritis , transcriptome sequencing, HUVECs, U937 cells 16. SECURITY CLASSIFICATION OF: 17. LIMITATION OF ABSTRACT 18. NUMBER OF...aims at identifying novel players that are critically involved in the progression of rheumatoid arthritis (RA). The identification of these factors

  8. A new test of computational protein design: predicting posttranslational modification specificity for the enzyme SMYD2.

    PubMed

    Reynolds, Kimberly A

    2015-01-06

    In this issue of Structure, Lanouette and colleagues use a combination of computation and experiment to define a specificity motif for the lysine methyltransferase SMYD2. Using this motif, they predict and experimentally verify four new SMYD2 substrates.

  9. Aquatic Plant Control Research Program. Biological Control of Hydrilla verticillata (L.f.) Royle with Lytic Enzyme-Producing Microorganisms.

    DTIC Science & Technology

    1985-09-01

    Lytic enzyme-producing microorganisms Biocontrol Mycoherbicides Hydrilla Induced pathogenicity 20. ASTRACT (Coartinue G rev’wm eft if n*..eeam7 mod...However, no natural enemies of hydrilla have yet been imported that are promising biocontrol candidates. Therefore, a less conventional approach was...of microorganisms that function in the decay process. These microorganisms pro- duce enzymes capable of lysing specific plant components such as

  10. Cardiolipin: a stereospecifically spin-labeled analogue and its specific enzymic hydrolysis.

    PubMed Central

    Cable, M B; Jacobus, J; Powell, G L

    1978-01-01

    The spin-labeled cardiolipin 1-(3-sn-phosphatidyl)-3-[1-acyl-2-(16-doxylstearoyl)glycero(3)phosphol]-sn-glycerol has been prepared. The stereoselective synthesis makes use of the monolysocardiolipin 1-(3-sn-phosphatidyl)-3-[1-acyl-2-lyso-sn-glycero(3)phospho]-sn-glycerol, available from the stereospecific hydrolysis of cardiolipin by phospholipase A2 (phosphatide 2-acylhydrolase, EC 3.1.1.4) of Trimeresurus flavoviridis. The results of treatment of the spin-labeled cardiolipin with the cardiolipin-specific phospholipase D (phosphatidylcholine phosphatidohydrolase, EC 3.1.4.4) (Hemophilus parainfluenzae) of known specificity and with phospholipase C (phosphatidylcholine cholinephosphohydrolase, EC 3.1.4.3) of Bacillus cereus are consistent with the assigned structure. The spin-labeled cardiolipin is further characterized and the unique features of this diastereomer are discussed in the context of the unusual stereochemistry of the natural phospholipid. PMID:274715

  11. Identification of the Elusive Mammalian Enzyme Phosphatidylcholine-Specific Phospholipase C

    DTIC Science & Technology

    2014-07-01

    processes involved in progression of rheumatoid arthritis (RA). Thus, the main scopes of this proposal are: 1. to identify the PC-PLC gene and protein...of PC-PLC. 15. SUBJECT TERMS Phosphatidycholine-specific phospholipase C, lipopolisaccharide, oxidized lipoproteins, serum, rheumatoid arthritis ...present proposal aims at identifying novel players that are critically involved in the progression of rheumatoid arthritis (RA). The identification of

  12. Novel substrate specificity of glutathione synthesis enzymes from Streptococcus agalactiae and Clostridium acetobutylicum

    SciTech Connect

    Kino, Kuniki . E-mail: kkino@waseda.jp; Kuratsu, Shoko; Noguchi, Atsushi; Kokubo, Masahiro; Nakazawa, Yuji; Arai, Toshinobu; Yagasaki, Makoto; Kirimura, Kohtaro

    2007-01-12

    Glutathione (GSH) is synthesized by {gamma}-glutamylcysteine synthetase ({gamma}-GCS) and glutathione synthetase (GS) in living organisms. Recently, bifunctional fusion protein, termed {gamma}-GCS-GS catalyzing both {gamma}-GCS and GS reactions from gram-positive firmicutes Streptococcus agalactiae, has been reported. We revealed that in the {gamma}-GCS activity, S. agalactiae {gamma}-GCS-GS had different substrate specificities from those of Escherichia coli {gamma}-GCS. Furthermore, S. agalactiae {gamma}-GCS-GS synthesized several kinds of {gamma}-glutamyltripeptide, {gamma}-Glu-X{sub aa}-Gly, from free three amino acids. In Clostridium acetobutylicum, the genes encoding {gamma}-GCS and putative GS were found to be immediately adjacent by BLAST search, and had amino acid sequence homology with S. agalactiae {gamma}-GCS-GS, respectively. We confirmed that the proteins expressed from each gene showed {gamma}-GCS and GS activity, respectively. C. acetobutylicum GS had broad substrate specificities and synthesized several kinds of {gamma}-glutamyltripeptide, {gamma}-Glu-Cys-X{sub aa}. Whereas the substrate specificities of {gamma}-GCS domain protein and GS domain protein of S. agalactiae {gamma}-GCS-GS were the same as those of S. agalactiae {gamma}-GCS-GS.

  13. Tumor-Specific Formation of Enzyme-Instructed Supramolecular Self-Assemblies as Cancer Theranostics

    PubMed Central

    Huang, Peng; Gao, Yuan; Lin, Jing; Hu, Hao; Liao, Hsien-Shun; Yan, Xuefeng; Tang, Yuxia; Jin, Albert; Song, Jibin; Niu, Gang; Zhang, Guofeng; Horkay, Ferenc; Chen, Xiaoyuan

    2017-01-01

    Despite the effort of developing various nanodelivery systems, most of them suffer from undesired high uptakes by the reticuloendothelial system, such as liver and spleen. Herein we develop an endogenous phosphatase-triggered coassembly strategy to form tumor-specific indocyanine green (ICG)-doped nanofibers (5) for cancer theranostics. Based on coordinated intermolecular interactions, 5 significantly altered near-infrared absorbance of ICG, which improves the critical photoacoustic and photothermal properties. The phosphatase-instructed coassembly process, as well as its theranostic capability, was successfully conducted at different levels ranging from in vitro, living cell, tissue mimic, to in vivo. Specifically, the tumor uptake of ICG was markedly increased to 15.05 ± 3.78%ID/g, which was 25-fold higher than that of free ICG (0.59 ± 0.24%ID/g) at 4 h after intravenous injection. The resulting ultrahigh T/N ratios (>15) clearly differentiated tumors from the surrounding normal tissue. Complete tumor elimination with high therapeutic accuracy has been successfully achieved upon laser irradiation (0.8 W/cm2, 5 min) within 24–48 h postinjection. As the first example, in vivo formation of tumor-specific ICG-doped nanofiber for PTT theranostics owns the immense potential for clinical translation of personalized nanomedicine with targeted drug delivery as well as for cancer theranostics. PMID:26301492

  14. 41 CFR 101-29.221 - Federal Specifications, Standards and Commercial Item Description Program (Federal...

    Code of Federal Regulations, 2010 CFR

    2010-07-01

    ..., Standards and Commercial Item Description Program (Federal Standardization Program). 101-29.221 Section 101... Standardization Program). The Federal Specifications, Standards and Commercial Item Description Program is a standardization program developed under authority of the Federal Property and Administrative Services Act of...

  15. Enzyme-Linked Immunosorbent Assay for Specific Identification and Enumeration of Azospirillum brasilense Cd. in Cereal Roots †

    PubMed Central

    Levanony, Hanna; Bashan, Yoav; Kahana, Zvi E.

    1987-01-01

    The enzyme-linked immunosorbent assay is suggested as a reliable, sensitive, and highly specific method for the identification and enumeration of Azospirillum brasilense Cd. As few as 105 CFU/ml can be practically identified by this method. At higher bacterial numbers, sensitivity increased linearly up to 5 × 108 CFU/ml, yielding useful standard curves. No cross-reaction was found either with different closely related Azospirillum strains or with other rhizosphere bacteria. The method allows for a specific identification of A. brasilense Cd. both in pure cultures and in mixtures with other bacterial species, even when the colony morphology is variable. The method was successfully applied to assess the degree of root colonization on various cereals by A. brasilense Cd. PMID:16347284

  16. Piezoelectric tuning fork probe for atomic force microscopy imaging and specific recognition force spectroscopy of an enzyme and its ligand.

    PubMed

    Makky, Ali; Viel, Pascal; Chen, Shu-wen Wendy; Berthelot, Thomas; Pellequer, Jean-Luc; Polesel-Maris, Jérôme

    2013-11-01

    Piezoelectric quartz tuning fork has drawn the attention of many researchers for the development of new atomic force microscopy (AFM) self-sensing probes. However, only few works have been done for soft biological materials imaging in air or aqueous conditions. The aim of this work was to demonstrate the efficiency of the AFM tuning fork probe to perform high-resolution imaging of proteins and to study the specific interaction between a ligand and its receptor in aqueous media. Thus, a new kind of self-sensing AFM sensor was introduced to realize imaging and biochemical specific recognition spectroscopy of glucose oxidase enzyme using a new chemical functionalization procedure of the metallic tips based on the electrochemical reduction of diazonium salt. This scanning probe as well as the functionalization strategy proved to be efficient respectively for the topography and force spectroscopy of soft biological materials in buffer conditions.

  17. Detection of Leptospira-Specific Antibodies Using a Recombinant Antigen-Based Enzyme-Linked Immunosorbent Assay

    PubMed Central

    Chen, Hua-Wei; Zhang, Zhiwen; Halsey, Eric S.; Guevara, Carolina; Canal, Enrique; Hall, Eric; Maves, Ryan; Tilley, Drake H.; Kochel, Tadeusz J.; Ching, Wei-Mei

    2013-01-01

    We produced three highly purified recombinant antigens rLipL32, rLipL41, and rLigA-Rep (leptospiral immunoglobulin-like A repeat region) for the detection of Leptospira-specific antibodies in an enzyme-linked immunosorbent assay (ELISA). The performance of these recombinant antigens was evaluated using 121 human sera. Among them, 63 sera were microscopic agglutination test (MAT)-confirmed positive sera from febrile patients in Peru, 22 sera were indigenous MAT-negative febrile patient sera, and 36 sera were from patients with other febrile diseases from Southeast Asia, where leptospirosis is also endemic. Combining the results of immunoglobulin M (IgM) and IgG detection from these three antigens, the overall sensitivity is close to 90% based on the MAT. These results suggest that an ELISA using multiple recombinant antigens may be used as an alternative method for the detection of Leptospira-specific antibodies. PMID:24166046

  18. Muscle enzyme and fiber type-specific sarcomere protein increases in serum after inertial concentric-eccentric exercise.

    PubMed

    Carmona, G; Guerrero, M; Cussó, R; Padullés, J M; Moras, G; Lloret, M; Bedini, J L; Cadefau, J A

    2015-12-01

    Muscle damage induced by inertial exercise performed on a flywheel device was assessed through the serum evolution of muscle enzymes, interleukin 6, and fiber type-specific sarcomere proteins such as fast myosin (FM) and slow myosin (SM). We hypothesized that a model of muscle damage could be constructed by measuring the evolution of serum concentration of muscle proteins following inertial exercise, according to their molecular weight and the fiber compartment in which they are located. Moreover, by measuring FM and SM, the type of fibers that are affected could be assessed. Serum profiles were registered before and 24, 48, and 144 h after exercise in 10 healthy and recreationally active young men. Creatine kinase (CK) and CK-myocardial band isoenzyme increased in serum early (24 h) and returned to baseline values after 48 h. FM increased in serum late (48 h) and remained elevated 144 h post-exercise. The increase in serum muscle enzymes suggests increased membrane permeability of both fast and slow fibers, and the increase in FM reveals sarcomere disruption as well as increased membrane permeability of fast fibers. Consequently, FM could be adopted as a fiber type-specific biomarker of muscle damage.

  19. A new sensitive and specific enzyme-linked immunosorbent assay for IgD.

    PubMed

    Mosedale, David E; Sandhu, Manjinder S; Luan, Jian'an; Goodall, Margaret; Grainger, David J

    2006-06-30

    We have developed a new highly specific ELISA for IgD, and then used it to measure levels of circulating IgD in the serum of 480 un-selected patients from the East Anglia region of UK. The assay is both extremely sensitive and specific, with a minimum detected IgD concentration of 30 pg/ml and more than 10,000-fold specificity for IgD over all other human immunoglobulins. The assay shows linear dilution characteristics with both purified IgD and human serum, and spiking of purified IgD into either purified immunoglobulins or human serum shows c. 100% recovery. Furthermore, intra-assay and inter-assay coefficients of variation for repeated measurements of the same samples are below 10% and 15% respectively. Measurement of IgD levels on the un-selected patient population showed levels to range from <300 pg/ml to over 100 microg/ml, with a geometric mean of 8 microg/ml. The distribution is approximately normal after log transformation. Levels of circulating IgD were higher in men than in women. There was a significant negative correlation between levels of IgD and age in women, but not in men. Moreover, after adjustment for age and sex, there were statistically significantly higher levels of circulating IgD in male (but not female) smokers, compared to their non-smoking counterparts. These results highlight the care that needs to be taken to control for age, sex and cigarette smoking when examining levels of circulating IgD in future studies.

  20. Heparin/heparan sulfate 6-O-sulfatase from Flavobacterium heparinum: integrated structural and biochemical investigation of enzyme active site and substrate specificity.

    PubMed

    Myette, James R; Soundararajan, Venkataramanan; Shriver, Zachary; Raman, Rahul; Sasisekharan, Ram

    2009-12-11

    Heparin and heparan sulfate glycosaminoglycans (HSGAGs) comprise a chemically heterogeneous class of sulfated polysaccharides. The development of structure-activity relationships for this class of polysaccharides requires the identification and characterization of degrading enzymes with defined substrate specificity and enzymatic activity. Toward this end, we report here the molecular cloning and extensive structure-function analysis of a 6-O-sulfatase from the Gram-negative bacterium Flavobacterium heparinum. In addition, we report the recombinant expression of this enzyme in Escherichia coli in a soluble, active form and identify it as a specific HSGAG sulfatase. We further define the mechanism of action of the enzyme through biochemical and structural studies. Through the use of defined substrates, we investigate the kinetic properties of the enzyme. This analysis was complemented by homology-based molecular modeling studies that sought to rationalize the substrate specificity of the enzyme and mode of action through an analysis of the active-site topology of the enzyme including identifying key enzyme-substrate interactions and assigning key amino acids within the active site of the enzyme. Taken together, our structural and biochemical studies indicate that 6-O-sulfatase is a predominantly exolytic enzyme that specifically acts on N-sulfated or N-acetylated 6-O-sulfated glucosamines present at the non-reducing end of HSGAG oligosaccharide substrates. This requirement for the N-acetyl or N-sulfo groups on the glucosamine substrate can be explained through eliciting favorable interactions with key residues within the active site of the enzyme. These findings provide a framework that enables the use of 6-O-sulfatase as a tool for HSGAG structure-activity studies as well as expand our biochemical and structural understanding of this important class of enzymes.

  1. Deadlock and fictitiousness problem in parallel program specifications

    SciTech Connect

    Panfilenko, V.P.

    1995-05-01

    One of the directions of modern programming based on algebraic methods takes its origin in V.M. Glushkov`s theory of systems of algorithmic algebras (SAA). The SAA apparatus with appropriately interpreted operations is used for program design and allows compact structured representation of program schemas in the form of algebraic formulas. Modified systems of algorithmic algebras (SAA-M) additionally represent parallelism description tools.

  2. As-built design specification for segment map (Sgmap) program

    NASA Technical Reports Server (NTRS)

    Tompkins, M. A. (Principal Investigator)

    1981-01-01

    The segment map program (SGMAP), which is part of the CLASFYT package, is described in detail. This program is designed to output symbolic maps or numerical dumps from LANDSAT cluster/classification files or aircraft ground truth/processed ground truth files which are in 'universal' format.

  3. Non-hydrolyzable Diubiquitin Probes Reveal Linkage-Specific Reactivity of Deubiquitylating Enzymes Mediated by S2 Pockets

    PubMed Central

    Flierman, Dennis; van der Heden van Noort, Gerbrand J.; Ekkebus, Reggy; Geurink, Paul P.; Mevissen, Tycho E.T.; Hospenthal, Manuela K.; Komander, David; Ovaa, Huib

    2016-01-01

    Summary Ubiquitin chains are important post-translational modifications that control a large number of cellular processes. Chains can be formed via different linkages, which determines the type of signal they convey. Deubiquitylating enzymes (DUBs) regulate ubiquitylation status by trimming or removing chains from attached proteins. DUBs can contain several ubiquitin-binding pockets, which confer specificity toward differently linked chains. Most tools for monitoring DUB specificity target binding pockets on opposing sides of the active site; however, some DUBs contain additional pockets. Therefore, reagents targeting additional pockets are essential to fully understand linkage specificity. We report the development of active site-directed probes and fluorogenic substrates, based on non-hydrolyzable diubiquitin, that are equipped with a C-terminal warhead or a fluorogenic activity reporter moiety. We demonstrate that various DUBs in lysates display differential reactivity toward differently linked diubiquitin probes, as exemplified by the proteasome-associated DUB USP14. In addition, OTUD2 and OTUD3 show remarkable linkage-specific reactivity with our diubiquitin-based reagents. PMID:27066941

  4. A comprehensive analysis of the geranylgeranylglyceryl phosphate synthase enzyme family identifies novel members and reveals mechanisms of substrate specificity and quaternary structure organization.

    PubMed

    Peterhoff, David; Beer, Barbara; Rajendran, Chitra; Kumpula, Esa-Pekka; Kapetaniou, Evangelia; Guldan, Harald; Wierenga, Rik K; Sterner, Reinhard; Babinger, Patrick

    2014-05-01

    Geranylgeranylglyceryl phosphate synthase (GGGPS) family enzymes catalyse the formation of an ether bond between glycerol-1-phosphate and polyprenyl diphosphates. They are essential for the biosynthesis of archaeal membrane lipids, but also occur in bacterial species, albeit with unknown physiological function. It has been known that there exist two phylogenetic groups (I and II) of GGGPS family enzymes, but a comprehensive study has been missing. We therefore visualized the variability within the family by applying a sequence similarity network, and biochemically characterized 17 representative GGGPS family enzymes regarding their catalytic activities and substrate specificities. Moreover, we present the first crystal structures of group II archaeal and bacterial enzymes. Our analysis revealed that the previously uncharacterized bacterial enzymes from group II have GGGPS activity like the archaeal enzymes and differ from the bacterial group I enzymes that are heptaprenylglyceryl phosphate synthases. The length of the isoprenoid substrate is determined in group II GGGPS enzymes by 'limiter residues' that are different from those in group I enzymes, as shown by site-directed mutagenesis. Most of the group II enzymes form hexamers. We could disrupt these hexamers to stable and catalytically active dimers by mutating a single amino acid that acts as an 'aromatic anchor'.

  5. Decreased H2B monoubiquitination and overexpression of ubiquitin-specific protease enzyme 22 in malignant colon carcinoma.

    PubMed

    Wang, Zijing; Zhu, Linlin; Guo, Tianjiao; Wang, Yiping; Yang, Jinlin

    2015-07-01

    This study aimed to evaluate the expression of H2B monoubiquitination enzyme (uH2B) and ubiquitin-specific protease enzyme 22 (USP22) in colon carcinoma and establish a correlation between the expression of these enzymes and clinicopathological parameters. The modification levels of uH2B and USP22 in 20 noncancerous and 129 cancerous colon samples were studied by immunohistochemistry. We used a dual-rated semiquantitative method to classify the expression according to 3 levels and analyzed these results. uH2B was abundant in the normal colon epithelium, but its expression was decreased in colon cancers (P < .001); the uH2B modification level correlated with tumor differentiation (P < .001), lymph node metastasis (P = .017), distant metastasis (P = .036), and tumor stage (P = .039). The USP22 expression in colon carcinoma was higher than that in normal tissues (P = .007) and negatively correlated with the degree of differentiation (P = .006), invasion (P = .025), lymph node metastasis (P = .026), and tumor stage (P = .044). uH2B and USP22 expression negatively correlated (r = -0.401, P < .001). Patients with uH2B-negative and USP22-positive staining were found to have lower survival rates (30.737 ± 2.866 versus 51.667 ± 2.286 months, P < .001). Positive uH2B and negative USP22 expression remained a statistically significant prognostic indicator in a multivariate Cox regression analysis (hazard ratio, 2.557; 95% confidence interval, 1.043-6.269; P = .04). We conclude that uH2B displays differential staining patterns according to progressive stages of colon cancer, indicating that uH2B may play an important inhibitory role in carcinogenesis. Increased USP22 expression in colon cancer correlated with reduced uH2B expression, and this expression pattern may contribute to tumor progression.

  6. Deciphering the Substrate Specificity of SbnA, the Enzyme Catalyzing the First Step in Staphyloferrin B Biosynthesis

    PubMed Central

    2016-01-01

    Staphylococcus aureus assembles the siderophore, staphyloferrin B, from l-2,3-diaminopropionic acid (l-Dap), α-ketoglutarate, and citrate. Recently, SbnA and SbnB were shown to produce l-Dap and α-ketoglutarate from O-phospho-l-serine (OPS) and l-glutamate. SbnA is a pyridoxal 5′-phosphate (PLP)-dependent enzyme with homology to O-acetyl-l-serine sulfhydrylases; however, SbnA utilizes OPS instead of O-acetyl-l-serine (OAS), and l-glutamate serves as a nitrogen donor instead of a sulfide. In this work, we examined how SbnA dictates substrate specificity for OPS and l-glutamate using a combination of X-ray crystallography, enzyme kinetics, and site-directed mutagenesis. Analysis of SbnA crystals incubated with OPS revealed the structure of the PLP-α-aminoacrylate intermediate. Formation of the intermediate induced closure of the active site pocket by narrowing the channel leading to the active site and forming a second substrate binding pocket that likely binds l-glutamate. Three active site residues were identified: Arg132, Tyr152, Ser185 that were essential for OPS recognition and turnover. The Y152F/S185G SbnA double mutant was completely inactive, and its crystal structure revealed that the mutations induced a closed form of the enzyme in the absence of the α-aminoacrylate intermediate. Lastly, l-cysteine was shown to be a competitive inhibitor of SbnA by forming a nonproductive external aldimine with the PLP cofactor. These results suggest a regulatory link between siderophore and l-cysteine biosynthesis, revealing a potential mechanism to reduce iron uptake under oxidative stress. PMID:26794841

  7. Deciphering the Substrate Specificity of SbnA, the Enzyme Catalyzing the First Step in Staphyloferrin B Biosynthesis.

    PubMed

    Kobylarz, Marek J; Grigg, Jason C; Liu, Yunan; Lee, Mathew S F; Heinrichs, David E; Murphy, Michael E P

    2016-02-16

    Staphylococcus aureus assembles the siderophore, staphyloferrin B, from l-2,3-diaminopropionic acid (l-Dap), α-ketoglutarate, and citrate. Recently, SbnA and SbnB were shown to produce l-Dap and α-ketoglutarate from O-phospho-l-serine (OPS) and l-glutamate. SbnA is a pyridoxal 5'-phosphate (PLP)-dependent enzyme with homology to O-acetyl-l-serine sulfhydrylases; however, SbnA utilizes OPS instead of O-acetyl-l-serine (OAS), and l-glutamate serves as a nitrogen donor instead of a sulfide. In this work, we examined how SbnA dictates substrate specificity for OPS and l-glutamate using a combination of X-ray crystallography, enzyme kinetics, and site-directed mutagenesis. Analysis of SbnA crystals incubated with OPS revealed the structure of the PLP-α-aminoacrylate intermediate. Formation of the intermediate induced closure of the active site pocket by narrowing the channel leading to the active site and forming a second substrate binding pocket that likely binds l-glutamate. Three active site residues were identified: Arg132, Tyr152, Ser185 that were essential for OPS recognition and turnover. The Y152F/S185G SbnA double mutant was completely inactive, and its crystal structure revealed that the mutations induced a closed form of the enzyme in the absence of the α-aminoacrylate intermediate. Lastly, l-cysteine was shown to be a competitive inhibitor of SbnA by forming a nonproductive external aldimine with the PLP cofactor. These results suggest a regulatory link between siderophore and l-cysteine biosynthesis, revealing a potential mechanism to reduce iron uptake under oxidative stress.

  8. Starch-branching enzyme I-deficient mutation specifically affects the structure and properties of starch in rice endosperm.

    PubMed

    Satoh, Hikaru; Nishi, Aiko; Yamashita, Kazuhiro; Takemoto, Yoko; Tanaka, Yasumasa; Hosaka, Yuko; Sakurai, Aya; Fujita, Naoko; Nakamura, Yasunori

    2003-11-01

    We have isolated a starch mutant that was deficient in starch-branching enzyme I (BEI) from the endosperm mutant stocks of rice (Oryza sativa) induced by the treatment of fertilized egg cells with N-methyl-N-nitrosourea. The deficiency of BEI in this mutant was controlled by a single recessive gene, tentatively designated as starch-branching enzyme mutant 1 (sbe1). The mutant endosperm exhibited the normal phenotype and contained the same amount of starch as the wild type. However, the mutation apparently altered the fine structure of amylopectin. The mutant amylopectin was characterized by significant decrease in both long chains with degree of polymerization (DP) > or = 37 and short chains with DP 12 to 21, marked increase in short chains with DP < or = 10 (A chains), and slight increase in intermediate chains with DP 24 to 34, suggesting that BEI specifically synthesizes B1 and B2-3 chains. The endosperm starch from the sbe1 mutant had a lower onset concentration for urea gelatinization and a lower onset temperature for thermo-gelatinization compared with the wild type, indicating that the genetic modification of amylopectin fine structure is responsible for changes in physicochemical properties of sbe1 starch.

  9. Tissue-specific variation in glycation of proteins in diabetes: evidence for a functional role of amadoriase enzymes.

    PubMed

    Brown, Sarah M; Smith, Della M; Alt, Nadja; Thorpe, Suzanne R; Baynes, John W

    2005-06-01

    The Amadori product fructoselysine (FL), an intermediate in the formation of many advanced glycation end products, may be deglycated by various pathways. These include spontaneous chemical degradation or enzymatic deglycation by amadoriases. This study was designed to compare changes in FL in various tissues in response to changes in glycemia, thereby testing tissue-specific deglycation. FL content in skin collagen, red cell hemoglobin, and total muscle, liver, and brain protein was analyzed by isotope dilution gas chromatography-mass spectrometry. Mean blood glucose increased over fourfold in diabetic versus control rats, whereas changes in glycation of proteins varied from fivefold in collagen to no change in the liver and brain. These results suggest significant differences among tissues in the activity of deglycating enzymes and/or protein turnover.

  10. Comparison of measles virus-specific antibody titres as measured by enzyme-linked immunosorbent assay and virus neutralisation assay.

    PubMed

    van den Hof, Susan; van Gageldonk-Lafeber, Arianne B; van Binnendijk, Robert S; van Gageldonk, Pieter G M; Berbers, Guy A M

    2003-10-01

    We assessed whether measles virus-specific antibody levels in the Dutch population as estimated by an enzyme-linked immunosorbent assay (ELISA) were comparable with estimates by virus neutralisation assay (NT), prompted by a relatively low ELISA seroprevalence in the 10-21-year-old group. We tested 791 sera from individuals aged 2-49 years both in ELISA and NT. Seroprevalence in the 10-21-year-old group was 93.4% (95% confidence interval (CI) 89.5-97.2%) in ELISA versus 97.2% (CI 94.7-99.6%) in NT. There was good agreement between NT and ELISA seroprevalences in the vaccinated 2-9-year-olds and the unvaccinated 22-49-year-olds.

  11. Effect of hapten structures on specific and sensitive enzyme-linked immunosorbent assays for N-methylcarbamate insecticide metolcarb.

    PubMed

    Zhang, Qi; Wu, Yunru; Wang, Limin; Hu, Baishi; Li, Peiwu; Liu, Fengquan

    2008-09-05

    Five different haptens of the N-methylcarbamate insecticide metolcarb were designed and synthesized. All of the haptens were conjugated with ovalbumin (OVA) for the coating antigen, and one hapten containing all of the structure of metolcarb was conjugated with bovine serum albumin (BSA) for the immunogen. Two polyclonal antisera were raised against the BSA conjugate, and ten antibody/coating conjugate combinations were selected for studies of assay sensitivity and specificity for metolcarb. A class-specific combination was found, with the I50 of the assay ranged from 0.64 to 20.98 microg mL(-1) for seven tested N-methylcarbamate insecticides except for pirimicarb. Considering titer, I50 and cross-reactivity of all combinations of antibody/coating conjugate, a competitive indirect enzyme-linked immunosorbent assay (ELISA) in a homologous system, whose limit of detection (LoD) reached 1.4 ng mL(-1), was presented. The results of competitive ELISAs indicated that coating hapten structure can significantly affect not only assay sensitivity but also its specificity.

  12. Evaluation of enzyme-linked immunosorbent assays for detection of Mycoplasma bovis-specific antibody in bison sera.

    PubMed

    Register, Karen B; Sacco, Randy E; Olsen, Steven C

    2013-09-01

    Mycoplasma bovis has recently emerged as a significant and costly infectious disease problem in bison. A method for the detection of M. bovis-specific serum antibodies is needed in order to establish prevalence and transmission patterns. Enzyme-linked immunosorbent assays (ELISAs) validated for the detection of M. bovis-specific serum IgG in cattle are commercially available, but their suitability for bison sera has not been determined. A collection of bison sera, most from animals with a known history of infection or vaccination with M. bovis, was tested for M. bovis-specific IgG using commercially available kits as well as an in-house ELISA in which either cattle or bison M. bovis isolates were used as a source of antigen. Comparison of the results demonstrates that ELISAs optimized for cattle sera may not be optimal for the identification of bison seropositive for M. bovis, particularly those with low to moderate antibody levels. The reagent used for the detection of bison IgG and the source of the antigen affect the sensitivity of the assay. Optimal performance was obtained when the capture antigen was derived from bison isolates rather than cattle isolates and when a protein G conjugate rather than an anti-bovine IgG conjugate was used for the detection of bison IgG.

  13. Neonatal handling affects learning, reversal learning and antioxidant enzymes activities in a sex-specific manner in rats.

    PubMed

    Noschang, Cristie; Krolow, Rachel; Arcego, Danusa Mar; Toniazzo, Ana Paula; Huffell, Ana Paula; Dalmaz, Carla

    2012-06-01

    Early life experiences have profound influences on behavior and neurochemical parameters in adult life. The aim of this study is to verify neonatal handling-induced sex specific differences on learning and reversal learning as well as oxidative stress parameters in the prefrontal cortex and striatum of adult rats. Litters of rats were non-handled or handled (10 min/day, days 1-10 after birth). In adulthood, learning and reversal learning were evaluated using a Y maze associated with palatable food in male and female rats. Morris water maze reversal learning was verified in males. Oxidative stress parameters were evaluated in both genders. Male neonatal handled animals had a worse performance in the Y maze reversal learning compared to non-handled ones and no difference was observed in the water maze reversal learning task. Regarding females, neonatal handled rats had a better performance during the Y maze learning phase compared to non-handled ones. In addition, neonatal handled female animals showed a decreased SOD/CAT ratio in the PFC compared to non-handled females. We conclude that neonatal handling effects on learning and memory in adult rats are sex and task specific. The sex specific differences are also observed in the evaluation of antioxidant enzymes activities with neonatal handling affecting only females.

  14. 78 FR 17943 - Draft Program-Specific Guidance About Fixed Gauge Licenses

    Federal Register 2010, 2011, 2012, 2013, 2014

    2013-03-25

    ... safety culture, security of radioactive materials, protection of sensitive information, and changes in... NUREG-1556, Volume 4, Revision 1, ``Consolidated Guidance About Materials Licenses: Program-Specific... Materials and Environmental Management Programs; U.S. Nuclear Regulatory Commission, Washington, DC...

  15. Aeronautics research and technology program and specific objectives

    NASA Technical Reports Server (NTRS)

    1981-01-01

    Aeronautics research and technology program objectives in fluid and thermal physics, materials and structures, controls and guidance, human factors, multidisciplinary activities, computer science and applications, propulsion, rotorcraft, high speed aircraft, subsonic aircraft, and rotorcraft and high speed aircraft systems technology are addressed.

  16. 47 CFR 76.75 - Specific EEO program requirements.

    Code of Federal Regulations, 2014 CFR

    2014-10-01

    ... employment opportunity program to job applicants, employees, and those with whom it regularly does business... opportunity notices, where appropriate; (2) Placing a notice in bold type on the employment application... shall be undertaken by each employment unit: (1) Recruit for every full-time job vacancy in...

  17. 47 CFR 76.75 - Specific EEO program requirements.

    Code of Federal Regulations, 2012 CFR

    2012-10-01

    ... employment opportunity program to job applicants, employees, and those with whom it regularly does business... opportunity notices, where appropriate; (2) Placing a notice in bold type on the employment application... shall be undertaken by each employment unit: (1) Recruit for every full-time job vacancy in...

  18. 47 CFR 76.75 - Specific EEO program requirements.

    Code of Federal Regulations, 2013 CFR

    2013-10-01

    ... employment opportunity program to job applicants, employees, and those with whom it regularly does business... opportunity notices, where appropriate; (2) Placing a notice in bold type on the employment application... shall be undertaken by each employment unit: (1) Recruit for every full-time job vacancy in...

  19. 47 CFR 76.75 - Specific EEO program requirements.

    Code of Federal Regulations, 2011 CFR

    2011-10-01

    ... employment opportunity program to job applicants, employees, and those with whom it regularly does business... opportunity notices, where appropriate; (2) Placing a notice in bold type on the employment application... shall be undertaken by each employment unit: (1) Recruit for every full-time job vacancy in...

  20. 47 CFR 76.75 - Specific EEO program requirements.

    Code of Federal Regulations, 2010 CFR

    2010-10-01

    ... employment opportunity program to job applicants, employees, and those with whom it regularly does business... opportunity notices, where appropriate; (2) Placing a notice in bold type on the employment application... shall be undertaken by each employment unit: (1) Recruit for every full-time job vacancy in...

  1. 78 FR 71987 - Programs for Specific Positions and Examinations (Miscellaneous)

    Federal Register 2010, 2011, 2012, 2013, 2014

    2013-12-02

    ... a final rule to eliminate the licensure requirement for incumbent administrative law judges. DATES... (CFR), to revise the Administrative Law Judge Program. These revisions included a requirement for incumbent administrative law judges (ALJs) to ``. . . possess a professional license to practice law and...

  2. 75 FR 61998 - Programs for Specific Positions and Examinations (Miscellaneous)

    Federal Register 2010, 2011, 2012, 2013, 2014

    2010-10-07

    ... licensure requirements for incumbent administrative law judges who are covered under the Administrative Law... published a final rule at 72 FR 12947, to revise the Administrative Law Judge Program. These revisions included a requirement for incumbent administrative law judges (ALJs) to ``possess a professional...

  3. Program Requirements and Design Specifications for the Quincy School Complex.

    ERIC Educational Resources Information Center

    Quincy School Community Council, MA.

    The Quincy School Complex is a unique community facility. A high level of cooperation has led to the planning of this facility whose operation and ownership is divided among several agencies and groups. The introduction to the report on the school complex provides background material for the program itself, and attempts to define the "why" of the…

  4. Gerontology-specific graduate programs in Brazil and Colombia.

    PubMed

    Bos, Angelo J G; Padilha, Dalva Maria Pereira; Bos, Antonio M G; Gómez, Fernando

    2007-01-01

    Every year the proportion of elderly people increases at a greater rate compared with other age groups, changing the population structure of most countries. Latin America has been internationally known for its higher percentage of young compared with elderly persons. The United Nations predicts that the proportion of elderly persons in Latin America and the Caribbean will be more similar to world figures in 2020 and even higher in 2040. The increasing elderly population in Latin America has increased the demand for advanced degree professionals with gerontology training. Nevertheless, in spite of training efforts during the last decade, the number of gerontology professionals is still insufficient. In total, the authors were able to locate only ten gerontology programs in Latin America (four in Brazil, two in Argentina, and one each in Uruguay, Peru, Cuba, and Colombia). The programs currently available in Brazil and Colombia are described in an effort to share information on the common characteristics of Master's and PhD degree programs in gerontology in Latin America. The authors concluded that, in Latin America, programs focused exclusively on gerontology are scarce.

  5. Angiotensin-converting enzyme-2 (ACE2): comparative modeling of the active site, specificity requirements, and chloride dependence.

    PubMed

    Guy, Jodie L; Jackson, Richard M; Acharya, K Ravi; Sturrock, Edward D; Hooper, Nigel M; Turner, Anthony J

    2003-11-18

    Angiotensin-converting enzyme 2 (ACE2), a homologue of ACE, represents a new and potentially important target in cardio-renal disease. A model of the active site of ACE2, based on the crystal structure of testicular ACE, has been developed and indicates that the catalytic mechanism of ACE2 resembles that of ACE. Structural differences exist between the active site of ACE (dipeptidyl carboxypeptidase) and ACE2 (carboxypeptidase) that are responsible for the differences in specificity. The main differences occur in the ligand-binding pockets, particularly at the S2' subsite and in the binding of the peptide carboxy-terminus. The model explains why the classical ACE inhibitor lisinopril is unable to bind to ACE2. On the basis of the ability of ACE2 to cleave a variety of biologically active peptides, a consensus sequence of Pro-X-Pro-hydrophobic/basic for the protease specificity of ACE2 has been defined that is supported by the ACE2 model. The dipeptide, Pro-Phe, completely inhibits ACE2 activity at 180 microM with angiotensin II as the substrate. As with ACE, the chloride dependence of ACE2 is substrate-specific such that the hydrolysis of angiotensin I and the synthetic peptide substrate, Mca-APK(Dnp), are activated in the presence of chloride ions, whereas the cleavage of angiotensin II is inhibited. The ACE2 model is also suggestive of a possible mechanism for chloride activation. The structural insights provided by these analyses for the differences in inhibition pattern and substrate specificity among ACE and its homologue ACE2 and for the chloride dependence of ACE/ACE2 activity are valuable in understanding the function and regulation of ACE2.

  6. Detection of HLA class I-specific antibodies by the QuikScreen enzyme-linked immunosorbent assay.

    PubMed Central

    Lucas, D P; Paparounis, M L; Myers, L; Hart, J M; Zachary, A A

    1997-01-01

    The GTI QuikScreen test is an enzyme-linked immunosorbent assay (ELISA) that uses soluble HLA class I antigens as targets. In tests of 5,893 human serum specimens, we evaluated the reliability, sensitivity, and utility of the GTI QuikScreen test for detecting HLA class I-specific antibody. We found that the test could reliably detect HLA-specific antibodies of the immunoglobulin G (IgG) but not the IgM class. The degree of correlation with lymphocytotoxicity testing varied among the different serum sources, with the best correlation achieved with sera from renal transplant candidates (r > 0.7) and the poorest with sera from patients with end-stage liver disease (r = 0.26), possibly because of elevated alkaline phosphatase levels in the liver patients. Test reproducibility was high (96%), and test failure rate was low (1.7%). The test sensitivity is comparable to that of the antiglobulin cytotoxicity and, possibly, even flow cytometric tests. There was a highly significant (P < 0.001) correlation between the optical densities obtained in the ELISA and the percent panel reactive antibody determined by cytotoxicity testing. Therefore, although designed only to determine the presence or absence of HLA-specific antibody, GTI QuikScreen test results also provided an indication of the extent of sensitization. The test is one of the most effective and efficient ways to determine if antibodies producing a positive result in crossmatch tests are specific for HLA class I antigens. As an adjunct to serum screening by cytotoxicity testing, the GTI QuikScreen test can produce a substantial savings of time and effort that reduces the cost to the laboratory and to the patient. PMID:9144358

  7. Indirect enzyme-linked immunosorbent assay for detection of Brucella melitensis-specific antibodies in goat milk.

    PubMed

    Funk, N D; Tabatabai, L B; Elzer, P H; Hagius, S D; Martin, B M; Hoffman, L J

    2005-02-01

    Brucella melitensis is the cause of brucellosis in sheep and goats, which often results in abortion. Few cases of B. melitensis infection in goats have occurred in the United States over the last 25 years. However, vigilance must be maintained, as it is for the bovine milk industry, to ensure that brucellosis is not introduced into the U.S. goat population. The objective of this study was to develop a sensitive and specific indirect enzyme-linked immunosorbent assay (iELISA) for the detection of B. melitensis-specific antibodies in goat milk. Brucella salt-extractable protein extract was employed as an antigen, and a horseradish peroxidase-labeled polyclonal anti-goat antibody was used as an anti-species conjugate. Thirteen of 13 (100%) individual infected goat milk samples tested positive and 134 of 134 (100%) uninfected bulk milk samples tested negative by the developed iELISA. Three positive milk samples with high, medium, and low absorbance values were used to simulate one positive animal in an otherwise negative herd. By this estimation, one high-titer animal could be detected in a herd of >1,600 animals. Detection estimates for medium- and low-titer animals were one positive animal per herd of <200 and 50 animals, respectively. Based on this estimation, it is recommended that herds be sampled in groups of 50 animals or less for bulk milk testing. The iELISA developed for this study was found to be sensitive and specific and shows potential for use as a bulk milk test for the detection of B. melitensis-specific antibodies in goat milk.

  8. Aeronautics Research and Technology Program and specific objectives, fiscal year 1982

    NASA Technical Reports Server (NTRS)

    Olstad, W. B.

    1981-01-01

    The Aeronautics Research and Technology program is broken down into two program areas (research and technology base, and systems technology programs) which are further broken down into succeedingly more detailed activities to form a work breakdown structure for the aeronautics program: program area, program/discipline objective, specific objective, and research and technology objective and plan (RTOP). A detailed view of this work breakdown structure down to the specific objective level is provided, and goals or objectives at each of these levels are set forth. What is to be accomplished and why are addressed, but not how. The letter falls within the domain of the RTOP.

  9. Affinity electrophoresis as a method for determining substrate-binding specificity of carbohydrate-active enzymes for soluble polysaccharides.

    PubMed

    Moraïs, Sarah; Lamed, Raphael; Bayer, Edward A

    2012-01-01

    Affinity electrophoresis is a simple and rapid tool for the analysis of protein-binding affinities to soluble polysaccharides. This approach is particularly suitable for the characterization of the carbohydrate-active enzymes that contain a carbohydrate-binding module and for their mutants and chimeras. Knowledge of the binding characteristics of these enzymes can be the first step to elucidate the enzymatic activity of a putative enzyme; moreover in some cases, enzymes are able to bind polysaccharides targets other than their specified substrate, and this knowledge can be essential to understand the basics of the intrinsic mechanism of these enzymes in their natural environment.

  10. The mRNA capping enzyme of Saccharomyces cerevisiae has dual specificity to interact with CTD of RNA Polymerase II

    PubMed Central

    Bharati, Akhilendra Pratap; Singh, Neha; Kumar, Vikash; Kashif, Md.; Singh, Amit Kumar; Singh, Priyanka; Singh, Sudhir Kumar; Siddiqi, Mohammad Imran; Tripathi, Timir; Akhtar, Md. Sohail

    2016-01-01

    RNA Polymerase II (RNAPII) uniquely possesses an extended carboxy terminal domain (CTD) on its largest subunit, Rpb1, comprising a repetitive Tyr1Ser2Pro3Thr4 Ser5Pro6Ser7 motif with potential phosphorylation sites. The phosphorylation of the CTD serves as a signal for the binding of various transcription regulators for mRNA biogenesis including the mRNA capping complex. In eukaryotes, the 5 prime capping of the nascent transcript is the first detectable mRNA processing event, and is crucial for the productive transcript elongation. The binding of capping enzyme, RNA guanylyltransferases to the transcribing RNAPII is known to be primarily facilitated by the CTD, phosphorylated at Ser5 (Ser5P). Here we report that the Saccharomyces cerevesiae RNA guanylyltransferase (Ceg1) has dual specificity and interacts not only with Ser5P but also with Ser7P of the CTD. The Ser7 of CTD is essential for the unconditional growth and efficient priming of the mRNA capping complex. The Arg159 and Arg185 of Ceg1 are the key residues that interact with the Ser5P, while the Lys175 with Ser7P of CTD. These interactions appear to be in a specific pattern of Ser5PSer7PSer5P in a tri-heptad CTD (YSPTSPPS YSPTSPSP YSPTSPPS) and provide molecular insights into the Ceg1-CTD interaction for mRNA transcription. PMID:27503426

  11. Detection of specific antibody by enzyme-linked immunosorbent assay and antigenemia by counterimmunoelectrophoresis in humans infected with Pneumocystis carinii.

    PubMed

    Maddison, S E; Hayes, G V; Slemenda, S B; Norman, L G; Ivey, M H

    1982-06-01

    A urea-soluble extract of cyst-rich material from rat lung heavily infected with Pneumocystis carinii was evaluated in an enzyme-linked immunosorption assay for antibody in 461 human sera. The highest level of reactivity occurred in sera submitted for serodiagnosis from proved or highly suspect cases. However, the range of reactivities in these groups, many of whom were on immunosuppressive therapy, was very wide. A more restricted lower range of reactivity was observed in both hospital-family contacts and healthy Serum Bank donors. Because of the overlap in levels of reactivity between the pneumocystosis and control groups, no concise cutoff value to separate infected from noninfected individuals could be made. Specificity of the reactions was shown by absorption of patients' and control sera with uninfected and P. carinii-infected human and rat lung tissue. The data support the concept that P. carinii is highly prevalent as a latent agent in the general population and is provoked to cause clinically manifest disease in the compromised host. Detection of circulating antigen appeared to be specific and possibly a useful adjunct to diagnosis, as 10 of the 14 proved or highly suspect patients with antigenemia did not have measurable antibody to P. carinii.

  12. Tissue-specific accumulation and regulation of zeaxanthin epoxidase in Arabidopsis reflect the multiple functions of the enzyme in plastids.

    PubMed

    Schwarz, Nadine; Armbruster, Ute; Iven, Tim; Brückle, Lena; Melzer, Michael; Feussner, Ivo; Jahns, Peter

    2015-02-01

    The enzyme zeaxanthin epoxidase (ZEP) catalyzes the conversion of zeaxanthin to violaxanthin, a key reaction for ABA biosynthesis and the xanthophyll cycle. Both processes are important for acclimation to environmental stress conditions, in particular drought (ABA biosynthesis) and light (xanthophyll cycle) stress. Hence, both ZEP functions may require differential regulation to optimize plant fitness. The key to understanding the function of ZEP in both stress responses might lie in its spatial and temporal distribution in plant tissues. Therefore, we analyzed the distribution of ZEP in plant tissues and plastids under drought and light stress by use of a ZEP-specific antibody. In addition, we determined the pigment composition of the plant tissues and chloroplast membrane subcompartments in response to these stresses. The ZEP protein was detected in all plant tissues (except flowers) concomitant with xanthophylls. The highest levels of ZEP were present in leaf chloroplasts and root plastids. Within chloroplasts, ZEP was localized predominantly in the thylakoid membrane and stroma, while only a small fraction was bound by the envelope membrane. Light stress affected neither the accumulation nor the relative distribution of ZEP in chloroplasts, while drought stress led to an increase of ZEP in roots and to a degradation of ZEP in leaves. However, drought stress-induced increases in ABA were similar in both tissues. These data support a tissue- and stress-specific accumulation of the ZEP protein in accordance with its different functions in ABA biosynthesis and the xanthophyll cycle.

  13. Fluoroimmunoassay for detection of rubella-specific immunoglobulin M: comparison with indirect enzyme immunoassay and mu-chain capture.

    PubMed Central

    Echevarria, J M; de Ory, F; Najera, R

    1985-01-01

    The performance of a commercially-available method of fluoroimmunoassay (Rubella M FIAX, International Diagnostic Technology, Santa Clara, Calif.), designed for the detection of rubella-specific immunoglobulin M, was tested with 137 selected sera, including 52 from cases of primary rubella, 29 from healthy pregnant women, 21 containing rheumatoid factor, and 35 from cases of infectious mononucleosis (IM) caused by Epstein-Barr virus. The results were compared with those obtained by commercial indirect enzyme immunoassay (EIA) and EIA anti-mu chain capture tests. The fluoroimmunoassay technique showed a satisfactory level of sensitivity, but low values had to be interpreted with caution as false-positive results were detected with sera with rheumatoid factor and from IM cases, even after preliminary treatment of sera with the anti-human immunoglobulin G antisera provided in the kit. On the other hand, no false-positive results in the analysis of IM sera were seen in the EIA anti-mu chain capture method. Because of its sensitivity and specificity, we recommend the use of the latter technique for the diagnosis of primary rubella. PMID:2995439

  14. Elucidation of Xylem-Specific Transcription Factors and Absolute Quantification of Enzymes Regulating Cellulose Biosynthesis in Populus trichocarpa.

    PubMed

    Loziuk, Philip L; Parker, Jennifer; Li, Wei; Lin, Chien-Yuan; Wang, Jack P; Li, Quanzi; Sederoff, Ronald R; Chiang, Vincent L; Muddiman, David C

    2015-10-02

    Cellulose, the main chemical polymer of wood, is the most abundant polysaccharide in nature.1 The ability to perturb the abundance and structure of cellulose microfibrils is of critical importance to the pulp and paper industry as well as for the textile, wood products, and liquid biofuels industries. Although much has been learned at the transcript level about the biosynthesis of cellulose, a quantitative understanding at the proteome level has yet to be established. The study described herein sought to identify the proteins directly involved in cellulose biosynthesis during wood formation in Populus trichocarpa along with known xylem-specific transcription factors involved in regulating these key proteins. Development of an effective discovery proteomic strategy through a combination of subcellular fractionation of stem differentiating xylem tissue (SDX) with recently optimized FASP digestion protocols, StageTip fractionation, as well as optimized instrument parameters for global proteomic analysis using the quadrupole-orbitrap mass spectrometer resulted in the deepest proteomic coverage of SDX protein from P. trichocarpa with 9,146 protein groups being identified (1% FDR). Of these, 20 cellulosic/hemicellulosic enzymes and 43 xylem-specific transcription factor groups were identified. Finally, selection of surrogate peptides led to an assay for absolute quantification of 14 cellulosic proteins in SDX of P. trichocarpa.

  15. Directed evolution of rubisco in Escherichia coli reveals a specificity-determining hydrogen bond in the form II enzyme.

    PubMed

    Mueller-Cajar, Oliver; Morell, Matthew; Whitney, Spencer M

    2007-12-11

    Ribulose 1,5-bisphosphate carboxylase/oxygenase (Rubisco) occupies a critical position in photosynthetic CO2-fixation and consequently has been the focus of intense study. Crystal-structure-guided site-directed mutagenesis studies have met with limited success in engineering kinetic improvements to Rubisco, highlighting our inadequate understanding of structural constraints at the atomic level that dictate the enzyme's catalytic chemistry. Bioselection provides an alternative random mutagenic approach that is useful for identifying and elucidating imperceptible structure-function relationships. Using the dimeric Form II Rubisco from Rhodospirillum rubrum, its gene (rbcM) was randomly mutated and introduced under positive selection into Escherichia coli cells metabolically engineered to be dependent on Rubisco to detoxify its substrate ribulose 1,5-bisphosphate. Thirteen colonies displaying improved fitness were isolated, and all were found to harbor mutations in rbcM at one of two codons, histidine-44 or aspartate-117, that are structurally adjacent amino acids located about 10 A from the active site. Biochemical characterization of the mutant enzymes showed the mutations reduced their CO2/O2 specificity by 40% and decreased their carboxylation turnover rate by 20-40%. Structural analyses showed histidine-44 and aspartate-117 form a hydrogen bond in R. rubrum Rubisco and that the residues are conserved among other Form II Rubiscos. This study demonstrated the utility of directed evolution in E. coli for identifying catalytically relevant residues (in particular nonobvious residues disconnected from active site residues) and their potential molecular interactions that influence Rubisco's catalytic chemistry.

  16. As-built design specification for the CLASFYG program

    NASA Technical Reports Server (NTRS)

    Horton, C. L. (Principal Investigator)

    1981-01-01

    This program produces a file with a Universal-formatted header and data records in a nonstandard format. Trajectory coefficients are calculated from 5 to 8 acquisitions of radiance values in the training field corresponding to an agricultural product. These coefficients are then used to calculate a time of emergence and corresponding trajectory coefficients for each pixel in the test field. The time of emergence, two of the coefficients, and the sigma value for each pixel are written to the file.

  17. Tad1p, a yeast tRNA-specific adenosine deaminase, is related to the mammalian pre-mRNA editing enzymes ADAR1 and ADAR2.

    PubMed Central

    Gerber, A; Grosjean, H; Melcher, T; Keller, W

    1998-01-01

    We have identified an RNA-specific adenosine deaminase (termed Tad1p/scADAT1) from Saccharomyces cerevisiae that selectively converts adenosine at position 37 of eukaryotic tRNAAla to inosine. The activity of purified recombinant Tad1p depends on the conformation of its tRNA substrate and the enzyme was found to be inactive on all other types of RNA tested. Mutant strains in which the TAD1 gene is disrupted are viable but lack Tad1p enzyme activity and their tRNAAla is not modified at position A37. Transformation of the mutant cells with the TAD1 gene restored enzyme activity. Tad1p has significant sequence similarity with the mammalian editing enzymes which act on specific precursor-mRNAs and on long double-stranded RNA. These findings suggest an evolutionary link between pre-mRNA editing and tRNA modification. PMID:9707437

  18. Stool diagnosis of giardiasis using a commercially available enzyme immunoassay to detect Giardia-specific antigen 65 (GSA 65).

    PubMed

    Rosoff, J D; Sanders, C A; Sonnad, S S; De Lay, P R; Hadley, W K; Vincenzi, F F; Yajko, D M; O'Hanley, P D

    1989-09-01

    A commercially available enzyme immunoassay for the diagnosis of giardiasis was evaluated in a clinical trial. The ProSpecT/Giardia diagnostic test (Alexon, Inc., Mountain View, Calif.) was compared with the standard ova and parasite (O&P) microscopic examination. Additionally, several widely used stool fixatives and a commonly used transport medium were assessed for compatibility with the immunoassay. A total of 325 stool specimens were collected and used to evaluate assay performance. Of those, 93 specimens were collected from symptomatic Giardia O&P-positive patients and 232 specimens were randomly collected from patients as part of a routine health screening procedure. All 93 Giardia O&P-positive stool specimens were strongly positive by visual and spectrophotometric examination using the immunoassay. Of the 232 randomly collected specimens, 16 were positive by O&P examination and immunoassay, 6 were negative by O&P examination but positive by immunoassay, and 1 was positive by O&P examination and negative by immunoassay. There was substantial supportive evidence that indicated that the six immunoassay-positive, O&P-negative specimens were true-positives. When these six specimens were accepted as true-positives, the immunoassay detected almost 30% more cases of Giardia infection than did O&P examination. Its sensitivity and specificity were 96 and 100%, respectively, while the sensitivity and specificity of O&P examination were 74 and 100%, respectively. The immunoassay also performed well on specimens treated with 10% neutral Formalin, sodium acetate-Formalin fixative, and Cary-Blair transport medium. However, the test was not compatible with polyvinyl alcohol-treated specimens. Overall, the ProSpecT/Giardia test was a sensitive, specific immunoassay which was easy to run and interpret. It offers a simple solution to traditional difficulties encountered in diagnosing Giardia infection.

  19. Development of a flatfish-specific enzyme-linked immunosorbent assay for Fsh using a recombinant chimeric gonadotropin.

    PubMed

    Chauvigné, François; Verdura, Sara; Mazón, María José; Boj, Mónica; Zanuy, Silvia; Gómez, Ana; Cerdà, Joan

    2015-09-15

    In flatfishes with asynchronous and semicystic spermatogenesis, such as the Senegalese sole (Solea senegalensis), the specific roles of the pituitary gonadotropins during germ cell development, particularly of the follicle-stimulating hormone (Fsh), are still largely unknown in part due to the lack of homologous immunoassays for this hormone. In this study, an enzyme-linked immunosorbent assay (ELISA) for Senegalese sole Fsh was developed by generating a rabbit antiserum against a recombinant chimeric single-chain Fsh molecule (rFsh-C) produced by the yeast Pichia pastoris. The rFsh-C N- and C-termini were formed by the mature sole Fsh β subunit (Fshβ) and the chicken glycoprotein hormone common α subunit (CGA), respectively. Depletion of the antiserum to remove anti-CGA antibodies further enriched the sole Fshβ-specific antibodies, which were used to develop the ELISA using the rFsh-C for the standard curve. The sensitivity of the assay was 10 and 50 pg/ml for Fsh measurement in plasma and pituitary, respectively, and the cross-reactivity with a homologous recombinant single-chain luteinizing hormone was 1%. The standard curve for rFsh-C paralleled those of serially diluted plasma and pituitary extracts of other flatfishes, such as the Atlantic halibut, common sole and turbot. In Senegalese sole males, the highest plasma Fsh levels were found during early spermatogenesis but declined during enhanced spermiation, as found in teleosts with cystic spermatogenesis. In pubertal males, however, the circulating Fsh levels were as high as in adult spermiating fish, but interestingly the Fsh receptor in the developing testis containing only spermatogonia was expressed in Leydig cells but not in the primordial Sertoli cells. These results indicate that a recombinant chimeric Fsh can be used to generate specific antibodies against the Fshβ subunit and to develop a highly sensitive ELISA for Fsh measurements in diverse flatfishes.

  20. Immunocapture-based fluorometric assay for the measurement of neprilysin-specific enzyme activity in brain tissue homogenates and cerebrospinal fluid.

    PubMed

    Miners, James Scott; Verbeek, Marcel M; Rikkert, Marcel Olde; Kehoe, Patrick Gavin; Love, Seth

    2008-01-30

    Neprilysin, a zinc-metalloendopeptidase, has important roles in the physiology and pathology of many diseases such as hypertension, cancer and Alzheimer's disease. We have developed an immunocapture assay to measure the specific enzyme activity of neprilysin in brain tissue homogenates and cerebrospinal fluid (CSF). The assay uses a neprilysin-specific antibody, previously used in a commercially available ELISA kit, to isolate and immobilise NEP from brain homogenates and CSF, prior to the addition of a fluorogenic peptide substrate (Mca-RPPGFSAFK(Dnp)). This fluorogenic substrate is ordinarily cleaved by multiple enzymes. We have shown that without the immunocapture phase, even under reaction conditions reported to be specific for neprilysin - i.e. in the presence of thiorphan, at pH above 7 - the fluorogenic peptide substrate does not allow neprilysin activity in brain homogenates and CSF to be discriminated from that of other closely related enzymes. The specificity of the immunocapture enzyme activity assay was confirmed by >80% inhibition of substrate cleavage in brain homogenates and CSF in the presence of thiorphan. The assay allows high-throughput analysis and, critically, also ensures a high level of enzyme specificity even when assaying crude tissue homogenates or CSF.

  1. Enzymes, Industrial

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Enzymes serve key roles in numerous biotechnology processes and products that are commonly encountered in the forms of food and beverages, cleaning supplies, clothing, paper products, transportation fuels, pharmaceuticals, and monitoring devices. Enzymes can display regio- and stereo-specificity, p...

  2. Application Specific Electronic Module Program (ASEM), Final Technical Report.

    DTIC Science & Technology

    1994-12-14

    Highlights and Accomplishments Associated with the Establishment of a Merchant MCM Foundry and Infrastructure. 14. S5U• ECT TERMS 15. NUM’•BER Of PAGESI...at IBM Microelectronics who built, assembled, and tested the demonstration vehicles 5 These people were invaluable in steering the activity and making ...2.1.1.1.3) Requirements Trade off tools are used to help make early decisions about specific alternatives and their impact to product objectives, even

  3. The External Quality Assurance Oversight Laboratory (EQAPOL) proficiency program for IFN-gamma enzyme-linked immunospot (IFN-γ ELISpot) assay.

    PubMed

    Sanchez, Ana M; Rountree, Wes; Berrong, Mark; Garcia, Ambrosia; Schuetz, Alexandra; Cox, Josephine; Frahm, Nicole; Manak, Mark; Sarzotti-Kelsoe, Marcella; D'Souza, M Patricia; Denny, Thomas; Ferrari, Guido

    2014-07-01

    The interferon-gamma enzyme-linked immunospot (IFN-γ ELISpot) assay has been developed and used as an end-point assay in clinical trials for infectious diseases and cancer to detect the magnitude of antigen-specific immune responses. The ability to compare data generated by different laboratories across organizations is pivotal to understand the relative potency of different therapeutic and vaccine strategies. We developed an external proficiency program for the IFN-γ ELISpot assay that evaluates laboratory performance based on five parameters: timeliness for data reporting; ability to handle cellular samples; detection of background (non-specific) responses; accuracy to consensus of the results; and precision of the measurements. Points are awarded for each criterion, and the sum of the points is used to determine a numeric and adjectival performance rating. Importantly, the evaluation of the accuracy to the consensus mean for the detection of antigen-specific responses using laboratory-specific procedures informs each laboratory and its sponsor on the degree of concordance of its results with those obtained by other laboratories. This study will ultimately provide the scientific community with information on how to organize and implement an external proficiency program to evaluate longitudinally the performance of the participating laboratories and, therefore, fulfill the requirements of the GCLP guidelines for laboratories performing end-point IFN-γ ELISpot assay for clinical trials.

  4. A Functional-Notional Approach for English for Specific Purposes (ESP) Programs.

    ERIC Educational Resources Information Center

    Kim, Young-Min

    English for Specific Purposes (ESP) programs, characterized by the special needs of the language learners, are described and a review of the literature on a functional-notional approach to the syllabus design of ESP programs is presented. It is suggested that effective ESP programs should teach the language skills necessary to function and perform…

  5. High Performance Computing - Power Application Programming Interface Specification.

    SciTech Connect

    Laros, James H.,; Kelly, Suzanne M.; Pedretti, Kevin; Grant, Ryan; Olivier, Stephen Lecler; Levenhagen, Michael J.; DeBonis, David

    2014-08-01

    Measuring and controlling the power and energy consumption of high performance computing systems by various components in the software stack is an active research area [13, 3, 5, 10, 4, 21, 19, 16, 7, 17, 20, 18, 11, 1, 6, 14, 12]. Implementations in lower level software layers are beginning to emerge in some production systems, which is very welcome. To be most effective, a portable interface to measurement and control features would significantly facilitate participation by all levels of the software stack. We present a proposal for a standard power Application Programming Interface (API) that endeavors to cover the entire software space, from generic hardware interfaces to the input from the computer facility manager.

  6. Northwest Energy Efficient Manufactured Housing Program Specification Development

    SciTech Connect

    Hewes, T.; Peeks, B.

    2013-02-01

    The Hood River Passive Project was developed by Root Design Build of Hood River Oregon using the Passive House Planning Package (PHPP) to meet all of the requirements for certification under the European Passive House standards. The Passive House design approach has been gaining momentum among residential designers for custom homes and BEopt modeling indicates that these designs may actually exceed the goal of the U.S. Department of Energy's (DOE) Building America program to reduce home energy use by 30%-50% (compared to 2009 energy codes for new homes). This report documents the short term test results of the Shift House and compares the results of PHPP and BEopt modeling of the project.

  7. Effects of Two Different Weight Training Programs on Swimming Performance and Muscle Enzyme Activities and Fiber Type.

    PubMed

    Belfry, Glen R; Noble, Earl G; Taylor, Albert W

    2016-02-01

    The effects of 2 different weight training programs incorporating bench press (BP) and pullover (PO) exercises on swimming performance, power, enzyme activity, and fiber type distribution were studied on 16 men (age = 23 ± 4 years). A 30-second group (n = 6) performed up to 20 repetitions of BP and PO in 30 seconds. The 2-minute group (n = 6) performed a maximum of 80 repetitions of BP and PO in 2 minutes. As participants reached the prescribed 20 or 80 repetitions, the weight was increased 4.5 kg. A third group (n = 4) served as nontraining controls. Exercise groups trained 3 times per week for 6 weeks. Maximal effort swims of 50 and 200 yd were performed before and after training. Training resulted in increases in work on both exercises in both groups pre- to post-training (BP 30 seconds, 722 ± 236-895 ± 250 kg; PO 30 seconds, 586 ± 252-1,090 ± 677 kg; and BP 2 minutes, 1,530 ± 414-1,940 ± 296; PO 2 minutes, 1,212 ± 406-2,348 ± 194, p ≤ 0.05). Swim performances of the 30-second group improved for both the 50-yd (32.0 ± 6.9 seconds, 30.0 ± 5.9 seconds, p ≤ 0.05) and 200-yd swims 200.0 ± 54 seconds, 182 ± 45.1 seconds (p ≤ 0.05), whereas 2-minute training improved only the 200-yd swim (198.3 ± 32.3 seconds, 186.2 ± 32.2 seconds). No changes in swim performance were observed for the control group. Triceps muscle succinate dehydrogenase activities increased (pre 3.48 ± 1.1 μmol · g(-1) wet weight per minute, post 6.25 ± 1.5 μmoles · g(-1) wet weight per minute, p ≤ 0.05) in only the 30-second training group, whereas phosphofructokinase activities and fiber type distribution did not change in either training group. This study has demonstrated that a 30-second 20-repetition weight training program, specific to the swimming musculature without concurrent swim training, improves swimming performances at both 50- and 200-yd distances.

  8. Employer Specific Training Program for Program Year 1988-89. Annual Report to the Governor and Legislature.

    ERIC Educational Resources Information Center

    New York State Education Dept., Albany.

    The Employer Specific Skills Training Program helps build the superior work force called for by the National Alliance of Business and other significant employer, union, government, and educational groups. Through a combination of state and federal funds, the New York State Department of Education has crafted a flexible and responsible program.…

  9. Characterization of a novel debranching enzyme from Nostoc punctiforme possessing a high specificity for long branched chains

    SciTech Connect

    Choi, Ji-Hye; Lee, Heeseob; Kim, Young-Wan; Park, Jong-Tae; Woo, Eui-Jeon; Kim, Myo-Jeong; Lee, Byong-Hoon

    2009-01-09

    A novel debranching enzyme from Nostoc punctiforme PCC73102 (NPDE) exhibits hydrolysis activity toward both {alpha}-(1,6)- and {alpha}-(1,4)-glucosidic linkages. The action patterns of NPDE revealed that branched chains are released first, and the resulting maltooligosaccharides are then hydrolyzed. Analysis of the reaction with maltooligosaccharide substrates labeled with {sup 14}C-glucose at the reducing end shows that NPDE specifically liberates glucose from the reducing end. Kinetic analyses showed that the hydrolytic activity of NPDE is greatly affected by the length of the substrate. The catalytic efficiency of NPDE increased considerably upon using substrates that can occupy at least eight glycone subsites such as maltononaose and maltooctaosyl-{alpha}-(1,6)-{beta}-cyclodextrin. These results imply that NPDE has a unique subsite structure consisting of -8 to +1 subsites. Given its unique subsite structure, side chains shorter than maltooctaose in amylopectin were resistant to hydrolysis by NPDE, and the population of longer side chains was reduced.

  10. Site-directed Mutagenesis Switching a Dimethylallyl Tryptophan Synthase to a Specific Tyrosine C3-Prenylating Enzyme*

    PubMed Central

    Fan, Aili; Zocher, Georg; Stec, Edyta; Stehle, Thilo; Li, Shu-Ming

    2015-01-01

    The tryptophan prenyltransferases FgaPT2 and 7-DMATS (7-dimethylallyl tryptophan synthase) from Aspergillus fumigatus catalyze C4- and C7-prenylation of the indole ring, respectively. 7-DMATS was found to accept l-tyrosine as substrate as well and converted it to an O-prenylated derivative. An acceptance of l-tyrosine by FgaPT2 was also observed in this study. Interestingly, isolation and structure elucidation revealed the identification of a C3-prenylated l-tyrosine as enzyme product. Molecular modeling and site-directed mutagenesis led to creation of a mutant FgaPT2_K174F, which showed much higher specificity toward l-tyrosine than l-tryptophan. Its catalytic efficiency toward l-tyrosine was found to be 4.9-fold in comparison with that of non-mutated FgaPT2, whereas the activity toward l-tryptophan was less than 0.4% of that of the wild-type. To the best of our knowledge, this is the first report on an enzymatic C-prenylation of l-tyrosine as free amino acid and altering the substrate preference of a prenyltransferase by mutagenesis. PMID:25477507

  11. The double-domain cytidine deaminase APOBEC3G is a cellular site-specific RNA editing enzyme

    PubMed Central

    Sharma, Shraddha; Patnaik, Santosh K.; Taggart, Robert T.; Baysal, Bora E.

    2016-01-01

    APOBEC3G is a cytidine deaminase with two homologous domains and restricts retroelements and HIV-1. APOBEC3G deaminates single-stranded DNAs via its C-terminal domain, whereas the N-terminal domain is considered non-catalytic. Although APOBEC3G is known to bind RNAs, APOBEC3G-mediated RNA editing has not been observed. We recently discovered RNA editing by the single-domain enzyme APOBEC3A in innate immune cells. To determine if APOBEC3G is capable of RNA editing, we transiently expressed APOBEC3G in the HEK293T cell line and performed transcriptome-wide RNA sequencing. We show that APOBEC3G causes site-specific C-to-U editing of mRNAs from over 600 genes. The edited cytidines are often flanked by inverted repeats, but are largely distinct from those deaminated by APOBEC3A. We verified protein-recoding RNA editing of selected genes including several that are known to be involved in HIV-1 infectivity. APOBEC3G co-purifies with highly edited mRNA substrates. We find that conserved catalytic residues in both cytidine deaminase domains are required for RNA editing. Our findings demonstrate the novel RNA editing function of APOBEC3G and suggest a role for the N-terminal domain in RNA editing. PMID:27974822

  12. Development of an enzyme-linked immunosorbent assay (ELISA) using highly-specific monoclonal antibodies against plumbagin.

    PubMed

    Sakamoto, Seiichi; Putalun, Waraporn; Tsuchihashi, Ryota; Morimoto, Satoshi; Kinjo, Junei; Tanaka, Hiroyuki

    2008-01-21

    Plumbagin (PL; 5-hydroxy-2-methyl-1,4-naphthoquinone) is a natural compound mainly isolated from Plumbago zeylanica. This plant is distributed in Southeast Asia, and well known as Ayurvedic medicine in India for its medicinal properties. PL has been shown to have various pharmacological activities. We have successfully prepared monoclonal antibodies against PL, and developed an enzyme-linked immunosorbent assay (ELISA) system for determination of PL. 3-(5-Hydroxy-2-methyl-1,4-naphthoquinone-3-yl) propanoic acid was synthesized and purified to prepare PL-bovine serum albumin conjugate (PL-BSA), which was used as an immunogen. PL-BSA conjugate was administered into BALB/c male mice for production of monoclonal antibodies against PL. The monoclonal antibody against PL which is secreted from established hybridoma cell line 3A3 (MAb 3A3) has been proven to have highly-specific to PL resulting from cross-reactivities test. The range for calibration of PL by ELISA was 0.2-25 microg mL(-1). Based on validation analysis, this analytical method by ELISA is a precise, accurate, and sensitive method for the determination of PL in plant.

  13. The double-domain cytidine deaminase APOBEC3G is a cellular site-specific RNA editing enzyme.

    PubMed

    Sharma, Shraddha; Patnaik, Santosh K; Taggart, Robert T; Baysal, Bora E

    2016-12-15

    APOBEC3G is a cytidine deaminase with two homologous domains and restricts retroelements and HIV-1. APOBEC3G deaminates single-stranded DNAs via its C-terminal domain, whereas the N-terminal domain is considered non-catalytic. Although APOBEC3G is known to bind RNAs, APOBEC3G-mediated RNA editing has not been observed. We recently discovered RNA editing by the single-domain enzyme APOBEC3A in innate immune cells. To determine if APOBEC3G is capable of RNA editing, we transiently expressed APOBEC3G in the HEK293T cell line and performed transcriptome-wide RNA sequencing. We show that APOBEC3G causes site-specific C-to-U editing of mRNAs from over 600 genes. The edited cytidines are often flanked by inverted repeats, but are largely distinct from those deaminated by APOBEC3A. We verified protein-recoding RNA editing of selected genes including several that are known to be involved in HIV-1 infectivity. APOBEC3G co-purifies with highly edited mRNA substrates. We find that conserved catalytic residues in both cytidine deaminase domains are required for RNA editing. Our findings demonstrate the novel RNA editing function of APOBEC3G and suggest a role for the N-terminal domain in RNA editing.

  14. Development of an Enzyme-Linked Immunosorbent Assay Method Specific for the Detection of G-Group Aflatoxins.

    PubMed

    Li, Peiwu; Zhou, Qian; Wang, Ting; Zhou, Haiyan; Zhang, Wen; Ding, Xiaoxia; Zhang, Zhaowei; Chang, Perng-Kuang; Zhang, Qi

    2015-12-28

    To detect and monitor G-group aflatoxins in agricultural products, we generated class-specific monoclonal antibodies that specifically recognized aflatoxins G₁ and G₂. Of the final three positive and stable hybridomas obtained, clone 2G6 produced a monoclonal antibody that had equal sensitivity to aflatoxins G₁ and G₂, and did not cross-react with aflatoxins B₁, B₂, or M₁. Its IC50 values for aflatoxins G₁ and G₂ were 17.18 ng·mL(-1) and 19.75 ng·mL(-1), respectively. Using this new monoclonal antibody, we developed a competitive indirect enzyme-linked immunosorbent assay (CI-ELISA); the method had a limit of detection of 0.06 ng·mL(-1). To validate this CI-ELISA, we spiked uncontaminated peanut samples with various amounts of aflatoxins G₁ and G₂ and compared recovery rates with those determined by a standard HPLC method. The recovery rates of the CI-ELISA ranging from 94% to 103% were comparable to those of the HPLC (92% to 102%). We also used both methods to determine the amounts of G-group aflatoxins in five peanut samples contaminated by aflatoxin B₁-positive, and their relative standard deviations ranged from 8.4% to 17.7% (under 20%), which demonstrates a good correlation between the two methods. We further used this CI-ELISA to assess the ability of 126 fungal strains isolated from peanuts or field soils to produce G-group aflatoxins. Among these, seven stains producing different amounts of G-group aflatoxins were identified. Our results showed that the monoclonal antibody 2 G6-based CI-ELISA was suitable for the detection of G-group aflatoxins present in peanuts and also those produced by fungi.

  15. Detection of Specific Solvent Rearrangement Regions of an Enzyme: NMR and ITC Studies with Aminoglycoside Phosphotransferase(3??)-IIIa

    SciTech Connect

    Ozen, C.; Norris, Adrianne; Land, Miriam L; Tjioe, Elina; Serpersu, Engin H

    2008-01-01

    This work describes differential effects of solvent in complexes of the aminoglycoside phosphotransferase(3¢)-IIIa (APH) with different aminoglycosides and the detection of change in solvent structure at specific sites away from substrates. Binding of kanamycins to APH occurs with a larger negative ¢H in H2O relative to D2O (¢¢H(H2O-D2O) < 0), while the reverse is true for neomycins. Unusually large negative ¢Cp values were observed for binding of aminoglycosides to APH. ¢Cp for the APHneomycin complex was -1.6 kcalâmol-1âdeg-1. A break at 30 C was observed in the APH-kanamycin complex yielding ¢Cp values of -0.7 kcalâmol-1âdeg-1 and -3.8 kcalâmol-1âdeg-1 below and above 30 C, respectively. Neither the change in accessible surface area (¢ASA) nor contributions from heats of ionization were sufficient to explain the large negative ¢Cp values. Most significantly, 15N-1H HSQC experiments showed that temperature-dependent shifts of the backbone amide protons of Leu 88, Ser 91, Cys 98, and Leu143 revealed a break at 30 C only in the APH-kanamycin complex in spectra collected between 21 C and 38 C. These amino acids represent solVent reorganization sites that experience a change in solvent structure in their immediate environment as structurally different ligands bind to the enzyme. These residues were away from the substrate binding site and distributed in three hydrophobic patches in APH. Overall, our results show that a large number of factors affect ¢Cp and binding of structurally different ligand groups cause different solvent structure in the active site as well as differentially affecting specific sites away from the ligand binding site.

  16. Sensitive and specific enzyme-linked immunosorbent assay for detecting serum antibodies against Mycobacterium avium subsp. paratuberculosis in fallow deer.

    PubMed

    Prieto, José M; Balseiro, Ana; Casais, Rosa; Abendaño, Naiara; Fitzgerald, Liam E; Garrido, Joseba M; Juste, Ramon A; Alonso-Hearn, Marta

    2014-08-01

    The enzyme-linked immunosorbent assay (ELISA) is the diagnostic test most commonly used in efforts to control paratuberculosis in domestic ruminants. However, commercial ELISAs have not been validated for detecting antibodies against Mycobacterium avium subsp. paratuberculosis in wild animals. In this study, we compared the sensitivities and specificities of five ELISAs using individual serum samples collected from 41 fallow deer with or without histopathological lesions consistent with paratuberculosis. Two target antigenic preparations were selected, an ethanol-treated protoplasmic preparation obtained from a fallow deer M. avium subsp. paratuberculosis isolate (ELISAs A and B) and a paratuberculosis protoplasmic antigen (PPA3) (ELISAs C and D). Fallow deer antibodies bound to the immobilized antigens were detected by using a horseradish peroxidase (HRP)-conjugated anti-fallow deer IgG antibody (ELISAs A and C) or HRP-conjugated protein G (ELISAs B and D). A commercially available assay, ELISA-E, which was designed to detect M. avium subsp. paratuberculosis antibodies in cattle, sheep, and goats, was also tested. Although ELISAs A, C, and E had the same sensitivity (72%), ELISAs A and C were more specific (100%) for detecting fallow deer with lesions consistent with paratuberculosis at necropsy than was the ELISA-E (87.5%). In addition, the ELISA-A was particularly sensitive for detecting fallow deer in the latent stages of infection (62.5%). The antibody responses detected with the ELISA-A correlated with both the severity of enteric lesions and the presence of acid-fast bacteria in gut tissue samples. In summary, our study shows that the ELISA-A can be a cost-effective diagnostic tool for preventing the spread of paratuberculosis among fallow deer populations.

  17. Development and evaluation of a Sarcocystis neurona-specific IgM capture enzyme-linked immunosorbent assay.

    PubMed

    Murphy, J E; Marsh, A E; Reed, S M; Meadows, C; Bolten, K; Saville, W J A

    2006-01-01

    Equine protozoal myeloencephalitis (EPM) is a serious neurologic disease of horses caused primarily by the protozoal parasite Sarcocystis neurona. Currently available antemortem diagnostic testing has low specificity. The hypothesis of this study was that serum and cerebrospinal fluid (CSF) of horses experimentally challenged with S neurona would have an increased S neurona-specific IgM (Sn-IgM) concentration after infection, as determined by an IgM capture enzyme linked immunoassay (ELISA). The ELISA was based on the S neurona low molecular weight protein SNUCD-1 antigen and the monoclonal antibody 2G5 labeled with horseradish peroxidase. The test was evaluated using serum and CSF from 12 horses experimentally infected with 1.5 million S neurona sporocysts and 16 horses experimentally infected with varying doses (100 to 100,000) of S neurona sporocysts, for which results of histopathologic examination of the central nervous system were available. For horses challenged with 1.5 million sporocysts, there was a significant increase in serum Sn-IgM concentrations compared with values before infection at weeks 2-6 after inoculation (P < .0001). For horses inoculated with lower doses of S neurona, there were significant increases in serum Sn-IgM concentration at various points in time after inoculation, depending on the challenge dose (P < .01). In addition, there was a significant increase between the CSF Sn-IgM concentrations before and after inoculation (P < .0001). These results support further evaluation of the assay as a diagnostic test during the acute phase of EPM.

  18. Stereo-specificity for pro-(R) hydrogen of NAD(P)H during enzyme-catalyzed hydride transfer to CL-20

    SciTech Connect

    Bhushan, Bharat; Halasz, Annamaria; Hawari, Jalal . E-mail: jalal.hawari@nrc.ca

    2005-12-02

    A dehydrogenase from Clostridium sp. EDB2 and a diaphorase from Clostridium kluyveri were reacted with CL-20 to gain insights into the enzyme-catalyzed hydride transfer to CL-20, and the enzyme's stereo-specificity for either pro-R or pro-S hydrogens of NAD(P)H. Both enzymes biotransformed CL-20 at rates of 18.5 and 24 nmol/h/mg protein, using NADH and NADPH as hydride-source, respectively, to produce a N-denitrohydrogenated product with a molecular weight of 393 Da. In enzyme kinetics studies using reduced deuterated pyridine nucleotides, we found a kinetic deuterium isotopic effect of 2-fold on CL-20 biotransformation rate using dehydrogenase enzyme against (R)NADD as a hydride-source compared to either (S)NADD or NADH. Whereas, in case of diaphorase, the kinetic deuterium isotopic effect of about 1.5-fold was observed on CL-20 biotransformation rate using (R)NADPD as hydride-source. In a comparative study with LC-MS, using deuterated and non-deuterated NAD(P)H, we found a positive mass-shift of 1 Da in the N-denitrohydrogenated product suggesting the involvement of a deuteride (D{sup -}) transfer from NAD(P)D. The present study thus revealed that both dehydrogenase and diaphorase enzymes from the two Clostridium species catalyzed a hydride transfer to CL-20 and showed stereo-specificity for pro-R hydrogen of NAD(P)H.

  19. NASIS data base management system: IBM 360 TSS implementation. Volume 4: Program design specifications

    NASA Technical Reports Server (NTRS)

    1973-01-01

    The design specifications for the programs and modules within the NASA Aerospace Safety Information System (NASIS) are presented. The purpose of the design specifications is to standardize the preparation of the specifications and to guide the program design. Each major functional module within the system is a separate entity for documentation purposes. The design specifications contain a description of, and specifications for, all detail processing which occurs in the module. Sub-models, reference tables, and data sets which are common to several modules are documented separately.

  20. Effect of formal specifications on program complexity and reliability: An experimental study

    NASA Technical Reports Server (NTRS)

    Goel, Amrit L.; Sahoo, Swarupa N.

    1990-01-01

    The results are presented of an experimental study undertaken to assess the improvement in program quality by using formal specifications. Specifications in the Z notation were developed for a simple but realistic antimissile system. These specifications were then used to develop 2 versions in C by 2 programmers. Another set of 3 versions in Ada were independently developed from informal specifications in English. A comparison of the reliability and complexity of the resulting programs suggests the advantages of using formal specifications in terms of number of errors detected and fault avoidance.

  1. Combined effect of improved cell yield and increased specific productivity enhances recombinant enzyme production in genome-reduced Bacillus subtilis strain MGB874.

    PubMed

    Manabe, Kenji; Kageyama, Yasushi; Morimoto, Takuya; Ozawa, Tadahiro; Sawada, Kazuhisa; Endo, Keiji; Tohata, Masatoshi; Ara, Katsutoshi; Ozaki, Katsuya; Ogasawara, Naotake

    2011-12-01

    Genome reduction strategies to create genetically improved cellular biosynthesis machineries for proteins and other products have been pursued by use of a wide range of bacteria. We reported previously that the novel Bacillus subtilis strain MGB874, which was derived from strain 168 and has a total genomic deletion of 874 kb (20.7%), exhibits enhanced production of recombinant enzymes. However, it was not clear how the genomic reduction resulted in elevated enzyme production. Here we report that deletion of the rocDEF-rocR region, which is involved in arginine degradation, contributes to enhanced enzyme production in strain MGB874. Deletion of the rocDEF-rocR region caused drastic changes in glutamate metabolism, leading to improved cell yields with maintenance of enzyme productivity. Notably, the specific enzyme productivity was higher in the reduced-genome strain, with or without the rocDEF-rocR region, than in wild-type strain 168. The high specific productivity in strain MGB874 is likely attributable to the higher expression levels of the target gene resulting from an increased promoter activity and plasmid copy number. Thus, the combined effects of the improved cell yield by deletion of the rocDEF-rocR region and the increased specific productivity by deletion of another gene(s) or the genomic reduction itself enhanced the production of recombinant enzymes in MGB874. Our findings represent a good starting point for the further improvement of B. subtilis reduced-genome strains as cell factories for the production of heterologous enzymes.

  2. Enzyme-free detection of sequence-specific microRNAs based on nanoparticle-assisted signal amplification strategy.

    PubMed

    Li, Ru-Dong; Wang, Qian; Yin, Bin-Cheng; Ye, Bang-Ce

    2016-03-15

    Developing direct and convenient methods for microRNAs (miRNAs) analysis is of great significance in understanding biological functions of miRNAs, and early diagnosis of cancers. We have developed a rapid, enzyme-free method for miRNA detection based on nanoparticle-assisted signal amplification coupling fluorescent metal nanoclusters as signal output. The proposed method involves two processes: target miRNA-mediated nanoparticle capture, which consists of magnetic microparticle (MMP) probe and CuO nanoparticle (NP) probe, and nanoparticle-mediated amplification for signal generation, which consists of fluorescent DNA-Cu/Ag nanocluster (NC) and 3-mercaptopropionic acid (MPA). In the presence of target miRNA, MMP probe and NP probe sandwich-capture the target miRNA via their respective complementary sequence. The resultant sandwich complex (MMP probe-miRNA-CuO NP probe) is separated using a magnetic field and further dissolved by acidolysis to turn CuO NP into a great amount of copper (II) ions (Cu(2+)). Cu(2+) could disrupt the interactions between thiol moiety of MPA and the fluorescent Cu/Ag NCs by preferentially reacting with MPA to form a disulfide compound as intermediate. By this way, the fluorescence emission of the DNA-Cu/Ag NCs in the presence of MPA increases upon the increasing concentration of Cu(2+), which is directly proportional to the amount of target miRNA. The proposed method allows quantitative detection of a liver-specific miR-221-5p in the range of 5 pM to 1000 pM with a detection limit of ~0.73 pM, and shows a good ability to discriminate single-base difference. Moreover, the detection assay can be applied to detect miRNA in cancerous cell lysates in excellent agreement with that from a commercial miRNA detection kit.

  3. Glutathione-dependent detoxifying enzymes in rainbow trout liver: Search for specific biochemical markers of chemical stress

    SciTech Connect

    Petrivalsky, M.; Machala, M.; Nezveda, K.; Piacka, V.; Svobodova, Z. |; Drabek, P.

    1997-07-01

    Activities of trout liver microsomal glutathione S-transferase (GST) and a series of cytosolic glutathione-dependent detoxifying enzymes were determined after a single intraperitoneal treatment with phenobarbital, 2,2-bis (p-chlorophenyl)-1,1-dichloroethane (p,p{prime}-DDE), 2,3-dimethoxynaphthoquinone (NQ), or 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). This study aimed to find xenobiotic-specific parameters applicable as biochemical markers of the impacts of the prototypal xenobiotics. The effects of xenobiotics on cytosolic GST activities were substrate dependent. The rate of conjugation of p-nitrobenzyl chloride was significantly induced by higher doses of p,p{prime}-DDE or NQ. The conjugation of ethacrynic acid was enhanced by phenobarbital, p,p{prime}-DDE, and NQ. The GST activity against 1,2-epoxy-3-(p-nitrophenoxy)propane was induced only by phenobarbital and by lower doses of p,p{prime}-DDE. The cytosolic GST activity, measured with 1-chloro-2,4-dinitrobenzene as a substrate, was only weakly increased by phenobarbital, TCDD, higher doses of p,p{prime}-DDE, or by NQ at the lowest dose of 1 mg/kg. Although the latter activity is frequently used as a biomarker in ecotoxicology, various factors (including its weak inducibility) indicate that this biochemical parameter is probably not a suitable indicator of contamination in fish. Similarly, cytosolic glutathione peroxidase was not affected by the prototypal xenobiotics and appeared to be an unsuitable bioindicator of oxidative impacts of the tested compounds. On the other hand, microsomal GST activity was nonspecifically increased by phenobarbital, NQ, TCDD, and high doses of p,p{prime}-DDE. Glutathione reductase, another potential biomarker of oxidative stress, was induced by phenobarbital, NQ, and, to a lesser extent, p,p{prime}-DDE; therefore it appeared to be a less sensitive indicator to the exposure to prototypal xenobiotics than the microsomal GST.

  4. The Structure of the Bacterial Oxidoreductase Enzyme DsbA in Complex with a Peptide Reveals a Basis for Substrate Specificity in the Catalytic Cycle of DsbA Enzymes

    SciTech Connect

    Paxman, Jason J.; Borg, Natalie A.; Horne, James; Thompson, Philip E.; Chin, Yanni; Sharma, Pooja; Simpson, Jamie S.; Wielens, Jerome; Piek, Susannah; Kahler, Charlene M.; Sakellaris, Harry; Pearce, Mary; Bottomley, Stephen P.; Rossjohn, Jamie; Scanlon, Martin J.

    2010-09-07

    Oxidative protein folding in Gram-negative bacteria results in the formation of disulfide bonds between pairs of cysteine residues. This is a multistep process in which the dithiol-disulfide oxidoreductase enzyme, DsbA, plays a central role. The structure of DsbA comprises an all helical domain of unknown function and a thioredoxin domain, where active site cysteines shuttle between an oxidized, substrate-bound, reduced form and a DsbB-bound form, where DsbB is a membrane protein that reoxidizes DsbA. Most DsbA enzymes interact with a wide variety of reduced substrates and show little specificity. However, a number of DsbA enzymes have now been identified that have narrow substrate repertoires and appear to interact specifically with a smaller number of substrates. The transient nature of the DsbA-substrate complex has hampered our understanding of the factors that govern the interaction of DsbA enzymes with their substrates. Here we report the crystal structure of a complex between Escherichia coli DsbA and a peptide with a sequence derived from a substrate. The binding site identified in the DsbA-peptide complex was distinct from that observed for DsbB in the DsbA-DsbB complex. The structure revealed details of the DsbA-peptide interaction and suggested a mechanism by which DsbA can simultaneously show broad specificity for substrates yet exhibit specificity for DsbB. This mode of binding was supported by solution nuclear magnetic resonance data as well as functional data, which demonstrated that the substrate specificity of DsbA could be modified via changes at the binding interface identified in the structure of the complex.

  5. The structure of the bacterial oxidoreductase enzyme DsbA in complex with a peptide reveals a basis for substrate specificity in the catalytic cycle of DsbA enzymes.

    PubMed

    Paxman, Jason J; Borg, Natalie A; Horne, James; Thompson, Philip E; Chin, Yanni; Sharma, Pooja; Simpson, Jamie S; Wielens, Jerome; Piek, Susannah; Kahler, Charlene M; Sakellaris, Harry; Pearce, Mary; Bottomley, Stephen P; Rossjohn, Jamie; Scanlon, Martin J

    2009-06-26

    Oxidative protein folding in Gram-negative bacteria results in the formation of disulfide bonds between pairs of cysteine residues. This is a multistep process in which the dithiol-disulfide oxidoreductase enzyme, DsbA, plays a central role. The structure of DsbA comprises an all helical domain of unknown function and a thioredoxin domain, where active site cysteines shuttle between an oxidized, substrate-bound, reduced form and a DsbB-bound form, where DsbB is a membrane protein that reoxidizes DsbA. Most DsbA enzymes interact with a wide variety of reduced substrates and show little specificity. However, a number of DsbA enzymes have now been identified that have narrow substrate repertoires and appear to interact specifically with a smaller number of substrates. The transient nature of the DsbA-substrate complex has hampered our understanding of the factors that govern the interaction of DsbA enzymes with their substrates. Here we report the crystal structure of a complex between Escherichia coli DsbA and a peptide with a sequence derived from a substrate. The binding site identified in the DsbA-peptide complex was distinct from that observed for DsbB in the DsbA-DsbB complex. The structure revealed details of the DsbA-peptide interaction and suggested a mechanism by which DsbA can simultaneously show broad specificity for substrates yet exhibit specificity for DsbB. This mode of binding was supported by solution nuclear magnetic resonance data as well as functional data, which demonstrated that the substrate specificity of DsbA could be modified via changes at the binding interface identified in the structure of the complex.

  6. Peroxidase from Catharanthus roseus (L.) G. Don and the biosynthesis of alpha-3',4'-anhydrovinblastine: a specific role for a multifunctional enzyme.

    PubMed

    Sottomayor, M; Ros Barceló, A

    2003-09-01

    We have characterized a basic peroxidase with alpha-3',4'-anhydrovinblastine (AVLB) synthase activity, which was purified from Catharanthus roseus leaves. This enzyme was the single peroxidase isoenzyme detected in C. roseus leaves, and the single AVLB synthase activity detected in C. roseus extracts. It was observed that the monomeric substrates of AVLB, vindoline and catharanthine, are both suitable electron donors for the oxidizing intermediates of the basic peroxidase, compounds I and II. Results also showed that the reaction proceeds by a radical-propagated mechanism. Substrate specificity studies of the enzyme revealed that it was also able to oxidize several common peroxidase substrates, indicating a broad range of substrate specificity that is characteristic of class III plant peroxidases. Cytochemical studies showed that the enzyme is localized in C. roseus mesophyll vacuoles, in individual spots at the inner surface of the tonoplast. This particular location suggests a meaningful spatial organization that led to the proposal of a metabolic channeling model for the peroxidase-mediated synthesis of AVLB. The importance of this type of mechanism in the regulation of peroxidase isoenzyme functions in vivo is discussed. In view of the results obtained it is concluded that the basic peroxidase present in C. roseus leaves fulfills all the requirements to be considered as an AVLB synthase, and it is proposed that this specific function of this multifunctional enzyme is determined by metabolic channeling resulting from specific protein-protein interactions.

  7. Highly broad-specific and sensitive enzyme-linked immunosorbent assay for screening sulfonamides: Assay optimization and application to milk samples

    Technology Transfer Automated Retrieval System (TEKTRAN)

    A broad-specific and sensitive immunoassay for the detection of sulfonamides was developed by optimizing the conditions of an enzyme-linked immunosorbent assay (ELISA) in regard to different monoclonal antibodies (MAbs), assay format, immunoreagents, and several physicochemical factors (pH, salt, de...

  8. Ligand-binding specificity and promiscuity of the main lignocellulolytic enzyme families as revealed by active-site architecture analysis

    PubMed Central

    Tian, Li; Liu, Shijia; Wang, Shuai; Wang, Lushan

    2016-01-01

    Biomass can be converted into sugars by a series of lignocellulolytic enzymes, which belong to the glycoside hydrolase (GH) families summarized in CAZy databases. Here, using a structural bioinformatics method, we analyzed the active site architecture of the main lignocellulolytic enzyme families. The aromatic amino acids Trp/Tyr and polar amino acids Glu/Asp/Asn/Gln/Arg occurred at higher frequencies in the active site architecture than in the whole enzyme structure. And the number of potential subsites was significantly different among different families. In the cellulase and xylanase families, the conserved amino acids in the active site architecture were mostly found at the −2 to +1 subsites, while in β-glucosidase they were mainly concentrated at the −1 subsite. Families with more conserved binding amino acid residues displayed strong selectivity for their ligands, while those with fewer conserved binding amino acid residues often exhibited promiscuity when recognizing ligands. Enzymes with different activities also tended to bind different hydroxyl oxygen atoms on the ligand. These results may help us to better understand the common and unique structural bases of enzyme-ligand recognition from different families and provide a theoretical basis for the functional evolution and rational design of major lignocellulolytic enzymes. PMID:27009476

  9. HAL/SM language specification. [programming languages and computer programming for space shuttles

    NASA Technical Reports Server (NTRS)

    Williams, G. P. W., Jr.; Ross, C.

    1975-01-01

    A programming language is presented for the flight software of the NASA Space Shuttle program. It is intended to satisfy virtually all of the flight software requirements of the space shuttle. To achieve this, it incorporates a wide range of features, including applications-oriented data types and organizations, real time control mechanisms, and constructs for systems programming tasks. It is a higher order language designed to allow programmers, analysts, and engineers to communicate with the computer in a form approximating natural mathematical expression. Parts of the English language are combined with standard notation to provide a tool that readily encourages programming without demanding computer hardware expertise. Block diagrams and flow charts are included. The semantics of the language is discussed.

  10. 75 FR 59058 - Competitive and Noncompetitive Non-Formula Federal Assistance Programs-Specific Administrative...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2010-09-27

    ... National Institute of Food and Agriculture 7 CFR Part 3430 RIN 0524-AA60 Competitive and Noncompetitive Non-Formula Federal Assistance Programs--Specific Administrative Provisions for the New Era Rural Technology Competitive Grants Program AGENCY: National Institute of Food and Agriculture, USDA. ACTION: Final...

  11. The Rise of International Relations Programs in the Brazilian Federal Universities: Curriculum Specificities and Current Challenges

    ERIC Educational Resources Information Center

    Ferreira, Marcos Alan S. V.

    2016-01-01

    The aim of this reflection is to study the new international relations (IR) programs introduced by Brazilian federal universities, looking comparatively at their curriculum specificities and current challenges. In recent years, Brazil has seen an increase of IR programs launched in several regions. Since 2003, the Ministry of Education is in the…

  12. Using the SCR Specification Technique in a High School Programming Course.

    ERIC Educational Resources Information Center

    Rosen, Edward; McKim, James C., Jr.

    1992-01-01

    Presents the underlying ideas of the Software Cost Reduction (SCR) approach to requirements specifications. Results of applying this approach to the teaching of programing to high school students indicate that students perform better in writing programs. An appendix provides two examples of how the method is applied to problem solving. (MDH)

  13. Lysine 190 is the catalytic base in MenF, the menaquinone-specific isochorismate synthase from Escherichia coli: implications for an enzyme family.

    PubMed

    Kolappan, Subramaniapillai; Zwahlen, Jacque; Zhou, Rong; Truglio, James J; Tonge, Peter J; Kisker, Caroline

    2007-01-30

    Menaquinone biosynthesis is initiated by the conversion of chorismate to isochorismate, a reaction that is catalyzed by the menaquinone-specific isochorismate synthase, MenF. The catalytic mechanism of MenF has been probed using a combination of structural and biochemical studies, including the 2.5 A structure of the enzyme, and Lys190 has been identified as the base that activates water for nucleophilic attack at the chorismate C2 carbon. MenF is a member of a larger family of Mg2+ dependent chorismate binding enzymes catalyzing distinct chorismate transformations. The studies reported here extend the mechanism recently proposed for this enzyme family by He et al.: He, Z., Stigers Lavoie, K. D., Bartlett, P. A., and Toney, M. D. (2004) J. Am. Chem. Soc. 126, 2378-85.

  14. Site-specific bioconjugation of an organometallic electron mediator to an enzyme with retained photocatalytic cofactor regenerating capacity and enzymatic activity.

    PubMed

    Lim, Sung In; Yoon, Sungho; Kim, Yong Hwan; Kwon, Inchan

    2015-04-07

    Photosynthesis consists of a series of reactions catalyzed by redox enzymes to synthesize carbohydrates using solar energy. In order to take the advantage of solar energy, many researchers have investigated artificial photosynthesis systems mimicking the natural photosynthetic enzymatic redox reactions. These redox reactions usually require cofactors, which due to their high cost become a key issue when constructing an artificial photosynthesis system. Combining a photosensitizer and an Rh-based electron mediator (RhM) has been shown to photocatalytically regenerate cofactors. However, maintaining the high concentration of cofactors available for efficient enzymatic reactions requires a high concentration of the expensive RhM; making this process cost prohibitive. We hypothesized that conjugation of an electron mediator to a redox enzyme will reduce the amount of electron mediators necessary for efficient enzymatic reactions. This is due to photocatalytically regenerated NAD(P)H being readily available to a redox enzyme, when the local NAD(P)H concentration near the enzyme becomes higher. However, conventional random conjugation of RhM to a redox enzyme will likely lead to a substantial loss of cofactor regenerating capacity and enzymatic activity. In order to avoid this issue, we investigated whether bioconjugation of RhM to a permissive site of a redox enzyme retains cofactor regenerating capacity and enzymatic activity. As a model system, a RhM was conjugated to a redox enzyme, formate dehydrogenase obtained from Thiobacillus sp. KNK65MA (TsFDH). A RhM-containing azide group was site-specifically conjugated to p-azidophenylalanine introduced to a permissive site of TsFDH via a bioorthogonal strain-promoted azide-alkyne cycloaddition and an appropriate linker. The TsFDH-RhM conjugate exhibited retained cofactor regenerating capacity and enzymatic activity.

  15. Sex-specific basal and hypoglycemic patterns of in vivo caudal dorsal vagal complex astrocyte glycogen metabolic enzyme protein expression.

    PubMed

    Tamrakar, Pratistha; Shrestha, Prem; Briski, Karen P

    2014-10-24

    Astrocytes contribute to neurometabolic stability through uptake, catabolism, and storage of glucose. These cells maintain the major brain glycogen reservoir, which is a critical fuel supply to neurons during glucose deficiency and increased brain activity. We used a combinatory approach incorporating immunocytochemistry, laser microdissection, and Western blotting to investigate the hypothesis of divergent expression of key enzymes regulating glycogen metabolism and glycolysis during in vivo normo- and/or hypoglycemia in male versus female hindbrain astrocytes. Glycogen synthase (GS) and glycogen phosphorylase (GP) levels were both enhanced in dorsal vagal complex astrocytes from vehicle-injected female versus male controls, with incremental increase in GS exceeding GP. Insulin-induced hypoglycemia (IIH) diminished GS and increased glycogen synthase kinase-3-beta (GSK3β) expression in both sexes, but decreased phosphoprotein phosphatase-1 (PP1) levels only in males. Astrocyte GP content was elevated by IIH in male, but not female rats. Data reveal sex-dependent sensitivity of these enzyme proteins to lactate as caudal hindbrain repletion of this energy substrate fully or incompletely reversed hypoglycemic inhibition of GS and prevented hypoglycemic augmentation of GSK3β and GP in females and males, respectively. Sex dimorphic patterns of glycogen branching and debranching enzyme protein expression were also observed. Levels of the rate-limiting glycolytic enzyme, phosphofructokinase, were unaffected by IIH with or without lactate repletion. Current data demonstrating sex-dependent basal and hypoglycemic patterns of hindbrain astrocyte glycogen metabolic enzyme expression imply that glycogen volume and turnover during glucose sufficiency and shortage may vary accordingly.

  16. A reversed genetic approach reveals the coenzyme specificity and other catalytic properties of three enzymes putatively involved in anaerobic oxidation of methane with sulfate.

    PubMed

    Kojima, Hisaya; Moll, Johanna; Kahnt, Jörg; Fukui, Manabu; Shima, Seigo

    2014-11-01

    Consortia of anaerobic methanotrophic (ANME) archaea and delta-proteobacteria anaerobically oxidize methane coupled to sulfate reduction to sulfide. The metagenome of ANME-1 archaea contains genes homologous to genes otherwise only found in methanogenic archaea, and transcription of some of these genes in ANME-1 cells has been shown. We now have heterologously expressed three of these genes in Escherichia coli, namely those homologous to genes for formylmethanofuran : tetrahydromethanopterin formyltransferase, methenyltetrahydromethanopterin cyclohydrolase (Mch) and coenzyme F420 -dependent methylenetetrahydromethanopterin dehydrogenase (Mtd), and have characterized the overproduced enzymes with respect to their coenzyme specificity and other catalytic properties. The three enzymes from ANME-1 were found to catalyse the same reactions and with similar specific activities using identical coenzymes as the respective enzymes in methanogenic archaea, the apparent Km for their substrates being in the same concentration range. The results support the proposal that anaerobic oxidation of methane to CO₂in ANME involves the same enzymes and coenzymes as CO₂reduction to methane in methanogenic archaea. Interestingly, the activity of Mch and the stability of Mtd from ANME-1 were found to be dependent on the presence of 0.5-1.0 M potassium phosphate, which suggested that ANME-1 archaea contain high concentrations of lyotropic salts, presumably as compatible solutes.

  17. Role of pectolytic enzymes in the programmed separation of cells from the root cap of higher plants. Final report

    SciTech Connect

    Hawes, M.C.

    1995-03-01

    The objective of this research was to develop a model system to study border cell separation in transgenic pea roots. In addition, the hypothesis that genes encoding pectolytic enzymes in the root cap play a role in the programmed separation of root border cells from the root tip was tested. The following objectives have been accomplished: (1) the use of transgenic hairy roots to study border cell separation has been optimized for Pisum sativum; (2) a cDNA encoding a root cap pectinmethylesterase (PME) has been cloned; (3) PME and polygalacturonase activities in cell walls of the root cap have been characterized and shown to be correlated with border cell separation. A fusion gene encoding pectate lyase has also been transformed into pea hairy root cells.

  18. Interaction of phospholipase C with liposome: A conformation transition of the enzyme is critical and specific to liposome composition for burst hydrolysis and fusion in concert

    NASA Astrophysics Data System (ADS)

    Patra, Samir Kumar; Sengupta, Dipta; Deb, Moonmoon; Kar, Swayamsiddha; Kausar, Chahat

    2017-02-01

    Phospholipase C (PLC)1 is known to help the pathogen B. cereus entry to the host cell and human PLC is over expressed in multiple cancers. Knowledge of dynamic activity of the enzyme PLC while in action on membrane lipids is essential and helpful to drug design and delivery. In view of this, interactions of PLC with liposome of various lipid compositions have been visualized by testing enzyme activity and microenvironments around the intrinsic fluorophores of the enzyme. Overall change of the protein's conformation has been monitored by fluorescence spectroscopy and circular dichroism (CD). Liposome aggregation and fusion were predicted by increase in turbidity and vesicle size. PLC in solution has high fluorescence and exhibit appreciable shift in its emission maxima, upon gradual change in excitation wavelength towards the red edge of the absorption band. REES fluorescence studies indicated that certain Trp fluorophores of inactive PLC are in motionally restricted compact/rigid environments in solution conformation. PLC fluorescence decreased in association with liposome and Trps loosed rigidity where liposome aggregation and fusion occurred. We argue that the structural flexibility is the cause of decrease of fluorescence, mostly to gain optimum conformation for maximum activity of the enzyme PLC. Further studies deciphered that the enzyme PLC undergoes change of conformation when mixed to LUVs prepared with specific lipids. CD data at the far-UV and near-UV regions of PLC in solution are in excellent agreement with the previous reports. CD analyses of PLC with LUVs, showed significant reduction of α-helices, increase of β-sheets; and confirmed dramatic change of orientations of Trps. In case of liposome composed of lipid raft like composition, the enzyme binds very fast, hydrolyze PC with higher rate, exhibit highest structural flexibility and promote vesicle fusion. These data strongly suggest marked differences in conformation transition induced PLC

  19. Interaction of phospholipase C with liposome: A conformation transition of the enzyme is critical and specific to liposome composition for burst hydrolysis and fusion in concert.

    PubMed

    Patra, Samir Kumar; Sengupta, Dipta; Deb, Moonmoon; Kar, Swayamsiddha; Kausar, Chahat

    2017-02-15

    Phospholipase C (PLC)(1) is known to help the pathogen B. cereus entry to the host cell and human PLC is over expressed in multiple cancers. Knowledge of dynamic activity of the enzyme PLC while in action on membrane lipids is essential and helpful to drug design and delivery. In view of this, interactions of PLC with liposome of various lipid compositions have been visualized by testing enzyme activity and microenvironments around the intrinsic fluorophores of the enzyme. Overall change of the protein's conformation has been monitored by fluorescence spectroscopy and circular dichroism (CD). Liposome aggregation and fusion were predicted by increase in turbidity and vesicle size. PLC in solution has high fluorescence and exhibit appreciable shift in its emission maxima, upon gradual change in excitation wavelength towards the red edge of the absorption band. REES fluorescence studies indicated that certain Trp fluorophores of inactive PLC are in motionally restricted compact/rigid environments in solution conformation. PLC fluorescence decreased in association with liposome and Trps loosed rigidity where liposome aggregation and fusion occurred. We argue that the structural flexibility is the cause of decrease of fluorescence, mostly to gain optimum conformation for maximum activity of the enzyme PLC. Further studies deciphered that the enzyme PLC undergoes change of conformation when mixed to LUVs prepared with specific lipids. CD data at the far-UV and near-UV regions of PLC in solution are in excellent agreement with the previous reports. CD analyses of PLC with LUVs, showed significant reduction of α-helices, increase of β-sheets; and confirmed dramatic change of orientations of Trps. In case of liposome composed of lipid raft like composition, the enzyme binds very fast, hydrolyze PC with higher rate, exhibit highest structural flexibility and promote vesicle fusion. These data strongly suggest marked differences in conformation transition induced PLC

  20. FRET microscopy demonstrates molecular association of non-specific lipid transfer protein (nsL-TP) with fatty acid oxidation enzymes in peroxisomes.

    PubMed Central

    Wouters, F S; Bastiaens, P I; Wirtz, K W; Jovin, T M

    1998-01-01

    The fate of fluorescently labeled pre-nsL-TP (Cy3-pre-nsL-TP) microinjected into BALB/c 3T3 fibroblasts was investigated by confocal laser scanning microscopy. The protein exhibited a distinct punctate fluorescence pattern and colocalized to a high degree with the immunofluorescence pattern for the peroxisomal enzyme acyl-CoA oxidase. Proteolytic removal of the C-terminal leucine of the putative peroxisomal targeting sequence (AKL) resulted in a diffuse cytosolic fluorescence. These results indicate that microinjected Cy3-pre-nsL-TP is targeted to peroxisomes. The association of nsL-TP with peroxisomal enzymes was investigated in cells by measuring fluorescence resonance energy transfer (FRET) between the microinjected Cy3-pre-nsL-TP and Cy5-labeled antibodies against the peroxisomal enzymes acyl-CoA oxidase, 3-ketoacyl-CoA thiolase, bifunctional enzyme, PMP70 and catalase. The technique of photobleaching digital imaging microscopy (pbDIM), used to quantitate the FRET efficiency on a pixel-by-pixel basis, revealed a specific association of nsL-TP with acyl-CoA oxidase, 3-ketoacyl-CoA thiolase and bifunctional enzyme in the peroxisomes. These observations were corroborated by subjecting a peroxisomal matrix protein fraction to affinity chromatography on Sepharose-immobilized pre-nsL-TP. Acyl-CoA oxidase was retained. These studies provide strong evidence for a role of nsL-TP in the regulation of peroxisomal fatty acid beta-oxidation, e.g. by facilitating the presentation of substrates and/or stabilization of the enzymes. PMID:9857175

  1. The isolation and purification of a specific "protector" protein which inhibits enzyme inactivation by a thiol/Fe(III)/O2 mixed-function oxidation system.

    PubMed

    Kim, K; Kim, I H; Lee, K Y; Rhee, S G; Stadtman, E R

    1988-04-05

    Mixed-function oxidation systems comprised of Fe3+, O2, and electron donors such as thiol compounds, ascorbate, NAD(P)H/NAD(P)H oxidase, and xanthine oxidase/hypoxanthine, catalyze the inactivation of many enzymes. This report describes the isolation and purification of a soluble protein from Saccharomyces cerevisiae, which specifically inhibits the inactivation of various enzymes by a nonenzymatic Fe3+/O2/thiol mixed-function oxidase system. When thiol is replaced with another electron donor (e.g. ascorbate), this specific protein no longer protects against iron (or copper)/O2-dependent radical-induced enzyme inactivation. Purification steps included a polyethylene glycol precipitation followed sequentially by a chromatography on DE52 and high pressure liquid chromatography on phenyl, DEAE, and gel-filtrated columns. The final gel filtration step yielded two protein peaks exhibiting protector activity and possessing a Mr of 500,000 and 90,000. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of these two fractions gave a single band at 27 kDa suggesting that these protein species simply represent different oligomeric structures. The protector protein did not possess catalase, glutathione peroxidase, superoxide dismutase, or iron chelation activities. Since the protection activity reported herein is specific for mixed-function oxidation systems containing thiols, we propose that the protector protein functions as a sulfur radical scavenger.

  2. NASA Broad Specification Fuels Combustion Technology program - Pratt and Whitney Aircraft Phase I results and status

    NASA Technical Reports Server (NTRS)

    Lohmann, R. P.; Fear, J. S.

    1982-01-01

    In connection with increases in the cost of fuels and the reduced availability of high quality petroleum crude, a modification of fuel specifications has been considered to allow acceptance of poorer quality fuels. To obtain the information upon which a selection of appropriate fuels for aircraft can be based, the Broad Specification Fuels Combustion Technology program was formulated by NASA. A description is presented of program-related investigations conducted by an American aerospace company. The specific objective of Phase I of this program has been to evaluate the impact of the use of broadened properties fuels on combustor design through comprehensive combustor rig testing. Attention is given to combustor concepts, experimental evaluation, results obtained with single stage combustors, the stage combustor concept, and the capability of a variable geometry combustor.

  3. Programmed death in a unicellular organism has species-specific fitness effects.

    PubMed

    Durand, Pierre M; Choudhury, Rajdeep; Rashidi, Armin; Michod, Richard E

    2014-02-01

    Programmed cell death (PCD) is an ancient phenomenon and its origin and maintenance in unicellular life is unclear. We report that programmed death provides differential fitness effects that are species specific in the model organism Chlamydomonas reinhardtii. Remarkably, PCD in this organism not only benefits others of the same species, but also has an inhibitory effect on the growth of other species. These data reveal that the fitness effects of PCD can depend upon genetic relatedness.

  4. [Comparative research into sensitivity and specificity of immune-enzyme analysis with chemiluminescence and colorimetric detection for detecting antigens and antibodies to avian influenza viruses and newcastle disease].

    PubMed

    Vitkova, O N; Kapustina, T P; Mikhailova, V V; Safonov, G A; Vlasova, N N; Belousova, R V

    2015-01-01

    The goal of this work was to demonstrate the results of the development of the enzyme-linked immunosorbent tests with chemiluminescence detection and colorimetric detection of specific viral antigens and antibodies for identifying the avian influenza and the Newcastle disease viruses: high sensitivity and specificity of the immuno- chemiluminescence assay, which are 10-50 times higher than those of the ELISA colorimetric method. The high effectiveness of the results and the automation of the process of laboratory testing (using a luminometer) allow these methods to be recommended for including in primary screening tests for these infectious diseases.

  5. Contribution of distinct homeodomain DNA binding specificities to Drosophila embryonic mesodermal cell-specific gene expression programs.

    PubMed

    Busser, Brian W; Gisselbrecht, Stephen S; Shokri, Leila; Tansey, Terese R; Gamble, Caitlin E; Bulyk, Martha L; Michelson, Alan M

    2013-01-01

    Homeodomain (HD) proteins are a large family of evolutionarily conserved transcription factors (TFs) having diverse developmental functions, often acting within the same cell types, yet many members of this family paradoxically recognize similar DNA sequences. Thus, with multiple family members having the potential to recognize the same DNA sequences in cis-regulatory elements, it is difficult to ascertain the role of an individual HD or a subclass of HDs in mediating a particular developmental function. To investigate this problem, we focused our studies on the Drosophila embryonic mesoderm where HD TFs are required to establish not only segmental identities (such as the Hox TFs), but also tissue and cell fate specification and differentiation (such as the NK-2 HDs, Six HDs and identity HDs (I-HDs)). Here we utilized the complete spectrum of DNA binding specificities determined by protein binding microarrays (PBMs) for a diverse collection of HDs to modify the nucleotide sequences of numerous mesodermal enhancers to be recognized by either no or a single subclass of HDs, and subsequently assayed the consequences of these changes on enhancer function in transgenic reporter assays. These studies show that individual mesodermal enhancers receive separate transcriptional input from both I-HD and Hox subclasses of HDs. In addition, we demonstrate that enhancers regulating upstream components of the mesodermal regulatory network are targeted by the Six class of HDs. Finally, we establish the necessity of NK-2 HD binding sequences to activate gene expression in multiple mesodermal tissues, supporting a potential role for the NK-2 HD TF Tinman (Tin) as a pioneer factor that cooperates with other factors to regulate cell-specific gene expression programs. Collectively, these results underscore the critical role played by HDs of multiple subclasses in inducing the unique genetic programs of individual mesodermal cells, and in coordinating the gene regulatory networks

  6. Crystal Structures of the Helicobacter pylori MTAN Enzyme Reveal Specific Interactions between S-Adenosylhomocysteine and the 5'-Alkylthio Binding Subsite

    SciTech Connect

    Mishra, Vidhi; Ronning, Donald R.

    2012-11-13

    The bacterial 5'-methylthioadenosine/S-adenosylhomocysteine nucleosidase (MTAN) enzyme is a multifunctional enzyme that catalyzes the hydrolysis of the N-ribosidic bond of at least four different adenosine-based metabolites: S-adenosylhomocysteine (SAH), 5'-methylthioadenosine (MTA), 5'-deoxyadenosine (5'-DOA), and 6-amino-6-deoxyfutalosine. These activities place the enzyme at the hub of seven fundamental bacterial metabolic pathways: S-adenosylmethionine (SAM) utilization, polyamine biosynthesis, the purine salvage pathway, the methionine salvage pathway, the SAM radical pathways, autoinducer-2 biosynthesis, and menaquinone biosynthesis. The last pathway makes MTAN essential for Helicobacter pylori viability. Although structures of various bacterial and plant MTANs have been described, the interactions between the homocysteine moiety of SAH and the 5'-alkylthiol binding site of MTAN have never been resolved. We have determined crystal structures of an inactive mutant form of H. pylori MTAN bound to MTA and SAH to 1.63 and 1.20 Å, respectively. The active form of MTAN was also crystallized in the presence of SAH, allowing the determination of the structure of a ternary enzyme–product complex resolved at 1.50 Å. These structures identify interactions between the homocysteine moiety and the 5'-alkylthiol binding site of the enzyme. This information can be leveraged for the development of species-specific MTAN inhibitors that prevent the growth of H. pylori.

  7. Screening of white-rot fungi manganese peroxidases: a comparison between the specific activities of the enzyme from different native producers

    PubMed Central

    2012-01-01

    In this study manganese peroxidase (MnP) enzymes from selected white-rot fungi were isolated and compared for potential future recombinant production. White-rot fungi were cultivated in small-scale in liquid media and a simplified process was established for the purification of extracellular enzymes. Five lignin degrading organisms were selected (Bjerkandera sp., Phanerochaete (P.) chrysosporium, Physisporinus (P.) rivulosus, Phlebia (P.) radiata and Phlebia sp. Nf b19) and studied for MnP production in small-scale. Extracellular MnP activity was followed and cultivations were harvested at proximity of the peak activity. The production of MnPs varied in different organisms but was clearly regulated by inducing liquid media components (Mn2+, veratryl alcohol and malonate). In total 8 different MnP isoforms were purified. Results of this study reinforce the conception that MnPs from distinct organisms differ substantially in their properties. Production of the extracellular enzyme in general did not reach a substantial level. This further suggests that these native producers are not suitable for industrial scale production of the enzyme. The highest specific activities were observed with MnPs from P. chrysosporium (200 U mg-1), Phlebia sp. Nf b19 (55 U mg-1) and P. rivulosus (89 U mg-1) and these MnPs are considered as the most potential candidates for further studies. The molecular weight of the purified MnPs was estimated to be between 45–50 kDa. PMID:23190610

  8. Site- and horizon-specific patterns of microbial community structure and enzyme activities in permafrost-affected soils of Greenland

    PubMed Central

    Gittel, Antje; Bárta, Jiří; Kohoutová, Iva; Schnecker, Jörg; Wild, Birgit; Čapek, Petr; Kaiser, Christina; Torsvik, Vigdis L.; Richter, Andreas; Schleper, Christa; Urich, Tim

    2014-01-01

    Permafrost-affected soils in the Northern latitudes store huge amounts of organic carbon (OC) that is prone to microbial degradation and subsequent release of greenhouse gasses to the atmosphere. In Greenland, the consequences of permafrost thaw have only recently been addressed, and predictions on its impact on the carbon budget are thus still highly uncertain. However, the fate of OC is not only determined by abiotic factors, but closely tied to microbial activity. We investigated eight soil profiles in northeast Greenland comprising two sites with typical tundra vegetation and one wet fen site. We assessed microbial community structure and diversity (SSU rRNA gene tag sequencing, quantification of bacteria, archaea and fungi), and measured hydrolytic and oxidative enzyme activities. Sampling site and thus abiotic factors had a significant impact on microbial community structure, diversity and activity, the wet fen site exhibiting higher potential enzyme activities and presumably being a hot spot for anaerobic degradation processes such as fermentation and methanogenesis. Lowest fungal to bacterial ratios were found in topsoils that had been relocated by cryoturbation (“buried topsoils”), resulting from a decrease in fungal abundance compared to recent (“unburied”) topsoils. Actinobacteria (in particular Intrasporangiaceae) accounted for a major fraction of the microbial community in buried topsoils, but were only of minor abundance in all other soil horizons. It was indicated that the distribution pattern of Actinobacteria and a variety of other bacterial classes was related to the activity of phenol oxidases and peroxidases supporting the hypothesis that bacteria might resume the role of fungi in oxidative enzyme production and degradation of phenolic and other complex substrates in these soils. Our study sheds light on the highly diverse, but poorly-studied communities in permafrost-affected soils in Greenland and their role in OC degradation. PMID

  9. Site- and horizon-specific patterns of microbial community structure and enzyme activities in permafrost-affected soils of Greenland.

    PubMed

    Gittel, Antje; Bárta, Jiří; Kohoutová, Iva; Schnecker, Jörg; Wild, Birgit; Capek, Petr; Kaiser, Christina; Torsvik, Vigdis L; Richter, Andreas; Schleper, Christa; Urich, Tim

    2014-01-01

    Permafrost-affected soils in the Northern latitudes store huge amounts of organic carbon (OC) that is prone to microbial degradation and subsequent release of greenhouse gasses to the atmosphere. In Greenland, the consequences of permafrost thaw have only recently been addressed, and predictions on its impact on the carbon budget are thus still highly uncertain. However, the fate of OC is not only determined by abiotic factors, but closely tied to microbial activity. We investigated eight soil profiles in northeast Greenland comprising two sites with typical tundra vegetation and one wet fen site. We assessed microbial community structure and diversity (SSU rRNA gene tag sequencing, quantification of bacteria, archaea and fungi), and measured hydrolytic and oxidative enzyme activities. Sampling site and thus abiotic factors had a significant impact on microbial community structure, diversity and activity, the wet fen site exhibiting higher potential enzyme activities and presumably being a hot spot for anaerobic degradation processes such as fermentation and methanogenesis. Lowest fungal to bacterial ratios were found in topsoils that had been relocated by cryoturbation ("buried topsoils"), resulting from a decrease in fungal abundance compared to recent ("unburied") topsoils. Actinobacteria (in particular Intrasporangiaceae) accounted for a major fraction of the microbial community in buried topsoils, but were only of minor abundance in all other soil horizons. It was indicated that the distribution pattern of Actinobacteria and a variety of other bacterial classes was related to the activity of phenol oxidases and peroxidases supporting the hypothesis that bacteria might resume the role of fungi in oxidative enzyme production and degradation of phenolic and other complex substrates in these soils. Our study sheds light on the highly diverse, but poorly-studied communities in permafrost-affected soils in Greenland and their role in OC degradation.

  10. Structural and functional analysis of tomato β-galactosidase 4: insight into the substrate specificity of the fruit softening-related enzyme.

    PubMed

    Eda, Masahiro; Matsumoto, Takashi; Ishimaru, Megumi; Tada, Toshiji

    2016-05-01

    Plant β-galactosidases hydrolyze cell wall β-(1,4)-galactans to play important roles in cell wall expansion and degradation, and turnover of signaling molecules, during ripening. Tomato β-galactosidase 4 (TBG4) is an enzyme responsible for fruit softening through the degradation of β-(1,4)-galactan in the pericarp cell wall. TBG4 is the only enzyme among TBGs 1-7 that belongs to the β-galactosidase/exo-β-(1,4)-galactanase subfamily. The enzyme can hydrolyze a wide range of plant-derived (1,4)- or 4-linked polysaccharides, and shows a strong ability to attack β-(1,4)-galactan. To gain structural insight into its substrate specificity, we determined crystal structures of TBG4 and its complex with β-d-galactose. TBG4 comprises a catalytic TIM barrel domain followed by three β-sandwich domains. Three aromatic residues in the catalytic site that are thought to be important for substrate specificity are conserved in GH35 β-galactosidases derived from bacteria, fungi and animals; however, the crystal structures of TBG4 revealed that the enzyme has a valine residue (V548) replacing one of the conserved aromatic residues. The V548W mutant of TBG4 showed a roughly sixfold increase in activity towards β-(1,6)-galactobiose, and ~0.6-fold activity towards β-(1,4)-galactobiose, compared with wild-type TBG4. Amino acid residues corresponding to V548 of TBG4 thus appear to determine the substrate specificities of plant β-galactosidases towards β-1,4 and β-1,6 linkages.

  11. Curve Generation for the RF Systems of the Antiproton Source Console Program Specification and Implementation

    SciTech Connect

    MacLachlan, J.A.

    1984-11-01

    The RF curves program is a PDP-11 console application to calculate the time dependence of amplitude, frequency, and phase for the RF systems of the Antiproton Source. The results of the calculation are formatted and scaled for the curve generator hardware. The user interface of the program is highly flexible with respect to the choice of parameters used to specify the desired curve. It consists of file management, plotting, editing, and hardware loading phases which are implemented as separate pages on the console display. This document provides the functional specification of the program and a discussion of the status of its implementation.

  12. SLS-SPEC-159 Cross-Program Design Specification for Natural Environments (DSNE) Revision D

    NASA Technical Reports Server (NTRS)

    Roberts, Barry C.

    2015-01-01

    This document is derived from the former National Aeronautics and Space Administration (NASA) Constellation Program (CxP) document CxP 70023, titled "The Design Specification for Natural Environments (DSNE), Revision C." The original document has been modified to represent updated Design Reference Missions (DRMs) for the NASA Exploration Systems Development (ESD) Programs. The DSNE completes environment-related specifications for architecture, system-level, and lower-tier documents by specifying the ranges of environmental conditions that must be accounted for by NASA ESD Programs. To assure clarity and consistency, and to prevent requirements documents from becoming cluttered with extensive amounts of technical material, natural environment specifications have been compiled into this document. The intent is to keep a unified specification for natural environments that each Program calls out for appropriate application. This document defines the natural environments parameter limits (maximum and minimum values, energy spectra, or precise model inputs, assumptions, model options, etc.), for all ESD Programs. These environments are developed by the NASA Marshall Space Flight Center (MSFC) Natural Environments Branch (MSFC organization code: EV44). Many of the parameter limits are based on experience with previous programs, such as the Space Shuttle Program. The parameter limits contain no margin and are meant to be evaluated individually to ensure they are reasonable (i.e., do not apply unrealistic extreme-on-extreme conditions). The natural environments specifications in this document should be accounted for by robust design of the flight vehicle and support systems. However, it is understood that in some cases the Programs will find it more effective to account for portions of the environment ranges by operational mitigation or acceptance of risk in accordance with an appropriate program risk management plan and/or hazard analysis process. The DSNE is not intended

  13. Computer Program Development Specification for IDAMST Operational Flight Programs. Addendum 3. Error Handling and Recovery Software.

    DTIC Science & Technology

    1976-11-01

    LABORATORYDT AIR FORCE SYSTEM COMMAND D I "pUNITED STATES AIR FORCE ELECTE WRIGHT-PATTERSON AFB, OHIO 45PR143903 80 4 15 0 60’ 1 FOREWORD This specification...OF FIGURES vi LIST OF TABLES vii ABBREVIATIONS viii 1.0 SCOPE 1 1.1 IDENTIFICATION 1 1.2 FUNCTIONAL SUMMARY 1 1.2.1 ORGANIZATION 1 2.0 APPLICABLE...LIST OF FIGURES FIGURE NO. TITLE Page 3.1- 1 IDAMST ERROR DETECTION MECHANISMS 6 3.1-2 EHAR PROCESSOR INTERNAL ERROR HANDLING BY

  14. Energy star product specification development framework: Using data and analysis to make program decisions

    SciTech Connect

    McWhinney, Marla; Fanara, Andrew; Clark, Robin; Hershberg, Craig; Schmeltz, Rachel; Roberson, Judy

    2003-09-12

    The Product Development Team (PD) in the US Environmental Protection Agency's ENERGY STAR Labeling Program fuels the long-term market transformation process by delivering new specifications. PD's goal is to expand the reach and visibility of ENERGY STAR as well as the market for new energy-efficient products. Since 2000, PD has launched nine new ENERGY STAR specifications and continues to evaluate new program opportunities. To evaluate the ENERGY STAR carbon savings potential for a diverse group of products, PD prepared a framework for developing new and updating existing specifications that rationalizes new product opportunities and draws upon the expertise and resources of other stakeholders, including manufacturers, utilities, environmental groups and other government agencies. By systematically reviewing the potential of proposed product areas, PD makes informed decisions as to whether or not to proceed with developing a specification. In support of this strategy, PD ensures that new product specifications are consistent with the ENERGY STAR guidelines and that these guidelines are effectively communicated to stakeholders during the product development process. To date, the framework has been successful in providing consistent guidance on collecting the necessary information on which to base sound program decisions. Through the application of this framework, PD increasingly recognizes that each industry has unique market and product characteristics that can require reconciliation with the ENERGY STAR guidelines. The new framework allows PD to identify where reconciliation is needed to justify program decisions.

  15. 2.3.7.8-Tetrachlorodibenzo-p-Dioxin Induced Immunosuppression: Its Possible Alteration by In Vivo Administration of Specific Hepatic Enzyme Inducers.

    DTIC Science & Technology

    1987-06-27

    England .2 LIST OF ACOREVIAT:CNS AND SYMBOLS TCDD =2.3.7.8-tetrachlorodibenzo-p-dioxtn TCDF = 2.3.7 8-IOetrachlo’dibenzofuran 3MC =3-methyicholanthrene...this research program was to clarify whether drugs that in vitro inhibit TCDD binding to the hepatic cytosolic Ah "rec3ptor" such as 3MC , PNF and TCDF...these enzymes has also been performed. The study of this year has been concentrated on the effects of only two inducers, that is TCDF and 3MC , since

  16. Targeted Metabolomics Connects Thioredoxin-interacting Protein (TXNIP) to Mitochondrial Fuel Selection and Regulation of Specific Oxidoreductase Enzymes in Skeletal Muscle*

    PubMed Central

    DeBalsi, Karen L.; Wong, Kari E.; Koves, Timothy R.; Slentz, Dorothy H.; Seiler, Sarah E.; Wittmann, April H.; Ilkayeva, Olga R.; Stevens, Robert D.; Perry, Christopher G. R.; Lark, Daniel S.; Hui, Simon T.; Szweda, Luke; Neufer, P. Darrell; Muoio, Deborah M.

    2014-01-01

    Thioredoxin-interacting protein (TXNIP) is an α-arrestin family member involved in redox sensing and metabolic control. Growing evidence links TXNIP to mitochondrial function, but the molecular nature of this relationship has remained poorly defined. Herein, we employed targeted metabolomics and comprehensive bioenergetic analyses to evaluate oxidative metabolism and respiratory kinetics in mouse models of total body (TKO) and skeletal muscle-specific (TXNIPSKM−/−) Txnip deficiency. Compared with littermate controls, both TKO and TXNIPSKM−/− mice had reduced exercise tolerance in association with muscle-specific impairments in substrate oxidation. Oxidative insufficiencies in TXNIP null muscles were not due to perturbations in mitochondrial mass, the electron transport chain, or emission of reactive oxygen species. Instead, metabolic profiling analyses led to the discovery that TXNIP deficiency causes marked deficits in enzymes required for catabolism of branched chain amino acids, ketones, and lactate, along with more modest reductions in enzymes of β-oxidation and the tricarboxylic acid cycle. The decrements in enzyme activity were accompanied by comparable deficits in protein abundance without changes in mRNA expression, implying dysregulation of protein synthesis or stability. Considering that TXNIP expression increases in response to starvation, diabetes, and exercise, these findings point to a novel role for TXNIP in coordinating mitochondrial fuel switching in response to nutrient availability. PMID:24482226

  17. Genomic-Based Restriction Enzyme Selection for Specific Detection of Piscirickettsia salmonis by 16S rDNA PCR-RFLP

    PubMed Central

    Mandakovic, Dinka; Glasner, Benjamín; Maldonado, Jonathan; Aravena, Pamela; González, Mauricio; Cambiazo, Verónica; Pulgar, Rodrigo

    2016-01-01

    The gram negative facultative bacterium P. salmonis is the etiological agent of Salmonid Rickettsial Septicaemia (SRS), a severe disease that causes important economic losses in the global salmon farmer industry. Despite efforts to control this disease, the high frequency of new epizootic events indicate that the vaccine and antibiotics treatments have limited effectiveness, therefore the preventive and diagnostic approaches must be improved. A comparison of several methodologies for SRS diagnostic indicate differences in their specificity and its capacity to detect other bacteria coexisting with P. salmonis in culture media (contamination) and fish samples (coinfection), aspects relevant for research, vaccine development and clinical diagnostic. By computer-simulation analyses, we identified a group of restriction enzymes that generate unique P. salmonis 16S rDNA band patterns, distinguishable from all other bacteria. From this information, we designed and developed a PCR-RFLP (Polymerase Chain Reaction—Restriction Fragment Length Polymorphism) assay, which was validated using 16S rDNA universal primers and restriction enzyme PmaCI for the amplification and digestion, respectively. Experimental validation was performed by comparing the restriction pattern of P. salmonis with the restriction patterns generated by bacteria that cohabit with P. salmonis (fish bacterial isolates and culture media contaminants). Our results indicate that the restriction enzyme selection pipeline was suitable to design a more specific, sensible, faster and cheaper assay than the currently used P. salmonis detection methodologies. PMID:27242682

  18. Selective restoration of male fertility in mice lacking angiotensin-converting enzymes by sperm-specific expression of the testicular isozyme.

    PubMed Central

    Ramaraj, P; Kessler, S P; Colmenares, C; Sen, G C

    1998-01-01

    Although angiotensin-converting enzyme (ACE) has been studied primarily in the context of its role in blood pressure regulation, this widely distributed enzyme has many other physiological functions. The ACE gene encodes two isozymes. The somatic isozyme is expressed in many tissues, including vascular endothelial cells, renal epithelial cells, and testicular Leydig cells, whereas the testicular or germinal angiotensin-converting enzyme is expressed only in sperm. The ACE gene knockout mice lack both isozymes and they exhibit low blood pressure, kidney dysfunctions, and male infertility. Here, we report the use of a sperm-specific promoter and interbreeding of transgenic and gene knockout mice for generating a mouse strain that expressed ACE only in sperm. The experimental mice maintained the kidney defects of ACE-/- mice, but unlike the knockout strain, the males were fertile. Thus, we established that the role of ACE in male fertility is completely dependent on its exclusive expression in sperm. Our study clearly demonstrated how transgenic and knockout techniques can be combined for ascribing a specific physiological function to the expression of a multifunctional protein in a given tissue. PMID:9664078

  19. Genomic-Based Restriction Enzyme Selection for Specific Detection of Piscirickettsia salmonis by 16S rDNA PCR-RFLP.

    PubMed

    Mandakovic, Dinka; Glasner, Benjamín; Maldonado, Jonathan; Aravena, Pamela; González, Mauricio; Cambiazo, Verónica; Pulgar, Rodrigo

    2016-01-01

    The gram negative facultative bacterium P. salmonis is the etiological agent of Salmonid Rickettsial Septicaemia (SRS), a severe disease that causes important economic losses in the global salmon farmer industry. Despite efforts to control this disease, the high frequency of new epizootic events indicate that the vaccine and antibiotics treatments have limited effectiveness, therefore the preventive and diagnostic approaches must be improved. A comparison of several methodologies for SRS diagnostic indicate differences in their specificity and its capacity to detect other bacteria coexisting with P. salmonis in culture media (contamination) and fish samples (coinfection), aspects relevant for research, vaccine development and clinical diagnostic. By computer-simulation analyses, we identified a group of restriction enzymes that generate unique P. salmonis 16S rDNA band patterns, distinguishable from all other bacteria. From this information, we designed and developed a PCR-RFLP (Polymerase Chain Reaction-Restriction Fragment Length Polymorphism) assay, which was validated using 16S rDNA universal primers and restriction enzyme PmaCI for the amplification and digestion, respectively. Experimental validation was performed by comparing the restriction pattern of P. salmonis with the restriction patterns generated by bacteria that cohabit with P. salmonis (fish bacterial isolates and culture media contaminants). Our results indicate that the restriction enzyme selection pipeline was suitable to design a more specific, sensible, faster and cheaper assay than the currently used P. salmonis detection methodologies.

  20. Computer Program Development Specification for IDAMST Operational Test Program, Software Type B5. Addendum 4.

    DTIC Science & Technology

    1976-07-30

    UNCLASSIFIED AFAL-TR 76-209ADO- NL " NONhEI/ENl MENhhE 0EIt lllEll/lllllEI AFAL-TR-76-209, Addendum #4 SPECIFICATION NUMBER SD 2043 A b)E r4DOKCODEMIENT PART 1 ...JU/ 7 I/n ¢-2.3. DTIC • E APPROVED FOR PUBLIC RELEASE: DISTRIBUTION UNLIMITED O඘ I i5 06 AIR FORCE AVIONICS LABORATORY AFAL/AAA- 1 WRIGHT-PATTERSON...AFB, OHIO 45433 TABLE OF CONTENTS PARAGRAPH PAGE 1.0 SCOPE 1 1.1 Identification 1 1.2 Functional Summary 1 2.0 APPLICABLE DOCUMENTS 1 3.0 REQUIREMENTS

  1. Global Gene Expression Profiling of Endothelium Exposed to Heme Reveals an Organ-Specific Induction of Cytoprotective Enzymes in Sickle Cell Disease

    PubMed Central

    Yu, Tianwei; Li, Yuhua; Adisa, Olufolake; Mosunjac, Mario; Ofori-Acquah, Solomon F.

    2011-01-01

    Background Sickle cell disease (SCD) is characterized by hemolysis, vaso-occlusion and ischemia reperfusion injury. These events cause endothelial dysfunction and vasculopathies in multiple systems. However, the lack of atherosclerotic lesions has led to the idea that there are adaptive mechanisms that protect the endothelium from major vascular insults in SCD patients. The molecular bases for this phenomenon are poorly defined. This study was designed to identify the global profile of genes induced by heme in the endothelium, and assess expression of the heme-inducible cytoprotective enzymes in major organs impacted by SCD. Methods and Findings Total RNA isolated from heme-treated endothelial monolayers was screened with the Affymetrix U133 Plus 2.0 chip, and the microarray data analyzed using multiple bioinformatics software. Hierarchical cluster analysis of significantly differentially expressed genes successfully segregated heme and vehicle-treated endothelium. Validation studies showed that the induction of cytoprotective enzymes by heme was influenced by the origin of endothelial cells, the duration of treatment, as well as the magnitude of induction of individual enzymes. In agreement with these heterogeneities, we found that induction of two major Nrf2-regulated cytoprotective enzymes, heme oxygenase-1 and NAD(P)H:quinone oxidoreductase-1 is organ-specific in two transgenic mouse models of SCD. This data was confirmed in the endothelium of post-mortem lung tissues of SCD patients. Conclusions Individual organ systems induce unique profiles of cytoprotective enzymes to neutralize heme in SCD. Understanding this heterogeneity may help to develop effective therapies to manage vasculopathies of individual systems. PMID:21483798

  2. Development of an enzyme-linked immunosorbent assay method specific for the detection of G-group aflatoxins

    Technology Transfer Automated Retrieval System (TEKTRAN)

    To detect and monitor G-group aflatoxins in agricultural products, we generated class-specific monoclonal antibodies that specifically recognized aflatoxins G1 and G2. Of the final three positive and stable hybridomas obtained, hybridoma 2G6 produced a monoclonal antibody that did not cross-react wi...

  3. Heterologous expression, purification, crystallization and preliminary X-ray analysis of raucaffricine glucosidase, a plant enzyme specifically involved in Rauvolfia alkaloid biosynthesis

    SciTech Connect

    Ruppert, Martin; Panjikar, Santosh; Barleben, Leif; Stöckigt, Joachim

    2006-03-01

    Raucaffricine glucosidase, an enzyme involved in the biosynthesis of monoterpenoid indole alkaloids in the plant Rauvolfia serpentina, was crystallized by the hanging-drop vapour-diffusion method using PEG4000 as precipitant. The crystals diffract to 2.3 Å resolution and belong to space group I222. Raucaffricine glucosidase (RG) is an enzyme that is specifically involved in the biosynthesis of indole alkaloids from the plant Rauvolfia serpentina. After heterologous expression in Escherichia coli cells, crystals of RG were obtained by the hanging-drop vapour-diffusion technique at 293 K with 0.3 M ammonium sulfate, 0.1 M sodium acetate pH 4.6 buffer and 11% PEG 4000 as precipitant. Crystals belong to space group I222 and diffract to 2.30 Å, with unit-cell parameters a = 102.8, b = 127.3, c = 215.8 Å.

  4. PpASCL, a moss ortholog of anther-specific chalcone synthase-like enzymes, is a hydroxyalkylpyrone synthase involved in an evolutionarily conserved sporopollenin biosynthesis pathway.

    PubMed

    Colpitts, Che C; Kim, Sung Soo; Posehn, Sarah E; Jepson, Christina; Kim, Sun Young; Wiedemann, Gertrud; Reski, Ralf; Wee, Andrew G H; Douglas, Carl J; Suh, Dae-Yeon

    2011-12-01

    Sporopollenin is the main constituent of the exine layer of spore and pollen walls. Recently, several Arabidopsis genes, including polyketide synthase A (PKSA), which encodes an anther-specific chalcone synthase-like enzyme (ASCL), have been shown to be involved in sporopollenin biosynthesis. The genome of the moss Physcomitrella patens contains putative orthologs of the Arabidopsis sporopollenin biosynthesis genes. We analyzed available P.patens expressed sequence tag (EST) data for putative moss orthologs of the Arabidopsis genes of sporopollenin biosynthesis and studied the enzymatic properties and reaction mechanism of recombinant PpASCL, the P.patens ortholog of Arabidopsis PKSA. We also generated structure models of PpASCL and Arabidopsis PKSA to study their substrate specificity. Physcomitrella patens orthologs of Arabidopsis genes for sporopollenin biosynthesis were found to be expressed in the sporophyte generation. Similarly to Arabidopsis PKSA, PpASCL condenses hydroxy fatty acyl-CoA esters with malonyl-CoA and produces hydroxyalkyl α-pyrones that probably serve as building blocks of sporopollenin. The ASCL-specific set of Gly-Gly-Ala residues predicted by the models to be located at the floor of the putative active site is proposed to serve as the opening of an acyl-binding tunnel in ASCL. These results suggest that ASCL functions together with other sporophyte-specific enzymes to provide polyhydroxylated precursors of sporopollenin in a pathway common to land plants.

  5. Synergistic binding of transcription factors to cell-specific enhancers programs motor neuron identity

    PubMed Central

    Mazzoni, Esteban O; Mahony, Shaun; Closser, Michael; Morrison, Carolyn A; Nedelec, Stephane; Williams, Damian J; An, Disi; Gifford, David K; Wichterle, Hynek

    2013-01-01

    Efficient transcriptional programming promises to open new frontiers in regenerative medicine. However, mechanisms by which programming factors transform cell fate are unknown, preventing more rational selection of factors to generate desirable cell types. Three transcription factors, Ngn2, Isl1 and Lhx3, were sufficient to program rapidly and efficiently spinal motor neuron identity when expressed in differentiating mouse embryonic stem cells. Replacement of Lhx3 by Phox2a led to specification of cranial, rather than spinal, motor neurons. Chromatin immunoprecipitation–sequencing analysis of Isl1, Lhx3 and Phox2a binding sites revealed that the two cell fates were programmed by the recruitment of Isl1-Lhx3 and Isl1-Phox2a complexes to distinct genomic locations characterized by a unique grammar of homeodomain binding motifs. Our findings suggest that synergistic interactions among transcription factors determine the specificity of their recruitment to cell type–specific binding sites and illustrate how a single transcription factor can be repurposed to program different cell types. PMID:23872598

  6. Crystal Structures of Physcomitrella patens AOC1 and AOC2: Insights into the Enzyme Mechanism and Differences in Substrate Specificity1[W][OA

    PubMed Central

    Neumann, Piotr; Brodhun, Florian; Sauer, Kristin; Herrfurth, Cornelia; Hamberg, Mats; Brinkmann, Jens; Scholz, Julia; Dickmanns, Achim; Feussner, Ivo; Ficner, Ralf

    2012-01-01

    In plants, oxylipins regulate developmental processes and defense responses. The first specific step in the biosynthesis of the cyclopentanone class of oxylipins is catalyzed by allene oxide cyclase (AOC) that forms cis(+)-12-oxo-phytodienoic acid. The moss Physcomitrella patens has two AOCs (PpAOC1 and PpAOC2) with different substrate specificities for C18- and C20-derived substrates, respectively. To better understand AOC’s catalytic mechanism and to elucidate the structural properties that explain the differences in substrate specificity, we solved and analyzed the crystal structures of 36 monomers of both apo and ligand complexes of PpAOC1 and PpAOC2. From these data, we propose the following intermediates in AOC catalysis: (1) a resting state of the apo enzyme with a closed conformation, (2) a first shallow binding mode, followed by (3) a tight binding of the substrate accompanied by conformational changes in the binding pocket, and (4) initiation of the catalytic cycle by opening of the epoxide ring. As expected, the substrate dihydro analog cis-12,13S-epoxy-9Z,15Z-octadecadienoic acid did not cyclize in the presence of PpAOC1; however, when bound to the enzyme, it underwent isomerization into the corresponding trans-epoxide. By comparing complex structures of the C18 substrate analog with in silico modeling of the C20 substrate analog bound to the enzyme allowed us to identify three major molecular determinants responsible for the different substrate specificities (i.e. larger active site diameter, an elongated cavity of PpAOC2, and two nonidentical residues at the entrance of the active site). PMID:22987885

  7. DNA Polymerases of Low-GC Gram-Positive Eubacteria: Identification of the Replication-Specific Enzyme Encoded by dnaE

    PubMed Central

    Barnes, Marjorie H.; Miller, Shelley D.; Brown, Neal C.

    2002-01-01

    dnaE, the gene encoding one of the two replication-specific DNA polymerases (Pols) of low-GC-content gram-positive bacteria (E. Dervyn et al., Science 294:1716-1719, 2001; R. Inoue et al., Mol. Genet. Genomics 266:564-571, 2001), was cloned from Bacillus subtilis, a model low-GC gram-positive organism. The gene was overexpressed in Escherichia coli. The purified recombinant product displayed inhibitor responses and physical, catalytic, and antigenic properties indistinguishable from those of the low-GC gram-positive-organism-specific enzyme previously named DNA Pol II after the polB-encoded DNA Pol II of E. coli. Whereas a polB-like gene is absent from low-GC gram-positive genomes and whereas the low-GC gram-positive DNA Pol II strongly conserves a dnaE-like, Pol III primary structure, it is proposed that it be renamed DNA polymerase III E (Pol III E) to accurately reflect its replicative function and its origin from dnaE. It is also proposed that DNA Pol III, the other replication-specific Pol of low-GC gram-positive organisms, be renamed DNA polymerase III C (Pol III C) to denote its origin from polC. By this revised nomenclature, the DNA Pols that are expressed constitutively in low-GC gram-positive bacteria would include DNA Pol I, the dispensable repair enzyme encoded by polA, and the two essential, replication-specific enzymes Pol III C and Pol III E, encoded, respectively, by polC and dnaE. PMID:12081953

  8. 24 CFR 200.936 - Supplementary specific procedural requirements under HUD building products certification program...

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ... 24 Housing and Urban Development 2 2012-04-01 2012-04-01 false Supplementary specific procedural... stoves. 200.936 Section 200.936 Housing and Urban Development Regulations Relating to Housing and Urban... HOUSING AND URBAN DEVELOPMENT GENERAL INTRODUCTION TO FHA PROGRAMS Minimum Property Standards §...

  9. 42 CFR 457.1170 - Program specific review process: Continuation of enrollment.

    Code of Federal Regulations, 2010 CFR

    2010-10-01

    ... 42 Public Health 4 2010-10-01 2010-10-01 false Program specific review process: Continuation of... review process: Continuation of enrollment. A State must ensure the opportunity for continuation of... decision to disenroll for failure to pay cost sharing....

  10. 43 CFR 1821.13 - What if the specific program regulations conflict with these regulations?

    Code of Federal Regulations, 2014 CFR

    2014-10-01

    ... conflict with these regulations? 1821.13 Section 1821.13 Public Lands: Interior Regulations Relating to Public Lands (Continued) BUREAU OF LAND MANAGEMENT, DEPARTMENT OF THE INTERIOR GENERAL MANAGEMENT (1000) APPLICATION PROCEDURES General Information § 1821.13 What if the specific program regulations conflict...

  11. 42 CFR 457.1140 - Program specific review process: Core elements of review.

    Code of Federal Regulations, 2010 CFR

    2010-10-01

    ... 42 Public Health 4 2010-10-01 2010-10-01 false Program specific review process: Core elements of... review process: Core elements of review. In adopting the procedures for review of matters described in... decisions are written; and (d) Applicants and enrollees have an opportunity to— (1) Represent themselves...

  12. 42 CFR 457.1160 - Program specific review process: Time frames.

    Code of Federal Regulations, 2011 CFR

    2011-10-01

    ... 42 Public Health 4 2011-10-01 2011-10-01 false Program specific review process: Time frames. 457... process: Time frames. (a) Eligibility or enrollment matter. A State must complete the review of a matter described in § 457.1130(a) within a reasonable amount of time. In setting time frames, the State...

  13. 42 CFR 457.1140 - Program specific review process: Core elements of review.

    Code of Federal Regulations, 2013 CFR

    2013-10-01

    ... 42 Public Health 4 2013-10-01 2013-10-01 false Program specific review process: Core elements of review. 457.1140 Section 457.1140 Public Health CENTERS FOR MEDICARE & MEDICAID SERVICES, DEPARTMENT OF... review process: Core elements of review. In adopting the procedures for review of matters described...

  14. 42 CFR 457.1140 - Program specific review process: Core elements of review.

    Code of Federal Regulations, 2011 CFR

    2011-10-01

    ... 42 Public Health 4 2011-10-01 2011-10-01 false Program specific review process: Core elements of review. 457.1140 Section 457.1140 Public Health CENTERS FOR MEDICARE & MEDICAID SERVICES, DEPARTMENT OF... review process: Core elements of review. In adopting the procedures for review of matters described...

  15. 42 CFR 457.1130 - Program specific review process: Matters subject to review.

    Code of Federal Regulations, 2010 CFR

    2010-10-01

    ... review process: Matters subject to review. (a) Eligibility or enrollment matter. A State must ensure that... services matter. A State must ensure that an enrollee has an opportunity for external review of a— (1... 42 Public Health 4 2010-10-01 2010-10-01 false Program specific review process: Matters subject...

  16. Exceptional Children Conference Papers: Specific Subject Programs for EMRs and TMRs.

    ERIC Educational Resources Information Center

    Council for Exceptional Children, Arlington, VA.

    Eight papers focus upon specific subject programs for educable and trainable mentally retarded (EMR and TMR) students. Three of the papers, concerning driver education and traffic safety education for EMR students, cover driver education guidelines and materials developed in a Michigan state institute involving teachers of EMR and teachers of…

  17. Structure of ALD1, a plant-specific homologue of the universal diaminopimelate aminotransferase enzyme of lysine biosynthesis.

    PubMed

    Sobolev, Vladimir; Edelman, Marvin; Dym, Orly; Unger, Tamar; Albeck, Shira; Kirma, Menny; Galili, Gad

    2013-02-01

    Diaminopimelate aminotransferase (DAP-AT) is an enzyme in the lysine-biosynthesis pathway. Conversely, ALD1, a close homologue of DAP-AT in plants, uses lysine as a substrate in vitro. Both proteins require pyridoxal-5'-phosphate (PLP) for their activity. The structure of ALD1 from the flowering plant Arabidopsis thaliana (AtALD1) was solved at a resolution of 2.3 Å. Comparison of AtALD1 with the previously solved structure of A. thaliana DAP-AT (AtDAP-AT) revealed similar interactions with PLP despite sequence differences within the PLP-binding site. However, sequence differences between the binding site of AtDAP-AT for malate, a purported mimic of substrate binding, and the corresponding site in AtALD1 led to different interactions. This suggests that either the substrate itself, or the substrate-binding mode, differs in the two proteins, supporting the known in vitro findings.

  18. Atmospheric, Magnetospheric and Plasmas in space (AMPS) spacelab payload definition study. Volume 4. Part 1, AMPS program specification

    NASA Technical Reports Server (NTRS)

    Keeley, J. T.

    1976-01-01

    The AMPS Program Specification delineates the AMPS Program requirements consistent with the resources defined in the AMPS Project Plan. All subsidiary specifications and requirements shall conform to the requirements presented. The requirements hierarchy for the AMPS program is illustrated. A brief description of each of the requirements documents and their intended use is provided.

  19. Protocatechuate 3,4-dioxygenase: a wide substrate specificity enzyme isolated from Stenotrophomonas maltophilia KB2 as a useful tool in aromatic acid biodegradation.

    PubMed

    Guzik, Urszula; Hupert-Kocurek, Katarzyna; Sitnik, Małgorzata; Wojcieszyńska, Danuta

    2014-01-01

    Protocatechuate 3,4-dioxygenases (P34Os) catalyze the reaction of the ring cleavage of aromatic acid derivatives. It is a key reaction in many xenobiotic metabolic pathways. P34Os characterize narrow substrate specificity. This property is an unfavorable feature in the biodegradation process because one type of pollution is rarely present in the environment. Thus, the following study aimed at the characterization of a P34O from Stenotrophomonas maltophilia KB2, being able to utilize a wide spectrum of aromatic carboxylic acids. A total of 3 mM vanillic acid and 4-hydroxybenzoate were completely degraded during 8 and 4.5 h, respectively. When cells of strain KB2 were grown on 9 mM 4-hydroxybenzoate, P34O was induced. Biochemical analysis revealed that the examined enzyme was similar to other known P34Os, but showed untypical wide substrate specificity. A high activity of P34O against 2,4- and 3,5-dihydroxybenzoate was observed. As these substrates do not possess ortho configuration hydroxyl groups, it is postulated that their cleavage could be connected with their monodentate binding of substrate to the active site. Since this enzyme characterizes untypical wide substrate specificity it makes it a useful tool in applications for environmental clean-up purposes.

  20. Enzyme-labeled Antigen Method: Development and Application of the Novel Approach for Identifying Plasma Cells Locally Producing Disease-specific Antibodies in Inflammatory Lesions

    PubMed Central

    Mizutani, Yasuyoshi; Shiogama, Kazuya; Onouchi, Takanori; Sakurai, Kouhei; Inada, Ken-ichi; Tsutsumi, Yutaka

    2016-01-01

    In chronic inflammatory lesions of autoimmune and infectious diseases, plasma cells are frequently observed. Antigens recognized by antibodies produced by the plasma cells mostly remain unclear. A new technique identifying these corresponding antigens may give us a breakthrough for understanding the disease from a pathophysiological viewpoint, simply because the immunocytes are seen within the lesion. We have developed an enzyme-labeled antigen method for microscopic identification of the antigen recognized by specific antibodies locally produced in plasma cells in inflammatory lesions. Firstly, target biotinylated antigens were constructed by the wheat germ cell-free protein synthesis system or through chemical biotinylation. Next, proteins reactive to antibodies in tissue extracts were screened and antibody titers were evaluated by the AlphaScreen method. Finally, with the enzyme-labeled antigen method using the biotinylated antigens as probes, plasma cells producing specific antibodies were microscopically localized in fixed frozen sections. Our novel approach visualized tissue plasma cells that produced 1) autoantibodies in rheumatoid arthritis, 2) antibodies against major antigens of Porphyromonas gingivalis in periodontitis or radicular cyst, and 3) antibodies against a carbohydrate antigen, Strep A, of Streptococcus pyogenes in recurrent tonsillitis. Evaluation of local specific antibody responses expectedly contributes to clarifying previously unknown processes in inflammatory disorders. PMID:27006517

  1. Development of a fibrinogen-specific sandwich enzyme-linked immunosorbent assay microarray assay for distinguishing between blood plasma and serum samples.

    PubMed

    Gonzalez, Rachel M; Zhang, Qibin; Zangar, Richard C; Smith, Richard D; Metz, Thomas O

    2011-07-01

    We have developed a fibrinogen-specific sandwich enzyme-linked immunosorbent assay (ELISA) microarray assay for use in qualitatively distinguishing between blood plasma and serum samples. Three capture antibodies (49D2, HPA001900, and F8512) were evaluated in conjunction with 1D6 as the detection antibody. The data show that 49D2 and (to a lesser extent) F8512 successfully identify previously unknown plasma and serum samples based on approximately a 28-fold difference in signal intensity between the sample types. This assay has utility in rapidly identifying previously archived clinical samples with incomplete annotation in a high-throughput manner prior to proteomic analyses.

  2. Compound- and enzyme-specific phosphodiester hydrolysis mechanisms revealed by δ18O of dissolved inorganic phosphate: Implications for marine P cycling

    NASA Astrophysics Data System (ADS)

    Liang, Yuhong; Blake, Ruth E.

    2009-07-01

    We have studied the oxygen isotope signature of inorganic phosphate (P i) generated by hydrolysis of nucleic acid phosphodiester (P-diester) compounds by cell-free enzymes (Deoxyribonuclease 1, Phosphodiesterase 1, Alkaline phosphatase) and microbial cultures at natural isotopic abundances. We demonstrate that the diesterase-catalyzed hydrolytic step leads to incorporation of at least one water O into released P i for a total of two O atoms from water incorporated into P i released from P-diesters. In the presence of Phosphodiesterase 1, 16O is preferentially incorporated into nucleotides released from DNA; whereas 18O is preferentially incorporated into nucleotides released from RNA. A strong consistency between predicted O-isotope regeneration signatures based on results of cell-free enzyme experiments and measured isotopic signatures from independent experiments with E. coli cultures was observed and confirms proposed models for phosphoester hydrolysis. Results from these studies made at natural 18O abundance levels provide a new tool, enzyme-specific O-isotope fractionation, for investigations of organophosphate metabolism and phosphorus cycling pathways in natural aquatic systems.

  3. A pH/enzyme-responsive polymer film consisting of Eudragit FS 30 D and arabinoxylane as a potential material formulation for colon-specific drug delivery system.

    PubMed

    Rabito, Mirela Fulgencio; Reis, Adriano Valim; Freitas, Adonilson dos Reis; Tambourgi, Elias Basile; Cavalcanti, Osvaldo Albuquerque

    2012-01-01

    Polymer film based on pH-dependent Eudragit FS 30 D acrylic polymer in association with arabinoxylane, a polysaccharide issued from gum psyllium, was produced by way of solvent casting. Physical-chemical characterization of the polymer film samples was performed by means of thermogravimetry (TGA), differential scanning calorimetry (DSC), and scanning electron microscopy (SEM). Furthermore, water-equilibrium swelling index (I(s)) and weight loss of the films in KCl buffer solution of pH 1.2, in KH(2)PO(4) buffer solution of pH 5.0, or in KH(2)PO(4) buffer solution of pH 5.0 consisting of 4% enzyme Pectinex 3X-L (w/v) were also carried out for the film characterization. No chemical interactions between the Eudragit FS 30 D and the arabinoxylane polymer chains were evidenced, thus suggesting that the film-forming polymer structure was obtained from a physical mixture of both polymers. The arabinoxylane-loader films showed a more pronounced weight loss after their immersion in buffer solution containing enzyme Pectinex 3X-L. The introduction of the arabinoxylane makes the film more susceptible to undergo an enzymatic degradation. This meant that the enzyme-dependent propriety issued from the arabinoxylane has been imprinted into the film formulation. This type of polymer film is an interesting system for applications in colon-specific drug delivery system.

  4. Evaluation of enzyme-linked immunosorbent assays for detection of Mycoplasma bovis-Specific antibody in bison sera

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Mycoplasma bovis has recently emerged as a significant and costly infectious disease problem in bison. This report demonstrates that ELISAs for detection of M. bovis-specific antibody in cattle are not optimal for identification of seropositive bison. An ELISA optimized for use with bison sera is ...

  5. The Importance of a Role-Specific, In-Hospital Ward Clerk Education Program.

    PubMed

    Kennedy, Maggie

    2016-01-01

    Ward clerks are essential members of the healthcare team, providing administrative and organizational support to acute care units and clinics. This role influences such matters as nurses' direct patient-care time, timeliness of patient discharges, and patient safety. To support ward clerks in the varying responsibilities and complex scope of this role, a formal orientation and ongoing education program is imperative. Whereas corporate orientation informs new employees of overall organizational processes, a ward clerk-specific workplace education program prepares individuals for the demands of the position, ultimately supporting the healthcare team and patient safety.

  6. Label-Free and Enzyme-Free Homogeneous Electrochemical Biosensing Strategy Based on Hybridization Chain Reaction: A Facile, Sensitive, and Highly Specific MicroRNA Assay.

    PubMed

    Hou, Ting; Li, Wei; Liu, Xiaojuan; Li, Feng

    2015-11-17

    Homogenous electrochemical biosensing strategies have attracted substantial attention, because of their advantages of being immobilization-free and having rapid response and improved recognition efficiency, compared to heterogeneous biosensors; however, the high cost of labeling and the strict reaction conditions of tool enzymes associated with current homogeneous electrochemical methods limit their potential applications. To address these issues, herein we reported, for the first time, a simple label-free and enzyme-free homogeneous electrochemical strategy based on hybridization chain reaction (HCR) for sensitive and highly specific detection of microRNA (miRNA). The target miRNA triggers the HCR of two species of metastable DNA hairpin probes, resulting in the formation of multiple G-quadruplex-incorporated long duplex DNA chains. Thus, with the electrochemical indicator Methylene Blue (MB) selectively intercalated into the duplex DNA chain and the multiple G-quadruplexes, a significant electrochemical signal drop is observed, which is dependent on the concentration of the target miRNA. Thus, using this "signal-off" mode, a simple, label-free and enzyme-free homogeneous electrochemical strategy for sensitive miRNA assay is readily realized. This strategy also exhibits excellent selectivity to distinguish even single-base mismatched miRNA. Furthermore, this method also exhibits additional advantages of simplicity and low cost, since both expensive labeling and sophisticated probe immobilization processes are avoided. Therefore, the as-proposed label-free and enzyme-free homogeneous electrochemical strategy may become an alternative method for simple, sensitive, and selective miRNA detection, and it has great potential to be applied in miRNA-related clinical diagnostics and biochemical research.

  7. Siddhartha: Domain-specific unit test generation for "low-testibility" programs

    NASA Astrophysics Data System (ADS)

    Reyes, Arthur Alexander

    This Dissertation validates the hypothesis that domain-specific language (DSL) methodology can provide essential automation support for specification-based testing (SBT) of computer program units expressed in difficult-to-test (i.e., "low-testability"), domain-specific design styles. This Dissertation presents Siddhartha, an extension to DSL methodology for development of program synthesizers to support SBT methods in novel application domains. Synthesizers map formal test data specifications (TestSpecs) into unit test driver procedures (Drivers). Both TestSpecs and Driver reference designs are represented via DSLs. Synthesizer development is iterative and example-driven. A Domain Designer applying the Siddhartha methodology in a novel application domain first selects a collection of general, example TestSpecs, then manually codes a collection of corresponding Drivers. Each Driver is expressed in a different reference design that specifically accommodates difficult-to-test, domain-specific program unit under test (UUT) design styles. After selecting the most promising Driver reference design, the set of (TestSpec, Driver) pairs become test cases for the synthesizer under development. The Domain Designer then designs a TestSpec→Driver translation function. The translation function maps TestSpecs to Driver kernels in the selected reference design. The translation function design simplifies DSL development by modularizing both TestSpec and Driver reference design DSLs into syntactic productions. This effectively optimizes DSL representations to support efficient TestSpec→Driver synthesis. This Dissertation validates the hypothesis by answering the question "What are the relative costs and benefits of applying DSL methods to generate requirements-based and regression Drivers for a flight control system expressed in Ada?" Costs and benefits are measured with respect to manually coded Drivers, which are arguably equivalent to Drivers generated by a general

  8. Correlation between antibodies to bisphenol A, its target enzyme protein disulfide isomerase and antibodies to neuron‐specific antigens

    PubMed Central

    Vojdani, Aristo

    2016-01-01

    Abstract Evidence continues to increase linking autoimmunity and other complex diseases to the chemicals commonly found in our environment. Bisphenol A (BPA) is a synthetic monomer used widely in many forms, from food containers to toys, medical products and many others. The potential for BPA to participate as a triggering agent for autoimmune diseases is likely due to its known immunological influences. The goal of this research was to determine if immune reactivity to BPA has any correlation with neurological antibodies. BPA binds to a target enzyme called protein disulfide isomerase (PDI). Myelin basic protein (MBP) and myelin oligodendrocyte glycoprotein (MOG) are neuronal antigens that are target sites for neuroinflammation and neuroautoimmunity. We determined the co‐occurrence of anti‐MBP and anti‐MOG antibodies with antibodies made against BPA bound to human serum albumin in 100 healthy human subjects. Correlation between BPA to PDI, BPA to MOG, BPA to MBP, PDI to MBP and PDI to MOG were all highly statistically significant (P < 0.0001). The outcome of our study suggests that immune reactivity to BPA‐human serum albumin and PDI has a high degree of statistical significance with substantial correlation with both MBP and MOG antibody levels. This suggests that BPA may be a trigger for the production of antibodies against PDI, MBP and MOG. Immune reactivity to BPA bound to human tissue proteins may be a contributing factor to neurological autoimmune disorders. Further research is needed to determine the exact relationship of these antibodies with neuroautoimmunities. Copyright © 2016 The Authors Journal of Applied Toxicology Published by John Wiley & Sons Ltd. PMID:27610592

  9. Indirect protein quantification of drug-transforming enzymes using peptide group-specific immunoaffinity enrichment and mass spectrometry

    PubMed Central

    Weiß, Frederik; Schnabel, Anke; Planatscher, Hannes; van den Berg, Bart H. J.; Serschnitzki, Bettina; Nuessler, Andreas K.; Thasler, Wolfgang E.; Weiss, Thomas S.; Reuss, Matthias; Stoll, Dieter; Templin, Markus F.; Joos, Thomas O.; Marcus, Katrin; Poetz, Oliver

    2015-01-01

    Immunoaffinity enrichment of proteotypic peptides, coupled with selected reaction monitoring, enables indirect protein quantification. However the lack of suitable antibodies limits its widespread application. We developed a method in which multi-specific antibodies are used to enrich groups of peptides, thus facilitating multiplexed quantitative protein assays. We tested this strategy in a pharmacokinetic experiment by targeting a group of homologous drug transforming proteins in human hepatocytes. Our results indicate the generic applicability of this method to any biological system. PMID:25737130

  10. Adult stem cells in the small intestine are intrinsically programmed with their location-specific function.

    PubMed

    Middendorp, Sabine; Schneeberger, Kerstin; Wiegerinck, Caroline L; Mokry, Michal; Akkerman, Ronald D L; van Wijngaarden, Simone; Clevers, Hans; Nieuwenhuis, Edward E S

    2014-05-01

    Differentiation and specialization of epithelial cells in the small intestine are regulated in two ways. First, there is differentiation along the crypt-villus axis of the intestinal stem cells into absorptive enterocytes, Paneth, goblet, tuft, enteroendocrine, or M cells, which is mainly regulated by WNT. Second, there is specialization along the cephalocaudal axis with different absorptive and digestive functions in duodenum, jejunum, and ileum that is controlled by several transcription factors such as GATA4. However, so far it is unknown whether location-specific functional properties are intrinsically programmed within stem cells or if continuous signaling from mesenchymal cells is necessary to maintain the location-specific identity of the small intestine. Using the pure epithelial organoid technique, we show that region-specific gene expression profiles are conserved throughout long-term cultures of both mouse and human intestinal stem cells and correlated with differential Gata4 expression. Furthermore, the human organoid culture system demonstrates that Gata4-regulated gene expression is only allowed in absence of WNT signaling. These data show that location-specific function is intrinsically programmed in the adult stem cells of the small intestine and that their differentiation fate is independent of location-specific extracellular signals. In light of the potential future clinical application of small intestine-derived organoids, our data imply that it is important to generate GATA4-positive and GATA4-negative cultures to regenerate all essential functions of the small intestine.

  11. Neural Progenitors Adopt Specific Identities by Directly Repressing All Alternative Progenitor Transcriptional Programs.

    PubMed

    Kutejova, Eva; Sasai, Noriaki; Shah, Ankita; Gouti, Mina; Briscoe, James

    2016-03-21

    In the vertebrate neural tube, a morphogen-induced transcriptional network produces multiple molecularly distinct progenitor domains, each generating different neuronal subtypes. Using an in vitro differentiation system, we defined gene expression signatures of distinct progenitor populations and identified direct gene-regulatory inputs corresponding to locations of specific transcription factor binding. Combined with targeted perturbations of the network, this revealed a mechanism in which a progenitor identity is installed by active repression of the entire transcriptional programs of other neural progenitor fates. In the ventral neural tube, sonic hedgehog (Shh) signaling, together with broadly expressed transcriptional activators, concurrently activates the gene expression programs of several domains. The specific outcome is selected by repressive input provided by Shh-induced transcription factors that act as the key nodes in the network, enabling progenitors to adopt a single definitive identity from several initially permitted options. Together, the data suggest design principles relevant to many developing tissues.

  12. Preferential labeling of alpha-amino N-terminal groups in peptides by biotin: application to the detection of specific anti-peptide antibodies by enzyme immunoassays.

    PubMed

    Sélo, I; Négroni, L; Créminon, C; Grassi, J; Wal, J M

    1996-12-15

    Experimental conditions (pH 6.5, 24 h reaction, peptide:biotin ratio 1:5) were defined for preferential incorporation of the biotin molecule in the N-terminal alpha-amino group of peptides. This strategy could be helpful in numerous applications when an entire peptide chain must remain accessible for antibody or receptor binding. We illustrate this advantage in a solid-phase enzyme immunoassay designed to detect antibodies specific for bovine beta-lactoglobulin present in rabbit or human sera. This test involves synthetic peptides biotinylated in different positions and immobilized on a solid phase. The use of biotin/streptavidin interactions permitted more efficient detection of specific anti-peptide antibodies than solid phases prepared using conventional passive-adsorption techniques. The highest levels of antibody binding were measured when biotinylation occurred at the N-terminal extremity of immobilized peptides.

  13. Specific Hsp100 Chaperones Determine the Fate of the First Enzyme of the Plastidial Isoprenoid Pathway for Either Refolding or Degradation by the Stromal Clp Protease in Arabidopsis

    PubMed Central

    Pulido, Pablo; Llamas, Ernesto; Llorente, Briardo; Ventura, Salvador; Wright, Louwrance P.; Rodríguez-Concepción, Manuel

    2016-01-01

    The lifespan and activity of proteins depend on protein quality control systems formed by chaperones and proteases that ensure correct protein folding and prevent the formation of toxic aggregates. We previously found that the Arabidopsis thaliana J-protein J20 delivers inactive (misfolded) forms of the plastidial enzyme deoxyxylulose 5-phosphate synthase (DXS) to the Hsp70 chaperone for either proper folding or degradation. Here we show that the fate of Hsp70-bound DXS depends on pathways involving specific Hsp100 chaperones. Analysis of individual mutants for the four Hsp100 chaperones present in Arabidopsis chloroplasts showed increased levels of DXS proteins (but not transcripts) only in those defective in ClpC1 or ClpB3. However, the accumulated enzyme was active in the clpc1 mutant but inactive in clpb3 plants. Genetic evidence indicated that ClpC chaperones might be required for the unfolding of J20-delivered DXS protein coupled to degradation by the Clp protease. By contrast, biochemical and genetic approaches confirmed that Hsp70 and ClpB3 chaperones interact to collaborate in the refolding and activation of DXS. We conclude that specific J-proteins and Hsp100 chaperones act together with Hsp70 to recognize and deliver DXS to either reactivation (via ClpB3) or removal (via ClpC1) depending on the physiological status of the plastid. PMID:26815787

  14. Characterization of the cloned full-length and a truncated human target of rapamycin: Activity, specificity, and enzyme inhibition as studied by a high capacity assay

    SciTech Connect

    Toral-Barza, Lourdes; Zhang Weiguo; Lamison, Craig; LaRocque, James; Gibbons, James; Yu, Ker . E-mail: yuk@wyeth.com

    2005-06-24

    The mammalian target of rapamycin (mTOR/TOR) is implicated in cancer and other human disorders and thus an important target for therapeutic intervention. To study human TOR in vitro, we have produced in large scale both the full-length TOR (289 kDa) and a truncated TOR (132 kDa) from HEK293 cells. Both enzymes demonstrated a robust and specific catalytic activity towards the physiological substrate proteins, p70 S6 ribosomal protein kinase 1 (p70S6K1) and eIF4E binding protein 1 (4EBP1), as measured by phosphor-specific antibodies in Western blotting. We developed a high capacity dissociation-enhanced lanthanide fluorescence immunoassay (DELFIA) for analysis of kinetic parameters. The Michaelis constant (K {sub m}) values of TOR for ATP and the His6-S6K substrate were shown to be 50 and 0.8 {mu}M, respectively. Dose-response and inhibition mechanisms of several known inhibitors, the rapamycin-FKBP12 complex, wortmannin and LY294002, were also studied in DELFIA. Our data indicate that TOR exhibits kinetic features of those shared by traditional serine/threonine kinases and demonstrate the feasibility for TOR enzyme screen in searching for new inhibitors.

  15. Genetic relationships within the Opisthorchis viverrini species complex with specific analysis of O. viverrini from Savannakhet, Lao PDR by multilocus enzyme electrophoresis.

    PubMed

    Kiatsopit, Nadda; Sithithaworn, Paiboon; Sithithaworn, Jiraporn; Boonmars, Thidarut; Tesana, Smarn; Pitaksakulrat, Opal; Saijuntha, Weerachai; Petney, Trevor N; Andrews, Ross H

    2011-01-01

    Recent systematic studies of Opisthorchis viverrini based on multilocus enzyme electrophoresis (MEE) have shown that there are at least five genetic groups and possibly two cryptic species occurring in Thailand and Lao PDR each associated with a specific wetland system. A study based on mitochondrial DNA (mtDNA) gene analyses of an O. viverrini population from Savannakhet (SV, Lao PDR) clustered with several isolates from Thailand and Lao PDR although they originated from different river wetland systems. We used MEE to re-examine whether O. viverrini from SV was similar genetically to isolates from Thailand and Lao PDR. The allelic profiles of O. viverrini from SV and five different wetlands representing defined genetic groups of O. viverrini were recorded at 24 enzyme loci as opposed to only two loci of mtDNA. Contrary to previous studies, O. viverrini from SV was found to have fixed genetic differences at six to eight of the 24 loci examined (24.50-35.42%). Allelic data indicated that O. viverrini from SV differed from isolates in the Nam Ngum River wetland in Lao PDR (29.33% fixed genetic differences) and clustered with O. viverrini from Nakhon Phanom and Sakon Nakhon within the Songkram River wetland in Thailand but had fixed genetic differences from these at 24.5% of loci examined. Our data confirm the association between genetic groups of O. viverrini and specific wetland systems, and raise important questions regarding the significance of the genetic differences and relationships of O. viverrini from these wetlands.

  16. Degenerate sequence recognition by the monomeric restriction enzyme: single mutation converts BcnI into a strand-specific nicking endonuclease

    PubMed Central

    Kostiuk, Georgij; Sasnauskas, Giedrius; Tamulaitiene, Giedre; Siksnys, Virginijus

    2011-01-01

    Unlike orthodox Type II restriction endonucleases that are homodimers and interact with the palindromic 4–8-bp DNA sequences, BcnI is a monomer which has a single active site but cuts both DNA strands within the 5′-CC↓CGG-3′/3′-GGG↓CC-5′ target site (‘↓’ designates the cleavage position). Therefore, after cutting the first strand, the BcnI monomer must re-bind to the target site in the opposite orientation; but in this case, it runs into a different central base because of the broken symmetry of the recognition site. Crystal-structure analysis shows that to accept both the C:G and G:C base pairs at the center of its target site, BcnI employs two symmetrically positioned histidines H77 and H219 that presumably change their protonation state depending on the binding mode. We show here that a single mutation of BcnI H77 or H219 residues restricts the cleavage activity of the enzyme to either the 5′-CCCGG-3′ or the 5′-CCGGG-3′ strand, thereby converting BcnI into a strand-specific nicking endonuclease. This is a novel approach for engineering of monomeric restriction enzymes into strand-specific nucleases. PMID:21227928

  17. Bromodomain-dependent stage-specific male genome programming by Brdt.

    PubMed

    Gaucher, Jonathan; Boussouar, Fayçal; Montellier, Emilie; Curtet, Sandrine; Buchou, Thierry; Bertrand, Sarah; Hery, Patrick; Jounier, Sylvie; Depaux, Arnaud; Vitte, Anne-Laure; Guardiola, Philippe; Pernet, Karin; Debernardi, Alexandra; Lopez, Fabrice; Holota, Hélène; Imbert, Jean; Wolgemuth, Debra J; Gérard, Matthieu; Rousseaux, Sophie; Khochbin, Saadi

    2012-10-03

    Male germ cell differentiation is a highly regulated multistep process initiated by the commitment of progenitor cells into meiosis and characterized by major chromatin reorganizations in haploid spermatids. We report here that a single member of the double bromodomain BET factors, Brdt, is a master regulator of both meiotic divisions and post-meiotic genome repackaging. Upon its activation at the onset of meiosis, Brdt drives and determines the developmental timing of a testis-specific gene expression program. In meiotic and post-meiotic cells, Brdt initiates a genuine histone acetylation-guided programming of the genome by activating essential genes and repressing a 'progenitor cells' gene expression program. At post-meiotic stages, a global chromatin hyperacetylation gives the signal for Brdt's first bromodomain to direct the genome-wide replacement of histones by transition proteins. Brdt is therefore a unique and essential regulator of male germ cell differentiation, which, by using various domains in a developmentally controlled manner, first drives a specific spermatogenic gene expression program, and later controls the tight packaging of the male genome.

  18. Protect your heart: a culture-specific multimedia cardiovascular health education program.

    PubMed

    Shah, Amy; Clayman, Marla L; Glass, Sara; Kandula, Namratha R

    2015-04-01

    South Asians, the second fastest growing racial/ethnic minority in the United States, have high rates of coronary heart disease. Few coronary heart disease prevention efforts target this population. The authors developed and tested a culture-specific, multimedia coronary heart disease prevention education program in English and Hindi for South Asians. Participants were recruited from community organizations in Chicago, Illinois, between June and October of 2011. Bilingual interviewers used questionnaires to assess participants' knowledge and perceptions before and after the patient education program. The change from pretest score to posttest score was calculated using a paired t test. Linear regression was used to determine the association between posttest scores and education and language. Participants' (N = 112) average age was 41 years, 67% had more than a high school education, and 50% spoke Hindi. Participants' mean pretest score was 15 (SD = 4). After the patient education program, posttest scores increased significantly among all participants (posttest score = 24, SD = 4), including those with limited English proficiency. Lower education was associated with a lower posttest score (β = -2.2, 95% CI [-0.68, -3.83]) in adjusted regression. A culture-specific, multimedia patient education program significantly improved knowledge and perceptions about coronary heart disease prevention among South Asian immigrants. Culturally salient multimedia education may be an effective and engaging way to deliver health information to diverse patient populations.

  19. Detection of beta-catenin mutations in paraffin-embedded sporadic desmoid-type fibromatosis by mutation-specific restriction enzyme digestion (MSRED): an ancillary diagnostic tool.

    PubMed

    Amary, Maria Fernanda C; Pauwels, Patrick; Meulemans, Els; Roemen, Guido M; Islam, Lily; Idowu, Bernadine; Bousdras, Konstantinos; Diss, Timothy C; O'Donnell, Paul; Flanagan, Adrienne M

    2007-09-01

    Desmoid-type fibromatosis is a locally aggressive deep soft tissue tumor. Some cases are associated with adenosis polyposis coli germline mutations whereas others harbor somatic beta-catenin point mutations mainly in exon 3, codons 41 and 45. These mutations result in stabilization of beta-catenin, and activation of the Wnt signaling pathway. The aim of this study was to determine the specificity and sensitivity of these 3 most common beta-catenin mutations in the diagnosis of desmoid-type fibromatosis using paraffin-embedded material. The results were compared with nuclear expression of beta-catenin. Mutation-specific restriction enzyme digestion methodology was employed to detect the 3 mutations. One hundred and thirty-three cases were analyzed, including 76 desmoid-type, and 18 superficial fibromatosis, in addition to a further 39 fibromatosis mimics. A restriction site was present for analysis of the codon 41 mutation. Mismatch primers were designed for the codon 45 mutations. Mutations were detected in 66 cases (87%) of 76 desmoid-type fibromatosis (71 extra-abdominal). Of these, 34 (45%) were in codon 45 (TCT>TTT), 27 (35%) in codon 41 (ACC>GCC), and 5 (7%) in codon 45 (TCT>CCT). No mutations were detected in the other lesions studied. All desmoid-type fibromatosis cases and 72% of the mimics tested showed nuclear positivity for beta-catenin indicating immunohistochemistry is a sensitive but not a specific test for desmoid-type fibromatosis. In contrast, to date, beta-catenin mutations have not been detected in any lesions which mimic desmoid-type fibromatosis. Mutation-specific restriction enzyme digestion, a simple and efficient means of detecting the common beta-catenin mutations in desmoid-type fibromatosis, complements light microscopy in reaching a diagnosis.

  20. Improved loop-mediated isothermal amplification for HLA-DRB1 genotyping using RecA and a restriction enzyme for enhanced amplification specificity.

    PubMed

    Mitsunaga, Shigeki; Shimizu, Sayoko; Okudaira, Yuko; Oka, Akira; Tanaka, Masafumi; Kimura, Minoru; Kulski, Jerzy K; Inoue, Ituro; Inoko, Hidetoshi

    2013-06-01

    Our aim was to test and develop the use of loop-mediated isothermal amplification (LAMP) for HLA-DRB1 genotyping. Initially, we found that the conventional LAMP protocols produced non-specific and variable amplification results depending on the sample DNA conditions. Experiments with different concentrations of DNase in the reaction mixture with and without T4 DNA ligase-treated samples suggested that the strand displacement activity of DNA polymerase in LAMP, at least in part, started from randomly existing nicks because T4 DNA ligase treatment of sample DNA resulted in no amplification. Such non-specific amplification due to the randomly existing nicks was improved specifically by the addition of RecA of Escherichia coli and a restriction enzyme, for example, PvuII, to the reaction mixture. We applied the modified LAMP (mLAMP) (1) to detect specific HLA-DRB1 alleles by using only specific primers for amplification or (2) for genotyping in multiple samples with a multi-probe typing system. In the latter case, HLA-DRB1 genotyping was developed by combining the mLAMP with amplicon capture using polymorphic region-specific probes fixed onto the bottom of the wells of a 96-well plate and the captured amplicons visualized as a black spot at the bottom of the well. The multi-probe human leukocyte antigen (HLA) typing method and the specific HLA allele detection method could be applied for point-of-care testing due to no requirement for specific and expensive instruments.

  1. Identification of specific antinuclear antibodies in dogs using a line immunoassay and enzyme-linked immunosorbent assay.

    PubMed

    Bremer, Hanna D; Lattwein, Erik; Renneker, Stefanie; Lilliehöök, Inger; Rönnelid, Johan; Hansson-Hamlin, Helene

    2015-12-15

    Circulating antinuclear antibodies (ANA) are commonly present in the systemic autoimmune disease Systemic Lupus Erythematosus (SLE) and in other systemic rheumatic diseases, in humans as well as in dogs. The indirect immunofluorescence (IIF)-ANA test is the standard method for detecting ANA. Further testing for specific ANA with immunoblot techniques or ELISAs is routinely performed in humans to aid in the diagnosis and monitoring of disease. Several specific ANA identified in humans have been identified also in suspected canine SLE but, in contrast to humans, investigation of autoantibodies in canine SLE is mainly restricted to the IIF-ANA test. Our aim was to identify both known and novel specific ANA in dogs and to investigate if different IIF-ANA patterns are associated with different specific ANA in dogs. Sera from 240 dogs with suspicion of autoimmune disease (210 IIF-ANA positive (ANA(pos)) and 30 IIF-ANA negative (ANA(neg))) as well as sera from 27 healthy controls were included. The samples were analysed with a line immunoassay, LIA (Euroline ANA Profile 5, Euroimmun, Lübeck, Germany) and four different ELISAs (Euroimmun). The ANA(pos) dogs were divided in two groups depending on the type of IIF-ANA pattern. Of the 210 ANA(pos) samples 68 were classified as ANA homogenous (ANA(H)) and 141 as ANA speckled (ANA(S)), one sample was not possible to classify. Dogs in the ANA(H) group had, compared to the other groups, most frequently high levels of anti-double stranded deoxyribonucleic acid (dsDNA) and anti-nucleosome ANA. Anti-dsDNA antibodies were confirmed in some dogs with the Crithidia luciliae indirect immunofluorescence test (CLIFT). The frequency of ANA(H) dogs with values above those observed in the healthy group was significantly higher compared to ANA(S) dogs for anti-dsDNA, anti-nucleosome, and anti-histone reactivity. Dogs in the ANA(S) group had, compared to the other groups, most frequently high levels of anti-ribonucleoproteins (RNP) and

  2. Substrate Specificities and Intracellular Distributions of Three N-glycan Processing Enzymes Functioning at a Key Branch Point in the Insect N-Glycosylation Pathway*

    PubMed Central

    Geisler, Christoph; Jarvis, Donald L.

    2012-01-01

    Man(α1–6)[GlcNAc(β1–2)Man(α1–3)]ManGlcNAc2 is a key branch point intermediate in the insect N-glycosylation pathway because it can be either trimmed by a processing β-N-acetylglucosaminidase (FDL) to produce paucimannosidic N-glycans or elongated by N-acetylglucosaminyltransferase II (GNT-II) to produce complex N-glycans. N-Acetylglucosaminyltransferase I (GNT-I) contributes to branch point intermediate production and can potentially reverse the FDL trimming reaction. However, there has been no concerted effort to evaluate the relationships among these three enzymes in any single insect system. Hence, we extended our previous studies on Spodoptera frugiperda (Sf) FDL to include GNT-I and -II. Sf-GNT-I and -II cDNAs were isolated, the predicted protein sequences were analyzed, and both gene products were expressed and their acceptor substrate specificities and intracellular localizations were determined. Sf-GNT-I transferred N-acetylglucosamine to Man5GlcNAc2, Man3GlcNAc2, and GlcNAc(β1–2)Man(α1–6)[Man(α1–3)]ManGlcNAc2, demonstrating its role in branch point intermediate production and its ability to reverse FDL trimming. Sf-GNT-II only transferred N-acetylglucosamine to Man(α1–6)[GlcNAc(β1–2)Man(α1–3)]ManGlcNAc2, demonstrating that it initiates complex N-glycan production, but cannot use Man3GlcNAc2 to produce hybrid or complex structures. Fluorescently tagged Sf-GNT-I and -II co-localized with an endogenous Sf Golgi marker and Sf-FDL co-localized with Sf-GNT-I and -II, indicating that all three enzymes are Golgi resident proteins. Unexpectedly, fluorescently tagged Drosophila melanogaster FDL also co-localized with Sf-GNT-I and an endogenous Drosophila Golgi marker, indicating that it is a Golgi resident enzyme in insect cells. Thus, the substrate specificities and physical juxtapositioning of GNT-I, GNT-II, and FDL support the idea that these enzymes function at the N-glycan processing branch point and are major factors determining the

  3. Crystal structure of Escherichia coli enterobactin-specific isochorismate synthase (EntC) bound to its reaction product isochorismate: implications for the enzyme mechanism and differential activity of chorismate-utilizing enzymes.

    PubMed

    Sridharan, Sudharsan; Howard, Nigel; Kerbarh, Olivier; Błaszczyk, Michał; Abell, Chris; Blundell, Tom L

    2010-03-19

    EntC, one of two isochorismate synthases in Escherichia coli, is specific to the biosynthesis of the siderophore enterobactin. Here, we report the crystal structure of EntC in complex with isochorismate and Mg(2+)at 2.3 A resolution, the first structure of a chorismate-utilizing enzyme with a non-aromatic reaction product. EntC exhibits a complex alpha+beta fold like the other chorismate-utilizing enzymes, such as salicylate synthase and anthranilate synthase. Comparison of active site structures allowed the identification of several residues, not discussed previously, that might be important for the isochorismate activity of the EntC. Although EntC, MenF and Irp9 all convert chorismate to isochorismate, only Irp9 subsequently exhibits isochorismate pyruvate lyase activity resulting in the formation of salicylate and pyruvate as the reaction products. With a view to understanding the roles of these amino acid residues in the conversion of chorismate to isochorismate and to obtaining clues about the pyruvate lyase activity of Irp9, several mutants of EntC were generated in which the selected residues in EntC were substituted for those of Irp9: these included A303T, L304A, F327Y, I346L and F359Q mutations. Biochemical analysis of these mutants indicated that the side chain of A303 in EntC may be crucial in the orientation of the carbonyl to allow formation of a hydrogen bond with isochorismate. Some mutations, such as L304A and F359Q, give rise to a loss of catalytic activity, whereas others, such as F327Y and I346L, show that subtle changes in the otherwise closely similar active sites influence activity. We did not find a combination of these residues that conferred pyruvate lyase activity.

  4. Identifying the emerging human pathogen Scedosporium prolificans by using a species-specific monoclonal antibody that binds to the melanin biosynthetic enzyme tetrahydroxynaphthalene reductase.

    PubMed

    Thornton, Christopher R; Ryder, Lauren S; Le Cocq, Kate; Soanes, Darren M

    2015-04-01

    The dematiaceous (melanized) fungus Scedosporium prolificans is an emerging and frequently fatal pathogen of immunocompromised humans and which, along with the closely related fungi Pseudallescheria boydii, Scedosporium apiospermum and S. aurantiacum in the Pseudallescheria-Scedosporium complex, is a contributing aetiology to tsunami lung and central nervous system infections in near-drowning victims who have aspirated water laden with spores. At present, the natural habitat of the fungus is largely unknown, and accurate detection methods are needed to identify environmental reservoirs of infectious propagules. In this study, we report the development of a monoclonal antibody (mAb) (CA4) specific to S. prolificans, which does not cross-react with closely related fungi in the Pseudallescheria-Scedosporium complex or with a wide range of mould and yeast species pathogenic to humans. Using genome sequencing of a soil isolate and targeted gene disruption of the CA4 antigen-encoding gene, we show that mAb CA4 binds to the melanin-biosynthetic enzyme tetrahydroxynaphthalene reductase. Enzyme-deficient mutants produce orange-brown or green-brown spore suspensions compared with the black spore suspension of the wild-type strain. Using mAb CA4 and a mAb (HG12) specific to the related fungi P. boydii, P. apiosperma, S. apiospermum and S. aurantiacum, we demonstrate how the mAbs can be used in combination with a semiselective isolation procedure to track these opportunistic pathogens in environmental samples containing mixed populations of human pathogenic fungi. Specificity of mAb CA4 was confirmed by sequencing of the internally transcribed spacer 1 (ITS1)-5.8S-ITS2 rRNA-encoding regions of fungi isolated from estuarine muds.

  5. The Vanadium Iodoperoxidase from the Marine Flavobacteriaceae Species Zobellia galactanivorans Reveals Novel Molecular and Evolutionary Features of Halide Specificity in the Vanadium Haloperoxidase Enzyme Family

    PubMed Central

    Fournier, Jean-Baptiste; Rebuffet, Etienne; Delage, Ludovic; Grijol, Romain; Meslet-Cladière, Laurence; Rzonca, Justyna; Potin, Philippe; Michel, Gurvan; Czjzek, Mirjam

    2014-01-01

    Vanadium haloperoxidases (VHPO) are key enzymes that oxidize halides and are involved in the biosynthesis of organo-halogens. Until now, only chloroperoxidases (VCPO) and bromoperoxidases (VBPO) have been characterized structurally, mainly from eukaryotic species. Three putative VHPO genes were predicted in the genome of the flavobacterium Zobellia galactanivorans, a marine bacterium associated with macroalgae. In a phylogenetic analysis, these putative bacterial VHPO were closely related to other VHPO from diverse bacterial phyla but clustered independently from eukaryotic algal VBPO and fungal VCPO. Two of these bacterial VHPO, heterogeneously produced in Escherichia coli, were found to be strictly specific for iodide oxidation. The crystal structure of one of these vanadium-dependent iodoperoxidases, Zg-VIPO1, was solved by multiwavelength anomalous diffraction at 1.8 Å, revealing a monomeric structure mainly folded into α-helices. This three-dimensional structure is relatively similar to those of VCPO of the fungus Curvularia inaequalis and of Streptomyces sp. and is superimposable onto the dimeric structure of algal VBPO. Surprisingly, the vanadate binding site of Zg-VIPO1 is strictly conserved with the fungal VCPO active site. Using site-directed mutagenesis, we showed that specific amino acids and the associated hydrogen bonding network around the vanadate center are essential for the catalytic properties and also the iodide specificity of Zg-VIPO1. Altogether, phylogeny and structure-function data support the finding that iodoperoxidase activities evolved independently in bacterial and algal lineages, and this sheds light on the evolution of the VHPO enzyme family. PMID:25261522

  6. Characterization of the third generation enzyme immunoassay IEA-HIV1/2-III for the detection of anti-HIV specific antibodies in human sera.

    PubMed

    Rayevskaya, G; Pilipenko, V G; Tkáciková, L; Spivak, N Y; Mikula, I; Chumak, R M

    2000-01-01

    The sensitivity and specificity of the developed anti-HIV1/2 third generation enzyme immunoassay, the IEA-HIV1/2-III, was examined. The test system for the detection of anti-HIV antibodies included peroxidase-conjugated HIV-specific recombinant Gag protein fragments (epitopes of p24 and p17 proteins), Env-1 (epitopes of p41 and p120 proteins), and Env-2 (p36 epitopes). Sensitivity was evaluated with 346 sera from HIV1-seropositive subjects, Anti-HIV1 Low Titer panels no. 10 and PRB-106 and seropositive panel PRB-931 in comparison with other third- and second-generation assays. The IEA-HIV1/2-III assays are characterized with high sensitivity comparable to the other third generation assays and the better sensitivity with respect to the second generation test-kit to determine HIV-specific antibodies in human sera. The specificity was determined using three hundred sixty-seven potentially cross-reactive samples (but negative for anti-HIV1/2). Only one specimen among them was reactive by IEA-HIV1/2-III.

  7. cAMP-Specific phosphodiesterase-4 enzymes in the cardiovascular system: a molecular toolbox for generating compartmentalized cAMP signaling.

    PubMed

    Houslay, Miles D; Baillie, George S; Maurice, Donald H

    2007-04-13

    Cyclic AMP regulates a vast number of distinct events in all cells. Early studies established that its hydrolysis by cyclic nucleotide phosphodiesterases (PDEs) controlled both the magnitude and the duration of its influence. Recent evidence shows that PDEs also act as coincident detectors linking cyclic-nucleotide- and non-cyclic-nucleotide-based cellular signaling processes and are tethered with great selectively to defined intracellular structures, thereby integrating and spatially restricting their cellular effects in time and space. Although 11 distinct families of PDEs have been defined, and cells invariably express numerous individual PDE enzymes, a large measure of our increased appreciation of the roles of these enzymes in regulating cyclic nucleotide signaling has come from studies on the PDE4 family. Four PDE4 genes encode more than 20 isoforms. Alternative mRNA splicing and the use of different promoters allows cells the possibility of expressing numerous PDE4 enzymes, each with unique amino-terminal-targeting and/or regulatory sequences. Dominant negative and small interfering RNA-mediated knockdown strategies have proven that particular isoforms can uniquely control specific cellular functions. Thus the protein kinase A phosphorylation status of the beta(2) adrenoceptor and, thereby, its ability to switch its signaling to extracellular signal-regulated kinase activation, is uniquely regulated by PDE4D5 in cardiomyocytes. We describe how cardiomyocytes and vascular smooth muscle cells selectively vary both the expression and the catalytic activities of PDE4 isoforms to regulate their various functions and how altered regulation of these processes can influence the development, or resolution, of cardiovascular pathologies, such as heart failure, as well as various vasculopathies.

  8. Exposure to ethanol during neurodevelopment modifies crucial offspring rat brain enzyme activities in a region-specific manner.

    PubMed

    Stolakis, Vasileios; Liapi, Charis; Zarros, Apostolos; Kalopita, Konstantina; Memtsas, Vassilios; Botis, John; Tsagianni, Anastasia; Kimpizi, Despoina; Varatsos, Alexios; Tsakiris, Stylianos

    2015-12-01

    The experimental simulation of conditions falling within "the fetal alcohol spectrum disorder" (FASD) requires the maternal exposure to ethanol (EtOH) during crucial neurodevelopmental periods; EtOH has been linked to a number of neurotoxic effects on the fetus, which are dependent upon the extent and the magnitude of the maternal exposure to EtOH and for which very little is known with regard to the exact mechanism(s) involved. The current study has examined the effects of moderate maternal exposure to EtOH (10 % v/v in the drinking water) throughout gestation, or gestation and lactation, on crucial 21-day-old offspring Wistar rat brain parameters, such as the activities of acetylcholinesterase (AChE) and two adenosine triphosphatases (Na(+),K(+)-ATPase and Mg(2+)-ATPase), in major offspring CNS regions (frontal cortex, hippocampus, hypothalamus, cerebellum and pons). The implemented experimental setting has provided a comparative view of the neurotoxic effects of maternal exposure to EtOH between gestation alone and a wider exposure timeframe that better covers the human third trimester-matching CNS neurodevelopment period (gestation and lactation), and has revealed a CNS region-specific susceptibility of the examined crucial neurochemical parameters to the EtOH exposure schemes attempted. Amongst these parameters, of particular importance is the recorded extensive stimulation of Na(+),K(+)-ATPase in the frontal cortex of the EtOH-exposed offspring that seems to be a result of the deleterious effect of EtOH during gestation. Although this stimulation could be inversely related to the observed inhibition of AChE in the same CNS region, its dependency upon the EtOH-induced modulation of other systems of neurotransmission cannot be excluded and must be further clarified in future experimental attempts aiming to simulate and to shed more light on the milder forms of the FASD-related pathophysiology.

  9. 25 CFR 39.137 - May schools operate a language development program without a specific appropriation from Congress?

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... 25 Indians 1 2010-04-01 2010-04-01 false May schools operate a language development program... Formula Language Development Programs § 39.137 May schools operate a language development program without a specific appropriation from Congress? Yes, a school may operate a language development...

  10. 25 CFR 39.137 - May schools operate a language development program without a specific appropriation from Congress?

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... 25 Indians 1 2011-04-01 2011-04-01 false May schools operate a language development program... Formula Language Development Programs § 39.137 May schools operate a language development program without a specific appropriation from Congress? Yes, a school may operate a language development...

  11. 25 CFR 39.137 - May schools operate a language development program without a specific appropriation from Congress?

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ... 25 Indians 1 2012-04-01 2011-04-01 true May schools operate a language development program without... Formula Language Development Programs § 39.137 May schools operate a language development program without a specific appropriation from Congress? Yes, a school may operate a language development...

  12. 25 CFR 39.137 - May schools operate a language development program without a specific appropriation from Congress?

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    ... 25 Indians 1 2013-04-01 2013-04-01 false May schools operate a language development program... Formula Language Development Programs § 39.137 May schools operate a language development program without a specific appropriation from Congress? Yes, a school may operate a language development...

  13. 25 CFR 39.137 - May schools operate a language development program without a specific appropriation from Congress?

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ... 25 Indians 1 2014-04-01 2014-04-01 false May schools operate a language development program... Formula Language Development Programs § 39.137 May schools operate a language development program without a specific appropriation from Congress? Yes, a school may operate a language development...

  14. Do fallers and nonfallers equally benefit from balance specific exercise program? A pilot study.

    PubMed

    Rugelj, Darja; Tomšič, Marija; Sevšek, France

    2013-01-01

    The purpose of the study was to determine the sample size that would allow broad generalizability of the results. To investigate the differences in the responsiveness of fallers and nonfallers to a multicomponent functional balance specific program, 23 participating subjects (70.1 ± 6.6 years) were divided into nonfallers group (13) and fallers group (10). The components of the balance specific program were (1) changing of the center of gravity (CoG) in the vertical direction, (2) shifting of the CoG to the border of stability, (3) rotation of the head and body about the vertical axis, (4) standing and walking on soft surface, and (5) walking over obstacles or on a narrow path. At the end of eight months of the training program, there was no significant difference between the two groups regarding postural sway. The total center of pressure path length was used as the principal outcome measure for the sample size calculation. Based on these results the a priori sample size calculation yielded the estimate of 110 subjects required to be enrolled in order to get 20 subjects in fallers and 30 subjects in nonfallers group for the 80% power to detect the results as significant.

  15. Automated size-specific CT dose monitoring program: Assessing variability in CT dose

    SciTech Connect

    Christianson, Olav; Li Xiang; Frush, Donald; Samei, Ehsan

    2012-11-15

    Purpose: The potential health risks associated with low levels of ionizing radiation have created a movement in the radiology community to optimize computed tomography (CT) imaging protocols to use the lowest radiation dose possible without compromising the diagnostic usefulness of the images. Despite efforts to use appropriate and consistent radiation doses, studies suggest that a great deal of variability in radiation dose exists both within and between institutions for CT imaging. In this context, the authors have developed an automated size-specific radiation dose monitoring program for CT and used this program to assess variability in size-adjusted effective dose from CT imaging. Methods: The authors radiation dose monitoring program operates on an independent health insurance portability and accountability act compliant dosimetry server. Digital imaging and communication in medicine routing software is used to isolate dose report screen captures and scout images for all incoming CT studies. Effective dose conversion factors (k-factors) are determined based on the protocol and optical character recognition is used to extract the CT dose index and dose-length product. The patient's thickness is obtained by applying an adaptive thresholding algorithm to the scout images and is used to calculate the size-adjusted effective dose (ED{sub adj}). The radiation dose monitoring program was used to collect data on 6351 CT studies from three scanner models (GE Lightspeed Pro 16, GE Lightspeed VCT, and GE Definition CT750 HD) and two institutions over a one-month period and to analyze the variability in ED{sub adj} between scanner models and across institutions. Results: No significant difference was found between computer measurements of patient thickness and observer measurements (p= 0.17), and the average difference between the two methods was less than 4%. Applying the size correction resulted in ED{sub adj} that differed by up to 44% from effective dose estimates

  16. Development of an efficient signal amplification strategy for label-free enzyme immunoassay using two site-specific biotinylated recombinant proteins.

    PubMed

    Tang, Jin-Bao; Tang, Ying; Yang, Hong-Ming

    2015-02-15

    Constructing a recombinant protein between a reporter enzyme and a detector protein to produce a homogeneous immunological reagent is advantageous over random chemical conjugation. However, the approach hardly recombines multiple enzymes in a difunctional fusion protein, which results in insufficient amplification of the enzymatic signal, thereby limiting its application in further enhancement of analytical signal. In this study, two site-specific biotinylated recombinant proteins, namely, divalent biotinylated alkaline phosphatase (AP) and monovalent biotinylated ZZ domain, were produced by employing the Avitag-BirA system. Through the high streptavidin (SA)-biotin interaction, the divalent biotinylated APs were clustered in the SA-biotin complex and then incorporated with the biotinylated ZZ. This incorporation results in the formation of a functional macromolecule that involves numerous APs, thereby enhancing the enzymatic signal, and in the production of several ZZ molecules for the interaction with immunoglobulin G (IgG) antibody. The advantage of this signal amplification strategy is demonstrated through ELISA, in which the analytical signal was substantially enhanced, with a 32-fold increase in the detection sensitivity compared with the ZZ-AP fusion protein approach. The proposed immunoassay without chemical modification can be an alternative strategy to enhance the analytical signals in various applications involving immunosensors and diagnostic chips, given that the label-free IgG antibody is suitable for the ZZ protein.

  17. The distribution of serotype-specific plasmids among different subgroups of strains of Salmonella enterica serotype Enteritidis: characterization of molecular variants by restriction enzyme fragmentation patterns.

    PubMed Central

    Rankin, S. C.; Benson, C. E.; Platt, D. J.

    1995-01-01

    Four hundred and thirty-four isolates of Salmonella enterica serotype Enteritidis were studied. They were grouped into five subsets defined by either the collection criteria or the parameter which formed the basis for subsequent analysis. Seventy-seven per cent harboured the serotype-specific plasmid (SSP). In 55% of the isolates this was the sole plasmid. Molecular variation in the SSP was detected in 17 (5%) of the isolates on the basis of restriction enzyme fragmentation pattern (REFP) analysis using Pst I and Sma I. The SSP variants were further characterized using additional restriction enzymes chosen to optimize the information content and analysed using a coefficient of similarity. A variant SSP designated pOG690 showed greater resemblance to the SSP of Salmonella enterica serotype Typhimurium than Enteritidis; 89% and 68% respectively for Pst I and 79% and 55% respectively for Sma I. In respect of the Pst I data pOG690 shared at least 55 kb of DNA with the Typhimurium SSP and 37 kb with the SSP of Enteritidis. This variant was associated with poultry (duck, goose, chicken) and all isolates belonged to phage type 9b. Other variants were associated with phage types 4, 6, 6a, 9a, 11, 15 and 24. The epidemiological implications of these results are discussed. Images Fig. 1 PMID:7867741

  18. Biophysical Characterization of Fluorotyrosine Probes Site-Specifically Incorporated into Enzymes: E. coli Ribonucleotide Reductase As an Example

    PubMed Central

    2016-01-01

    Fluorinated tyrosines (FnY’s, n = 2 and 3) have been site-specifically incorporated into E. coli class Ia ribonucleotide reductase (RNR) using the recently evolved M. jannaschii Y-tRNA synthetase/tRNA pair. Class Ia RNRs require four redox active Y’s, a stable Y radical (Y·) in the β subunit (position 122 in E. coli), and three transiently oxidized Y’s (356 in β and 731 and 730 in α) to initiate the radical-dependent nucleotide reduction process. FnY (3,5; 2,3; 2,3,5; and 2,3,6) incorporation in place of Y122-β and the X-ray structures of each resulting β with a diferric cluster are reported and compared with wt-β2 crystallized under the same conditions. The essential diferric-FnY· cofactor is self-assembled from apo FnY-β2, Fe2+, and O2 to produce ∼1 Y·/β2 and ∼3 Fe3+/β2. The FnY· are stable and active in nucleotide reduction with activities that vary from 5% to 85% that of wt-β2. Each FnY·-β2 has been characterized by 9 and 130 GHz electron paramagnetic resonance and high-field electron nuclear double resonance spectroscopies. The hyperfine interactions associated with the 19F nucleus provide unique signatures of each FnY· that are readily distinguishable from unlabeled Y·’s. The variability of the abiotic FnY pKa’s (6.4 to 7.8) and reduction potentials (−30 to +130 mV relative to Y at pH 7.5) provide probes of enzymatic reactions proposed to involve Y·’s in catalysis and to investigate the importance and identity of hopping Y·’s within redox active proteins proposed to protect them from uncoupled radical chemistry. PMID:27276098

  19. Production of Group Specific Monoclonal Antibody to Aflatoxins and its Application to Enzyme-linked Immunosorbent Assay

    PubMed Central

    Kim, Sung-Hee; Cha, Sang-Ho; Karyn, Bischoff; Park, Sung-Won; Son, Seong-Wan

    2011-01-01

    Through the present study, we produced a monoclonal antibody against aflatoxin B1 (AFB1) using AFB1- carboxymethoxylamine BSA conjugates. One clone showing high binding ability was selected and it was applied to develop a direct competitive ELISA system. The epitope densities of AFB1-CMO against BSA and KLH were about 1 : 6 and 1 : 545, respectively. The monoclonal antibody (mAb) from cloned hybridoma cell was the IgG1 subclass with λ-type light chains. The IC50s of the monoclonal antibody developed for AFB1, AFB2, AFG1 and AFG2 were 4.36, 7.22, 6.61 and 29.41 ng/ml, respectively, based on the AFB1-KLH coated ELISA system and 15.28, 26.62, 32.75 and 56.67 ng/ml, respectively, based on the mAb coated ELISA. Cross-relativities of mAb to AFB1 for AFB2, AFG1 and AFG2 were 60.47, 65.97 and 14.83% in the AFB1-KLH coated ELISA, and 59.41, 46.66 and 26.97% in the mAb coated ELISA, respectively. Quantitative calculations for AFB1 from the AFB1-Ab ELISA and AFB1-Ag ELISA ranged from 0.25 to 25 ng/ml (R2 > 0.99) and from 1 to 100 ng/ml (R2 > 0.99), respectively. The intra- and inter-assay precision CVs were < 10% in both ELISA assay, representing good reproducibility of developed assay. Recoveries ranged from 79.18 to 91.27%, CVs ranged from 3.21 to 7.97% after spiking AFB1 at concentrations ranging from 5 to 50 ng/ml and following by extraction with 70% methanol solution in the Ab-coated ELISA. In conclusion, we produced a group specific mAb against aflatoxins and developed two direct competitive ELISAs for the detection of AFB1 in feeds based on a monoclonal antibody developed. PMID:24278561

  20. Novel IgG-Degrading Enzymes of the IgdE Protease Family Link Substrate Specificity to Host Tropism of Streptococcus Species.

    PubMed

    Spoerry, Christian; Hessle, Pontus; Lewis, Melanie J; Paton, Lois; Woof, Jenny M; von Pawel-Rammingen, Ulrich

    2016-01-01

    Recently we have discovered an IgG degrading enzyme of the endemic pig pathogen S. suis designated IgdE that is highly specific for porcine IgG. This protease is the founding member of a novel cysteine protease family assigned C113 in the MEROPS peptidase database. Bioinformatical analyses revealed putative members of the IgdE protease family in eight other Streptococcus species. The genes of the putative IgdE family proteases of S. agalactiae, S. porcinus, S. pseudoporcinus and S. equi subsp. zooepidemicus were cloned for production of recombinant protein into expression vectors. Recombinant proteins of all four IgdE family proteases were proteolytically active against IgG of the respective Streptococcus species hosts, but not against IgG from other tested species or other classes of immunoglobulins, thereby linking the substrate specificity to the known host tropism. The novel IgdE family proteases of S. agalactiae, S. pseudoporcinus and S. equi showed IgG subtype specificity, i.e. IgdE from S. agalactiae and S. pseudoporcinus cleaved human IgG1, while IgdE from S. equi was subtype specific for equine IgG7. Porcine IgG subtype specificities of the IgdE family proteases of S. porcinus and S. pseudoporcinus remain to be determined. Cleavage of porcine IgG by IgdE of S. pseudoporcinus is suggested to be an evolutionary remaining activity reflecting ancestry of the human pathogen to the porcine pathogen S. porcinus. The IgG subtype specificity of bacterial proteases indicates the special importance of these IgG subtypes in counteracting infection or colonization and opportunistic streptococci neutralize such antibodies through expression of IgdE family proteases as putative immune evasion factors. We suggest that IgdE family proteases might be valid vaccine targets against streptococci of both human and veterinary medical concerns and could also be of therapeutic as well as biotechnological use.

  1. Evolutionary dynamics of enzymes.

    PubMed

    Demetrius, L

    1995-08-01

    This paper codifies and rationalizes the large diversity in reaction rates and substrate specificity of enzymes in terms of a model which postulates that the kinetic properties of present-day enzymes are the consequence of the evolutionary force of mutation and selection acting on a class of primordial enzymes with poor catalytic activity and broad substrate specificity. Enzymes are classified in terms of their thermodynamic parameters, activation enthalpy delta H* and activation entropy delta S*, in their kinetically significant transition states as follows: type 1, delta H* > 0, delta S* < 0; type 2, delta H* < or = 0, delta S* < or = 0; type 3, delta H* > 0, delta S* > 0. We study the evolutionary dynamics of these three classes of enzymes subject to mutation, which acts at the level of the gene which codes for the enzyme and selection, which acts on the organism that contains the enzyme. Our model predicts the following evolutionary trends in the reaction rate and binding specificity for the three classes of molecules. In type 1 enzymes, evolution results in random, non-directional changes in the reaction rate and binding specificity. In type 2 and 3 enzymes, evolution results in a unidirectional increase in both the reaction rate and binding specificity. We exploit these results in order to codify the diversity in functional properties of present-day enzymes. Type 1 molecules will be described by intermediate reaction rates and broad substrate specificity. Type 2 enzymes will be characterized by diffusion-controlled rates and absolute substrate specificity. The type 3 catalysts can be further subdivided in terms of their activation enthalpy into two classes: type 3a (delta H* small) and type 3b (delta H* large). We show that type 3a will be represented by the same functional properties that identify type 2, namely, diffusion-controlled rates and absolute substrate specificity, whereas type 3b will be characterized by non-diffusion-controlled rates and absolute

  2. NASA/General Electric broad-specification fuels combustion technology program - Phase I results and status

    NASA Technical Reports Server (NTRS)

    Dodds, W. J.; Ekstedt, E. E.; Bahr, D. W.; Fear, J. S.

    1982-01-01

    A program is being conducted to develop the technology required to utilize fuels with broadened properties in aircraft gas turbine engines. The first phase of this program consisted of the experimental evaluation of three different combustor concepts to determine their potential for meeting several specific emissions and performance goals, when operated on broadened property fuels. The three concepts were a single annular combustor; a double annular combustor; and a short single annular combustor with variable geometry. All of these concepts were sized for the General Electric CF6-80 engine. A total of 24 different configurations of these concepts were evaluated in a high pressure test facility, using four test fuels having hydrogen contents between 11.8 and 14%. Fuel effects on combustor performance, durability and emissions, and combustor design features to offset these effects were demonstrated.

  3. The role of renal proximal tubule P450 enzymes in chloroform-induced nephrotoxicity: Utility of renal specific P450 reductase knockout mouse models

    SciTech Connect

    Liu, Senyan; Yao, Yunyi; Lu, Shijun; Aldous, Kenneth; Ding, Xinxin; Mei, Changlin; Gu, Jun

    2013-10-01

    The kidney is a primary target for numerous toxic compounds. Cytochrome P450 enzymes (P450) are responsible for the metabolic activation of various chemical compounds, and in the kidney are predominantly expressed in proximal tubules. The aim of this study was to test the hypothesis that renal proximal tubular P450s are critical for nephrotoxicity caused by chemicals such as chloroform. We developed two new mouse models, one having proximal tubule-specific deletion of the cytochrome P450 reductase (Cpr) gene (the enzyme required for all microsomal P450 activities), designated proximal tubule-Cpr-null (PTCN), and the other having proximal tubule-specific rescue of CPR activity with the global suppression of CPR activity in all extra-proximal tubular tissues, designated extra-proximal tubule-Cpr-low (XPT-CL). The PTCN, XPT-CL, Cpr-low (CL), and wild-type (WT) mice were treated with a single oral dose of chloroform at 200 mg/kg. Blood, liver and kidney samples were obtained at 24 h after the treatment. Renal toxicity was assessed by measuring BUN and creatinine levels, and by pathological examination. The blood and tissue levels of chloroform were determined. The severity of toxicity was less in PTCN and CL mice, compared with that of WT and XPT-CL mice. There were no significant differences in chloroform levels in the blood, liver, or kidney, between PTCN and WT mice, or between XPT-CL and CL mice. These findings indicate that local P450-dependent activities play an important role in the nephrotoxicity induced by chloroform. Our results also demonstrate the usefulness of these novel mouse models for studies of chemical-induced kidney toxicity. - Highlights: • New mouse models were developed with varying P450 activities in the proximal tubule. • These mouse models were treated with chloroform, a nephrotoxicant. • Studies showed the importance of local P450s in chloroform-induced nephrotoxicity.

  4. Insights into substrate specificity of geranylgeranyl reductases revealed by the structure of digeranylgeranylglycerophospholipid reductase from Thermoplasma acidophilum, an essential enzyme in the biosynthesis of archaeal membrane lipids

    PubMed Central

    Xu, Qingping; Eguchi, Tadashi; Mathews, Irimpan I.; Rife, Christopher L.; Chiu, Hsiu-Ju; Farr, Carol L.; Feuerhelm, Julie; Jaroszewski, Lukasz; Klock, Heath E.; Knuth, Mark W.; Miller, Mitchell D.; Weekes, Dana; Elsliger, Marc-André; Deacon, Ashley M.; Godzik, Adam; Lesley, Scott A.; Wilson, Ian A.

    2010-01-01

    Archaeal membrane lipids consist of branched, saturated hydrocarbons distinct from those found in bacteria and eukaryotes. Digeranylgeranylglycerophospholipid reductase (DGGR) catalyzes the hydrogenation process that converts unsaturated 2,3-di-O-geranylgeranylglyceryl phosphate to saturated 2,3-di-O-phytanylglyceryl phosphate as a critical step in the biosynthesis of archaeal membrane lipids. The saturation of hydrocarbon chains confers the ability to resist hydrolysis and oxidation and helps archaea withstand extreme conditions. DGGR is a member of the geranylgeranyl reductase (GGR) family that is also widely distributed in bacteria and plants, where the family members are involved in the biosynthesis of photosynthetic pigments. We have determined the crystal structure of DGGR from the thermophilic heterotrophic archaea Thermoplasma acidophilum at 1.6 Å resolution, in complex with FAD and a bacterial lipid. The DGGR structure can be assigned to the well-studied, para-hydroxybenzoate hydroxylase (PHBH) SCOP superfamily of flavoproteins that include many aromatic hydroxylases and other enzymes with diverse functions. In the DGGR complex, FAD adopts the IN conformation (closed) previously observed in other PHBH flavoproteins. DGGR contains a large substrate-binding site that extends across the entire ligand-binding domain. Electron density corresponding to a bacterial lipid was found within this cavity. The cavity consists of a large opening that tapers down to two narrow curved tunnels that closely mimic the shape of the preferred substrate. We identified a sequence motif, PxxYxWxFP, that defines a specificity pocket in the structure and precisely aligns the double bond of the geranyl group with respect to the FAD cofactor, thus providing a structural basis for the substrate specificity of GGRs. DGGR is likely to share a common mechanism with other PHBH enzymes in which FAD switches between two conformations that correspond to the reductive and oxidative half

  5. A Hybrid Method to Estimate Specific Differential Phase and Rainfall With Linear Programming and Physics Constraints

    DOE PAGES

    Huang, Hao; Zhang, Guifu; Zhao, Kun; ...

    2016-10-20

    A hybrid method of combining linear programming (LP) and physical constraints is developed to estimate specific differential phase (KDP) and to improve rain estimation. Moreover, the hybrid KDP estimator and the existing estimators of LP, least squares fitting, and a self-consistent relation of polarimetric radar variables are evaluated and compared using simulated data. Our simulation results indicate the new estimator's superiority, particularly in regions where backscattering phase (δhv) dominates. Further, a quantitative comparison between auto-weather-station rain-gauge observations and KDP-based radar rain estimates for a Meiyu event also demonstrate the superiority of the hybrid KDP estimator over existing methods.

  6. Seroconversion risk perception among jail populations: a call for gender-specific HIV prevention programming.

    PubMed

    Alarid, Leanne Fiftal; Hahl, Jeannie M

    2014-04-01

    The prevalence of HIV/AIDS infection among prisoners is 3 to 4 times higher than in the U.S. population. Given that one in seven HIV-positive Americans pass through a correctional facility every year, the criminal justice system is in an ideal position to aggressively implement effective HIV education, treatment, and prevention. This study examines barriers to the effective delivery of these services and evaluates differences in risk perception among nearly 600 female and male inmates. The results underscore gender differences in Perceived Risk of Seroconversion and Exposure to HIV Education, suggesting that jails should implement gender-specific HIV prevention programming.

  7. Evaluation of the sensitivity and specificity of an enzyme-linked immunosorbent assay for diagnosing brucellosis in African buffalo (Syncerus caffer).

    PubMed

    Gorsich, Erin E; Bengis, Roy G; Ezenwa, Vanessa O; Jolles, Anna E

    2015-01-01

    Brucellosis is a disease of veterinary and public health importance worldwide. In sub-Saharan Africa, where the bacterium Brucella abortus has been identified in several free-ranging wildlife species, successful disease control may be dependent on accurate detection in wildlife reservoirs, including African buffalo (Syncerus caffer). We estimated the sensitivity and specificity of a commercial enzyme-linked immunosorbent assay (ELISA) (IDEXX Brucellosis Serum Ab test, IDEXX Laboratories, Westbrook, Maine, USA) for B. abortus based on a data set of 571 serum samples from 258 buffalo in the Kruger National Park, South Africa. We defined a pseudogold standard test result as those buffalo that were consistently positive or negative on two additional serologic tests, namely, the rose bengal test (RBT) and the complement fixation test (CFT). The ELISA's cutoff value was selected using receiver operating characteristics analysis, the pseudogold standard, and a threshold criterion that maximizes the total sensitivity and specificity. Then, we estimated the sensitivity and specificity of all three tests using Bayesian inference and latent class analysis. The ELISA had an estimated sensitivity of 0.928 (95% Bayesian posterior credibility interval [95% BCI] = 0.869-0.974) and specificity of 0.870 (95% BCI = 0.836-0.900). Compared with the ELISA, the RBT had a higher estimated sensitivity of 0.986 (95% BCI = 0.928-0.999), and both the RBT and CFT had higher specificities, estimated to be 0.992 (95% BCI = 0.971-0.996) and 0.998 (95% BCI = 0.992-0.999), respectively. Therefore, no single serologic test perfectly detected the antibody. However, after adjustment of cutoff values for South African conditions, the IDEXX Brucellosis Serum Ab Test may be a valuable additional screening test for brucellosis in Kruger National Park's African buffalo.

  8. Enzyme-Linked Immunosorbent Assay with Conserved Immunoreactive Glycoproteins gp36 and gp19 Has Enhanced Sensitivity and Provides Species-Specific Immunodiagnosis of Ehrlichia canis Infection▿

    PubMed Central

    Cárdenas, Ana Maria; Doyle, C. Kuyler; Zhang, Xiaofeng; Nethery, Kimberly; Corstvet, Richard E.; Walker, David H.; McBride, Jere W.

    2007-01-01

    Ehrlichia canis is the primary etiologic agent of canine monocytic ehrlichiosis, a globally distributed and potentially fatal disease of dogs. We previously reported on the identification of two conserved major immunoreactive antigens, gp36 and gp19, which are the first proteins to elicit an E. canis-specific antibody response, and gp200 and p28, which elicit strong antibody responses later in the acute phase of the infection. In this report, the sensitivities and specificities of five recombinant E. canis proteins for the immunodiagnosis of E. canis infection by an enzyme-linked immunosorbent assay (ELISA) were evaluated. Recombinant polypeptides gp36, gp19, and gp200 (N and C termini) exhibited 100% sensitivity and specificity for immunodiagnosis by the recombinant glycoprotein ELISA compared with the results obtained by an indirect fluorescent-antibody assay (IFA) for the detection of antibodies in dogs that were naturally infected with E. canis. Moreover, the enhanced sensitivities of gp36 and gp19 for immunodiagnosis by the recombinant glycoprotein ELISA compared to those obtained by IFA were demonstrated with dogs experimentally infected with E. canis, in which antibodies were detected as much as 2 weeks earlier, on day 14 postinoculation. gp36 and gp19 were not cross-reactive with antibodies in sera from E. chaffeensis-infected dogs and thus provided species-specific serologic discrimination between E. canis and E. chaffeensis infections. This is the first demonstration of the improved detection capability of the recombinant protein technology compared to the capability of the “gold standard” IFA and may eliminate the remaining obstacles associated with the immunodiagnosis of E. canis infections, including species-specific identification and the lack of sensitivity associated with low antibody titers early in the acute phase of the infection. PMID:17151186

  9. Using Space Weather Variability in Evaluating the Radiation Environment Design Specifications for NASA's Constellation Program

    NASA Technical Reports Server (NTRS)

    Coffey, Victoria N.; Blackwell, William C.; Minow, Joseph I.; Bruce, Margaret B.; Howard, James W.

    2007-01-01

    NASA's Constellation program, initiated to fulfill the Vision for Space Exploration, will create a new generation of vehicles for servicing low Earth orbit, the Moon, and beyond. Space radiation specifications for space system hardware are necessarily conservative to assure system robustness for a wide range of space environments. Spectral models of solar particle events and trapped radiation belt environments are used to develop the design requirements for estimating total ionizing radiation dose, displacement damage, and single event effects for Constellation hardware. We first describe the rationale using the spectra chosen to establish the total dose and single event design environmental specifications for Constellation systems. We then compare variability of the space environment to the spectral design models to evaluate their applicability as conservative design environments and potential vulnerabilities to extreme space weather events

  10. Effects of a Diabetes Prevention Program on Weight-Specific Quality of Life in Latino Youth

    PubMed Central

    Brito, Elizabeth; Patrick, Donald L.; Konopken, Yolanda P.; Keller, Colleen S.; Barroso, Cristina S.; Shaibi, Gabriel Q.

    2014-01-01

    Objective To examine the effects of a diabetes prevention program on weight-specific Quality of Life (QOL) in obese Latino youth. Design and Methods Fifteen obese Latino adolescents (BMI%=96.4±1.2;age=15.0±1.0) completed a 12-week culturally-grounded, community-based intervention designed to improve physical and psychosocial health. Weight-specific QOL was assessed by the Youth Quality of Life–Weight module and compared to age, sex, and ethnicity-matched lean youth. Results At baseline, intervention youth exhibited significantly lower weight-specific QOL compared to lean youth (70.8±5.4 vs. 91.2±2.2, p=0.002). However following the intervention, total weight-specific QOL increased by 21.8% among obese youth (70.8±5.4 to 86.2±4.3, p<0.001) and was no longer different from lean controls. Significant increases in weight-specific QOL were noted across all sub-domains including Self (45.7%), Social (11.9%), and Environmental (36.2%) despite the fact that weight did not change (90.6±6.8 to 89.9±7.2, p=0.44). The improvements in QOL were maintained for up to 12-months after the intervention. Conclusion Weight-specific QOL among obese Latino youth can be improved through lifestyle interventions to a level similar to lean peers. Further, weight-loss may not be necessary to observe improvements in QOL. PMID:24903526

  11. The Effects of Goal Specificity and Scaffolding on Programming Performance and Self-Regulation in Game Design

    ERIC Educational Resources Information Center

    Feng, Chia-Yen; Chen, Ming-Puu

    2014-01-01

    The purpose of this study was to investigate the influence of goal specificity and scaffolding on the programming performance and self-regulation of elementary students engaged in learning game design. This study recruited 232 students for the experimental activities. Two levels of goal specificity were employed: specific and nonspecific.…

  12. Endothelial, epithelial, and fibroblast cells exhibit specific splicing programs independently of their tissue of origin

    PubMed Central

    Mallinjoud, Pierre; Villemin, Jean-Philippe; Mortada, Hussein; Polay Espinoza, Micaela; Desmet, François-Olivier; Samaan, Samaan; Chautard, Emilie; Tranchevent, Léon-Charles; Auboeuf, Didier

    2014-01-01

    Alternative splicing is the main mechanism of increasing the proteome diversity coded by a limited number of genes. It is well established that different tissues or organs express different splicing variants. However, organs are composed of common major cell types, including fibroblasts, epithelial, and endothelial cells. By analyzing large-scale data sets generated by The ENCODE Project Consortium and after extensive RT-PCR validation, we demonstrate that each of the three major cell types expresses a specific splicing program independently of its organ origin. Furthermore, by analyzing splicing factor expression across samples, publicly available splicing factor binding site data sets (CLIP-seq), and exon array data sets after splicing factor depletion, we identified several splicing factors, including ESRP1 and 2, MBNL1, NOVA1, PTBP1, and RBFOX2, that contribute to establishing these cell type–specific splicing programs. All of the analyzed data sets are freely available in a user-friendly web interface named FasterDB, which describes all known splicing variants of human and mouse genes and their splicing patterns across several dozens of normal and cancer cells as well as across tissues. Information regarding splicing factors that potentially contribute to individual exon regulation is also provided via a dedicated CLIP-seq and exon array data visualization interface. To the best of our knowledge, FasterDB is the first database integrating such a variety of large-scale data sets to enable functional genomics analyses at exon-level resolution. PMID:24307554

  13. Young Finnish Unemployed Men's Experiences of Having Participated in a Specific Active Labor Market Program.

    PubMed

    Björklund, Ove; Häggström, Elisabeth; Nyström, Lisbet

    2015-09-07

    The purpose of the present study was to describe young Finnish unemployed men's experiences of having participated in a specific active labor market program, intended to fight unemployment and offered at a resource center. Fifteen young unemployed Finnish men in the age range 18 to 27 years were interviewed face-to-face. Purposive sampling was used to increase the variation among informants. The interview texts were analyzed using both manifest and latent qualitative content analysis. The present results reported that the young men felt that they, thanks to the program at the resource center, had acquired daily routines and could ultimately believe in the future. The young men described how they now had a structure, economic support, and that they could return to their daily life. The informants also described how they could see new possibilities and believe in oneself. There is a lack of empirical studies assessing the possible impact of active labor market programs on the unemployed based on participants' own experiences. Further research is needed to describe and elucidate in more detail the effects of targeted support measures and the needs of unemployed men of different ages and living in different contexts.

  14. Autonomous regulation of sex-specific developmental programming in mouse fetal germ cells.

    PubMed

    Iwahashi, Kazuhiro; Yoshioka, Hirotaka; Low, Eleanor W; McCarrey, John R; Yanagimachi, Ryuzo; Yamazaki, Yukiko

    2007-10-01

    In mice, unique events regulating epigenetic programming (e.g., genomic imprinting) and replication state (mitosis versus meiosis) occur during fetal germ cell development. To determine whether these processes are autonomously programmed in fetal germ cells or are dependent upon ongoing instructive interactions with surrounding gonadal somatic cells, we isolated male and female germ cells at 13.5 days postcoitum (dpc) and maintained them in culture for 6 days, either alone or in the presence of feeder cells or gonadal somatic cells. We examined allele-specific DNA methylation in the imprinted H19 and Snrpn genes, and we also determined whether these cells remained mitotic or entered meiosis. Our results show that isolated male germ cells are able to establish a characteristic "paternal" methylation pattern at imprinted genes in the absence of any support from somatic cells. On the other hand, cultured female germ cells maintain a hypomethylated status at these loci, characteristic of the normal "maternal" methylation pattern in endogenous female germ cells before birth. Further, the surviving female germ cells entered first meiotic prophase and reached the pachytene stage, whereas male germ cells entered mitotic arrest. These results indicate that mechanisms controlling both epigenetic programming and replication state are autonomously regulated in fetal germ cells that have been exposed to the genital ridge prior to 13.5 dpc.

  15. Highly-sensitive and specific enzyme-linked immunosorbent assays for GAD65 autoantibodies using a thioredoxin-GAD65 fusion antigen.

    PubMed

    Papouchado, M L; Valdez, S N; Ermácora, M R; Gañan, S; Poskus, E

    1997-09-24

    Autoantibodies against glutamic acid decarboxylase (GAD65) are present in the sera of most patients with recently diagnosed insulin-dependent diabetes mellitus (IDDM). These antibodies appear years before the clinical symptoms, and they are considered to be early markers of the disease. To detect GAD65 autoantibodies (GADA), we developed new enzyme-linked immunosorbent assays (ELISA) with a fusion protein thioredoxin-GAD65 (Trx-GAD65) produced in E. coli as the antigen. These assays were compared with the reference radiobinding assay (RBA). Since most GADA are directed against native epitopes, and adsorption of GAD65 to plastic may cause disruption of its native conformation, the new assays rely on the following immobilization procedures: (a) capture ELISA (c-ELISA) with Trx-GAD65 (protocol A) or biotin-Trx-GAD (protocol B) indirectly immobilized by a non-adsorptive process; (b) ELISA with antigen-antibody preincubation in solution (p-ELISA) in which GADA were reacted first with Trx-GAD65 (protocol C) or biotin-Trx-GAD (protocol D) and the free antigen was determined by conventional ELISA. The results obtained with 42 newly diagnosed IDDM patients and 30 normal individuals were as follows: RBA had 79% sensitivity (percentage of IDDM patients detected) and 97% specificity (100% minus the percentage of false positives). c-ELISA showed low sensitivity (36 and 50%, respectively for protocols A and B), and high specificity (100 and 97%, respectively). p-ELISA were highly-sensitive (74 and 79%, respectively) and specific (97 and 93% for protocols C and D, respectively). Thus, protocols C and D had a performance similar to the reference method. The results reported here provide the basis for simple, highly-sensitive, specific, and widely-applicable tests for GADA that eliminate many of the drawbacks of the radioactive methods.

  16. Enzyme-linked immunosorbent assay using recombinant SAG1 antigen to detect Toxoplasma gondii-specific immunoglobulin G antibodies in human sera and saliva.

    PubMed

    Chahed Bel-Ochi, Nouha; Bouratbine, Aïda; Mousli, Mohamed

    2013-04-01

    Serologic detection of Toxoplasma gondii IgG antibodies is widely accepted as a means to determine immune status and susceptibility to Toxoplasma infection during pregnancy. However, current commercial kits present some drawbacks, such as a requirement for whole-parasite antigen preparation or interassay variability. To address these problems, the purpose of this study was to produce a whole sequence of the recombinant T. gondii SAG1 antigen (rSAG1) to assess its diagnostic performance in Toxoplasma IgG screening and to explore a saliva-based method as a noninvasive alternative to serum-based testing. rSAG1 was expressed in recombinant bacteria as inclusion bodies, purified through one-step affinity chromatography, and refolded in native form by dialysis. A large amount was obtained, and the specific antigen immunoreactivity was confirmed by immunoblotting. Two rSAG1-based enzyme-linked immunosorbent assays (ELISAs) applied to paired serum and saliva samples were designed. The rSAG1-based ELISA evaluation consisted of testing intrinsic sensitivity and specificity of 49 serum samples from patients immune to toxoplasmosis and 42 serum samples from nonimmune controls identified by routinely used kits. To assess agreement between serum-based and saliva-based tests, the positive percent agreement (PPA) and negative percent agreement (NPA) between the 2 tests were estimated. The rSAG1 serum-based ELISA detected specific IgG with 100% sensitivity and specificity. The PPA and NPA between the serum-based and saliva-based tests varied according to the selected optical density threshold in saliva. Thus, for a selected cutoff of 0.14, the PPA was 100% and the NPA was 88.1%, whereas for a selected cutoff of 0.29, the PPA was 67.3% and the NPA was 100%.

  17. SARS hCoV papain-like protease is a unique Lys48 linkage-specific di-distributive deubiquitinating enzyme.

    PubMed

    Békés, Miklós; Rut, Wioletta; Kasperkiewicz, Paulina; Mulder, Monique P C; Ovaa, Huib; Drag, Marcin; Lima, Christopher D; Huang, Tony T

    2015-06-01

    Ubiquitin (Ub) and the Ub-like (Ubl) modifier interferon-stimulated gene 15 (ISG15) participate in the host defence of viral infections. Viruses, including the severe acute respiratory syndrome human coronavirus (SARS hCoV), have co-opted Ub-ISG15 conjugation pathways for their own advantage or have evolved effector proteins to counter pro-inflammatory properties of Ub-ISG15-conjugated host proteins. In the present study, we compare substrate specificities of the papain-like protease (PLpro) from the recently emerged Middle East respiratory syndrome (MERS) hCoV to the related protease from SARS, SARS PLpro. Through biochemical assays, we show that, similar to SARS PLpro, MERS PLpro is both a deubiquitinating (DUB) and a deISGylating enzyme. Further analysis of the intrinsic DUB activity of these viral proteases revealed unique differences between the recognition and cleavage specificities of polyUb chains. First, MERS PLpro shows broad linkage specificity for the cleavage of polyUb chains, whereas SARS PLpro prefers to cleave Lys48-linked polyUb chains. Secondly, MERS PLpro cleaves polyUb chains in a 'mono-distributive' manner (one Ub at a time) and SARS PLpro prefers to cleave Lys48-linked polyUb chains by sensing a di-Ub moiety as a minimal recognition element using a 'di-distributive' cleavage mechanism. The di-distributive cleavage mechanism for SARS PLpro appears to be uncommon among USP (Ub-specific protease)-family DUBs, as related USP family members from humans do not display such a mechanism. We propose that these intrinsic enzymatic differences between SARS and MERS PLpro will help to identify pro-inflammatory substrates of these viral DUBs and can guide in the design of therapeutics to combat infection by coronaviruses.

  18. Synthetic glycosylation of proteins using N-(beta-saccharide) iodoacetamides: applications in site-specific glycosylation and solid-phase enzymic oligosaccharide synthesis.

    PubMed Central

    Wong, S Y; Guile, G R; Dwek, R A; Arsequell, G

    1994-01-01

    A simple and efficient synthetic glycosylation method suitable for use in solid-phase enzymic oligosaccharide synthesis and site-specific glycosylation of recombinant proteins to produce defined glycoforms is described. This strategy utilizes N-(beta-saccharide) haloacetamides for attaching oligosaccharides specifically to cysteine residues of proteins in solution to form neoglycoproteins. The alkylation reaction was tested using N-(beta-chitotriose) bromoacetamide and an unprotected synthetic hexapeptide containing a single cysteine residue. The glycosylated product was confirmed by amino acid and hexosamine analyses as well as laser desorption mass spectrometry. Similarly N-(beta-chitotriose) iodoacetamide was covalently linked to non-reduced BSA to produce a defined glycoform of this protein. The specific attachment of chitotriose at the single cysteine residue in non-reduced serum albumin was suggested by Ellman's assay for free thiols. This was verified by amino acid sequencing of tryptic glycopeptide derived from this neoglycoprotein. Multiple sugar attachment was accomplished using fully reduced serum albumin as demonstrated by the formation of two neoglycoproteins using iodoacetamide derivatives of galactose beta 1-3-N-acetylgalactosamine (Gal beta 1-3GalNAc) and the major xylose/fucose-class plant-type oligosaccharide of horseradish peroxidase. These two neoglycoproteins with an average of 18-21 sugar residues attached were assayed positively for binding to peanut agglutinin and a sugar-specific anti-(horseradish peroxidase) monoclonal antibody YZ1/2.23 respectively. Sialylation of the neoglycoprotein containing Gal beta 1-3GalNAc was accomplished using alpha-2,3-sialyltransferase and radiolabelled CMP-N-acetylneuraminic acid. Significantly, glycan attachment using this conjugation method is reversible as demonstrated by the release of oligosaccharides from these two neoglycoproteins using hydrazinolysis. Therefore this method could provide invaluable

  19. Differentiation between human coronaviruses NL63 and 229E using a novel double-antibody sandwich enzyme-linked immunosorbent assay based on specific monoclonal antibodies.

    PubMed

    Sastre, Patricia; Dijkman, Ronald; Camuñas, Ana; Ruiz, Tamara; Jebbink, Maarten F; van der Hoek, Lia; Vela, Carmen; Rueda, Paloma

    2011-01-01

    Human coronaviruses (HCoVs) are responsible for respiratory tract infections ranging from common colds to severe acute respiratory syndrome. HCoV-NL63 and HCoV-229E are two of the four HCoVs that circulate worldwide and are close phylogenetic relatives. HCoV infections can lead to hospitalization of children, elderly individuals, and immunocompromised patients. Globally, approximately 5% of all upper and lower respiratory tract infections in hospitalized children are caused by HCoV-229E and HCoV-NL63. The latter virus has recently been associated with the childhood disease croup. Thus, differentiation between the two viruses is relevant for epidemiology studies. The aim of this study was to develop a double-antibody sandwich enzyme-linked immunosorbent assay (DAS-ELISA) as a potential tool for identification and differentiation between HCoV-NL63 and HCoV-229E. The nucleocapsid (N) proteins of HCoV-NL63 and HCoV-229E were expressed in an Escherichia coli system and used to immunize mice in order to obtain monoclonal antibodies (MAbs) specific for each virus. Three specific MAbs to HCoV-NL63, one MAb specific to HCoV-229E, and four MAbs that recognized both viruses were obtained. After their characterization, three MAbs were selected in order to develop a differential DAS-ELISA. The described assay could detect up to 3 ng/ml of N protein and 50 50% tissue culture infective doses/ml of virus stock. No cross-reactivity with other human coronaviruses or closely related animal coronaviruses was found. The newly developed DAS-ELISA was species specific, and therefore, it could be considered a potential tool for detection and differentiation of HCoV-NL63 and HCoV-229E infections.

  20. Development and implementation of a general compiler to produce specific programs for minicomputer control of physiological experiments in real time.

    PubMed

    Tzikoni, E; Feldman, S; Kedem, J

    1981-01-01

    A method for automatic generation of specific FORTRAN programs to control physiological experiments with a computer was developed. The general real-time software package is built into a high level language (MAB = MAcro Basic). From this package, the scientist can automatically generate for him/herself specific programs for controlling his own experiments by a simple procedure. The generated programs contain only the relevant code, adjusted dimensions of arrays, names of parameters, and formatting for printing of tables and graphics for the particular experiment. Therefore, the resulting program is efficient both in terms of memory utilization and in execution time.

  1. Au nanoparticles/hollow molybdenum disulfide microcubes based biosensor for microRNA-21 detection coupled with duplex-specific nuclease and enzyme signal amplification.

    PubMed

    Shuai, Hong-Lei; Huang, Ke-Jing; Chen, Ying-Xu; Fang, Lin-Xia; Jia, Meng-Pei

    2017-03-15

    An ultrasensitive electrochemical biosensor for detecting microRNAs is fabricated based on hollow molybdenum disulfide (MoS2) microcubes. Duplex-specific nuclease, enzyme and electrochemical-chemical-chemical redox cycling are used for signal amplification. Hollow MoS2 microcubes constructed by ultrathin nanosheets are synthesized by a facile template-assisted strategy and used as supporting substrate. For biosensor assembling, biotinylated ssDNA capture probes are first immobilized on Au nanoparticles (AuNPs)/MoS2 modified electrode in order to combine with streptavidin-conjugated alkaline phosphatase (SA-ALP). When capture probes hybridize with miRNAs, duplex-specific nuclease cleaves the formative duplexes. At the moment, the biotin group strips from the electrode surface and SA-ALP is incapacitated to attach onto electrode. Then, ascorbic acids induce the electrochemical-chemical-chemical redox cycling to produce electrochemical response in the presence of ferrocene methanol and tris (2-carboxyethyl) phosphine. Under optimum conditions, the proposed biosensor shows a good linear relationship between the current variation and logarithm of the microRNAs concentration ranging from 0.1fM to 0.1pM with a detection limit of 0.086fM (S/N=3). Furthermore, the biosensor is successfully applied to detect target miRNA-21 in human serum samples.

  2. Nucleoprotein-based indirect enzyme-linked immunosorbent assay (indirect ELISA) for detecting antibodies specific to Ebola virus and Marbug virus.

    PubMed

    Huang, Yi; Zhu, Youjie; Yang, Mengshi; Zhang, Zhenqing; Song, Donglin; Yuan, Zhiming

    2014-12-01

    Full-length nucleoproteins from Ebola and Marburg viruses were expressed as His-tagged recombinant proteins in Escherichia coli and nucleoprotein-based enzyme-linked immunosorbent assays (ELISAs) were established for the detection of antibodies specific to Ebola and Marburg viruses. The ELISAs were evaluated by testing antisera collected from rabbit immunized with Ebola and Marburg virus nucleoproteins. Although little cross-reactivity of antibodies was observed in anti-Ebola virus nucleoprotein rabbit antisera, the highest reactions to immunoglobulin G (IgG) were uniformly detected against the nucleoprotein antigens of homologous viruses. We further evaluated the ELISA's ability to detect antibodies to Ebola and Marburg viruses using human sera samples collected from individuals passing through the Guangdong port of entry. With a threshold set at the mean plus three standard deviations of average optical densities of sera tested, the ELISA systems using these two recombinant nucleoproteins have good sensitivity and specificity. These results demonstrate the usefulness of ELISA for diagnostics as well as ecological and serosurvey studies of Ebola and Marburg virus infection.

  3. Phloem-Specific Expression of Yang Cycle Genes and Identification of Novel Yang Cycle Enzymes in Plantago and Arabidopsis[C][W

    PubMed Central

    Pommerrenig, Benjamin; Feussner, Kirstin; Zierer, Wolfgang; Rabinovych, Valentyna; Klebl, Franz; Feussner, Ivo; Sauer, Norbert

    2011-01-01

    The 5-methylthioadenosine (MTA) or Yang cycle is a set of reactions that recycle MTA to Met. In plants, MTA is a byproduct of polyamine, ethylene, and nicotianamine biosynthesis. Vascular transcriptome analyses revealed phloem-specific expression of the Yang cycle gene 5-METHYLTHIORIBOSE KINASE1 (MTK1) in Plantago major and Arabidopsis thaliana. As Arabidopsis has only a single MTK gene, we hypothesized that the expression of other Yang cycle genes might also be vascular specific. Reporter gene studies and quantitative analyses of mRNA levels for all Yang cycle genes confirmed this hypothesis for Arabidopsis and Plantago. This includes the Yang cycle genes 5-METHYLTHIORIBOSE-1-PHOSPHATE ISOMERASE1 and DEHYDRATASE-ENOLASE-PHOSPHATASE-COMPLEX1. We show that these two enzymes are sufficient for the conversion of methylthioribose-1-phosphate to 1,2-dihydroxy-3-keto-5-methylthiopentene. In bacteria, fungi, and animals, the same conversion is catalyzed in three to four separate enzymatic steps. Furthermore, comparative analyses of vascular and nonvascular metabolites identified Met, S-adenosyl Met, and MTA preferentially or almost exclusively in the vascular tissue. Our data represent a comprehensive characterization of the Yang cycle in higher plants and demonstrate that the Yang cycle works primarily in the vasculature. Finally, expression analyses of polyamine biosynthetic genes suggest that the Yang cycle in leaves recycles MTA derived primarily from polyamine biosynthesis. PMID:21540433

  4. An Application of Outer Membrane Protein P6-Specific Enzyme-Linked Immunosorbent Assay for Detection of Haemophilus influenzae in Middle Ear Fluids and Nasopharyngeal Secretions

    PubMed Central

    Hotomi, Muneki; Togawa, Akihisa; Kono, Masamitsu; Sugita, Gen; Sugita, Rinya; Fujimaki, Yutaka; Kamide, Yosuke; Uchizono, Akihiro; Kanesada, Keiko; Sawada, Shoichi; Okitsu, Naohiro; Masuda, Hisayo; Tanaka, Hideaki; Tanaka, Yumi; Yamanaka, Noboru

    2013-01-01

    An enzyme-linked immunosorbent assay specific to outer membrane protein P6 (P6-ELISA) was applied for detecting Haemophilus influenzae in middle ear fluids (MEFs) from acute otitis media (AOM) patients and in nasopharyngeal secretions (NPSs) from acute rhinosinusitis patients. P6-ELISA had a sensitivity of 83.3% for MEFs and 71.5% for NPSs and a specificity of 85.6% for MEFs and 92.5% for NPSs, respectively. Real-time PCR exhibited significant differences in the number of ompP1 gene copies among samples determined by P6-ELISA to be positive and negative for H. influenzae. However, because the P6-ELISA test has the reactivity in Haemophilus species include two commensals H. haemolyticus and H. parainfluenzae, it is thus a weak method in order to detect only NTHi correctly. Consequently, diagnosis using the P6-ELISA should be based on an overall evaluation, including the results of other related examinations and clinical symptoms to prevent misleading conclusions in clinical setting. PMID:24015192

  5. Diagnosis of amebic dysentery by detection of Entamoeba histolytica fecal antigen by an invasive strain-specific, monoclonal antibody-based enzyme-linked immunosorbent assay.

    PubMed Central

    Gonzalez-Ruiz, A; Haque, R; Rehman, T; Aguirre, A; Hall, A; Guhl, F; Warhurst, D C; Miles, M A

    1994-01-01

    An invasive strain-specific monoclonal antibody against Entamoeba histolytica has been used in a capture enzyme-linked immunosorbent assay (ELISA) for the detection of invasive E. histolytica fecal antigen in clinical specimens and for the diagnosis of amebic dysentery in patients from Bangladesh. The fecal antigen capture ELISA (FAC-ELISA) did not cross-react with other parasite species in the clinical specimens or with noninvasive E. histolytica present in those specimens and in experimentally seeded stools. The limit of detection of the assay for invasive E. histolytica crude antigen diluted in phosphate-buffered saline or in stools was 0.58 and 3.9 micrograms/ml, respectively, which is the equivalent of approximately 72 and 487 E. histolytica trophozoites per well, respectively. The sensitivity, specificity, and efficiency of the FAC-ELISA were 87, 100, and 98%, respectively, for the detection of invasive E. histolytica antigens and 100, 100, and 100%, respectively, for the diagnosis of amebic dysentery. The FAC-ELISA is a potential alternative for the field diagnosis of amebic dysentery and for epidemiological studies to define the distribution of invasive E. histolytica. PMID:8027351

  6. Human fibroblast-derived molecules as antigens in enzyme-linked immunosorbent assay for coeliac disease-specific IgA.

    PubMed

    Marttinen, A; Sulkanen, S; Mäki, M

    1997-02-01

    We have recently shown that cultured human fibroblasts synthesize and secrete protein molecules that bind to IgA-class anti-reticulin and anti-endomysium antibodies but not to anti-gliadin antibodies in coeliac disease patient sera. In the present report, we describe a reproducible method for purification of these antigen molecules from fibroblast culture medium. Using reversed-phase chromatography as the final purification step, four different protein molecules reacting with coeliac disease patient sera IgA were obtained. In enzyme-linked immunosorbent assay (ELISA) for coeliac disease-specific IgA, a mixture of 0.5 microgram of the four reversed-phase-separated molecules was used as antigen. The optical density values in ELISA of the sera from newly diagnosed coeliac disease patients (n = 34) were 0.740-3.400 (mean 1.830) and in control patients (n = 66) 0.090-0.850 (mean 0.320). Using an arbitrary cut-off level of 0.700, the sensitivity of the present autoantibody test was 100%, specificity 91% and positive predictive value 85%. Our identified autoantigens may generate the production of the classical tissue antibodies, known as anti-reticulin and anti-endomysium antibodies, and may be used as antigen in an immunoassay for the antibodies.

  7. Mutagenesis of a specificity-determining residue in tyrosine hydroxylase establishes that the enzyme is a robust phenylalanine hydroxylase but a fragile tyrosine hydroxylase.

    PubMed

    Daubner, S Colette; Avila, Audrey; Bailey, Johnathan O; Barrera, Dimitrios; Bermudez, Jaclyn Y; Giles, David H; Khan, Crystal A; Shaheen, Noel; Thompson, Janie Womac; Vasquez, Jessica; Oxley, Susan P; Fitzpatrick, Paul F

    2013-02-26

    The aromatic amino acid hydroxylases tyrosine hydroxylase (TyrH) and phenylalanine hydroxylase (PheH) have essentially identical active sites; however, PheH is nearly incapable of hydroxylating tyrosine, while TyrH can readily hydroxylate both tyrosine and phenylalanine. Previous studies have indicated that Asp425 of TyrH is important in determining the substrate specificity of that enzyme [Daubner, S. C., Melendez, J., and Fitzpatrick, P. F. (2000) Biochemistry 39, 9652-9661]. Alanine-scanning mutagenesis of amino acids 423-427, a mobile loop containing Asp425, shows that only mutagenesis of Asp425 alters the activity of the enzyme significantly. Saturation mutagenesis of Asp425 results in large (up to 10(4)) decreases in the V(max) and V(max)/K(tyr) values for tyrosine hydroxylation, but only small decreases or even increases in the V(max) and V(max)/K(phe) values for phenylalanine hydroxylation. The decrease in the tyrosine hydroxylation activity of the mutant proteins is due to an uncoupling of tetrahydropterin oxidation from amino acid hydroxylation with tyrosine as the amino acid substrate. In contrast, with the exception of the D425W mutant, the extent of coupling of tetrahydropterin oxidation and amino acid hydroxylation is unaffected or increases with phenylalanine as the amino acid substrate. The decrease in the V(max) value with tyrosine as the substrate shows a negative correlation with the hydrophobicity of the amino acid residue at position 425. The results are consistent with a critical role of Asp425 being to prevent a hydrophobic interaction that results in a restricted active site in which hydroxylation of tyrosine does not occur.

  8. Enzyme assays.

    PubMed

    Reymond, Jean-Louis; Fluxà, Viviana S; Maillard, Noélie

    2009-01-07

    Enzyme assays are analytical tools to visualize enzyme activities. In recent years a large variety of enzyme assays have been developed to assist the discovery and optimization of industrial enzymes, in particular for "white biotechnology" where selective enzymes are used with great success for economically viable, mild and environmentally benign production processes. The present article highlights the aspects of fluorogenic and chromogenic substrates, sensors, and enzyme fingerprinting, which are our particular areas of interest.

  9. ASSIST - THE ABSTRACT SEMI-MARKOV SPECIFICATION INTERFACE TO THE SURE TOOL PROGRAM (VAX VMS VERSION)

    NASA Technical Reports Server (NTRS)

    Johnson, S. C.

    1994-01-01

    ASSIST, the Abstract Semi-Markov Specification Interface to the SURE Tool program, is an interface that will enable reliability engineers to accurately design large semi-Markov models. The user describes the failure behavior of a fault-tolerant computer system in an abstract, high-level language. The ASSIST program then automatically generates a corresponding semi-Markov model. The abstract language allows efficient description of large, complex systems; a one-page ASSIST-language description may result in a semi-Markov model with thousands of states and transitions. The ASSIST program also includes model-reduction techniques to facilitate efficient modeling of large systems. Instead of listing the individual states of the Markov model, reliability engineers can specify the rules governing the behavior of a system, and these are used to automatically generate the model. ASSIST reads an input file describing the failure behavior of a system in an abstract language and generates a Markov model in the format needed for input to SURE, the semi-Markov Unreliability Range Evaluator program, and PAWS/STEM, the Pade Approximation with Scaling program and Scaled Taylor Exponential Matrix. A Markov model consists of a number of system states and transitions between them. Each state in the model represents a possible state of the system in terms of which components have failed, which ones have been removed, etc. Within ASSIST, each state is defined by a state vector, where each element of the vector takes on an integer value within a defined range. An element can represent any meaningful characteristic, such as the number of working components of one type in the system, or the number of faulty components of another type in use. Statements representing transitions between states in the model have three parts: a condition expression, a destination expression, and a rate expression. The first expression is a Boolean expression describing the state space variable values of states

  10. ASSIST - THE ABSTRACT SEMI-MARKOV SPECIFICATION INTERFACE TO THE SURE TOOL PROGRAM (SUN VERSION)

    NASA Technical Reports Server (NTRS)

    Johnson, S. C.

    1994-01-01

    ASSIST, the Abstract Semi-Markov Specification Interface to the SURE Tool program, is an interface that will enable reliability engineers to accurately design large semi-Markov models. The user describes the failure behavior of a fault-tolerant computer system in an abstract, high-level language. The ASSIST program then automatically generates a corresponding semi-Markov model. The abstract language allows efficient description of large, complex systems; a one-page ASSIST-language description may result in a semi-Markov model with thousands of states and transitions. The ASSIST program also includes model-reduction techniques to facilitate efficient modeling of large systems. Instead of listing the individual states of the Markov model, reliability engineers can specify the rules governing the behavior of a system, and these are used to automatically generate the model. ASSIST reads an input file describing the failure behavior of a system in an abstract language and generates a Markov model in the format needed for input to SURE, the semi-Markov Unreliability Range Evaluator program, and PAWS/STEM, the Pade Approximation with Scaling program and Scaled Taylor Exponential Matrix. A Markov model consists of a number of system states and transitions between them. Each state in the model represents a possible state of the system in terms of which components have failed, which ones have been removed, etc. Within ASSIST, each state is defined by a state vector, where each element of the vector takes on an integer value within a defined range. An element can represent any meaningful characteristic, such as the number of working components of one type in the system, or the number of faulty components of another type in use. Statements representing transitions between states in the model have three parts: a condition expression, a destination expression, and a rate expression. The first expression is a Boolean expression describing the state space variable values of states

  11. Dendritic cell maturation: functional specialization through signaling specificity and transcriptional programming

    PubMed Central

    Dalod, Marc; Chelbi, Rabie; Malissen, Bernard; Lawrence, Toby

    2014-01-01

    Dendritic cells (DC) are key regulators of both protective immune responses and tolerance to self-antigens. Soon after their discovery in lymphoid tissues by Steinman and Cohn, as cells with the unique ability to prime naïve antigen-specific T cells, it was realized that DC can exist in at least two distinctive states characterized by morphological, phenotypic and functional changes—this led to the description of DC maturation. It is now well appreciated that there are several subsets of DC in both lymphoid and non-lymphoid tissues of mammals, and these cells show remarkable functional specialization and specificity in their roles in tolerance and immunity. This review will focus on the specific characteristics of DC subsets and how their functional specialization may be regulated by distinctive gene expression programs and signaling responses in both steady-state and in the context of inflammation. In particular, we will highlight the common and distinctive genes and signaling pathways that are associated with the functional maturation of DC subsets. PMID:24737868

  12. Comprehensive Tissue-Specific Transcriptome Analysis Reveals Distinct Regulatory Programs during Early Tomato Fruit Development.

    PubMed

    Pattison, Richard J; Csukasi, Fabiana; Zheng, Yi; Fei, Zhangjun; van der Knaap, Esther; Catalá, Carmen

    2015-08-01

    Fruit formation and early development involve a range of physiological and morphological transformations of the various constituent tissues of the ovary. These developmental changes vary considerably according to tissue type, but molecular analyses at an organ-wide level inevitably obscure many tissue-specific phenomena. We used laser-capture microdissection coupled to high-throughput RNA sequencing to analyze the transcriptome of ovaries and fruit tissues of the wild tomato species Solanum pimpinellifolium. This laser-capture microdissection-high-throughput RNA sequencing approach allowed quantitative global profiling of gene expression at previously unobtainable levels of spatial resolution, revealing numerous contrasting transcriptome profiles and uncovering rare and cell type-specific transcripts. Coexpressed gene clusters linked specific tissues and stages to major transcriptional changes underlying the ovary-to-fruit transition and provided evidence of regulatory modules related to cell division, photosynthesis, and auxin transport in internal fruit tissues, together with parallel specialization of the pericarp transcriptome in stress responses and secondary metabolism. Analysis of transcription factor expression and regulatory motifs indicated putative gene regulatory modules that may regulate the development of different tissues and hormonal processes. Major alterations in the expression of hormone metabolic and signaling components illustrate the complex hormonal control underpinning fruit formation, with intricate spatiotemporal variations suggesting separate regulatory programs.

  13. Pancancer modelling predicts the context-specific impact of somatic mutations on transcriptional programs

    PubMed Central

    Osmanbeyoglu, Hatice U.; Toska, Eneda; Chan, Carmen; Baselga, José; Leslie, Christina S.

    2017-01-01

    Pancancer studies have identified many genes that are frequently somatically altered across multiple tumour types, suggesting that pathway-targeted therapies can be deployed across diverse cancers. However, the same ‘actionable mutation' impacts distinct context-specific gene regulatory programs and signalling networks—and interacts with different genetic backgrounds of co-occurring alterations—in different cancers. Here we apply a computational strategy for integrating parallel (phospho)proteomic and mRNA sequencing data across 12 TCGA tumour data sets to interpret the context-specific impact of somatic alterations in terms of functional signatures such as (phospho)protein and transcription factor (TF) activities. Our analysis predicts distinct dysregulated transcriptional regulators downstream of somatic alterations in different cancers, and we validate the context-specific differential activity of TFs associated to mutant PIK3CA in isogenic cancer cell line models. These results have implications for the pancancer use of targeted drugs and potentially for the design of combination therapies. PMID:28139702

  14. Enzyme-linked immunospot assay for detection of human respiratory syncytial virus f protein-specific gamma interferon-producing T cells.

    PubMed

    Patton, Kathryn; Aslam, Shahin; Lin, Jim; Yu, Li; Lambert, Stacie; Dawes, Glenn; Esser, Mark T; Woo, Jennifer; Janetzki, Sylvia; Cherukuri, Anu

    2014-05-01

    Respiratory syncytial virus (RSV) causes significant disease in elderly adults, and we have previously reported that individuals 65 years of age and older have reduced RSV F protein-specific gamma interferon (IFN-γ)-producing T cells compared to healthy younger adults. To measure RSV F-specific memory T cell responses in the elderly following infection or vaccination, we optimized and qualified an IFN-γ enzyme-linked immunospot (ELISPOT) assay. Since peripheral blood mononuclear cells (PBMC) from the elderly could be more fragile, we established optimal cryopreservation techniques and minimal viability acceptance criteria. The number of cells per well, types and concentrations of stimulation antigens, and incubation times were evaluated to maximize assay sensitivity and precision. The optimized assay uses 300,000 cells/well, 2 μg/ml of an RSV F peptide pool (RSV Fpp), and incubation for 22 ± 2 h in serum-free CTL-Test medium. The assay was qualified by 3 analysts using 3 RSV F-responding donor PBMC samples (high, medium, and low responders) tested on 5 different assay days. The assay sensitivity or limit of detection (LOD) was determined to be 21 spot-forming cells (SFC) per 10(6) PBMC, and the lower limit of quantitation (LLOQ) was estimated to be 63 SFC/10(6) PBMC. The intra- and interassay percent coefficients of variation (CV) were <10.5% and <31%, respectively. The results of the qualification study demonstrate that a robust, precise, and sensitive IFN-γ ELISPOT assay has been developed that is fit for measuring RSV F-specific IFN-γ T cell responses in subjects enrolled in a vaccine clinical trial or in epidemiology studies.

  15. Sensitive and Specific Serodiagnosis of Lyme Disease by Enzyme-Linked Immunosorbent Assay with a Peptide Based on an Immunodominant Conserved Region of Borrelia burgdorferi VlsE

    PubMed Central

    Liang, Fang Ting; Steere, Allen C.; Marques, Adriana R.; Johnson, Barbara J. B.; Miller, James N.; Philipp, Mario T.

    1999-01-01

    VlsE, the variable surface antigen of Borrelia burgdorferi, contains an immunodominant conserved region named IR6. In the present study, the diagnostic performance of a peptide enzyme-linked immunosorbent assay (ELISA) based on a 26-mer synthetic peptide (C6) with the IR6 sequence was explored. Sensitivity was assessed with serum samples (n = 210) collected from patients with clinically defined Lyme disease at the acute (early localized or early disseminated disease), convalescent, or late disease phase. The sensitivities for acute-, convalescent-, and late-phase specimens were 74% (29 of 39), 85 to 90% (34 of 40 to 35 of 39), and 100% (59 of 59), respectively. Serum specimens from early neuroborreliosis patients were 95% positive (19 of 20), and those from an additional group of patients with posttreatment Lyme disease syndrome yielded a sensitivity of 62% (8 of 13). To assess the specificity of the peptide ELISA, 77 serum samples from patients with other spirochetal or chronic infections, autoimmune diseases, or neurologic diseases and 99 serum specimens from hospitalized patients in an area where Lyme disease is not endemic were examined. Only two potential false positives from the hospitalized patients were found, and the overall specificity was 99% (174 of 176). Precision, which was assessed with a panel of positive and negative serum specimens arranged in blinded duplicates, was 100%. Four serum samples with very high anti-OspA antibody titers obtained from four monkeys given the OspA vaccine did not react with the C6 peptide. This simple, sensitive, specific, and precise ELISA may contribute to alleviate some of the remaining problems in Lyme disease serodiagnosis. Because of its synthetic peptide base, it will be inexpensive to manufacture. It also will be applicable to serum specimens from OspA-vaccinated subjects. PMID:10565920

  16. [Reconstruction of muscle glycogen phosphorylase b from an apoenzyme and pyridoxal-5'-phosphate and its analogs. Interaction of apophosphorylase and the reconstructed enzyme with specific ligands].

    PubMed

    Chebotareva, N A; Sugrobova, N P; Bulanova, L N; Poznanskaia, A A; Kurganov, B I; Gunar, V I

    1995-12-01

    Sedimentation methods were used to study the effects of modification of the pyridoxal-5'-phosphate (PLP) molecule at the 5th position on the affinity of reconstituted muscle glycogen phosphorylase b for the substrate (glycogen) and the allosteric inhibitor (FMN) as well as on the enzyme capacity to association induced by AMP. Reconstituted phosphorylase b was obtained with PLP analogs containing at the 5th position -CH2-CH2-COOH (analog I), trans-CH=CH-COOH (analog II) or -C identical to COOH (analog III) residues. Reconstitution of phosphorylase b is accompanied by the recovery of the enzyme quaternary structure. Phosphorylase b reconstituted with PLP or analogs I, II and III is not distinguished practically from the native enzyme in its affinity for glycogen. Substitution of the native coenzyme in the phosphorylase molecule with any tested PLP analog leads to lower enzyme affinity for FMN. Microscopic dissociation constants of the FMN-enzyme complexes increase in the following order: enzyme.I < enzyme.II < enzyme.III. Phosphorylase b reconstituted with analogs I, II and III differs substantially from the native enzyme in its capacity to association in the presence of 1 mM AMP: the reconstituted enzyme is represented practically by only the tetrameric form.

  17. Structures of enzyme-intermediate complexes of yeast Nit2: insights into its catalytic mechanism and different substrate specificity compared with mammalian Nit2.

    PubMed

    Liu, Hejun; Gao, Yongxiang; Zhang, Mengying; Qiu, Xiaoting; Cooper, Arthur J L; Niu, Liwen; Teng, Maikun

    2013-08-01

    The Nit (nitrilase-like) protein subfamily constitutes branch 10 of the nitrilase superfamily. Nit proteins are widely distributed in nature. Mammals possess two members of the Nit subfamily, namely Nit1 and Nit2. Based on sequence similarity, yeast Nit2 (yNit2) is a homologue of mouse Nit1, a tumour-suppressor protein whose substrate specificity is not yet known. Previous studies have shown that mammalian Nit2 (also a putative tumour suppressor) is identical to ω-amidase, an enzyme that catalyzes the hydrolysis of α-ketoglutaramate (α-KGM) and α-ketosuccinamate (α-KSM) to α-ketoglutarate (α-KG) and oxaloacetate (OA), respectively. In the present study, crystal structures of wild-type (WT) yNit2 and of WT yNit2 in complex with α-KG and with OA were determined. In addition, the crystal structure of the C169S mutant of yNit2 (yNit2-C169S) in complex with an endogenous molecule of unknown structure was also solved. Analysis of the structures revealed that α-KG and OA are covalently bound to Cys169 by the formation of a thioester bond between the sulfhydryl group of the cysteine residue and the γ-carboxyl group of α-KG or the β-carboxyl group of OA, reflecting the presumed reaction intermediates. However, an enzymatic assay suggests that α-KGM is a relatively poor substrate of yNit2. Finally, a ligand was found in the active site of yNit2-C169S that may be a natural substrate of yNit2 or an endogenous regulator of enzyme activity. These crystallographic analyses provide information on the mode of substrate/ligand binding at the active site of yNit2 and insights into the catalytic mechanism. These findings suggest that yNit2 may have broad biological roles in yeast, especially in regard to nitrogen homeostasis, and provide a framework for the elucidation of the substrate specificity and biological role of mammalian Nit1.

  18. Identifying patient, community and program specific barriers to free specialty care utilization by uninsured patients in East Baltimore.

    PubMed

    Handy, Catherine; Ma, Sai; Block, Lauren; de la Torre, Desiree; Langley, Anne; Cook, Barbara

    2013-05-01

    Uninsured individuals face multiple barriers to accessing specialty care. The Access Partnership (TAP) offers free specialty care and care coordination to qualified uninsured patients at an urban academic medical center for a small program entry fee (waived for financial hardship). In the program's first year, 104 eligible patients (31%) did not enroll. To understand why, we investigated demographic, referral, personal, and program-specific factors. After adjusting for age, gender, and ZIP code, diagnostic and therapeutic referrals were more likely to be completed than ancillary referrals (OR=8.56, p=.001; OR 3.53, p=.03). There was no difference between pain related and ancillary referrals (OR=2.80, p=.139). Eighteen patients were surveyed and reported program and patient-specific barriers. While removing costs is necessary to improve access to specialty care for underserved patients, it is insufficient. Improving communication from program coordinators and enrollment strategies may help to improve utilization of free care programs by the uninsured.

  19. A Hybrid Method to Estimate Specific Differential Phase and Rainfall With Linear Programming and Physics Constraints

    SciTech Connect

    Huang, Hao; Zhang, Guifu; Zhao, Kun; Giangrande, Scott E.

    2016-10-20

    A hybrid method of combining linear programming (LP) and physical constraints is developed to estimate specific differential phase (KDP) and to improve rain estimation. Moreover, the hybrid KDP estimator and the existing estimators of LP, least squares fitting, and a self-consistent relation of polarimetric radar variables are evaluated and compared using simulated data. Our simulation results indicate the new estimator's superiority, particularly in regions where backscattering phase (δhv) dominates. Further, a quantitative comparison between auto-weather-station rain-gauge observations and KDP-based radar rain estimates for a Meiyu event also demonstrate the superiority of the hybrid KDP estimator over existing methods.

  20. An across-the-bay flight support structure designed specifically for the Spartan program

    NASA Technical Reports Server (NTRS)

    Rast, J. J.

    1986-01-01

    The preliminary design of an across-the-bay Flight Support Structure optimized to the specific requirements of the Spartan program has been completed. Although approximately 15 percent heavier than the initial goal, preliminary analysis indicate very low stress levels and significant reduction in weight should be possible in the final design. This structure is capable of supporting two Spartan 200-class spacecraft or attached payloads of equivalent total weight. The inherent structural efficiency of monocoque type construction is utilized. The minimization of fabrication and assembly costs and provision for simple and versatile payload interfacing where given significant considerations. Modularity of this structure allows it to be readily adapted for use as the primary structure of an Aries-class Spartan free-flyer utilizing the Orbit trunning latch mechanisms, thus eliminating the need for a flight support structure.

  1. Legal and definitional issues affecting the identification and education of adults with specific learning disabilities in adult education programs.

    PubMed

    Taymans, Juliana M

    2012-01-01

    Although the exact prevalence is not determined, a noticeable subset of individuals who enroll in adult education and training programs have either diagnosed or undiagnosed specific learning disabilities (SLD). Understanding SLD is important basic information for adult educators to inform program policies as well as determine effective instructional practices. This article discusses the development of definitions of SLD and current agreement on the nature of SLD relevant to working with adults. It concludes with implications for adult education programs.

  2. Promoting Health-Related Fitness for Elementary Students with Intellectual Disabilities through a Specifically Designed Activity Program

    ERIC Educational Resources Information Center

    Davis, Kathryn; Zhang, Guili; Hodson, Patricia

    2011-01-01

    The Motivate, Adapt, and Play Program was specifically designed as an adapted physical activity (PA) program for students with intellectual disabilities (ID) to meet required school PA policies to combat childhood obesity. The policies commonly require a minimum of 30 min of PA per school day. A study was undertaken to test the efficacy of the…

  3. What Did I Do? A Scenario-Based Program To Assist Specific Learning Disabled Adolescents in Understanding Legal Issues.

    ERIC Educational Resources Information Center

    McDougall, Donna M.

    This practicum was designed to train eight adolescents with specific learning disabilities (SLD) about their legal rights and responsibilities, through a scenario-based program presented in the classroom as part of a transition program. The practicum involved the development of 22 scenarios, a pretest and posttest, and discussions and role-playing…

  4. Alcohol-Specific Parenting within a Cluster-Randomized Effectiveness Trial of a Swedish Primary Prevention Program

    ERIC Educational Resources Information Center

    Strandberg, Anna K.; Bodin, Maria C.

    2011-01-01

    Purpose: Within the framework of an ongoing cluster-randomized effectiveness trial of a parental prevention program, the aim of the present study is to investigate attitudes towards under-age drinking and use of program components, i.e. alcohol-specific parenting behaviors, in parents who did and did not take part in the programme.…

  5. Discovering rules for protein-ligand specificity using support vector inductive logic programming.

    PubMed

    Kelley, Lawrence A; Shrimpton, Paul J; Muggleton, Stephen H; Sternberg, Michael J E

    2009-09-01

    Structural genomics initiatives are rapidly generating vast numbers of protein structures. Comparative modelling is also capable of producing accurate structural models for many protein sequences. However, for many of the known structures, functions are not yet determined, and in many modelling tasks, an accurate structural model does not necessarily tell us about function. Thus, there is a pressing need for high-throughput methods for determining function from structure. The spatial arrangement of key amino acids in a folded protein, on the surface or buried in clefts, is often the determinants of its biological function. A central aim of molecular biology is to understand the relationship between such substructures or surfaces and biological function, leading both to function prediction and to function design. We present a new general method for discovering the features of binding pockets that confer specificity for particular ligands. Using a recently developed machine-learning technique which couples the rule-discovery approach of inductive logic programming with the statistical learning power of support vector machines, we are able to discriminate, with high precision (90%) and recall (86%) between pockets that bind FAD and those that bind NAD on a large benchmark set given only the geometry and composition of the backbone of the binding pocket without the use of docking. In addition, we learn rules governing this specificity which can feed into protein functional design protocols. An analysis of the rules found suggests that key features of the binding pocket may be tied to conformational freedom in the ligand. The representation is sufficiently general to be applicable to any discriminatory binding problem. All programs and data sets are freely available to non-commercial users at http://www.sbg.bio.ic.ac.uk/svilp_ligand/.

  6. ENCOMPASS: A SAGA based environment for the compositon of programs and specifications, appendix A

    NASA Technical Reports Server (NTRS)

    Terwilliger, Robert B.; Campbell, Roy H.

    1985-01-01

    ENCOMPASS is an example integrated software engineering environment being constructed by the SAGA project. ENCOMPASS supports the specification, design, construction and maintenance of efficient, validated, and verified programs in a modular programming language. The life cycle paradigm, schema of software configurations, and hierarchical library structure used by ENCOMPASS is presented. In ENCOMPASS, the software life cycle is viewed as a sequence of developments, each of which reuses components from the previous ones. Each development proceeds through the phases planning, requirements definition, validation, design, implementation, and system integration. The components in a software system are modeled as entities which have relationships between them. An entity may have different versions and different views of the same project are allowed. The simple entities supported by ENCOMPASS may be combined into modules which may be collected into projects. ENCOMPASS supports multiple programmers and projects using a hierarchical library system containing a workspace for each programmer; a project library for each project, and a global library common to all projects.

  7. Atmospheric Radiation Measurement (ARM) Data from Specific Instruments Used in the ARM Program

    DOE Data Explorer

    ARM is known for its comprehensive set of world-class, and in some cases, unique, instruments available for use by the global scientific community. In addition to the ARM instruments, the ARM Climate Research Facility identifies and acquires a wide variety of data including model, satellite, and surface data, from "external instruments," to augment the data being generated within the program. External instruments belong to organizations that are outside of the ARM Program. Field campaign instruments are another source of data used to augment routine observations. The huge archive of ARM data can be organized by instrument categories into twelve "collections:" Aerosols, Airborne Observations, Atmospheric Carbon, Atmospheric Profiling, Cloud Properties, Derived Quantities and Models, Ocean Observations, Radiometric, Satellite Observations, Surface Meteorology, Surface/Subsurface Properties, and Other. Clicking on one of the instrument categories leads to a page that breaks that category down into sub-categories. For example, "Atmospheric Profiling" is broken down into ARM instruments (with 11 subsets), External Instruments (with 6 subsets), and Field Campaign Instruments (with 42 subsets). Each of the subset links, in turn, leads to detailed information pages and links to specific data streams. Users will be requested to create a password, but the data files are free for viewing and downloading.

  8. Enzyme-linked immunosorbent assay (ELISA) using a specific monoclonal antibody as a new tool to detect Sudan dyes and Para red.

    PubMed

    Ju, Chunmei; Tang, Yong; Fan, Huiying; Chen, Jinding

    2008-07-28

    To set up an immunoassay-based method to detect Sudan dyes and Para red, we generated a monoclonal antibody (Mab) using a specially designed carboxyl derivative of Sudan I (CSD I) as the immunogen. CSD I was synthesized by azocoupling reaction using 2-naphthol and diazotised 4-aminobenzoic acid. The antibody was obtained from a hybridoma, which was derived from the fusion of the mouse myeloma SP2/0 cells and the splenocytes from the mice immunized with the CSD I-bovine serum albumin (BSA) conjugate. In addition, we showed that the Mab was highly specific for Sudan I, III and Para red. The limit of detection was approximately 0.01ngmL(-1) in phosphate-buffered saline (PBS) buffer and 0.5ngg(-1) in chilli tomato sauce. The recoveries of Sudan I, III and Para red for the chilli tomato sauce were from 84% to 99% and coefficients of variation were from 14.9% to 33.3%. Thus, the enzyme-linked immunosorbent assay (ELISA) method is a rapid and high throughput screening tool to detect Sudan dyes and Para red in food products.

  9. Antioxidant Defense Enzyme Genes and Asthma Susceptibility: Gender-Specific Effects and Heterogeneity in Gene-Gene Interactions between Pathogenetic Variants of the Disease

    PubMed Central

    Polonikov, Alexey V.; Ivanov, Vladimir P.; Bogomazov, Alexey D.; Freidin, Maxim B.; Illig, Thomas; Solodilova, Maria A.

    2014-01-01

    Oxidative stress resulting from an increased amount of reactive oxygen species and an imbalance between oxidants and antioxidants plays an important role in the pathogenesis of asthma. The present study tested the hypothesis that genetic susceptibility to allergic and nonallergic variants of asthma is determined by complex interactions between genes encoding antioxidant defense enzymes (ADE). We carried out a comprehensive analysis of the associations between adult asthma and 46 single nucleotide polymorphisms of 34 ADE genes and 12 other candidate genes of asthma in Russian population using set association analysis and multifactor dimensionality reduction approaches. We found for the first time epistatic interactions between ADE genes underlying asthma susceptibility and the genetic heterogeneity between allergic and nonallergic variants of the disease. We identified GSR (glutathione reductase) and PON2 (paraoxonase 2) as novel candidate genes for asthma susceptibility. We observed gender-specific effects of ADE genes on the risk of asthma. The results of the study demonstrate complexity and diversity of interactions between genes involved in oxidative stress underlying susceptibility to allergic and nonallergic asthma. PMID:24895604

  10. Podocyte-specific deletion of NDST1, a key enzyme in the sulfation of heparan sulfate glycosaminoglycans, leads to abnormalities in podocyte organization in vivo.

    PubMed

    Sugar, Terrel; Wassenhove-McCarthy, Deborah J; Esko, Jeffrey D; van Kuppevelt, Toin H; Holzman, Lawrence; McCarthy, Kevin J

    2014-02-01

    Heparan sulfate proteoglycans have been shown to modulate podocyte adhesion to--and pedicel organization on--the glomerular basement membrane. Recent studies showed that foot process effacement developed in a mutant mouse model whose podocytes were unable to assemble heparan sulfate glycosaminoglycan chains. This study, a further refinement, explored the role of heparan N-sulfation on podocyte behavior. A novel mutant mouse (Ndst1(-/-)) was developed, having podocyte-specific deletion of Ndst1, the enzyme responsible for N-sulfation of heparan sulfate chains. Podocytes having this mutation had foot process effacement and abnormal adhesion to Bowman's capsule. Although glomerular hypertrophy did develop in the kidneys of mutant animals, mesangial expansion was not seen. The lack of heparan N-sulfation did not affect the expression of agrin or perlecan proteoglycan core proteins. Loss of N-sulfation did not result in significant proteinuria, but the increase in the albumin/creatinine ratio was coincident with the development of the enlarged lysosomes in the proximal tubules. Thus, although the renal phenotype of the Ndst1(-/-) mouse is mild, the data show that heparan chain N-sulfation plays a key role in podocyte organization.

  11. Detection of antibodies specific for sheeppox and goatpox viruses using recombinant capripoxvirus antigens in an indirect enzyme-linked immunosorbent assay.

    PubMed

    Bowden, Timothy R; Coupar, Barbara E; Babiuk, Shawn L; White, John R; Boyd, Victoria; Duch, Christine J; Shiell, Brian J; Ueda, Norihito; Parkyn, Geoff R; Copps, John S; Boyle, David B

    2009-10-01

    Viruses in the genus Capripoxvirus, family Poxviridae, cause sheeppox, goatpox and lumpy skin disease, which are the most serious poxvirus diseases of production animals. Despite the considerable threat that these viruses pose to livestock production and global trade in sheep, goats, cattle and their products, convenient and effective serodiagnostic tools are not readily available. To develop a more effective antibody detection capability, selected open reading frames from capripoxvirus DNA were amplified and expressed in Escherichia coli as His-tagged fusion proteins. By screening 42 candidate antigens, two sheeppox virus virion core proteins that were expressed efficiently, purified readily using affinity chromatography and reactive against capripoxvirus immune sera in an indirect enzyme-linked immunosorbent assay (ELISA) were identified. The ELISA performed favourably when sera from sheep and goats infected experimentally with virulent capripoxvirus isolates were tested, with sensitivity and diagnostic specificity ranging between 95 and 97%, but it was unable to detect antibodies reliably in vaccinated sheep or goats. Furthermore, no cross-reactivity with antibodies against orf virus was detected. This assay offers the prospect of a convenient and standardised ELISA-based serodiagnostic test, with no requirement for infectious reagents, that is well suited to high-throughput capripoxvirus surveillance on a flock or herd basis.

  12. Studies on some specific Ap4A-degrading enzymes with the use of various methylene analogues of P1P4-bis-(5',5'''-adenosyl) tetraphosphate.

    PubMed Central

    Guranowski, A; Starzyńska, E; Taylor, G E; Blackburn, G M

    1989-01-01

    Six new methylenephosphonate analogues of P1P4-bis-(5',5'''-adenosyl) tetraphosphate, Ap4A, having P2-P3 carbon bridges CF2, CCl2 and CH2CH2 or P1-P2 and P3-P4 carbon bridges CF2, CCl2 and CH2CH2 in the tetraphosphate chain, were examined as substrates or inhibitors for two specific Ap4A-degrading enzymes: (asymmetrical) Ap4A hydrolase (EC 3.6.1.17) from yellow-lupin seeds and (symmetrical) Ap4A hydrolase (EC 3.6.1.41) from Escherichia coli. All analogues in which the central oxygen atom was replaced by a stable carbon bridge were hydrolysed by the asymmetrical hydrolase (CF2 greater than CCl2 greater than O greater than CHBr greater than CH2 greater than CH2CH2). As expected, these analogues were not hydrolysed by the symmetrical hydrolase, which was also unable to act on analogues having P1-P2 and P3-P4 carbon bridges. PMID:2554885

  13. Expression pattern and sub-cellular distribution of phosphoinositide specific phospholipase C enzymes after treatment with U-73122 in rat astrocytoma cells.

    PubMed

    Lo Vasco, Vincenza Rita; Fabrizi, Cinzia; Panetta, Barbara; Fumagalli, Lorenzo; Cocco, Lucio

    2010-07-01

    Phosphoinositide specific phospholipase C (PI-PLC) enzymes interfere with the metabolism of inositol phospholipids (PI), molecules involved in signal transduction, a complex process depending on various components. Many evidences support the hypothesis that, in the glia, isoforms of PI-PLC family display different expression and/or sub cellular distribution under non-physiological conditions such as the rat astrocytes activation during neurodegeneration, the tumoural progression of some neoplasms and the inflammatory cascade activation after lipopolysaccharide administration, even if their role remains not completely elucidated. Treatment of a cultured established glioma cell line (C6 rat astrocytoma cell line) induces a modification in the pattern of expression and of sub cellular distribution of PI-PLCs compared to untreated cells. Special attention require PI-PLC beta3 and PI-PLC gamma2 isoforms, whose expression and sub cellular localization significantly differ after U-73122 treatment. The meaning of these modifications is unclear, also because the use of this N-aminosteroid compound remains controversial, inasmuch it has further actions which might contribute to the global effect recorded on the treated cells.

  14. A simple, specific high-throughput enzyme-linked immunosorbent assay (ELISA) for quantitative determination of melatonin in cell culture medium.

    PubMed

    Li, Ye; Cassone, Vincent M

    2015-09-01

    A simple, specific, high-throughput enzyme-linked immunosorbent assay (ELISA) for quantitative determination of melatonin was developed for directly measuring melatonin in cell culture medium with 10% FBS. This assay adopts a commercial monoclonal melatonin antibody and melatonin-HRP conjugate, so it can be applied in multiple labs rapidly with low cost compared with commercial RIA and ELISA kits. In addition, the procedure is much simpler with only four steps: 1) sample/conjugate incubation, 2) plate washing, 3) TMB color reaction and 4) reading of results. The standards of the assay cover a wide working range from 100 pg/mL to 10 ng/mL. The sensitivity was 68 pg/mL in cell culture medium with 10% FBS and 26 pg/mL in PBS with as little as 25 μL sample volume. The recovery of melatonin from cell culture medium was 101.0%. The principal cross-reacting compound was 5-methoxytryptophol (0.1%). The variation coefficients of the assay, within and between runs, ranged between 6.68% and 15.76% in cell culture medium. The mean linearity of a series diluted cell culture medium sample was 105% (CV=5%), ranging between 98% and 111%, y=5.5263x+0.0646, R(2)=0.99. The assay enables small research and teaching labs to reliably measure this important neurohormone.

  15. RandSpg: An open-source program for generating atomistic crystal structures with specific spacegroups

    NASA Astrophysics Data System (ADS)

    Avery, Patrick; Zurek, Eva

    2017-04-01

    A new algorithm, RANDSPG, that can be used to generate trial crystal structures with specific space groups and compositions is described. The program has been designed for systems where the atoms are independent of one another, and it is therefore primarily suited towards inorganic systems. The structures that are generated adhere to user-defined constraints such as: the lattice shape and size, stoichiometry, set of space groups to be generated, and factors that influence the minimum interatomic separations. In addition, the user can optionally specify if the most general Wyckoff position is to be occupied or constrain select atoms to specific Wyckoff positions. Extensive testing indicates that the algorithm is efficient and reliable. The library is lightweight, portable, dependency-free and is published under a license recognized by the Open Source Initiative. A web interface for the algorithm is publicly accessible at http://xtalopt.openmolecules.net/randSpg/randSpg.html. RANDSPG has also been interfaced with the XTALOPT evolutionary algorithm for crystal structure prediction, and it is illustrated that the use of symmetric lattices in the first generation of randomly created individuals decreases the number of structures that need to be optimized to find the global energy minimum.

  16. Site-specific emergency response concept plans for the Chemical Stockpile Disposal Program

    SciTech Connect

    Carnes, S.A.

    1989-12-01

    Site-specific emergency response concept plans were developed to help initiate enhanced emergency preparedness for continued storage of the stockpile and the Chemical Stockpile Disposal Program (CSDP) at the eight army installations storing the unitary chemical stockpile -- Aberdeen Proving Ground, Anniston Army Depot, Lexington-Blue Grass Army Depot, Newport Army Ammunition Plant, Pine Bluff Arsenal, Pueblo Depot Activity, Tooele Army Depot, and Umatilla Depot Activity. This document summarizes the emergency response plans for all the sites and highlights similarities and differences among them. Section 2 summarizes site-specific differences in stockpile hazard and risk by showing differences in planning-basis accident categories and distributions of topographical features, meteorological conditions, and populations at risk. Section 3 presents a summary of the methodology used to identify the emergency planning zones for each site and the actual recommended boundaries of those zones for the eight sites. Section 4 identifies feasible and recommended protective actions for the sites and explains reasons for differences in them. Finally, Section 5 notes the dependence of protective action effectiveness on the development and implementation of command and control and warning systems that can be implemented in a timely manner, it also identifies the differences in recommended lead times (i.e., from the onset of an accidental release) needed at the sites for effective implementation of protective actions. 17 refs., 11 figs. , 12 tabs.

  17. Steady-state kinetic studies reveal that the anti-cancer target Ubiquitin-Specific Protease 17 (USP17) is a highly efficient deubiquitinating enzyme.

    PubMed

    Hjortland, Nicole M; Mesecar, Andrew D

    2016-12-15

    USP17 is a deubiquitinating enzyme that is upregulated in numerous cancers and therefore a drug target. We developed a robust expression, purification, and assay system for USP17 enabling its enzymatic and structural characterization. USP17 was expressed in E. coli as inclusion bodies and then solubilized, refolded, and purified using affinity and size-exclusion chromatography. Milligram quantities of pure USP17 can be produced that is catalytically more efficient (kcat/Km = 1500 (x10(3)) M(-1)sec(-1)) than other human USPs studied to date. Analytical size-exclusion chromatography, analytical ultracentrifugation, and dynamic light scattering studies suggest that the quaternary structure of USP17 is a monomer. Steady-state kinetic studies show that USP17 efficiently hydrolyzes both ubiquitin-AMC (kcat = 1.5 sec(-1) and Km = 1.0 μM) and ubiquitin-rhodamine110 (kcat = 1.8 sec(-1) and Km = 2.0 μM) substrates. Ubiquitin chain cleavage assays reveal that USP17 efficiently cleaves di-ubiquitin chains with Lys(11), Lys(33), Lys(48) and Lys(63) linkages and tetra-ubiquitin chains with Lys(11), Lys(48) and Lys(63) linkages but is inefficient in cleaving di-ubiquitin chains with Lys(6), Lys(27), or Lys(29) linkages or linear ubiquitin chains. The substrate specificity of USP17 is most similar to that of USP1, where both USPs display higher specificity than other characterized members of the USP family.

  18. Engineering the substrate specificity of cytochrome P450 CYP102A2 by directed evolution: production of an efficient enzyme for bioconversion of fine chemicals.

    PubMed

    Axarli, Irene; Prigipaki, Ariadne; Labrou, Nikolaos E

    2005-06-01

    The P450 cytochromes constitute a large family of hemoproteins that catalyze the monooxygenation of a diversity of hydrophobic substrates. CYP102A2 is a catalytically self-sufficient cytoplasmic enzyme from Bacillus subtilis, containing both a monooxygenase domain and a reductase domain on a single polypeptide chain. CYP102A2 was subjected to error-prone PCR to generate mutants with enhanced activity with fatty acids and other aromatic substrates. The library of CYP102A2 mutants was expressed in BL21(DE3) Escherichia coli cells and screened for their ability to oxidize different substrates by means of an activity assay. After a single round of error-prone PCR, the variant Pro15Ser exhibiting modified substrate specificity was generated. This variant showed approximately 6- to 9-fold increased activity with SDS, lauric acid and 1,4-naphthoquinone, and enhanced activity for other substrates such as ethacrynic acid and epsilon-amino-n-caproic acid. Molecular modeling of the CYP102A2 monooxygenase domain suggested that Pro15 is located in a short helical segment and is involved in extensive interactions between the N-terminal domain and the beta2 sheet, which contribute to the formation of the substrate binding site. Thus, Pro15 appears to affect substrate binding and catalysis indirectly. These results clearly demonstrate the importance of remote residues, not readily predicted by rational design, for the determination of substrate specificity. In addition, we report here that the Pro15Ser variant of CYP102A2 can be efficiently immobilized on epoxy-activated Sepharose at pH 8.5 and 4 degrees C. The immobilized variant of CYP102A2 retains most of its activity (81%) and shows improved stability at 37 degrees C. The approach offers the possibility of designing a P450 bioreactor that can be operated over a long period of time with high efficiency and which can be used in fine chemical synthesis.

  19. A Chimeric LysK-Lysostaphin Fusion Enzyme Lysing Staphylococcus aureus Cells: a Study of Both Kinetics of Inactivation and Specifics of Interaction with Anionic Polymers.

    PubMed

    Filatova, Lyubov Y; Donovan, David M; Ishnazarova, Nadiya T; Foster-Frey, Juli A; Becker, Stephen C; Pugachev, Vladimir G; Balabushevich, Nadezda G; Dmitrieva, Natalia F; Klyachko, Natalia L

    2016-10-01

    A staphylolytic fusion protein (chimeric enzyme K-L) was created, harboring three unique lytic activities composed of the LysK CHAP endopeptidase, and amidase domains, and the lysostaphin glycyl-glycine endopeptidase domain. To assess the potential of possible therapeutic applications, the kinetic behavior of chimeric enzyme K-L was investigated. As a protein antimicrobial, with potential antigenic properties, the biophysical effect of including chimeric enzyme K-L in anionic polymer matrices that might help reduce the immunogenicity of the enzyme was tested. Chimeric enzyme K-L reveals a high lytic activity under the following optimal (opt) conditions: pHopt 6.0-10.0, topt 20-30 °C, NaClopt 400-800 mM. At the working temperature of 37 °C, chimeric enzyme K-L is inactivated by a monomolecular mechanism and possesses a high half-inactivation time of 12.7 ± 3.0 h. At storage temperatures of 22 and 4 °C, a complex mechanism (combination of monomolecular and bimolecular mechanisms) is involved in the chimeric enzyme K-L inactivation. The optimal storage conditions under which the enzyme retains 100 % activity after 140 days of incubation (4 °C, the enzyme concentration of 0.8 mg/mL, pH 6.0 or 7.5) were established. Chimeric enzyme K-L is included in complexes with block-copolymers of poly-L-glutamic acid and polyethylene glycol, while the enzyme activity and stability are retained, thus suggesting methods to improve the application of this fusion as an effective antimicrobial agent.

  20. Computer program design specifications for the Balloon-borne Ultraviolet Stellar Spectrometer (BUSS) science data decommutation program (BAPS48)

    NASA Technical Reports Server (NTRS)

    Rodriguez, R. M.

    1975-01-01

    The Balloon-Borne Ultraviolet Stellar Spectrometer (BUSS) Science Data Docummutation Program (BAPS48) is a pulse code modulation docummutation program that will format the BUSS science data contained on a one inch PCM tracking tape into a seven track serial bit stream formatted digital tape.

  1. Aliskiren Administration during Early Postnatal Life Sex-Specifically Alleviates Hypertension Programmed by Maternal High Fructose Consumption

    PubMed Central

    Hsu, Chien-Ning; Wu, Kay L. H.; Lee, Wei-Chia; Leu, Steve; Chan, Julie Y. H.; Tain, You-Lin

    2016-01-01

    Key points summary Maternal high-fructose (HF) induces programmed hypertension in adult offspring.Early aliskiren administration prevents HF-induced hypertension in both sexes of adult offspring.HF regulates RAS components in the offspring kidney in a sex-specific manner.HF alters renal transcriptome, with female offspring being more sensitive.Deprogramming strategy to prevent hypertension might be sex-specific. Background: Maternal high fructose (HF) intake induced renal programming and hypertension in male adult offspring. We examined whether maternal HF intake causes programmed hypertension and whether aliskiren administration confers protection against the process in a sex-specific manner, with a focus on the transcriptome changes in the kidney using next-generation RNA sequencing (NGS) technology and renin-angiotensin system (RAS). Methods: Pregnant Sprague—Dawley rats received regular chow or chow supplemented with 60% fructose throughout pregnancy and lactation. Offspring were assigned to six groups: male control, male HF (MHF), MHF+Aliskiren, female control, female HF (FHF), and FHF+Aliskiren. Oral aliskiren 10 mg/kg/day was administered via gastric gavage between 2 and 4 weeks of age. Rats were sacrificed at 12 weeks of age. Results: Maternal HF intake induced programmed hypertension in 12-week-old offspring of both sexes. HF regulated renal transcriptome and RAS components in the offspring kidney in a sex-specific manner. Aliskiren administration prevented HF-induced programmed hypertension in both sexes of adult offspring. Aliskiren administration increased ACE2 and MAS protein levels in female kidneys exposed to maternal HF intake. Conclusion: Maternal HF induced programmed hypertension in both sexes of adult offspring, which was sex-specifically mitigated by early aliskiren administration. Better understanding of the sex-dependent mechanisms that underlie maternal HF-induced renal programming will help develop a novel sex-specific strategy to prevent

  2. Varicella-Zoster Virus-Specific Enzyme-Linked Immunospot Assay Responses and Zoster-Associated Pain in Herpes Zoster Subjects

    PubMed Central

    Tyring, Stephen K.; Stek, Jon E.; Smith, Jeffrey G.; Xu, Jin; Pagnoni, Marco; Chan, Ivan S. F.; Silber, Jeffrey L.; Levin, Myron J.

    2012-01-01

    Varicella-zoster virus (VZV)-specific cell-mediated immunity (CMI) responses were compared over time following an episode of herpes zoster (HZ) with those of age-, race-, and gender-matched healthy controls (HC) without HZ, using a validated gamma interferon (IFN-γ) enzyme-linked immunospot (ELISPOT) assay. The zoster brief-pain inventory (ZBPI) was used to assess zoster-associated pain. HZ patients (n = 140) had significantly higher IFN-γ ELISPOT responses to VZV antigen than did HC (n = 140). ELISPOT geometric mean count (GMC) responses (with 95% confidence intervals [CI]) for subjects who presented within 72 h were as follows: for HZ patients ≥ 60 years of age, at day 0 the GMC was 110 and at week 2 the GMC was 235; for HZ patients 21 to 59 years of age, at day 0 the GMC was 111 and at week 2 the GMC was 198; for HC ≥ 60 years of age, at day 0 the GMC was 19 and at week 2 the GMC was 18; and for HC 21 to 59 years of age, at day 0 the GMC was 59 and at week 2 the GMC was 56. The mean pain score (95% CI) across age groups at 1 week postrash (n = 106) was 6.0 (5.5, 6.5) and at 2 weeks postrash (n = 119) was 3.5 (2.9, 4.0). The percentage of HZ patients with substantial pain (score ≥ 3) at 6 weeks postrash increased with age from 8% for patients 21 to 49 years of age to 16% for patients 50 to 59 years of age to 22% for patients ≥ 60 years of age. The VZV-specific CMI response was substantially boosted by an episode of HZ, as measured by ELISPOT results. Older adults had lower VZV-specific cellular immunity than younger subjects at baseline, but the boosting effect of HZ was substantial for all age groups. HZ patients experienced considerable zoster-associated acute (1 to 2 weeks after rash) pain across age groups, while chronic pain increased with age. PMID:22787198

  3. Varicella-zoster virus-specific enzyme-linked immunospot assay responses and zoster-associated pain in herpes zoster subjects.

    PubMed

    Tyring, Stephen K; Stek, Jon E; Smith, Jeffrey G; Xu, Jin; Pagnoni, Marco; Chan, Ivan S F; Silber, Jeffrey L; Parrino, Janie; Levin, Myron J

    2012-09-01

    Varicella-zoster virus (VZV)-specific cell-mediated immunity (CMI) responses were compared over time following an episode of herpes zoster (HZ) with those of age-, race-, and gender-matched healthy controls (HC) without HZ, using a validated gamma interferon (IFN-γ) enzyme-linked immunospot (ELISPOT) assay. The zoster brief-pain inventory (ZBPI) was used to assess zoster-associated pain. HZ patients (n = 140) had significantly higher IFN-γ ELISPOT responses to VZV antigen than did HC (n = 140). ELISPOT geometric mean count (GMC) responses (with 95% confidence intervals [CI]) for subjects who presented within 72 h were as follows: for HZ patients ≥ 60 years of age, at day 0 the GMC was 110 and at week 2 the GMC was 235; for HZ patients 21 to 59 years of age, at day 0 the GMC was 111 and at week 2 the GMC was 198; for HC ≥ 60 years of age, at day 0 the GMC was 19 and at week 2 the GMC was 18; and for HC 21 to 59 years of age, at day 0 the GMC was 59 and at week 2 the GMC was 56. The mean pain score (95% CI) across age groups at 1 week postrash (n = 106) was 6.0 (5.5, 6.5) and at 2 weeks postrash (n = 119) was 3.5 (2.9, 4.0). The percentage of HZ patients with substantial pain (score ≥ 3) at 6 weeks postrash increased with age from 8% for patients 21 to 49 years of age to 16% for patients 50 to 59 years of age to 22% for patients ≥ 60 years of age. The VZV-specific CMI response was substantially boosted by an episode of HZ, as measured by ELISPOT results. Older adults had lower VZV-specific cellular immunity than younger subjects at baseline, but the boosting effect of HZ was substantial for all age groups. HZ patients experienced considerable zoster-associated acute (1 to 2 weeks after rash) pain across age groups, while chronic pain increased with age.

  4. Comparison of Chain-Length Preferences and Glucan Specificities of Isoamylase-Type α-Glucan Debranching Enzymes from Rice, Cyanobacteria, and Bacteria

    PubMed Central

    Utsumi, Yoshinori; Fujita, Naoko; Umeda, Kazuhiro; Sawada, Takayuki; Kubo, Akiko; Abe, Jun-ichi; Colleoni, Christophe; Ball, Steven

    2016-01-01

    It has been believed that isoamylase (ISA)-type α-glucan debranching enzymes (DBEs) play crucial roles not only in α-glucan degradation but also in the biosynthesis by affecting the structure of glucans, although molecular basis on distinct roles of the individual DBEs has not fully understood. In an attempt to relate the roles of DBEs to their chain-length specificities, we analyzed the chain-length distribution of DBE enzymatic reaction products by using purified DBEs from various sources including rice, cyanobacteria, and bacteria. When DBEs were incubated with phytoglycogen, their chain-length specificities were divided into three groups. First, rice endosperm ISA3 (OsISA3) and Eschericia coli GlgX (EcoGlgX) almost exclusively debranched chains having degree of polymerization (DP) of 3 and 4. Second, OsISA1, Pseudomonas amyloderamosa ISA (PsaISA), and rice pullulanase (OsPUL) could debranch a wide range of chains of DP≧3. Third, both cyanobacteria ISAs, Cyanothece ATCC 51142 ISA (CytISA) and Synechococcus elongatus PCC7942 ISA (ScoISA), showed the intermediate chain-length preference, because they removed chains of mainly DP3-4 and DP3-6, respectively, while they could also react to chains of DP5-10 and 7–13 to some extent, respectively. In contrast, all these ISAs were reactive to various chains when incubated with amylopectin. In addition to a great variation in chain-length preferences among various ISAs, their activities greatly differed depending on a variety of glucans. Most strikingly, cyannobacteria ISAs could attack branch points of pullulan to a lesser extent although no such activity was found in OsISA1, OsISA3, EcoGlgX, and PsaISA. Thus, the present study shows the high possibility that varied chain-length specificities of ISA-type DBEs among sources and isozymes are responsible for their distinct functions in glucan metabolism. PMID:27309534

  5. Transition of Patients with Esophageal Atresia to Adult Care: Results of a Transition-Specific Education Program.

    PubMed

    Dingemann, Jens; Szczepanski, Rüdiger; Ernst, Gundula; Thyen, Ute; Ure, Benno; Goll, Melanie; Menrath, Ingo

    2017-02-01

    Aim of the Study A history of esophageal atresia (EA) may result in chronic morbidity. The transition of patients from pediatric to adult care has been recognized as an important factor to maintain disease-specific follow-up and prevent exacerbation of chronic disease. Patient education is recognized as a necessary part of transition programs for children with chronic diseases. Structured education programs for patients with EA have not yet been developed. We aimed to evaluate the efficacy of a transition-specific educational program in adolescents with a history of EA. Methods An ethical approval was obtained. Patients with a history of EA (age 14-21 years) and their parents were invited to participate in a 2-day transition-specific educational program. Overall, 29 patients and 25 parents were recruited out of whom 10/7 were allocated to the intervention group (program) and 19/18 to the control group (no program). Subjective satisfaction (ZUF-8) and expected effects of the program on the future course of the disease, transition-specific knowledge (standardized questionnaire addressing organizational and health-related aspects of transition), health-related quality of life (DISABKIDS), and confidence for self-management (Patient Activation Measure-13) were measured with appropriate psychological instruments. Nonparametric tests were used for statistical analysis. Main Results Subjects participating were highly satisfied with the program (patients 26/32 points, parents 25/32; ZUF-8). Overall, 90% of the patients and 67% of the parents anticipated a positive effect on the future course of the disease. Patient's transition-specific knowledge was low before the program (36% correct answers). It improved by 18% after the intervention (56% correct answers; p = 0.004). It did not change in the control group (54 vs. 52%; n.s.). Parent's transition-specific knowledge did not change after the intervention (66 vs. 67% correct answers; ns). In patients, there were no

  6. Staphylococcal phosphoenolpyruvate-dependent phosphotransferase system: purification and characterization of the mannitol-specific enzyme III/sup mtl/ of Staphylococcus aureus and Staphylococcus carnosus and homology with the enzyme II/sup mtl/ of Escherichia coli

    SciTech Connect

    Reiche, B.; Frank, R.; Deutscher, J.; Meyer, N.; Hengstenberg, W.

    1988-08-23

    Enzyme III/sup mtl/ is part of the mannitol phosphotransferase system of Staphylococcus aureus and Staphylococcus carnosus and is phosphorylated by phosphoenolpyruvate in a reaction sequence requiring enzyme I (phosphoenolpyruvate-protein phosphotransferase) and the histidine-containing protein HPr. In this paper, the authors report the isolation of III/sup mtl/ from both S. aureus and S. carnosus and the characterization of the active center. After phosphorylation of III/sup mtl/ with (/sup 32/P)PEP, enzyme I, and HPr, the phosphorylated protein was cleaved with endoproteinase GLu(C). The amino acid sequence of the S. aureus peptide carrying the phosphoryl group was found to be Gln-Val-Val-Ser-Thr-Phe-Met-Gly-Asn-Gly-Leu-Ala-Ile-Pro-His-Gly-Thr-Asp-Asp. The corresponding peptide from S. carnosus shows an equal sequence except that the first residue is Ala instead of Gln. These peptides both contain a single histidyl residue which they assume to carry the phosphoryl group. All proteins of the PTS so far investigated indeed carry the phosphoryl group attached to a histidyl residue. According to sodium dodecyl sulfate gels, the molecular weight of the III/sup mtl/ proteins was found to be 15,000. They have also determined the N-terminal sequence of both proteins. Comparison of the III/sup mtl/ peptide sequences and the C-terminal part of the enzyme II/sup mtl/ of Escherichia coli reveals considerable sequence homology, which supports the suggestion that II/sup mtl/ of E. coli is a fusion protein of a soluble III protein with a membrane-bound enzyme II.

  7. Development of an analytical protocol for a fast, sensitive and specific protein recognition in paintings by enzyme-linked immunosorbent assay (ELISA).

    PubMed

    Palmieri, M; Vagnini, Manuela; Pitzurra, L; Rocchi, P; Brunetti, B G; Sgamellotti, A; Cartechini, L

    2011-03-01

    Enzyme-linked immunosorbent assay (ELISA) analysis of proteins offers a particularly promising approach for investigations in cultural heritage on account of its appreciated properties of being highly specific, sensitive, relatively fast, and cost-affordable with respect to other conventional techniques. In spite of that, it has never been fully exploited for routine analyses of painting materials in consideration of several analytical issues that inhibited its diffusion in conservation science: limited sample dimensions, decrease of binder solubility and reduced availability of antibody bonding sites occurring with protein degradation. In this study, an ELISA analytical protocol suited for the identification of aged denatured proteins in ancient painting micro-samples has been developed. We focused on the detection of bovine β-casein and chicken ovalbumin as markers of bovine milk (or casein) and chicken albumen, respectively. A systematic experimentation of the ELISA protocol has been carried out on mock-ups of mural and easel painting prepared with 13 different pigments to assess limits and strengths of the method when applied for the identification of proteins in presence of a predominant inorganic matrix. The analytical procedure has been optimized with respect to protein extraction, antibodies' concentrations, incubation time and temperature; it allows the detection of the investigated proteins with sensitivity down to nanograms. The optimized protocol was then tested on artificially aged painting models. Analytical results were very encouraging and demonstrated that ELISA allows for protein analysis also in degraded painting samples. To address the feasibility of the developed ELISA methodology, we positively investigated real painting samples and results have been cross-validated by gas chromatography-mass spectrometry.

  8. Identification of programmed cell death in situ via specific labeling of nuclear DNA fragmentation

    PubMed Central

    1992-01-01

    Programmed cell death (PCD) plays a key role in developmental biology and in maintenance of the steady state in continuously renewing tissues. Currently, its existence is inferred mainly from gel electrophoresis of a pooled DNA extract as PCD was shown to be associated with DNA fragmentation. Based on this observation, we describe here the development of a method for the in situ visualization of PCD at the single-cell level, while preserving tissue architecture. Conventional histological sections, pretreated with protease, were nick end labeled with biotinylated poly dU, introduced by terminal deoxy- transferase, and then stained using avidin-conjugated peroxidase. The reaction is specific, only nuclei located at positions where PCD is expected are stained. The initial screening includes: small and large intestine, epidermis, lymphoid tissues, ovary, and other organs. A detailed analysis revealed that the process is initiated at the nuclear periphery, it is relatively short (1-3 h from initiation to cell elimination) and that PCD appears in tissues in clusters. The extent of tissue-PCD revealed by this method is considerably greater than apoptosis detected by nuclear morphology, and thus opens the way for a variety of studies. PMID:1400587

  9. [Prenatal sex-specific programming and chronic diseases or Finis Ab Orígine Pendet].

    PubMed

    Arck, P C; Hecher, K

    2014-09-01

    An increasing incidence of chronic immune diseases such as allergies, multiple sclerosis, and type 2 diabetes, as well as obesity and cardiovascular and psychiatric disorders has been reported over the last five decades. Since the human genome has not altered significantly over this period of time, gene-environment interactions are suspected to be responsible for these increased disease incidences. In this context, the prenatal period is believed to significantly contribute to altered disease susceptibilities, which could be associated with environmental factors to which pregnant women were exposed to. This observation has led to a concept entitled 'developmental origin of health and disease', a topic that is enjoying much attention in clinical and basic science research. The aim of these research endeavors is to postulate guidelines for primary disease prevention. Whilst the emerging insights from this field of research provide significant pieces of the puzzle, one area is still largely neglected: the clear identification of a sex-specific programming effect. Thus it is essential that such an approach becomes fully integrated in future research goals.

  10. Enzymic formation of riboflavin 4',5'-cyclic phosphate from FAD: evidence for a specific low-Km FMN cyclase in rat liver1.

    PubMed Central

    Fraiz, F J; Pinto, R M; Costas, M J; Aavalos, M; Canales, J; Cabezas, A; Cameselle, J C

    1998-01-01

    An enzyme activity splitting FAD to AMP and riboflavin 4',5'-cyclic phosphate (4',5'-cFMN), with a Km of 6-8 microM, was partially purified from the cytosolic fraction of rat liver homogenates. 4', 5'-cFMN was characterized by enzyme, HPLC, UV-visible and NMR spectroscopic analyses. The data suggest that a novel enzyme, tentatively named FAD-AMP lyase (cyclizing) or FMN cyclase, is involved. Also, 4',5'-cFMN was hydrolysed to 5'-FMN by a rat liver cyclic phosphodiesterase. The results indicate a novel enzymic pathway for flavins in mammals, and support the biological relevance of 4',5'-cFMN, perhaps as a flavocoenzyme or a regulatory signal. PMID:9480905

  11. Effects of a Task-specific Exercise Program on Balance, Mobility, and Muscle Strength in the Elderly.

    PubMed

    Seo, Hyung-Seok; Lee, Jung-Ho; Park, Young-Han

    2014-11-01

    [Purpose] The aim of this study was to investigate the effects of a task-specific exercise program based on motor learning on balance ability and strength of the lower extremity in the elderly with/without falling experiences. [Subjects and Methods] Individuals who had experiences of falling over 2 times within the past 6 months were included in the falling group. The task-specific exercise program consisted of 3 stages (weeks 1-2, 3-4, and 5-6) and was conducted according to the level of difficulty in this study. [Results] The scores of the Korean version of the Activities-Specific Balance Confidence Scale and Performance-Oriented Mobility Assessment were significantly changed in both the falling group and non-falling group after the task-specific exercise program. In comparisons between the falling group and non-falling group, there were also significant differences in the Korean version of the Activities-Specific Balance Confidence Scale and muscle strength of the semitendinosus and gastrocnemius. [Conclusion] The task-specific exercise program has a positive effect on balance ability and muscle strength related to falls in the elderly.

  12. Effects of a Task-specific Exercise Program on Balance, Mobility, and Muscle Strength in the Elderly

    PubMed Central

    Seo, Hyung-Seok; Lee, Jung-Ho; Park, Young-Han

    2014-01-01

    [Purpose] The aim of this study was to investigate the effects of a task-specific exercise program based on motor learning on balance ability and strength of the lower extremity in the elderly with/without falling experiences. [Subjects and Methods] Individuals who had experiences of falling over 2 times within the past 6 months were included in the falling group. The task-specific exercise program consisted of 3 stages (weeks 1–2, 3–4, and 5–6) and was conducted according to the level of difficulty in this study. [Results] The scores of the Korean version of the Activities-Specific Balance Confidence Scale and Performance-Oriented Mobility Assessment were significantly changed in both the falling group and non-falling group after the task-specific exercise program. In comparisons between the falling group and non-falling group, there were also significant differences in the Korean version of the Activities-Specific Balance Confidence Scale and muscle strength of the semitendinosus and gastrocnemius. [Conclusion] The task-specific exercise program has a positive effect on balance ability and muscle strength related to falls in the elderly. PMID:25435679

  13. Revisiting the Nucleotide and Aminoglycoside Substrate Specificity of the Bifunctional Aminoglycoside Acetyltransferase(6′)-Ie/Aminoglycoside Phosphotransferase(2″)-Ia Enzyme*

    PubMed Central

    Frase, Hilary; Toth, Marta; Vakulenko, Sergei B.

    2012-01-01

    The bifunctional aminoglycoside-modifying enzyme aminoglycoside acetyltransferase(6′)-Ie/aminoglycoside phosphotransferase(2″)-Ia, or AAC(6′)-Ie/APH(2″)-Ia, is the major source of aminoglycoside resistance in Gram-positive bacterial pathogens. In previous studies, using ATP as the cosubstrate, it was reported that the APH(2″)-Ia domain of this enzyme is unique among aminoglycoside phosphotransferases, having the ability to inactivate an unusually broad spectrum of aminoglycosides, including 4,6- and 4,5-disubstituted and atypical. We recently demonstrated that GTP, and not ATP, is the preferred cosubstrate of this enzyme. We now show, using competition assays between ATP and GTP, that GTP is the exclusive phosphate donor at intracellular nucleotide levels. In light of these findings, we reevaluated the substrate profile of the phosphotransferase domain of this clinically important enzyme. Steady-state kinetic characterization using the phosphate donor GTP demonstrates that AAC(6′)-Ie/APH(2″)-Ia phosphorylates 4,6-disubstituted aminoglycosides with high efficiency (kcat/Km = 105-107 m−1 s−1). Despite this proficiency, no resistance is conferred to some of these antibiotics by the enzyme in vivo. We now show that phosphorylation of 4,5-disubstituted and atypical aminoglycosides are negligible and thus these antibiotics are not substrates. Instead, these aminoglycosides tend to stimulate an intrinsic GTPase activity of the enzyme. Taken together, our data show that the bifunctional enzyme efficiently phosphorylates only 4,6-disubstituted antibiotics; however, phosphorylation does not necessarily result in bacterial resistance. Hence, the APH(2″)-Ia domain of the bifunctional AAC(6′)-Ie/APH(2″)-Ia enzyme is a bona fide GTP-dependent kinase with a narrow substrate profile, including only 4,6-disubstituted aminoglycosides. PMID:23115238

  14. Enzyme Kinetics.

    ERIC Educational Resources Information Center

    Moe, Owen; Cornelius, Richard

    1988-01-01

    Conveys an appreciation of enzyme kinetic analysis by using a practical and intuitive approach. Discusses enzyme assays, kinetic models and rate laws, the kinetic constants (V, velocity, and Km, Michaels constant), evaluation of V and Km from experimental data, and enzyme inhibition. (CW)

  15. Rational enzyme redesign

    SciTech Connect

    Ornstein, R.L.

    1994-05-01

    Protein engineering is first a means of elucidating structure-function relations in an enzyme, and second, a means of changing a protein to make it serve a different, but generally related, purpose. In principle, one may change the functional characteristics of an enzyme by altering its substrate specificity, kinetics, optimum range of activity, and chemical mechanism. Obviously one cannot make all possible combinations of amino acid changes for even the smallest enzyme, so the essential question is which changes to make. The intent of rational protein/enzyme redesign is to alter a protein/enzyme in a timely and premeditated fashion. This article provides an outline of the process of rational enzyme redesign.

  16. Recommendations for Guidelines for Environment-Specific Magnetic-Field Measurements, Rapid Program Engineering Project #2

    SciTech Connect

    Electric Research and Management, Inc.; IIT Research Institute; Magnetic Measurements; Survey Research Center, University of California; T. Dan Bracken, Inc.

    1997-03-11

    The purpose of this project was to document widely applicable methods for characterizing the magnetic fields in a given environment, recognizing the many sources co-existing within that space. The guidelines are designed to allow the reader to follow an efficient process to (1) plan the goals and requirements of a magnetic-field study, (2) develop a study structure and protocol, and (3) document and carry out the plan. These guidelines take the reader first through the process of developing a basic study strategy, then through planning and performing the data collection. Last, the critical factors of data management, analysis reporting, and quality assurance are discussed. The guidelines are structured to allow the researcher to develop a protocol that responds to specific site and project needs. The Research and Public Information Dissemination Program (RAPID) is based on exposure to magnetic fields and the potential health effects. Therefore, the most important focus for these magnetic-field measurement guidelines is relevance to exposure. The assumed objective of an environment-specific measurement is to characterize the environment (given a set of occupants and magnetic-field sources) so that information about the exposure of the occupants may be inferred. Ideally, the researcher seeks to obtain complete or "perfect" information about these magnetic fields, so that personal exposure might also be modeled perfectly. However, complete data collection is not feasible. In fact, it has been made more difficult as the research field has moved to expand the list of field parameters measured, increasing the cost and complexity of performing a measurement and analyzing the data. The guidelines address this issue by guiding the user to design a measurement protocol that will gather the most exposure-relevant information based on the locations of people in relation to the sources. We suggest that the "microenvironment" become the base unit of area in a study, with

  17. Role of the Substrate Specificity-Defining Residues of Human SIRT5 in Modulating the Structural Stability and Inhibitory Features of the Enzyme.

    PubMed

    Yu, Junru; Haldar, Manas; Mallik, Sanku; Srivastava, D K

    2016-01-01

    Sirtuins are emerging as the key regulators of metabolism and aging, and their potential activators and inhibitors are being explored as therapeutics for improving health and treating associated diseases. Despite the global structural similarity among all seven isoforms of sirtuins (of which most of them catalyze the deacetylation reaction), SIRT5 is the only isoform that catalyzes the cleavage of negatively charged acylated substrates, and the latter feature appears to be encoded by the presence of Tyr102 and Arg105 residues at the active site pocket of the enzyme. To determine the contributions of the above residues in SIRT5 (vis a vis the corresponding residues of SIRT1) on substrate selectivity, inhibition by EX527 and nicotinamide, secondary structural features and thermal stability of the enzymes, we created single and double mutations (viz. Y102A, R105l, and Y102A/R105I) in SIRT5. The kinetic data revealed that while Y102A mutant enzyme catalyzed both deacetylation and desuccinylation reactions with comparable efficiencies, R105I and Y102A/R105I mutant enzymes favored the deacetylase reaction. Like SIRT1, the nicotinamide inhibition of SIRT5 double mutant (Y102A/R105I) exhibited the mixed non-competitive behavior. On the other hand, the desuccinylation reaction of both wild-type and Y102A mutant enzymes conformed to the competitive inhibition model. The inhibitory potency of EX527 progressively increased from Y102A, R105I, to Y102A/R105 mutant enzymes in SIRT5, but it did not reach to the level obtained with SIRT1. The CD spectroscopic data for the wild-type and mutant enzymes revealed changes in the secondary structural features of the enzymes, and such changes were more pronounced on examining their thermal denaturation patterns. A cumulative account of our experimental data reveal mutual cooperation between Y102 and R105 residues in promoting the desuccinylation versus deacetylation reaction in SIRT5, and the overall catalytic feature of the enzyme is

  18. Role of the Substrate Specificity-Defining Residues of Human SIRT5 in Modulating the Structural Stability and Inhibitory Features of the Enzyme

    PubMed Central

    Yu, Junru; Haldar, Manas; Mallik, Sanku; Srivastava, D. K.

    2016-01-01

    Sirtuins are emerging as the key regulators of metabolism and aging, and their potential activators and inhibitors are being explored as therapeutics for improving health and treating associated diseases. Despite the global structural similarity among all seven isoforms of sirtuins (of which most of them catalyze the deacetylation reaction), SIRT5 is the only isoform that catalyzes the cleavage of negatively charged acylated substrates, and the latter feature appears to be encoded by the presence of Tyr102 and Arg105 residues at the active site pocket of the enzyme. To determine the contributions of the above residues in SIRT5 (vis a vis the corresponding residues of SIRT1) on substrate selectivity, inhibition by EX527 and nicotinamide, secondary structural features and thermal stability of the enzymes, we created single and double mutations (viz. Y102A, R105l, and Y102A/R105I) in SIRT5. The kinetic data revealed that while Y102A mutant enzyme catalyzed both deacetylation and desuccinylation reactions with comparable efficiencies, R105I and Y102A/R105I mutant enzymes favored the deacetylase reaction. Like SIRT1, the nicotinamide inhibition of SIRT5 double mutant (Y102A/R105I) exhibited the mixed non-competitive behavior. On the other hand, the desuccinylation reaction of both wild-type and Y102A mutant enzymes conformed to the competitive inhibition model. The inhibitory potency of EX527 progressively increased from Y102A, R105I, to Y102A/R105 mutant enzymes in SIRT5, but it did not reach to the level obtained with SIRT1. The CD spectroscopic data for the wild-type and mutant enzymes revealed changes in the secondary structural features of the enzymes, and such changes were more pronounced on examining their thermal denaturation patterns. A cumulative account of our experimental data reveal mutual cooperation between Y102 and R105 residues in promoting the desuccinylation versus deacetylation reaction in SIRT5, and the overall catalytic feature of the enzyme is

  19. Transcript profiling of the ruminant liver indicates a unique program of transcriptional regulation of ketogenic enzymes during food restriction.

    PubMed

    Doelman, John; Cao, Honghe; Purdie, Norman G; Kim, Julie J M; Swanson, Kendall C; Osborne, Vernon R; Tey, Jasper; Ali, Ayesha; Feng, Zeny; Karrow, Niel A; Cant, John P

    2012-09-01

    Ruminants absorb little glucose and rely on hepatic gluconeogenesis and ketogenesis in the fed state to convert short-chain fatty acids produced during digestion into glucose and ketone bodies, respectively. In contrast to the non-ruminant response, fluxes through gluconeogenic and ketogenic pathways decrease during food restriction. Transcriptional regulation responsible for these unique food restriction responses has not been established. To determine the hepatic transcriptional response of ruminants to an acute drop in dietary nutrient supply, 102 yearling heifers were assigned to either ad libitum feeding or 24 h of food withdrawal in a randomized block design. Liver biopsies were obtained for microarray and quantitative real-time PCR analyses of gene expression. Plasma concentrations of non-esterified fatty acids were higher in food restricted heifers, while levels of β-hydroxybutyrate, triacylglycerol, and glucose were decreased. Despite a decline in substrate supply and a lower hepatic production of glucose, expression of the key gluconeogenic enzymes pyruvate carboxylase, phosphoenolpyruvate carboxykinase and fructose-1,6-bisphosphatase was upregulated as in non-ruminants. Downregulation of cholesterolgenic genes and upregulation of fatty acid oxidative genes were consistent with SREBP-2 and PPARα control, respectively. Ketogenesis from short-chain fatty acids was downregulated, contrary to the non-ruminant response to food restriction. Short-chain fatty acids may exert transcriptional control in the ruminant liver similar to that demonstrated in the large intestine of non-ruminants.

  20. Enzyme Molar Fractions: A Powerful Tool for Understanding Enzyme Kinetics.

    ERIC Educational Resources Information Center

    Serra, Juan L.; And Others

    1986-01-01

    Deduces the relationship between reduced velocity and molar fractions for productive enzyme complexes; obtains the mathematical expression of molar fractions for an enzyme with two specific binding sites per molecule; and proposes a useful plot to follow the dependence of enzyme molar fractions with the concentration of one of its ligands. (JN)

  1. To What Extent Is Criminal Justice Content Specifically Addressed in MSW Programs?

    ERIC Educational Resources Information Center

    Epperson, Matthew W.; Roberts, Leslie E.; Ivanoff, Andre; Tripodi, Stephen J.; Gilmer, Christy N.

    2013-01-01

    This study examined the extent to which criminal justice content is addressed in all CSWE-accredited MSW programs in the United States ("N"?=?192). Criminal justice content was measured in three areas: (1) dual or joint degree programs, (2) concentrations or specializations, and (3) coursework. Excluding social work and law classes, 22%…

  2. Predicting the Academic Success of Community College Students in Specific Programs of Study.

    ERIC Educational Resources Information Center

    Yess, James P.

    The intent of this study was to determine the influence of selected independent variables on the graduating grade point average (GPA) of community college students in various programs of study. A sample of 483 students from one community college represented seven programs of study: Business Administration-General, Business Administration-Transfer,…

  3. Experiences of Students with Specific Learning Disorder (Including ADHD) in Online College Degree Programs: A Phenomenological Study

    ERIC Educational Resources Information Center

    Bunch, Seleta LeAnn

    2016-01-01

    Enrollment in online degree programs is rapidly expanding due to the convenience and affordability offered to students and improvements in technology. The purpose of this hermeneutical phenomenological study was to understand the shared experiences of students with documented specific learning disorders (including Attention-Deficit/Hyperactivity…

  4. Specific and Optional Curriculum: An Experience in the Undergraduate Program of Chemical Engineering in Cienfuegos University, Cuba

    ERIC Educational Resources Information Center

    Martínez, Yolanda García; Velázquez, Claudia Alvarado; Castillo, Rolando Delgado

    2016-01-01

    This paper pursues to define the pillars for designing the specific (SC) and optional curricula (OC) of Unit Operations and Processes (UOP) Discipline in the Chemical Engineering Program. To achieve this objective a methodology was developed, which was characterized by the participation of every member in the educational process: professors,…

  5. Educational Specifications for Pearl Harbor Heights High School, Developed from the Program Delineation Study, January-April 1961.

    ERIC Educational Resources Information Center

    Hawaii State Dept. of Education, Honolulu.

    The report is a discussion of the curriculum and supportive educational facilities for the state of Hawaii. An administrative view of guidance services and teacher programing is included. Supportive facilities are sketched, showing their relationship to such specific instructional areas as music, shop, home economics, drawing and painting,…

  6. Insights from Smart Meters. Identifying Specific Actions, Behaviors and Characteristics that drive savings in Behavior-Based Programs

    SciTech Connect

    Todd, Annika; Perry, Michael; Smith, Brian; Sullivan, Michael; Cappers, Peter; Goldman, Charles A.

    2014-12-01

    In this report, we use smart meter data to analyze specific actions, behaviors, and characteristics that drive energy savings in a behavior-based (BB) program. Specifically, we examine a Home Energy Report (HER) program. These programs typically obtain 1% to 3% annual savings, and recent studies have shown hourly savings of between 0.5% and 3%. But what is driving these savings? What types of households tend to be “high-savers”, and what behaviors are they adopting? There are several possibilities: one-time behaviors (e.g., changing thermostat settings); reoccurring habitual behaviors (e.g., turning off lights); and equipment purchase behaviors (e.g., energy efficient appliances), and these may vary across households, regions, and over time.

  7. Insights from Smart Meters: Identifying Specific Actions, Behaviors, and Characteristics That Drive Savings in Behavior-Based Programs

    SciTech Connect

    Todd, A.; Perry, M.; Smith, B.; Sullivan, M.; Cappers, P.; Goldman, C.

    2014-12-01

    In this report, we use smart meter data to analyze specific actions, behaviors, and characteristics that drive energy savings in a BB program. Specifically, we examine a Home Energy Report (HER) program. These programs typically obtain 1% to 3% annual savings, and recent studies have shown hourly savings of between 0.5% and 3%.1 But what is driving these savings? What types of households tend to be “high-savers,” and what behaviors are they adopting? There are several possibilities: one-time behaviors (e.g., changing thermostat settings), reoccurring habitual behaviors (e.g., turning off lights), and equipment purchase behaviors (e.g., energy efficient appliances); these may vary across households, regions, and over time.

  8. Characterizations of substrate and enzyme specificity of glucoamylase assays of mucosal starch digestion with determinations of group and single biopsy reference values

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Carbohydrate digesting enzyme activities are measured in duodenal biopsies to detect deficiencies of lactase and sucrase activities, however glucoamylase (GA) assays for starch digestion are not included. Because food starch represents half of energy intake in the human diet, assays for starch diges...

  9. THE IMPORTANCE OF OBTAINING INFORMATION ON THE SPECIFIC CONTENT OF TISSUE ENZYMES METABOLIZING ORGANOPHOSPHORUS PESTICIDES, PRIOR TO DETERMINE VMAX, KM VALUES FOR USE IN PBPK MODELS

    EPA Science Inventory

    Physiological pharmacokinetic/pharmacodynamic models require Vmax, Km values for the metabolism of OPs by tissue enzymes. Current literature values cannot be easily used in OP PBPK models (i.e., parathion and chlorpyrifos) because standard methodologies were not used in their ...

  10. THE IMPORTANCE OF OBTAINING INFORMATION ON THE SPECIFIC CONTENT OF TISSUE ENZYMES METABOLIZING ORGANOPHOSPHORUS PESTICIDES, PRIOR TO DETERMINING VMAX, KM VALUES FOR USE IN PBPK MODELS

    EPA Science Inventory

    Physiological pharmacokinetic\\pharmacodynamic models require Vmax, Km values for the metabolism of OPs by tissue enzymes. Current literature values cannot be easily used in OP PBPK models (i.e., parathion and chlorpyrifos) because standard methodologies were not used in their ...

  11. Marine enzymes.

    PubMed

    Debashish, Ghosh; Malay, Saha; Barindra, Sana; Joydeep, Mukherjee

    2005-01-01

    Marine enzyme biotechnology can offer novel biocatalysts with properties like high salt tolerance, hyperthermostability, barophilicity, cold adaptivity, and ease in large-scale cultivation. This review deals with the research and development work done on the occurrence, molecular biology, and bioprocessing of marine enzymes during the last decade. Exotic locations have been accessed for the search of novel enzymes. Scientists have isolated proteases and carbohydrases from deep sea hydrothermal vents. Cold active metabolic enzymes from psychrophilic marine microorganisms have received considerable research attention. Marine symbiont microorganisms growing in association with animals and plants were shown to produce enzymes of commercial interest. Microorganisms isolated from sediment and seawater have been the most widely studied, proteases, carbohydrases, and peroxidases being noteworthy. Enzymes from marine animals and plants were primarily studied for their metabolic roles, though proteases and peroxidases have found industrial applications. Novel techniques in molecular biology applied to assess the diversity of chitinases, nitrate, nitrite, ammonia-metabolizing, and pollutant-degrading enzymes are discussed. Genes encoding chitinases, proteases, and carbohydrases from microbial and animal sources have been cloned and characterized. Research on the bioprocessing of marine-derived enzymes, however, has been scanty, focusing mainly on the application of solid-state fermentation to the production of enzymes from microbial sources.

  12. Longitudinal and transverse magnetic field program procedure and detailed specification for Sigma 5

    NASA Technical Reports Server (NTRS)

    Wang, C. K.

    1980-01-01

    A computer program and procedure for plotting the contour of the data transferred from the Marshall Space Flight Center solar magnetography is presented. The plotted data then can be easily compared with solar data from other sources, such as the Solar Maximum Mission. From the data file for circular polarization the longitudinal program plots the contours for filtered longitudinal plot and intensity plot by choosing the positive and negative contour levels, intensity levels, and also X,Y plotting ranges which need to be used. In a similar manner the transverse program generates the transverse contour plot, azimuth plot, and intensity plot from the linear polarization data files.

  13. The deubiquitinating enzyme mUBPy interacts with the sperm-specific molecular chaperone MSJ-1: the relation with the proteasome, acrosome, and centrosome in mouse male germ cells.

    PubMed

    Berruti, Giovanna; Martegani, Enzo

    2005-01-01

    The mouse USP8/mUBPy gene codifies a deubiquitinating enzyme expressed preferentially in testis and brain. While the ubiquitin-specific processing proteases (UBPs) are known to be important for the early development in invertebrate organisms, their specific functions remain still unclear in mammals. Using specific antibodies, raised against a recombinant mUBPy protein, we studied mUBPy in mouse testis. The mUBPy is expressed exclusively by the germ cell component and is maintained in epididymal spermatozoa. The enzyme is functionally active, being able to detach ubiquitin moieties from endogenous protein substrates. Protein interaction assays showed that sperm UBPy interacts with MSJ-1, the sperm-specific DnaJ protein evolutionarily conserved for spermiogenesis. Immunocytochemistry revealed that mUBPy shares with MSJ-1 the intracellular localization during spermatid cell differentiation; intriguingly, we show here that the proteasomes also locate in mUBPy/MSJ-1-positive sites, such as the cytoplasmic surface of the developing acrosome and the centrosomal region. These colocalization sites are maintained in epididymal spermatozoa. The demonstration of a protein interaction between a deubiquitinating enzyme and a molecular chaperone and the documentation on the proteasomes in both differentiating and mature mouse male germ cells suggest that members of the chaperone and ubiquitin/proteasome systems could cooperate in the fine control of protein quality to yield functional spermatozoa.

  14. The Influence of Marriage on Community College Student Achievement in Specific Programs of Study.

    ERIC Educational Resources Information Center

    Yess, James P.

    1981-01-01

    A study is reported that found marital status to be an important predictor of community college graduating quality point average in the following programs of study: Business Administration General, Business Administration Transfer, Executive Secretarial, and Nursing Education. (Author/MLW)

  15. Specific features of after-school program quality: associations with children's functioning in middle childhood.

    PubMed

    Pierce, Kim M; Bolt, Daniel M; Vandell, Deborah Lowe

    2010-06-01

    This longitudinal study examined associations between three after-school program quality features (positive staff-child relations, available activities, programming flexibility) and child developmental outcomes (reading and math grades, work habits, and social skills with peers) in Grade 2 and then Grade 3. Participants (n = 120 in Grade 2, n = 91 in Grade 3) attended after-school programs more than 4 days per week, on average. Controlling for child and family background factors and children's prior functioning on the developmental outcomes, positive staff-child relations in the programs were positively associated with children's reading grades in both Grades 2 and 3, and math grades in Grade 2. Positive staff-child relations also were positively associated with social skills in Grade 2, for boys only. The availability of a diverse array of age-appropriate activities at the programs was positively associated with children's math grades and classroom work habits in Grade 3. Programming flexibility (child choice of activities) was not associated with child outcomes.

  16. Beyond primary prevention of alcohol use: a culturally specific secondary prevention program for Mexican heritage adolescents.

    PubMed

    Marsiglia, Flavio F; Ayers, Stephanie; Gance-Cleveland, Bonnie; Mettler, Kathleen; Booth, Jaime

    2012-06-01

    Classroom-based primary prevention programs with adolescents are effective in inhibiting the onset of drug use, but these programs are not designed to directly address the unique needs of adolescents at higher risk of use or already using alcohol and other drugs. This article describes the initial efficacy evaluation of a companion psychosocial small group program which aims at addressing the needs of Mexican heritage students identified by their teachers as being at higher risk for substance use or already experimenting with alcohol and other drugs. The adolescent (7th grade) small group curricula, REAL Groups, is a secondary prevention program which supplements the primary classroom-based substance use prevention program, keepin' it REAL. Following a mutual aid approach, a total of 109 7th grade students were referred by their teachers and participated in the REAL Groups. The remaining 252 7th grade students who did not participate served as the control group. To account for biased selection into REAL Groups, propensity score matching (PSM) was employed. The estimated average treatment effect for participants' use of alcohol was calculated at the end of the 8th grade. Results indicate that alcohol use decreased among students who participated in the REAL Groups relative to matched students who did not participate. These findings suggest that REAL Groups may be an effective secondary prevention program for higher-risk Mexican heritage adolescents.

  17. Flood Catastrophe Model for Designing Optimal Flood Insurance Program: Estimating Location-Specific Premiums in the Netherlands.

    PubMed

    Ermolieva, T; Filatova, T; Ermoliev, Y; Obersteiner, M; de Bruijn, K M; Jeuken, A

    2017-01-01

    As flood risks grow worldwide, a well-designed insurance program engaging various stakeholders becomes a vital instrument in flood risk management. The main challenge concerns the applicability of standard approaches for calculating insurance premiums of rare catastrophic losses. This article focuses on the design of a flood-loss-sharing program involving private insurance based on location-specific exposures. The analysis is guided by a developed integrated catastrophe risk management (ICRM) model consisting of a GIS-based flood model and a stochastic optimization procedure with respect to location-specific risk exposures. To achieve the stability and robustness of the program towards floods with various recurrences, the ICRM uses stochastic optimization procedure, which relies on quantile-related risk functions of a systemic insolvency involving overpayments and underpayments of the stakeholders. Two alternative ways of calculating insurance premiums are compared: the robust derived with the ICRM and the traditional average annual loss approach. The applicability of the proposed model is illustrated in a case study of a Rotterdam area outside the main flood protection system in the Netherlands. Our numerical experiments demonstrate essential advantages of the robust premiums, namely, that they: (1) guarantee the program's solvency under all relevant flood scenarios rather than one average event; (2) establish a tradeoff between the security of the program and the welfare of locations; and (3) decrease the need for other risk transfer and risk reduction measures.

  18. Investigation of restriction-modification enzymes from M. varians RFL19 with a new type of specificity toward modification of substrate.

    PubMed Central

    Butkus, V; Klimasauskas, S; Kersulyte, D; Vaitkevicius, D; Lebionka, A; Janulaitis, A

    1985-01-01

    The characterization of MvaI restriction-modification enzymes, isolated from Micrococcus varians RFL19, is reported. Both enzymes recognize the 5'CC decreases (A/T)GG nucleotide sequence. The endonuclease cleaves the sequence at the position indicated by the arrow, whereas the methylase modifies the internal cytosine, yielding N4-methylcytosine. This type of modification protects the substrate from R.MvaI cleavage. 5-Methylcytosine in the same position of the recognition sequence does not protect the substrate from R.MvaI cleavage. R.MvaI proved to be the first example of a restriction endonuclease differentiating the position of the methyl group in the heterocyclic ring of cytosine, located in the same site of the recognition sequence. M.MvaI modifies DNA dcm+ in vitro yielding N4,5-dimethylcytosine. N4-methylcytosine cannot be differentiated from cytosine using the Maxam-Gilbert DNA sequencing procedure. Images PMID:2994011

  19. Substrate specificity and kinetic properties of enzymes belonging to the hormone-sensitive lipase family: comparison with non-lipolytic and lipolytic carboxylesterases.

    PubMed

    Chahinian, Henri; Ali, Yassine Ben; Abousalham, Abdelkarim; Petry, Stefan; Mandrich, Luigi; Manco, Guiseppe; Canaan, Stephane; Sarda, Louis

    2005-12-30

    We have studied the kinetics of hydrolysis of triacylglycerols, vinyl esters and p-nitrophenyl butyrate by four carboxylesterases of the HSL family, namely recombinant human hormone-sensitive lipase (HSL), EST2 from Alicyclobacillus acidocaldarius, AFEST from Archeoglobus fulgidus, and protein RV1399C from Mycobacterium tuberculosis. The kinetic properties of enzymes of the HSL family have been compared to those of a series of lipolytic and non-lipolytic carboxylesterases including human pancreatic lipase, guinea pig pancreatic lipase related protein 2, lipases from Mucor miehei and Thermomyces lanuginosus, cutinase from Fusarium solani, LipA from Bacillus subtilis, porcine liver esterase and Esterase A from Aspergilus niger. Results indicate that human HSL, together with other lipolytic carboxylesterases, are active on short chain esters and hydrolyze water insoluble trioctanoin, vinyl laurate and olive oil, whereas the action of EST2, AFEST, protein RV1399C and non-lipolytic carboxylesterases is restricted to solutions of short chain substrates. Lipolytic and non-lipolytic carboxylesterases can be differentiated by their respective value of K(0.5) (apparent K(m)) for the hydrolysis of short chain esters. Among lipolytic enzymes, those possessing a lid domain display higher activity on tributyrin, trioctanoin and olive oil suggesting, then, that the lid structure contributes to enzyme binding to triacylglycerols. Progress reaction curves of the hydrolysis of p-nitrophenyl butyrate by lipolytic carboxylesterases with lid domain show a latency phase which is not observed with human HSL, non-lipolytic carboxylesterases, and lipolytic enzymes devoid of a lid structure as cutinase.

  20. Germacrene A synthase in yarrow (Achillea millefolium) is an enzyme with mixed substrate specificity: gene cloning, functional characterization and expression analysis

    PubMed Central

    Pazouki, Leila; Memari, Hamid R.; Kännaste, Astrid; Bichele, Rudolf; Niinemets, Ülo

    2015-01-01

    Terpenoid synthases constitute a highly diverse gene family producing a wide range of cyclic and acyclic molecules consisting of isoprene (C5) residues. Often a single terpene synthase produces a spectrum of molecules of given chain length, but some terpene synthases can use multiple substrates, producing products of different chain length. Only a few such enzymes has been characterized, but the capacity for multiple-substrate use can be more widespread than previously thought. Here we focused on germacrene A synthase (GAS) that is a key cytosolic enzyme in the sesquiterpene lactone biosynthesis pathway in the important medicinal plant Achillea millefolium (AmGAS). The full length encoding gene was heterologously expressed in Escherichia coli BL21 (DE3), functionally characterized, and its in vivo expression was analyzed. The recombinant protein catalyzed formation of germacrene A with the C15 substrate farnesyl diphosphate (FDP), while acyclic monoterpenes were formed with the C10 substrate geranyl diphosphate (GDP) and cyclic monoterpenes with the C10 substrate neryl diphosphate (NDP). Although monoterpene synthesis has been assumed to be confined exclusively to plastids, AmGAS can potentially synthesize monoterpenes in cytosol when GDP or NDP become available. AmGAS enzyme had high homology with GAS sequences from other Asteraceae species, suggesting that multi-substrate use can be more widespread among germacrene A synthases than previously thought. Expression studies indicated that AmGAS was expressed in both autotrophic and heterotrophic plant compartments with the highest expression levels in leaves and flowers. To our knowledge, this is the first report on the cloning and characterization of germacrene A synthase coding gene in A. millefolium, and multi-substrate use of GAS enzymes. PMID:25784918

  1. Germacrene A synthase in yarrow (Achillea millefolium) is an enzyme with mixed substrate specificity: gene cloning, functional characterization and expression analysis.

    PubMed

    Pazouki, Leila; Memari, Hamid R; Kännaste, Astrid; Bichele, Rudolf; Niinemets, Ülo

    2015-01-01

    Terpenoid synthases constitute a highly diverse gene family producing a wide range of cyclic and acyclic molecules consisting of isoprene (C5) residues. Often a single terpene synthase produces a spectrum of molecules of given chain length, but some terpene synthases can use multiple substrates, producing products of different chain length. Only a few such enzymes has been characterized, but the capacity for multiple-substrate use can be more widespread than previously thought. Here we focused on germacrene A synthase (GAS) that is a key cytosolic enzyme in the sesquiterpene lactone biosynthesis pathway in the important medicinal plant Achillea millefolium (AmGAS). The full length encoding gene was heterologously expressed in Escherichia coli BL21 (DE3), functionally characterized, and its in vivo expression was analyzed. The recombinant protein catalyzed formation of germacrene A with the C15 substrate farnesyl diphosphate (FDP), while acyclic monoterpenes were formed with the C10 substrate geranyl diphosphate (GDP) and cyclic monoterpenes with the C10 substrate neryl diphosphate (NDP). Although monoterpene synthesis has been assumed to be confined exclusively to plastids, AmGAS can potentially synthesize monoterpenes in cytosol when GDP or NDP become available. AmGAS enzyme had high homology with GAS sequences from other Asteraceae species, suggesting that multi-substrate use can be more widespread among germacrene A synthases than previously thought. Expression studies indicated that AmGAS was expressed in both autotrophic and heterotrophic plant compartments with the highest expression levels in leaves and flowers. To our knowledge, this is the first report on the cloning and characterization of germacrene A synthase coding gene in A. millefolium, and multi-substrate use of GAS enzymes.

  2. The enzyme profiles in the connective tissue attaching pin bones to the surrounding tissue is specific in farmed salmon (Salmo salar) and cod (Gadus morhua L.).

    PubMed

    Vuong, Tram T; Rønning, Sissel B; Kolset, Svein O; Pedersen, Mona E

    2017-02-01

    Post mortem storage is a necessary process for removal of pin bones without destruction of fillets, thereby avoiding volume and economic loss. However, the enzymes involved in loosening pin bones during storage have not been studied to a great extent. In this study, the activities and localization of MMPs in the connective tissue (CT) of pin bones dissected from fillet of salmon and cod were investigated. Interestingly, the enzyme activity profile in these two species was different during post mortem storage of fish fillets. Adding MMP inhibitor (GM6001) and serine protease inhibitor (Pefabloc) revealed different effects in the two species, suggesting different regulations in salmon and cod. In situ zymography with the same inhibitors verified MMP and serine protease activity in CT close to pin bone at early post mortem (6 h) in salmon. However, MMP inhibition was not evident in cod in this area at that time point. Immunohistochemistry further revealed MMP9 and MMP13 were located more to the outer rim of CT, facing the pin bone and adipose tissue, while MMP7 was more randomly distributed within CT in salmon. In contrast, all these three MMPs were randomly distributed in CT in cod. In summary, our study reveals different MMP enzyme profiles in salmon and cod in the pin bone area, influenced by serine proteases, and suggests that MMPs and serine proteases must be taken in consideration when studying the conditions for early pin bone removal.

  3. Differentiation of Moraxella nonliquefaciens, M. lacunata, and M. bovis by using multilocus enzyme electrophoresis and hybridization with pilin-specific DNA probes.

    PubMed Central

    Tønjum, T; Caugant, D A; Bøvre, K

    1992-01-01

    Genetic relationships among strains of Moraxella nonliquefaciens, M. lacunata, and M. bovis were studied by using multilocus enzyme electrophoresis and DNA-DNA hybridization. The 74 isolates analyzed for electrophoretic variation at 12 enzyme loci were assigned to 59 multilocus genotypes. The multilocus genotypes were grouped in four major clusters, one representing strains of M. nonliquefaciens, two representing strains of M. lacunata, and one comprising strains of M. bovis and the single strain of M. equi analyzed. DNA-DNA hybridization with total genomic probes also revealed four major distinctive entities that corresponded to those identified by multilocus enzyme electrophoresis. The two distinct clusters recognized among the M. lacunata strains apparently corresponded to the species previously designated M. lacunata and M. liquefaciens. Distinction of the four entities was improved by hybridization with polymerase chain reaction products of nonconserved parts of pilin genes as DNA probes. With these polymerase chain reaction probes, new isolates of M. nonliquefaciens, M. lacunata, M. liquefaciens, and M. bovis can be identified easily by hybridization. PMID:1452691

  4. Enzyme-substrate complexes of the quinate/shikimate dehydrogenase from Corynebacterium glutamicum enable new insights in substrate and cofactor binding, specificity, and discrimination.

    PubMed

    Höppner, Astrid; Schomburg, Dietmar; Niefind, Karsten

    2013-11-01

    Quinate dehydrogenase (QDH) catalyzes the reversible oxidation of quinate to 3-dehydroquinate by nicotineamide adenine dinucleotide (NADH) and is involved in the catabolic quinate metabolism required for the degradation of lignin. The enzyme is a member of the family of shikimate/quinate dehydrogenases (SDH/QDH) occurring in bacteria and plants. We characterized the dual-substrate quinate/shikimate dehydrogenase (QSDH) from Corynebacterium glutamicum (CglQSDH) kinetically and revealed a clear substrate preference of CglQSDH for quinate compared with shikimate both at the pH optimum and in a physiological pH range, which is a remarkable contrast to closely related SDH/QDH enzymes. With respect to the cosubstrate, CglQSDH is strictly NAD(H) dependent. These substrate and cosubstrate profiles correlate well with the details of three atomic resolution crystal structures of CglQSDH in different functional states we report here: with bound NAD+ (binary complex) and as ternary complexes with NADH plus either shikimate or quinate. The CglQSDH-NADH-quinate structure is the first complex structure of any member of the SDH/QDH family with quinate. Based on this novel structural information and systematic sequence and structure comparisons with closely related enzymes, we can explain the strict NAD(H) dependency of CglQSDH as well as its discrimination between shikimate and quinate.

  5. As-Built design specification for the CLASFYT program. [production of classification files - crop inventory

    NASA Technical Reports Server (NTRS)

    Horton, C. L. (Principal Investigator)

    1981-01-01

    The CLASFYT program is described in detail. The program produces a one-channel universal-formatted classification file. Trajectory coefficients and a composite set of tolerance values are calculated from five acquisitions of radiance values in each of the training fields corresponding to up to ten agricultural products. These coefficients and tolerance values are used to classify each pixel in the test field of the same segment to be the same agricultural product as one of the training fields, none of the products or a screened pixel.

  6. Flight instrumentation specification for parameter identification: Program user's guide. [instrument errors/error analysis

    NASA Technical Reports Server (NTRS)

    Mohr, R. L.

    1975-01-01

    A set of four digital computer programs is presented which can be used to investigate the effects of instrumentation errors on the accuracy of aircraft and helicopter stability-and-control derivatives identified from flight test data. The programs assume that the differential equations of motion are linear and consist of small perturbations about a quasi-steady flight condition. It is also assumed that a Newton-Raphson optimization technique is used for identifying the estimates of the parameters. Flow charts and printouts are included.

  7. Course Design and Delivery Specifications as a Tool for Ensuring Quality in an Online Training Program

    ERIC Educational Resources Information Center

    Docq, Françoise

    2015-01-01

    This case discusses the design, implementation, and regulation of a hybrid training program (60 credits over two years) organised by three business schools in Europe, and stretching over a five-year period. Following an incremental design process, the design team faced multiple challenges, from finding the added value of hybridization to choosing…

  8. Safety in the Chemical Laboratory: Safety in the Chemistry Laboratories: A Specific Program.

    ERIC Educational Resources Information Center

    Corkern, Walter H.; Munchausen, Linda L.

    1983-01-01

    Describes a safety program adopted by Southeastern Louisiana University. Students are given detailed instructions on laboratory safety during the first laboratory period and a test which must be completely correct before they are allowed to return to the laboratory. Test questions, list of safety rules, and a laboratory accident report form are…

  9. GirlPOWER! Strengthening Mentoring Relationships through a Structured, Gender-Specific Program

    ERIC Educational Resources Information Center

    Pryce, Julia M.; Silverthorn, Naida; Sanchez, Bernadette; DuBois, David L.

    2010-01-01

    The authors examine GirlPOWER! an innovative program that uses structure and group-based activities to enhance one-to-one mentoring relationships for young adolescent girls from the perspective of the focus, purpose, and authorship dimensions of mentoring relationships that Karcher and Nakkula described. The discussion draws on several sources of…

  10. 42 CFR 457.1150 - Program specific review process: Impartial review.

    Code of Federal Regulations, 2010 CFR

    2010-10-01

    ... matter under review. (b) Health services matter. The State must ensure that an enrollee has an... conducted by the State or a contractor other than the contractor responsible for the matter subject to... AND HUMAN SERVICES (CONTINUED) STATE CHILDREN'S HEALTH INSURANCE PROGRAMS (SCHIPs) ALLOTMENTS...

  11. 42 CFR 457.1160 - Program specific review process: Time frames.

    Code of Federal Regulations, 2010 CFR

    2010-10-01

    ... process: Time frames. (a) Eligibility or enrollment matter. A State must complete the review of a matter... services matter. The State must ensure that reviews are completed in accordance with the medical needs of... HUMAN SERVICES (CONTINUED) STATE CHILDREN'S HEALTH INSURANCE PROGRAMS (SCHIPs) ALLOTMENTS AND GRANTS...

  12. Ecosystem Services Research Program, Pollutant Specific Studies: Nitrogen Regulations Services Implementation Plan

    EPA Science Inventory

    The Ecosystem Services Research Program (ESRP) is a new, multi-year research initiative under development by the U.S. Environmental Protection Agency (EPA). The overall goal of the ESRP is to transform the way decision-makers understand and respond to environmental issues, making...

  13. 7 CFR 1755.522 - RUS general specification for digital, stored program controlled central office equipment.

    Code of Federal Regulations, 2013 CFR

    2013-01-01

    ... requirements for a digital telephone central office switching system, which is fully electronic and controlled by stored program processors. A digital switching system transfers information which is digitally encoded from any input port to a temporarily addressed exit port. The information may enter the system...

  14. 7 CFR 1755.522 - RUS general specification for digital, stored program controlled central office equipment.

    Code of Federal Regulations, 2014 CFR

    2014-01-01

    ... requirements for a digital telephone central office switching system, which is fully electronic and controlled by stored program processors. A digital switching system transfers information which is digitally encoded from any input port to a temporarily addressed exit port. The information may enter the system...

  15. 7 CFR 1755.522 - RUS general specification for digital, stored program controlled central office equipment.

    Code of Federal Regulations, 2012 CFR

    2012-01-01

    ... requirements for a digital telephone central office switching system, which is fully electronic and controlled by stored program processors. A digital switching system transfers information which is digitally encoded from any input port to a temporarily addressed exit port. The information may enter the system...

  16. GirlPOWER! Strengthening mentoring relationships through a structured, gender-specific program.

    PubMed

    Pryce, Julia M; Silverthorn, Naida; Sanchez, Bernadette; DuBois, David L

    2010-01-01

    The authors examine GirlPOWER! an innovative program that uses structure and group-based activities to enhance one-to-one mentoring relationships for young adolescent girls from the perspective of the focus, purpose, and authorship dimensions of mentoring relationships that Karcher and Nakkula described. The discussion draws on several sources of data that contributed to the development and ongoing refinement of the program. The authors highlight their efforts to design the program in a way that navigates the tensions they encountered in balancing attention to competing concerns associated with each dimension. Based on their analysis, they conclude that what may appear to be competing areas of emphasis in mentoring relationships, such as a focus on goals or relationship development, may in practice often prove to be mutually reinforcing and thus synergistic. Their experience underscores a need to complement program enhancements such as GirlPOWER! with individualized support that is geared to the unique backgrounds of mentors and the distinctive features of each mentoring relationship.

  17. Ada Integrated Environment III Computer Program Development Specification. Volume III. Ada Optimizing Compiler.

    DTIC Science & Technology

    1981-12-01

    Corporation , Order No. GY28-6800-5 (December 1971). [IBM72] FORTRAN IV (H) Compiler Program Logic Manual, IBM Corporation , Order No. GH28-6642-5...RAVC ptans6 and excutes~ ’Leseatc, devetopment, .te,~t and 4etected acquisition p)LopLamn in -sappo~~t o6 Command, Con-tt Commnications ~ and

  18. Specific and long-term effects of Nova Scotia's graduated licensing program.

    PubMed

    Mayhew, Daniel R; Simpson, Herbert M; Desmond, Katharine; Williams, Allan F

    2003-06-01

    A graduated licensing (GL) program was introduced in Nova Scotia, Canada, in October 1994. Previous research has shown that it reduced collisions in the short term. The present study examined the relative contribution of each stage of the program (i.e., learner and intermediate levels) and the program's impact after beginning drivers graduated to full licensure. The research focused on teenage beginning drivers (age 16-17), but the effects on older beginners also was examined. Per-driver crash rates of two groups of novices selected from driver records in Nova Scotia were compared. One group (pre-GL) received their learner's permits during the 2 years before the program was implemented, and the second group (GL) received their learner's permits during the 2 years after implementation. The findings clearly establish that most of the collision reduction in Nova Scotia's program occurred during the first year of the program, particularly during the first 6 months when the majority of novices were driving under supervision. The collision rate for 16 to 17-year-old GL novices was 50% lower than the rate for pre-GL novices during the 6 months after they received their learner's permits, and about 10% lower during their first 2 years of licensure when unsupervised driving from midnight to 5 A.M. was prohibited. Much of this improvement for 16 to 17-year-olds occurred during restricted night hours. Collision rates also were lower during nonrestricted hours in the initial 6 months of licensure. The 3-month "time discount" for driver education provided no safety benefit, and GL novices with driver education had collision rates that were not lower than pre-GL novices. There was no long-term effect found for the program after 16 to 17-year-olds graduated to full licensure. For older beginning drivers, crash rates during the first year after obtaining a learner's permit showed a 31% reduction. This effect diminished rapidly. There was only a 2% reduction during the first year

  19. Programming with non-heap memory in the real time specification for Java

    NASA Technical Reports Server (NTRS)

    Bollella, G.; Canham, T.; Carson, V.; Champlin, V.; Dvorak, D.; Giovannoni, B.; Indictor, M.; Meyer, K.; Reinholtz, A.; Murray, K.

    2003-01-01

    The Real-Time Specification for Java (RTSJ) provides facilities for deterministic, real-time execution in a language that is otherwise subject to variable latencies in memory allocation and garbage collection.

  20. Managing Challenging Situations in Practice: a new program developed to meet the specific needs of nursing students.

    PubMed

    Lyng, Colette; Cocoman, Angela; Ward, Emer; McGrath, Mary

    2012-11-01

    Health care workers, nurses, and nursing students face a high risk of workplace aggression and violence. Potential adverse consequences oblige health care providers and educators to protect the safety of everyone in the health care setting. It is broadly agreed that health care personnel should receive education and training in the management of work-related aggression and violence. However, there are no training programs designed to meet the specific needs of nursing students. In the absence of Irish or international policies or guidelines, an evidence-based training program for first-year undergraduate nursing students was developed. Its focus was to enable nursing students to recognize potential problems and develop the skills necessary to appropriately handle situations that may arise during their clinical nursing practice. This article outlines the development and delivery of a training program for first-year nursing students, entitled Managing Challenging Situations in Practice.

  1. Simplifying the construction of domain-specific automatic programming systems: The NASA automated software development workstation project

    NASA Technical Reports Server (NTRS)

    Allen, Bradley P.; Holtzman, Peter L.

    1987-01-01

    An overview is presented of the Automated Software Development Workstation Project, an effort to explore knowledge-based approaches to increasing software productivity. The project focuses on applying the concept of domain specific automatic programming systems (D-SAPSs) to application domains at NASA's Johnson Space Center. A version of a D-SAPS developed in Phase 1 of the project for the domain of space station momentum management is described. How problems encountered during its implementation led researchers to concentrate on simplifying the process of building and extending such systems is discussed. Researchers propose to do this by attacking three observed bottlenecks in the D-SAPS development process through the increased automation of the acquisition of programming knowledge and the use of an object oriented development methodology at all stages of the program design. How these ideas are being implemented in the Bauhaus, a prototype workstation for D-SAPS development is discussed.

  2. Simplifying the construction of domain-specific automatic programming systems: The NASA automated software development workstation project

    NASA Technical Reports Server (NTRS)

    Allen, Bradley P.; Holtzman, Peter L.

    1988-01-01

    An overview is presented of the Automated Software Development Workstation Project, an effort to explore knowledge-based approaches to increasing software productivity. The project focuses on applying the concept of domain specific automatic programming systems (D-SAPSs) to application domains at NASA's Johnson Space Flight Center. A version of a D-SAPS developed in Phase 1 of the project for the domain of space station momentum management is described. How problems encountered during its implementation led researchers to concentrate on simplifying the process of building and extending such systems is discussed. Researchers propose to do this by attacking three observed bottlenecks in the D-SAPS development process through the increased automation of the acquisition of programming knowledge and the use of an object oriented development methodology at all stages of the program design. How these ideas are being implemented in the Bauhaus, a prototype workstation for D-SAPS development is discussed.

  3. Tissue-specific contaminant accumulation and associated effects on hepatic serum analytes and cytochrome P450 enzyme activities in hooded seals (Cystophora cristata) from the Gulf of St. Lawrence.

    PubMed

    Wolkers, Hans; Boily, France; Fink-Gremmels, Johanna; van Bavel, Bert; Hammill, Mike O; Primicerio, Raul

    2009-02-01

    The current study aims to assess contaminant levels and tissue burdens in hooded seal (Cystophora cristata) blubber, liver, and blood in association with cytochrome P450 (CYP) enzymes (CYP1A and -3A) and serum analytes (hepatic enzymes like alanine aminotransferase [ALT], aspartate aminotransferase, alkaline phosphatase [AP], and gamma-glutamyltransferase [GGT], serum proteins, and creatine kinase). Contaminant accumulation levels and patterns of polychlorinated biphenyls, chlorinated pesticides, and polybrominated diphenyl ethers (PBDEs) differed between tissues and seal groups, with the highest levels in liver. Pups showed higher liver contaminant levels, especially for PBDEs, than adults. These high levels might be associated with the ingestion of large amounts of contaminated milk and subsequent accumulation in the liver. Adult males and females mainly differed in PBDE levels, which were higher in females, possibly due to a sex-specific diet. The association between blubber contaminant burdens and the diagnostic enzymes ALT, GGT, and AP, and serum albumin, was inconclusive. In contrast, several CYP isoenzymes showed a clear positive relationship with the overall blubber contaminant burden, indicating enzyme induction following exposure to polyhalogenated hydrocarbons. Therefore, liver CYP isoenzymes may serve as a sensitive biomarker for long-term exposure to polyhalogenated hydrocarbons.

  4. The tomato Fni3 lysine-63-specific ubiquitin-conjugating enzyme and suv ubiquitin E2 variant positively regulate plant immunity.

    PubMed

    Mural, Ravi V; Liu, Yao; Rosebrock, Tracy R; Brady, Jennifer J; Hamera, Sadia; Connor, Richard A; Martin, Gregory B; Zeng, Lirong

    2013-09-01

    The activation of an immune response in tomato (Solanum lycopersicum) against Pseudomonas syringae relies on the recognition of E3 ligase-deficient forms of AvrPtoB by the host protein kinase, Fen. To investigate the mechanisms by which Fen-mediated immunity is regulated, we characterize in this study a Fen-interacting protein, Fni3, and its cofactor, S. lycoperiscum Uev (Suv). Fni3 encodes a homolog of the Ubc13-type ubiquitin-conjugating enzyme that catalyzes exclusively Lys-63-linked ubiquitination, whereas Suv is a ubiquitin-conjugating enzyme variant. The C-terminal region of Fen was necessary for interaction with Fni3, and this interaction was required for cell death triggered by overexpression of Fen in Nicotiana benthamiana leaves. Fni3 was shown to be an active E2 enzyme, but Suv displayed no ubiquitin-conjugating activity; Fni3 and Suv together directed Lys-63-linked ubiquitination. Decreased expression of Fni3, another tomato Ubc13 homolog, Sl-Ubc13-2, or Suv in N. benthamiana leaves diminished cell death associated with Fen-mediated immunity and cell death elicited by several other resistance (R) proteins and their cognate effectors. We also discovered that coexpression of Fen and other R proteins/effectors with a Fni3 mutant that is compromised for ubiquitin-conjugating activity diminished the cell death. These results suggest that Fni3/Sl-Ubc13-2 and Suv regulate the immune response mediated by Fen and other R proteins through Lys-63-linked ubiquitination.

  5. Space Shuttle Program (SSP) Shock Test and Specification Experience for Reusable Flight Hardware Equipment

    NASA Technical Reports Server (NTRS)

    Larsen, Curtis E.

    2012-01-01

    As commercial companies are nearing a preliminary design review level of design maturity, several companies are identifying the process for qualifying their multi-use electrical and mechanical components for various shock environments, including pyrotechnic, mortar firing, and water impact. The experience in quantifying the environments consists primarily of recommendations from Military Standard-1540, Product Verification Requirement for Launch, Upper Stage, and Space Vehicles. Therefore, the NASA Engineering and Safety Center (NESC) formed a team of NASA shock experts to share the NASA experience with qualifying hardware for the Space Shuttle Program (SSP) and other applicable programs and projects. Several team teleconferences were held to discuss past experience and to share ideas of possible methods for qualifying components for multiple missions. This document contains the information compiled from the discussions

  6. Using Space Weather Variability in Evaluation the Radiation Environment Specifications for NASA's Constellation Program

    NASA Technical Reports Server (NTRS)

    Coffey, Victoria N.; Minow, Joseph I.; Bruce, Margaret; Howard, James W.

    2008-01-01

    Hardware design environments for NASA's Constellation Program-the Vision for Space Exploration program to design and build new vehicles for servicing low Earth orbit and the Moon and beyond-have been developed that are necessarily conservative in nature to assure robust hardware design and development required to build space systems which will meet operational goals in a wide range of space environments, This presentation will describe the rationale used to establish the space radiation and plasma design environments specified for a variety of applications including total ionizing radiation dose, dose rate effects, and spacecraft charging and will compare the design environments with "space weather" variability to evaluate the applicability of the design environments and potential vulnerabilities of the system to extreme space weather events.

  7. MFEprimer-2.0: a fast thermodynamics-based program for checking PCR primer specificity.

    PubMed

    Qu, Wubin; Zhou, Yang; Zhang, Yanchun; Lu, Yiming; Wang, Xiaolei; Zhao, Dongsheng; Yang, Yi; Zhang, Chenggang

    2012-07-01

    Evaluating the specificity of polymerase chain reaction (PCR) primers is an essential step in PCR primer design. The MFEprimer-2.0 server allows users to check primer specificity against genomic DNA and messenger RNA/complementary DNA sequence databases quickly and easily. MFEprimer-2.0 uses a k-mer index algorithm to accelerate the search process for primer binding sites and uses thermodynamics to evaluate binding stability between each primer and its DNA template. Several important characteristics, such as the sequence, melting temperature and size of each amplicon, either specific or non-specific, are reported on the results page. Based on these characteristics and the user-friendly output, users can readily draw conclusions about the specificity of PCR primers. Analyses for degenerate primers and multiple PCR primers are also supported in MFEprimer-2.0. In addition, the databases supported by MFEprimer-2.0 are comprehensive, and custom databases can also be supported on request. The MFEprimer-2.0 server does not require a login and is freely available at http://biocompute.bmi.ac.cn/CZlab/MFEprimer-2.0. More over, the MFEprimer-2.0 command-line version and local server version are open source and can be downloaded at https://github.com/quwubin/MFEprimer/wiki/Manual/.

  8. Program Design Language Architecture Specification for the AN/UYK-7 Central Processor

    DTIC Science & Technology

    1980-02-01

    description of the central processor architecture of the Navy Standard AN/UYK-7 computer.-i... architecture was described using a Program Design Language...Information Center Cameron Station Alexandria, VA 22314 (12) Defense Printing Service Washinqton Navy Yard Washinqton, DC 20374 Library of Conqress Washington...GCfIu G Sall Ditqo, r ’’ I 𔃼.17 NAVSEC Code 6122 Department of the Navy Washington, DC 20362 ATTN: Joe Mallonee NUSC Code 313 New London, CONN 06320

  9. Genomic selection needs to be carefully assessed to meet specific requirements in livestock breeding programs.

    PubMed

    Jonas, Elisabeth; de Koning, Dirk-Jan

    2015-01-01

    Genomic selection is a promising development in agriculture, aiming improved production by exploiting molecular genetic markers to design novel breeding programs and to develop new markers-based models for genetic evaluation. It opens opportunities for research, as novel algorithms and lab methodologies are developed. Genomic selection can be applied in many breeds and species. Further research on the implementation of genomic selection (GS) in breeding programs is highly desirable not only for the common good, but also the private sector (breeding companies). It has been projected that this approach will improve selection routines, especially in species with long reproduction cycles, late or sex-limited or expensive trait recording and for complex traits. The task of integrating GS into existing breeding programs is, however, not straightforward. Despite successful integration into breeding programs for dairy cattle, it has yet to be shown how much emphasis can be given to the genomic information and how much additional phenotypic information is needed from new selection candidates. Genomic selection is already part of future planning in many breeding companies of pigs and beef cattle among others, but further research is needed to fully estimate how effective the use of genomic information will be for the prediction of the performance of future breeding stock. Genomic prediction of production in crossbreeding and across-breed schemes, costs and choice of individuals for genotyping are reasons for a reluctance to fully rely on genomic information for selection decisions. Breeding objectives are highly dependent on the industry and the additional gain when using genomic information has to be considered carefully. This review synthesizes some of the suggested approaches in selected livestock species including cattle, pig, chicken, and fish. It outlines tasks to help understanding possible consequences when applying genomic information in breeding scenarios.

  10. Aquatic Plant Control Research Program: Host Specificity of Microbial Flora from Eurasian Watermilfoil

    DTIC Science & Technology

    1990-06-01

    IDENTIFICATION NUMBER ORGANIZATION (If applicable) US Army Corps of Engineers I 8c ADDRESS (rity, State, and ZIP Code) 10. SOURCE OF FUNDING NUMBERS PROGRAM...required microorganisms for testing, some fieldwork was necessary to isolate prospective microbes. However, the emphasis was on developing a screening...ability. 3. Study hypotheses were that microbes isolated from milfoil (or else- where) segregate by growth potential into colonists and noncolonists

  11. High-throughput profiling of off-target DNA cleavage reveals RNA-programmed Cas9 nuclease specificity.

    PubMed

    Pattanayak, Vikram; Lin, Steven; Guilinger, John P; Ma, Enbo; Doudna, Jennifer A; Liu, David R

    2013-09-01

    The RNA-programmable Cas9 endonuclease cleaves double-stranded DNA at sites complementary to a 20-base-pair guide RNA. The Cas9 system has been used to modify genomes in multiple cells and organisms, demonstrating its potential as a facile genome-engineering tool. We used in vitro selection and high-throughput sequencing to determine the propensity of eight guide-RNA:Cas9 complexes to cleave each of 10(12) potential off-target DNA sequences. The selection results predicted five off-target sites in the human genome that were confirmed to undergo genome cleavage in HEK293T cells upon expression of one of two guide-RNA:Cas9 complexes. In contrast to previous models, our results show that guide-RNA:Cas9 specificity extends past a 7- to 12-base-pair seed sequence. Our results also suggest a tradeoff between activity and specificity both in vitro and in cells as a shorter, less-active guide RNA is more specific than a longer, more-active guide RNA. High concentrations of guide-RNA:Cas9 complexes can cleave off-target sites containing mutations near or within the PAM that are not cleaved when enzyme concentrations are limiting.

  12. A competitive enzyme-linked immunosorbent assay specific for murine hepcidin-1: correlation with hepatic mRNA expression in established and novel models of dysregulated iron homeostasis.

    PubMed

    Gutschow, Patrick; Schmidt, Paul J; Han, Huiling; Ostland, Vaughn; Bartnikas, Thomas B; Pettiglio, Michael A; Herrera, Carolina; Butler, James S; Nemeth, Elizabeta; Ganz, Tomas; Fleming, Mark D; Westerman, Mark

    2015-02-01

    Mice have been essential for distinguishing the role of hepcidin in iron homeostasis. Currently, investigators monitor levels of murine hepatic hepcidin-1 mRNA as a surrogate marker for the bioactive hepcidin protein itself. Here, we describe and validate a competitive, enzyme-linked immunosorbent assay that quantifies hepcidin-1 in mouse serum and urine. The assay exhibits a biologically relevant lower limit of detection, high precision, and excellent linearity and recovery. We also demonstrate correlation between serum and urine hepcidin-1 values and validate the competitive enzyme-linked immunosorbent assay by analyzing plasma hepcidin response of mice to physiological challenges, including iron deficiency, iron overload, acute blood loss, and inflammation. Furthermore, we analyze multiple murine genetic models of iron dysregulation, including β-thalassemia intermedia (Hbb(th3/+)), hereditary hemochromatosis (Hfe(-/-), Hjv(-/-), and Tfr2(Y245X/Y245X)), hypotransferrinemia (Trf(hpx/hpx)), heterozygous transferrin receptor 1 deficiency (Tfrc(+/-)) and iron refractory iron deficiency anemia (Tmprss6(-/-) and Tmprss6(hem8/hem8)). Novel compound iron metabolism mutants were also phenotypically characterized here for the first time. We demonstrate that serum hepcidin concentrations correlate with liver hepcidin mRNA expression, transferrin saturation and non-heme liver iron. In some circumstances, serum hepcidin-1 more accurately predicts iron parameters than hepcidin mRNA, and distinguishes smaller, statistically significant differences between experimental groups.

  13. Experiment K304: Studies of specific hepatic enzymes and liver constituents involved in the conversion of carbohydrates to lipids in rats exposed to prolonged space flight

    NASA Technical Reports Server (NTRS)

    Abraham, S.; Klein, H. P.; Lin, C. Y.; Volkmann, C.; Tigranyan, R. A.; Vetrova, E. G.

    1981-01-01

    The effects of space flight on the activities of 26 enzymes concerned with carbohydrate and lipid metabolism in hepatic tissue taken from male Wistar rats are investigated. These activities were measured in the various hepatic cell compartments, i.e., cytosol, mitochondria and microsomes. In addition, the levels of glycogen, total lipids, phospholipids, triglycerides, cholesterol, cholesterol esters, and the fatty acid composition of the rat livers were also examined and quantified. A similar group of ground-based rats treated in an identical manner served as controls. Both flight and synchronous control rats were sacrificed at three time intervals: R+0, 7-11 hours after recovery; R+6, after 6 days; R+6(S), after 6 days (having undergone 2-5 hour periods of fixed stress in a "backupward" position on days 0, 3, 4, 5 and 6) and R+29, after 29 days post-flight. Although most of the enzyme activities and the amounts of liver constituents studied were unaffected by the period of weightlessness, some significant differences were observed.

  14. Heterodisaccharide 4-O-(N-acetyl-beta-D-glucosaminyl)-D-glucosamine is a specific inducer of chitinolytic enzyme production in Vibrios harboring chitin oligosaccharide deacetylase genes.

    PubMed

    Hirano, Takako; Kadokura, Kazunari; Ikegami, Takanori; Shigeta, Yuko; Kumaki, Yasuko; Hakamata, Wataru; Oku, Tadatake; Nishio, Toshiyuki

    2009-09-01

    Vibrio parahaemolyticus KN1699 produces 4-O-(N-acetyl-beta-d-glucosaminyl)-d-glucosamine (GlcNAc-GlcN) as a major end product from chitin using two extracellular hydrolases: glycoside hydrolase family 18 chitinase, which produces (GlcNAc)(2) from chitin, and carbohydrate esterase (CE) family 4 chitin oligosaccharide deacetylase (COD), which hydrolyzes the N-acetyl group at the reducing-end GlcNAc residue of (GlcNAc)(2). In this study, we clarified that this heterodisaccharide functions as an inducer of the production of the two above-mentioned chitinolytic enzymes, particularly chitinase. Similar results for chitinase production were obtained with other chitin-decomposing Vibrio strains harboring the CE family 4 COD gene; however, such an increase in chitinase production was not observed in chitinolytic Vibrio strains that did not harbor the COD gene. These results suggest that GlcNAc-GlcN is a unique inducer of chitinase production in Vibrio bacteria that have the COD-producing ability and that the COD involved in the synthesis of this signal compound is one of the key enzymes in the chitin catabolic cascade of these bacteria.

  15. Mod-5A wind turbine generator program design report. Volume 4: Drawings and specifications, book 5

    NASA Technical Reports Server (NTRS)

    1984-01-01

    The design, development and analysis of the 7.3 MW MOD-5A wind turbine generator is documented. There are four volumes. This volume contains the drawings and specifications that were developed in preparation for building the MOD-5A wind turbine generator. Detail drawings of several assemblies and subassemblies are given. This is the fifth book of volume 4.

  16. Chemotactic separation of enzymes.

    PubMed

    Dey, Krishna Kanti; Das, Sambeeta; Poyton, Matthew F; Sengupta, Samudra; Butler, Peter J; Cremer, Paul S; Sen, Ayusman

    2014-12-23

    We demonstrate a procedure for the separation of enzymes based on their chemotactic response toward an imposed substrate concentration gradient. The separation is observed within a two-inlet, five-outlet microfluidic network, designed to allow mixtures of active (ones that catalyze substrate turnover) and inactive (ones that do not catalyze substrate turnover) enzymes, labeled with different fluorophores, to flow through one of the inlets. Substrate solution prepared in phosphate buffer was introduced through the other inlet of the device at the same flow rate. The steady-state concentration profiles of the enzymes were obtained at specific positions within the outlets of the microchannel using fluorescence microscopy. In the presence of a substrate concentration gradient, active enzyme molecules migrated preferentially toward the substrate channel. The excess migration of the active enzyme molecules was quantified in terms of an enrichment coefficient. Experiments were carried out with different pairs of enzymes. Coupling the physics of laminar flow of liquid and molecular diffusion, multiphysics simulations were carried out to estimate the extent of the chemotactic separation. Our results show that, with appropriate microfluidic arrangement, molecular chemotaxis leads to spontaneous separation of active enzyme molecules from their inactive counterparts of similar charge and size.

  17. Data systems elements technology assessment and system specifications, issue no. 2. [nasa programs

    NASA Technical Reports Server (NTRS)

    1978-01-01

    The ability to satisfy the objectives of future NASA Office of Applications programs is dependent on technology advances in a number of areas of data systems. The hardware and software technology of end-to-end systems (data processing elements through ground processing, dissemination, and presentation) are examined in terms of state of the art, trends, and projected developments in the 1980 to 1985 timeframe. Capability is considered in terms of elements that are either commercially available or that can be implemented from commercially available components with minimal development.

  18. Formal semantic specifications as implementation blueprints for real-time programming languages

    NASA Technical Reports Server (NTRS)

    Feyock, S.

    1981-01-01

    Formal definitions of language and system semantics provide highly desirable checks on the correctness of implementations of programming languages and their runtime support systems. If these definitions can give concrete guidance to the implementor, major increases in implementation accuracy and decreases in implementation effort can be achieved. It is shown that of the wide variety of available methods the Hgraph (hypergraph) definitional technique (Pratt, 1975), is best suited to serve as such an implementation blueprint. A discussion and example of the Hgraph technique is presented, as well as an overview of the growing body of implementation experience of real-time languages based on Hgraph semantic definitions.

  19. DNA Adduct Formation from Metabolic 5'-Hydroxylation of the Tobacco-Specific Carcinogen N'-Nitrosonornicotine in Human Enzyme Systems and in Rats.

    PubMed

    Zarth, Adam T; Upadhyaya, Pramod; Yang, Jing; Hecht, Stephen S

    2016-03-21

    N'-Nitrosonornicotine (NNN) is carcinogenic in multiple animal models and has been evaluated as a human carcinogen. NNN can be metabolized by cytochrome P450s through two activation pathways: 2'-hydroxylation and 5'-hydroxylation. While most previous studies have focused on 2'-hydroxylation in target tissues of rats, available evidence suggests that 5'-hydroxylation is a major activation pathway in human enzyme systems, in nonhuman primates, and in target tissues of some other rodent carcinogenicity models. In the study reported here, we investigated DNA damage resulting from NNN 5'-hydroxylation by quantifying the adduct 2-(2-(3-pyridyl)-N-pyrrolidinyl)-2'-deoxyinosine (py-py-dI). In rats treated with NNN in the drinking water (7-500 ppm), py-py-dI was the major DNA adduct resulting from 5'-hydroxylation of NNN in vivo. Levels of py-py-dI in the lung and nasal cavity were the highest, consistent with the tissue distribution of CYP2A3. In rats treated with (S)-NNN or (R)-NNN, the ratios of formation of (R)-py-py-dI to (S)-py-py-dI were not the expected mirror image, suggesting that there may be a carrier for one of the unstable intermediates formed upon 5'-hydroxylation of NNN. Rat hepatocytes treated with (S)- or (R)-NNN or (2'S)- or (2'R)-5'-acetoxyNNN exhibited a pattern of adduct formation similar to that of live rats. In vitro studies with human liver S9 fraction or human hepatocytes incubated with NNN (2-500 μM) demonstrated that py-py-dI formation was greater than the formation of pyridyloxobutyl-DNA adducts resulting from 2'-hydroxylation of NNN. (S)-NNN formed more total py-py-dI adducts than (R)-NNN in human liver enzyme systems, which is consistent with the critical role of CYP2A6 in the 5'-hydroxylation of NNN in human liver. The results of this study demonstrate that the major DNA adduct resulting from NNN metabolism by human enzymes is py-py-dI and provide potentially important new insights into the metabolic activation of NNN in rodents and humans.

  20. Re-engineering the discrimination between the oxidized coenzymes NAD+ and NADP+ in clostridial glutamate dehydrogenase and a thorough reappraisal of the coenzyme specificity of the wild-type enzyme.

    PubMed

    Capone, Marina; Scanlon, David; Griffin, Joanna; Engel, Paul C

    2011-07-01

    Clostridial glutamate dehydrogenase mutants, designed to accommodate the 2'-phosphate of disfavoured NADPH, showed the expected large specificity shifts with NAD(P)H. Puzzlingly, similar assays with oxidized cofactors initially revealed little improvement with NADP(+) , although rates with NAD(+) were markedly diminished. This article reveals that the enzyme's discrimination in favour of NAD(+) and against NADP(+) had been greatly underestimated and has indeed been abated by a factor of > 16,000 by the mutagenesis. Initially, stopped-flow studies of the wild-type enzyme showed a burst increase of A(340) with NADP(+) but not NAD(+), with amplitude depending on the concentration of the coenzyme, rather than enzyme. Amplitude also varied with the commercial source of the NADP(+). FPLC, HPLC and mass spectrometry identified NAD(+) contamination ranging from 0.04 to 0.37% in different commercial samples. It is now clear that apparent rates of NADP(+) utilization mainly reflected the reduction of contaminating NAD(+), creating an entirely false view of the initial coenzyme specificity and also of the effects of mutagenesis. Purification of the NADP(+) eliminated the burst. With freshly purified NADP(+), the NAD(+) : NADP(+) activity ratio under standard conditions, previously estimated as 300 : 1, is 11,000. The catalytic efficiency ratio is even higher at 80,000. Retested with pure cofactor, mutants showed marked specificity shifts in the expected direction, for example, 16 200 fold change in catalytic efficiency ratio for the mutant F238S/P262S, confirming that the key structural determinants of specificity have been successfully identified. Of wider significance, these results underline that, without purification, even the best commercial coenzyme preparations are inadequate for such studies.

  1. [The effect of muscle load on the post-prandial content of blood serum hydrolytic enzymes in men with different levels and specificity of daily physical activity].

    PubMed

    Griaznykh, A V

    2014-01-01

    The article presents data on the effect of the combined action of food and muscular load on the level of hydrolytic enzymes in blood serum of healthy young people 18-22 years old, with various levels of adaptation to the effects of physical activity. The first group (n = 8) of the examined persons were high-qualified athletes developing their speed and power qualities in anaerobic energetic regime (Greco-Roman wrestling, sambo, judo). The second group (n = 8) were athletes developing endurance in aerobic energetic regime (skiers, track and field athletes--stayers, biathletes). The control group (n = 8) consists of non-athletes. The content of hydrolytic enzymes: pepsinogen-1, pepsinogen-2, the activity of pancreatic alpha-amylase, lipase were defined by ELISA. The content and activity of ferments were defined in blood serum, taken in the morning fasting and post-prandial period in dynamics after 15, 45, 75 and 105 min after administration of the test breakfast (100 g of ground boiled beef and 200 ml of unsweetened tea) in a state of relative physiological rest and after the veloergometric exercise muscular load (at the level of 60-70% of maximal oxygen consumption) during an hour (in 7-14 days). Multidirectional changes of concentration of investigated enzymes in the postprandial period among examined were defined in the conditions of relative physiological rest and under the action of the muscular tension. For groups of athletes higher alpha-amylase and lipase blood activity were characteristic both in a state of physiological rest and under the action of muscular load. It was also determined that after the muscular tension there was an increase in activity of alpha-amylase at 75 min and lipases at 15 min relative to background indicators at non-athletes. For the athletes from the second group the increase (p < 0.01) relative to background data of activity as alpha-amylase as lipase on an empty stomach was noted. However postprandial (15-45 min) alpha-amylase (p

  2. Telecommunication system specific to high temperature environment for JAXA Mercury exploration program

    NASA Astrophysics Data System (ADS)

    Toda, Tomoaki; Kamata, Yukio; Kawahara, Kousuke; Maejima, Hironori; Hayakawa, Hajime

    2014-02-01

    BepiColombo is the joint Mercury exploration program between JAXA (Japan Aerospace Exploration Agency) and ESA (European Space Agency). MMO (Mercury Magnetospheric Orbiter) is JAXA's satellite in this program. She requires a telecommunication system that survives a harsh heat environment surrounding Mercury. She will stay in a polar orbit circulating Mercury for a year for continuous observations of Mercury magnetosphere. MMO has an X-band telecommunication system. We newly developed a high gain antenna for the use of her daily operations and wider field of view antennas for critical events. They are ones directly exposed to a high temperature environment of Mercury. The remains of the telecommunication system such as a transponder and a power amplifier were selected from the heritage of our past deep space missions. These instruments are placed inside MMO where a milder environment is expected than the outside. The total telecommunication system has been designed so that it should work through the MMO mission lifetime from the launch in 2016 to the end of the mission in 2025 including an extra year of extension. The system has experienced thermal environmental tests and proved its excellent resistivity to predicted environments. We will discuss these technologies incorporated in MMO and her telecommunication system design.

  3. Characterization of Factors Affecting Attainment of Glycemic Control in Asian Americans With Diabetes in a Culturally Specific Program

    PubMed Central

    Le, Hung; Wong, Sophia; Iftikar, Tracy; Keenan, Hillary; King, George L.; Hsu, William C.

    2014-01-01

    Purpose The purpose of this study is to examine the effectiveness of a culturally specific pilot clinic for Asian Americans (AA) in reaching glycemic target and to characterize factors affecting the attainment of glycemic control in comparison with white counterparts. Methods This electronic health record review included all new AA patients with type 2 diabetes (n = 109) in a culturally specific program and a randomly selected sample of new white patients with type 2 diabetes (n = 218) in the adult clinic within the same time period and diabetes center. Results AA and whites had a comparable proportion of patients with A1C ≤7% (32.1%, 34.9%; P = .621) at baseline and after 12 months of care (48.6%, 56.0%; P = .210), with a similar A1C decline (−0.9% ± 1.6%, −0.8% ± 1.7%, P = .710) by 12 months. Factors associated with the lack of success in reaching target in AA but not in whites included older age, lower educational attainment, less likelihood of having health insurance, and a need for more educational visits. The percentage of AA reaching A1C ≤7%, as compared to whites, worsened among those with highest initial A1C when stratified by ascending quartiles (96.7% vs 85.2%, P = .101; 61.9% vs 58.9%, P = .813; 24.0% vs 37.7%, P = .230; 15.2% vs 35.4%, P = .044). Conclusion While a culturally specific diabetes program in a specialty setting achieved a similar glycemic outcome for AA compared with whites, reasons for not reaching glycemic target differed. The findings suggest that the elimination of diabetes disparities requires not only culturally and linguistically specific programs, but must also identify and address the socio-environmental differences unique to each population. PMID:23771841

  4. Sensitive electrochemical aptamer cytosensor for highly specific detection of cancer cells based on the hybrid nanoelectrocatalysts and enzyme for signal amplification.

    PubMed

    Sun, Duanping; Lu, Jing; Zhong, Yuwen; Yu, Yanyan; Wang, Yu; Zhang, Beibei; Chen, Zuanguang

    2016-01-15

    Human cancer is becoming a leading cause of death in the world and the development of a straightforward strategy for early detection of cancer is urgently required. Herein, a sandwich-type electrochemical aptamer cytosensor was developed for detection of human liver hepatocellular carcinoma cells (HepG2) based on the hybrid nanoelectrocatalysts and enzyme for signal amplification. The thiolated TLS11a aptamers were used as a selective bio-recognition element, attached to the gold nanoparticles (AuNPs) modified the glassy carbon electrode (GCE) surface. Meanwhile, the electrochemical nanoprobes were fabricated through the G-quadruplex/hemin/aptamer complexes and horseradish peroxidase (HRP) immobilized on the surfaces of Au@Pd core-shell nanoparticle-modified magnetic Fe3O4/MnO2 beads (Fe3O4/MnO2/Au@Pd). After the target cells were captured, the hybrid nanoprobes were further assembled to form an aptamer-cell-nanoprobes sandwich-like system on the electrode surface. Then, hybrid Fe3O4/MnO2/Au@Pd nanoelectrocatalysts, G-quadruplex/hemin HRP-mimicking DNAzymes and the natural HRP enzyme efficiently catalyzed the oxidation of hydroquinone (HQ) with H2O2, amplifying the electrochemical signals and improving the detection sensitivity. This electrochemical cytosensor delivered a wide detection range of 1×10(2)-1×10(7)cellsmL(-1), high sensitivity with a low detection limit of 15cellsmL(-1), good selectivity and repeatability. Finally, an electrochemical reductive desorption method was performed to break gold-thiol bond and desorb the components on the AuNPs/GCE for regenerating the cytosensor. These results have demonstrated that the electrochemical cytosensor has the potential to be a feasible tool for cost-effective cancer cell detection in early cancer diagnosis.

  5. Crystal Structure of Full-length Mycobacterium tuberculosis H37Rv Glycogen Branching Enzyme; Insights of N-Terminal [beta]-Sandwich in Sustrate Specifity and Enzymatic Activity

    SciTech Connect

    Pal, Kuntal; Kumar, Shiva; Sharma, Shikha; Garg, Saurabh Kumar; Alam, Mohammad Suhail; Xu, H. Eric; Agrawal, Pushpa; Swaminathan, Kunchithapadam

    2010-07-13

    The open reading frame Rv1326c of Mycobacterium tuberculosis (Mtb) H37Rv encodes for an {alpha}-1,4-glucan branching enzyme (MtbGlgB, EC 2.4.1.18, Uniprot entry Q10625). This enzyme belongs to glycoside hydrolase (GH) family 13 and catalyzes the branching of a linear glucose chain during glycogenesis by cleaving a 1 {yields} 4 bond and making a new 1 {yields} 6 bond. Here, we show the crystal structure of full-length MtbGlgB (MtbGlgBWT) at 2.33-{angstrom} resolution. MtbGlgBWT contains four domains: N1 {beta}-sandwich, N2 {beta}-sandwich, a central ({beta}/{alpha}){sub 8} domain that houses the catalytic site, and a C-terminal {beta}-sandwich. We have assayed the amylase activity with amylose and starch as substrates and the glycogen branching activity using amylose as a substrate for MtbGlgBWT and the N1 domain-deleted (the first 108 residues deleted) Mtb{Delta}108GlgB protein. The N1 {beta}-sandwich, which is formed by the first 105 amino acids and superimposes well with the N2 {beta}-sandwich, is shown to have an influence in substrate binding in the amylase assay. Also, we have checked and shown that several GH13 family inhibitors are ineffective against MtbGlgBWT and Mtb{Delta}108GlgB. We propose a two-step reaction mechanism, for the amylase activity (1 {yields} 4 bond breakage) and isomerization (1 {yields} 6 bond formation), which occurs in the same catalytic pocket. The structural and functional properties of MtbGlgB and Mtb{Delta}108GlgB are compared with those of the N-terminal 112-amino acid-deleted Escherichia coli GlgB (EC{Delta}112GlgB).

  6. Mod-5A wind turbine generator program design report. Volume 4: Drawings and specifications, book 3

    NASA Technical Reports Server (NTRS)

    1984-01-01

    The design, development and analysis of the 7.3 MW MOD-5A wind turbine generator is documented. This volume contains the drawings and specifications developed for the final design. This volume is divided into 5 books of which this is the third, containing drawings 47A380074 through 47A380126. A full breakdown parts listing is provided as well as a where used list.

  7. Effectiveness of Culturally Specific Approaches to Substance Abuse Prevention: Findings from CSAP's National Cross-Site Evaluation of High Risk Youth Programs

    ERIC Educational Resources Information Center

    Springer, J. Fred; Sale, Elizabeth; Kasim, Rafa; Winter, William; Sambrano, Soledad; Chipungu, Sandra

    2004-01-01

    This study assesses the degree to which culturally specific interventions enhance substance abuse prevention effectiveness for targeted cultural groups. A large and diverse (African American, Hispanic, American Indian, and Asian) sample of 10,500 youth across 48 programs was obtained. Youth participating in culturally specific programming showed…

  8. Micellar Polymer Encapsulation of Enzymes.

    PubMed

    Besic, Sabina; Minteer, Shelley D

    2017-01-01

    Although enzymes are highly efficient and selective catalysts, there have been problems incorporating them into fuel cells. Early enzyme-based fuel cells contained enzymes in solution rather than immobilized on the electrode surface. One problem utilizing an enzyme in solution is an issue of transport associated with long diffusion lengths between the site of bioelectrocatalysis and the electrode. This issue drastically decreases the theoretical overall power output due to the poor electron conductivity. On the other hand, enzymes immobilized at the electrode surface have eliminated the issue of poor electron conduction due to close proximity of electron transfer between electrode and the biocatalyst. Another problem is inefficient and short term stability of catalytic activity within the enzyme that is suspended in free flowing solution. Enzymes in solutions are only stable for hours to days, whereas immobilized enzymes can be stable for weeks to months and now even years. Over the last decade, there has been substantial research on immobilizing enzymes at electrode surfaces for biofuel cell and sensor applications. The most commonly used techniques are sandwich or wired. Sandwich techniques are powerful and successful for enzyme immobilization; however, the enzymes optimal activity is not retained due to the physical distress applied by the polymer limiting its applications as well as the non-uniform distribution of the enzyme and the diffusion of analyte through the polymer is slowed significantly. Wired techniques have shown to extend the lifetime of an enzyme at the electrode surface; however, this technique is very hard to master due to specific covalent bonding of enzyme and polymer which changes the three-dimensional configuration of enzyme and with that decreases the optimal catalytic activity. This chapter details encapsulation techniques where an enzyme will be immobilized within the pores/pockets of the hydrophobically modified micellar polymers such as

  9. Wheelchair Propulsion Biomechanics in Junior Basketball Players: A Method for the Evaluation of the Efficacy of a Specific Training Program

    PubMed Central

    Bergamini, Elena; Morelli, Francesca; Marchetti, Flavia; Vannozzi, Giuseppe; Polidori, Lorenzo; Paradisi, Francesco; Traballesi, Marco; Cappozzo, Aurelio; Delussu, Anna Sofia

    2015-01-01

    As participation in wheelchair sports increases, the need of quantitative assessment of biomechanical performance indicators and of sports- and population-specific training protocols has become central. The present study focuses on junior wheelchair basketball and aims at (i) proposing a method to identify biomechanical performance indicators of wheelchair propulsion using an instrumented in-field test and (ii) developing a training program specific for the considered population and assessing its efficacy using the proposed method. Twelve athletes (10 M, 2 F, age = 17.1 ± 2.7 years, years of practice = 4.5 ± 1.8) equipped with wheelchair- and wrist-mounted inertial sensors performed a 20-metre sprint test. Biomechanical parameters related to propulsion timing, progression force, and coordination were estimated from the measured accelerations and used in a regression model where the time to complete the test was set as dependent variable. Force- and coordination-related parameters accounted for 80% of the dependent variable variance. Based on these results, a training program was designed and administered for three months to six of the athletes (the others acting as control group). The biomechanical indicators proved to be effective in providing additional information about the wheelchair propulsion technique with respect to the final test outcome and demonstrated the efficacy of the developed program. PMID:26543852

  10. High Performance Computing - Power Application Programming Interface Specification Version 1.4

    SciTech Connect

    Laros III, James H.; DeBonis, David; Grant, Ryan; Kelly, Suzanne M.; Levenhagen, Michael J.; Olivier, Stephen Lecler; Pedretti, Kevin

    2016-10-01

    Measuring and controlling the power and energy consumption of high performance computing systems by various components in the software stack is an active research area [13, 3, 5, 10, 4, 21, 19, 16, 7, 17, 20, 18, 11, 1, 6, 14, 12]. Implementations in lower level software layers are beginning to emerge in some production systems, which is very welcome. To be most effective, a portable interface to measurement and control features would significantly facilitate participation by all levels of the software stack. We present a proposal for a standard power Application Programming Interface (API) that endeavors to cover the entire software space, from generic hardware interfaces to the input from the computer facility manager.

  11. Comparison of pregnancy-specific interventions to a traditional treatment program for cocaine-addicted pregnant women.

    PubMed

    Weisdorf, T; Parran, T V; Graham, A; Snyder, C

    1999-01-01

    Alcohol and drug use in pregnancy is a significant concern. There is a paucity of treatment programs for substance-abusing pregnant women, especially if indigent. Furthermore, treatment retention is compromised when the drug of choice is crack-cocaine. This paper reports the results of a study comparing treatment retention of cocaine-abusing indigent pregnant women before and after incorporating pregnancy-specific interventions. Audits were performed on 603 charts of women enrolled between 1988 and 1994 in either a traditional treatment program (n = 114) or in the Pregnancy Substance Abuse Program (PSAP) (n = 489). Differences in treatment retention were found between the two treatment groups. Drop-out rates from the inpatient component of treatment were significantly lower in the PSAP group than in the control group (11.3% vs. 38.6%, p < .001). There was a higher rate of completion of outpatient treatment in the PSAP compared to the control group (34.4% vs. 13.5%, p < .005). These results were achieved with a 2-day decreased inpatient stay. Treatment retention improved when specialized interventions were provided, at minimal additional cost. These results have implications for other publicly funded treatment programs.

  12. Mod-5A Wind Turbine Generator Program Design Report. Volume 4: Drawings and Specifications, Book 1

    NASA Technical Reports Server (NTRS)

    1984-01-01

    The design, development and analysis of the 7.3 MW MOD-5A wind turbine generator is documented. Volume 4 contains the drawings and specifications that were developed in preparation for building the MOD-5A wind turbine generator. This is the first of five books of volume four. It contains structural design criteria, generator step-up transformer specs, specs for design, fabrication and testing of the system, specs for the ground control enclosure, systems specs, slip ring specs, and control system specs.

  13. Mod-5A wind turbine generator program design report. Volume 4: Drawings and specifications, book 2

    NASA Technical Reports Server (NTRS)

    1984-01-01

    The design, development and analysis of the 7.3 MW MOD-5A wind turbine generator is documented. There are four volumes. This volume contains the drawings and specifications that were developed in preparation for building the MOD-5A wind turbine generator. This is the second book of volume four. Some of the items it contains are specs for the emergency shutdown panel, specs for the simulator software, simulator hardware specs, site operator terminal requirements, control data system requirements, software project management plan, elastomeric teeter bearing requirement specs, specs for the controls electronic cabinet, and specs for bolt pretensioning.

  14. Mod-5A wind turbine generator program design report. Volume 4: Drawings and specifications, book 4

    NASA Technical Reports Server (NTRS)

    1984-01-01

    The design, development and analysis of the 7.3 MW MOD-5A wind turbine generator are documented. There are four volumes. This volume contains the drawings and specifications that were developed in preparation for building the MOD-5A wind turbine generator. This volume contains 5 books of which this is the fourth, providing drawings 47A380128 through 47A387125. In addition to the parts listing and where-used list, the logic design of the controller software and the code listing of the controller software are provided. Also given are the aerodynamic profile coordinates.

  15. Mod-5A wind turbine generator program design report. Volume 4: Drawings and specifications, book 2

    NASA Astrophysics Data System (ADS)

    1984-08-01

    The design, development and analysis of the 7.3 MW MOD-5A wind turbine generator is documented. There are four volumes. This volume contains the drawings and specifications that were developed in preparation for building the MOD-5A wind turbine generator. This is the second book of volume four. Some of the items it contains are specs for the emergency shutdown panel, specs for the simulator software, simulator hardware specs, site operator terminal requirements, control data system requirements, software project management plan, elastomeric teeter bearing requirement specs, specs for the controls electronic cabinet, and specs for bolt pretensioning.

  16. Development of a model of machine hand eye coordination and program specifications for a topological machine vision system

    NASA Technical Reports Server (NTRS)

    1972-01-01

    A unified approach to computer vision and manipulation is developed which is called choreographic vision. In the model, objects to be viewed by a projected robot in the Viking missions to Mars are seen as objects to be manipulated within choreographic contexts controlled by a multimoded remote, supervisory control system on Earth. A new theory of context relations is introduced as a basis for choreographic programming languages. A topological vision model is developed for recognizing objects by shape and contour. This model is integrated with a projected vision system consisting of a multiaperture image dissector TV camera and a ranging laser system. System program specifications integrate eye-hand coordination and topological vision functions and an aerospace multiprocessor implementation is described.

  17. Specific NIST projects in support of the NIJ Concealed Weapon Detection and Imaging Program

    NASA Astrophysics Data System (ADS)

    Paulter, Nicholas G.

    1998-12-01

    The Electricity Division of the National Institute of Standards and Technology is developing revised performance standards for hand-held (HH) and walk-through (WT) metal weapon detectors, test procedures and systems for these detectors, and a detection/imaging system for finding concealed weapons. The revised standards will replace the existing National Institute of Justice (NIJ) standards for HH and WT devices and will include detection performance specifications as well as system specifications (environmental conditions, mechanical strength and safety, response reproducibility and repeatability, quality assurance, test reporting, etc.). These system requirements were obtained from the Law Enforcement and corrections Technology Advisory Council, an advisory council for the NIJ. Reproducible and repeatable test procedures and appropriate measurement systems will be developed for evaluating HH and WT detection performance. A guide to the technology and application of non- eddy-current-based detection/imaging methods (such as acoustic, passive millimeter-wave and microwave, active millimeter-wave and terahertz-wave, x-ray, etc.) Will be developed. The Electricity Division is also researching the development of a high- frequency/high-speed (300 GH to 1 THz) pulse-illuminated, stand- off, video-rate, concealed weapons/contraband imaging system.

  18. Serodiagnosis of sporotrichosis infection in cats by enzyme-linked immunosorbent assay using a specific antigen, SsCBF, and crude exoantigens.

    PubMed

    Fernandes, Geisa Ferreira; Lopes-Bezerra, Leila Maria; Bernardes-Engemann, Andréa Reis; Schubach, Tânia Maria Pacheco; Dias, Maria Adelaide Galvão; Pereira, Sandro Antonio; de Camargo, Zoilo Pires

    2011-01-27

    The main objective of this study is to standardize an ELISA for the diagnosis of feline sporotrichosis. Sporothrix schenckii is the etiological agent of human and animal sporotrichosis. Cats may act as reservoirs for S. schenckii and can transmit the infection to humans by a bite or scratch. There are few methods for the serological diagnosis of fungal diseases in animals. In this paper, an ELISA test for the diagnosis of cat sporotrichosis is proposed, which detects S. schenckii-specific antibodies in feline sera. Two different kinds of antigens were used: "SsCBF", a specific molecule from S. schenckii that consists of a Con A-binding fraction derived from a peptido-rhamnomannan component of the cell wall, and a S. schenckii crude exoantigen preparation. The ELISA was developed, optimized, and evaluated using sera from 30 cats with proven sporotrichosis (by culture isolation); 22 sera from healthy feral cats from a zoonosis center were used as negative controls. SsCBF showed 90% sensitivity and 96% specificity in ELISA; while crude exoantigens demonstrated 96% sensitivity and 98% specificity. The ELISA assay described here would be a valuable screening tool for the detection of specific S. schenckii antibodies in cats with sporotrichosis. The assay is inexpensive, quick to perform, easy to interpret, and permits the diagnosis of feline sporotrichosis.

  19. Acute intermittent porphyria: identification and expression of exonic mutations in the hydroxymethylbilane synthase gene. An initiation codon missense mutation in the housekeeping transcript causes "variant acute intermittent porphyria" with normal expression of the erythroid-specific enzyme.

    PubMed Central

    Chen, C H; Astrin, K H; Lee, G; Anderson, K E; Desnick, R J

    1994-01-01

    Acute intermittent porphyria (AIP), an autosomal dominant inborn error, results from the half-normal activity of the heme biosynthetic enzyme, hydroxymethylbilane synthase (EC 4.3.1.8). Diagnosis of AIP heterozygotes is essential to prevent acute, life-threatening neurologic attacks by avoiding various precipitating factors. Since biochemical diagnosis is problematic, the identification of hydroxymethylbilane synthase mutations has facilitated the detection of AIP heterozygotes. Molecular analyses of unrelated AIP patients revealed six exonic mutations: an initiating methionine to isoleucine substitution (M1I) in a patient with variant AIP, which precluded translation of the housekeeping, but not the erythroid-specific isozyme; four missense mutations in classical AIP patients, V93F, R116W, R201W, C247F; and a nonsense mutation W283X in a classi